Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Ca, H, M, Mk, Po, R, F||IF, IHC, IH(P), WB||M||Purified||Monoclonal Antibody|
|Presentation||Protein A Purified mouse immunoglobulin in 20 mM sodium phosphate, 250 mM NaCl, pH. 7.6, with 0.1% sodium azide as a preservative.|
|Safety Information according to GHS|
|Material Size||50 µg|
References | 165 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Antroquinonol Lowers Brain Amyloid-β Levels and Improves Spatial Learning and Memory in a Transgenic Mouse Model of Alzheimer's Disease. |
Chang, WH; Chen, MC; Cheng, IH
Scientific reports 5 15067 2015
Alzheimer's disease (AD) is the most common form of dementia. The deposition of brain amyloid-β peptides (Aβ), which are cleaved from amyloid precursor protein (APP), is one of the pathological hallmarks of AD. Aβ-induced oxidative stress and neuroinflammation play important roles in the pathogenesis of AD. Antroquinonol, a ubiquinone derivative isolated from Antrodia camphorata, has been shown to reduce oxidative stress and inflammatory cytokines via activating the nuclear transcription factor erythroid-2-related factor 2 (Nrf2) pathway, which is downregulated in AD. Therefore, we examined whether antroquinonol could improve AD-like pathological and behavioral deficits in the APP transgenic mouse model. We found that antroquinonol was able to cross the blood-brain barrier and had no adverse effects via oral intake. Two months of antroquinonol consumption improved learning and memory in the Morris water maze test, reduced hippocampal Aβ levels, and reduced the degree of astrogliosis. These effects may be mediated through the increase of Nrf2 and the decrease of histone deacetylase 2 (HDAC2) levels. These findings suggest that antroquinonol could have beneficial effects on AD-like deficits in APP transgenic mouse.
|Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease. |
Dewji, NN; Singer, SJ; Masliah, E; Rockenstein, E; Kim, M; Harber, M; Horwood, T
PloS one 10 e0122451 2015
β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.
|Brivaracetam, but not ethosuximide, reverses memory impairments in an Alzheimer's disease mouse model. |
Nygaard, HB; Kaufman, AC; Sekine-Konno, T; Huh, LL; Going, H; Feldman, SJ; Kostylev, MA; Strittmatter, SM
Alzheimer's research & therapy 7 25 2015
Recent studies have shown that several strains of transgenic Alzheimer's disease (AD) mice overexpressing the amyloid precursor protein (APP) have cortical hyperexcitability, and their results have suggested that this aberrant network activity may be a mechanism by which amyloid-β (Aβ) causes more widespread neuronal dysfunction. Specific anticonvulsant therapy reverses memory impairments in various transgenic mouse strains, but it is not known whether reduction of epileptiform activity might serve as a surrogate marker of drug efficacy for memory improvement in AD mouse models.Transgenic AD mice (APP/PS1 and 3xTg-AD) were chronically implanted with dural electroencephalography electrodes, and epileptiform activity was correlated with spatial memory function and transgene-specific pathology. The antiepileptic drugs ethosuximide and brivaracetam were tested for their ability to suppress epileptiform activity and to reverse memory impairments and synapse loss in APP/PS1 mice.We report that in two transgenic mouse models of AD (APP/PS1 and 3xTg-AD), the presence of spike-wave discharges (SWDs) correlated with impairments in spatial memory. Both ethosuximide and brivaracetam reduce mouse SWDs, but only brivaracetam reverses memory impairments in APP/PS1 mice.Our data confirm an intriguing therapeutic role of anticonvulsant drugs targeting synaptic vesicle protein 2A across AD mouse models. Chronic ethosuximide dosing did not reverse spatial memory impairments in APP/PS1 mice, despite reduction of SWDs. Our data indicate that SWDs are not a reliable surrogate marker of appropriate target engagement for reversal of memory dysfunction in APP/PS1 mice.
|Characterization of a Novel Mouse Model of Alzheimer's Disease--Amyloid Pathology and Unique β-Amyloid Oligomer Profile. |
Liu, P; Paulson, JB; Forster, CL; Shapiro, SL; Ashe, KH; Zahs, KR
PloS one 10 e0126317 2015
Amyloid plaques composed of β-amyloid (Aβ) protein are a pathological hallmark of Alzheimer's disease. We here report the generation and characterization of a novel transgenic mouse model of Aβ toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APPNLI) linked to familial Alzheimer's disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APPNLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aβ plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology--such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation--was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aβ production at varied ages of mice, and we showed that mice produced primarily fibrillar Aβ assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aβ*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aβ assemblies in situ.
|Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation. |
Kegler, K; Spitzbarth, I; Imbschweiler, I; Wewetzer, K; Baumgärtner, W; Seehusen, F
PloS one 10 e0133916 2015
Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide evidence for regeneration promoting cells recruitable for therapeutic purposes. There is evidence that neurotrophin receptor p75 (p75NTR)-expressing cells emerge in the lesioned CNS. However, the phenotype and identity of these cells, and signals triggering their in situ generation under normal conditions and certain pathological situations has remained enigmatic. In the present study, we used a spontaneous, idiopathic and inflammatory CNS condition in dogs with prominent lympho-histiocytic infiltration as a model to study the phenotype of Schwann cells and their relation to Schwann cell remyelination within the CNS. Furthermore, the phenotype of p75NTR-expressing cells within the injured CNS was compared to their counter-part in control sciatic nerve and after peripheral nerve injury. In addition, organotypic slice cultures were used to further elucidate the origin of p75NTR-positive cells. In cerebral and cerebellar white and grey matter lesions as well as in the brain stem, p75NTR-positive cells co-expressed the transcription factor Sox2, but not GAP-43, GFAP, Egr2/Krox20, periaxin and PDGFR-α. Interestingly, and contrary to the findings in control sciatic nerves, p75NTR-expressing cells only co-localized with Sox2 in degenerative neuropathy, thus suggesting that such cells might represent dedifferentiated Schwann cells both in the injured CNS and PNS. Moreover, effective Schwann cell remyelination represented by periaxin- and P0-positive mature myelinating Schwann cells, was strikingly associated with the presence of p75NTR/Sox2-expressing Schwann cells. Intriguingly, the emergence of dedifferentiated Schwann cells was not affected by astrocytes, and a macrophage-dominated inflammatory response provided an adequate environment for Schwann cells plasticity within the injured CNS. Furthermore, axonal damage was reduced in brain stem areas with p75NTR/Sox2-positive cells. This study provides novel insights into the involvement of Schwann cells in CNS remyelination under natural occurring CNS inflammation. Targeting p75NTR/Sox2-expressing Schwann cells to enhance their differentiation into competent remyelinating cells appears to be a promising therapeutic approach for inflammatory/demyelinating CNS diseases.
|Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence. |
Morancho, B; Martínez-Barriocanal, Á; Villanueva, J; Arribas, J
Breast cancer research : BCR 17 106 2015
Cellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogene-induced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown.We analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatase-tagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescence-associated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells.Using breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo.Considering these findings, we conclude that ectodomain shedding is tightly regulated in oncogene-induced senescent cells by integrating transcription of the shedding substrates with limiting ADAM17 activity. The remaining activity of ADAM17 contributes to the non-cell autonomous protumorigenic effects of p95HER2-induced senescent cells. Because ADAM17 is druggable, these results represent an approximation to the pharmacological regulation of the senescence secretome.
|Alzheimer's Disease-Related Protein Expression in the Retina of Octodon degus. |
Du, LY; Chang, LY; Ardiles, AO; Tapia-Rojas, C; Araya, J; Inestrosa, NC; Palacios, AG; Acosta, ML
PloS one 10 e0135499 2015
New studies show that the retina also undergoes pathological changes during the development of Alzheimer's disease (AD). While transgenic mouse models used in these previous studies have offered insight into this phenomenon, they do not model human sporadic AD, which is the most common form. Recently, the Octodon degus has been established as a sporadic model of AD. Degus display age-related cognitive impairment associated with Aβ aggregates and phosphorylated tau in the brain. Our aim for this study was to examine the expression of AD-related proteins in young, adult and old degus retina using enzyme-linked or fluorescence immunohistochemistry and to quantify the expression using slot blot and western blot assays. Aβ4G8 and Aβ6E10 detected Aβ peptides in some of the young animals but the expression was higher in the adults. Aβ peptides were observed in the inner and outer segment of the photoreceptors, the nerve fiber layer (NFL) and ganglion cell layer (GCL). Expression was higher in the central retinal region than in the retinal periphery. Using an anti-oligomer antibody we detected Aβ oligomer expression in the young, adult and old retina. Immunohistochemical labeling showed small discrete labeling of oligomers in the GCL that did not resemble plaques. Congo red staining did not result in green birefringence in any of the animals analyzed except for one old (84 months) animal. We also investigated expression of tau and phosphorylated tau. Expression was seen at all ages studied and in adults it was more consistently observed in the NFL-GCL. Hyperphosphorylated tau detected with AT8 antibody was significantly higher in the adult retina and it was localized to the GCL. We confirm for the first time that Aβ peptides and phosphorylated tau are expressed in the retina of degus. This is consistent with the proposal that AD biomarkers are present in the eye.
|Localization of reelin signaling pathway components in murine midbrain and striatum. |
Sharaf, A; Rahhal, B; Spittau, B; Roussa, E
Cell and tissue research 359 393-407 2015
We investigated the distribution patterns of the extracellular matrix protein Reelin and of crucial Reelin signaling components in murine midbrain and striatum. The cellular distribution of the Reelin receptors VLDLr and ApoER2, the intracellular downstream mediator Dab1, and the alternative Reelin receptor APP were analyzed at embryonic day 16, at postnatal stage 15 (P15), and in 3-month-old mice. Reelin was expressed intracellularly and extracellularly in midbrain mesencephalic dopaminergic (mDA) neurons of newborns. In the striatum, Calbindin D-28k(+) neurons exhibited Reelin intracellularly at E16 and extracellularly at P15 and 3 months. ApoER2 and VLDLr were expressed in mDA neurons at E16 and P15 and in oligodendrocytes at 3 months, whereas Dab1 and APP immunoreactivity was observed in mDA at all stages analyzed. In the striatum, Calbindin D-28k(+)/GAD67(+) inhibitory neurons expressed VLDLr, ApoER2, and Dab1 at P15, but only Dab1 at E16 and 3 months. APP was always expressed in mouse striatum in which it colocalized with Calbindin D-28k. Our data underline the importance of Reelin signalling during embryonic development and early postnatal maturation of the mesostriatal and mesocorticolimbic system, and suggest that the striatum and not the midbrain is the primary source of Reelin for midbrain neurons. The loss of ApoER2 and VLDLr expression in the mature midbrain and striatum implies that Reelin functions are restricted to migratory events and early postnatal maturation and are dispensable for the maintenance of dopaminergic neurons.
|The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1). |
von Einem, B; Wahler, A; Schips, T; Serrano-Pozo, A; Proepper, C; Boeckers, TM; Rueck, A; Wirth, T; Hyman, BT; Danzer, KM; Thal, DR; von Arnim, CA
PloS one 10 e0129047 2015
Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer's disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.
|Experimental microembolism induces localized neuritic pathology in guinea pig cerebrum. |
Li, JM; Cai, Y; Liu, F; Yang, L; Hu, X; Patrylo, PR; Cai, H; Luo, XG; Xiao, D; Yan, XX
Oncotarget 6 10772-85 2015
Microbleeds are a common finding in aged human brains. In Alzheimer's disease (AD), neuritic plaques composed of β-amyloid (Aβ) deposits and dystrophic neurites occur frequently around cerebral vasculature, raising a compelling question as to whether, and if so, how, microvascular abnormality and amyloid/neuritic pathology might be causally related. Here we used a guinea pig model of cerebral microembolism to explore a potential inductive effect of vascular injury on neuritic and amyloid pathogenesis. Brains were examined 7-30 days after experimental microvascular embolization occupying ~0.5% of total cortical area. Compared to sham-operated controls, glial fibrillary acidic protein immunoreactivity was increased in the embolized cerebrum, evidently around intracortical vasculature. Swollen/sprouting neurites exhibiting increased reactivity of nicotinamide adenine dinucleotide phosphate diaphorase, parvalbumin, vesicular glutamate transporter 1 and choline acetyltransferase appeared locally in the embolized brains in proximity to intracortical vasculature. The embolization-induced swollen/sprouting neurites were also robustly immunoreactive for β-amyloid precursor protein and β-secretase-1, the substrate and initiating enzyme for Aβ genesis. These experimental data suggest that microvascular injury can induce multisystem neuritic pathology associated with an enhanced amyloidogenic potential in wild-type mammalian brain.
|PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons. |
Dilsizoglu Senol, A; Tagliafierro, L; Huguet, L; Gorisse-Hussonnois, L; Chasseigneaux, S; Allinquant, B
BMC neuroscience 16 10 2015
The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate.We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling.These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.
|Two cases of multinodular and vacuolating neuronal tumour. |
Bodi, I; Curran, O; Selway, R; Elwes, R; Burrone, J; Laxton, R; Al-Sarraj, S; Honavar, M
Acta neuropathologica communications 2 7 2014
An unusual multinodular and vacuolating neuronal tumour (MVNT) has been described in the cerebral hemispheres of ten patients with adult-onset seizures. We report the findings in two cases with similar features, a surgical resection and the other an autopsy specimen.Case 1, a 34-year-old female, underwent surgical resection for a multinodular non-enhancing frontal white matter lesion causing intractable epilepsy. Case 2, presented with motor neurone disease (MND) at the age of 71 and MRI scanning revealed extensive multinodular non-enhancing white matter lesions in the temporal lobe. There was no history of epilepsy and post mortem histology confirmed MND.Macroscopically multiple small grey well-formed, discrete and coalescent nodules were seen in the deep cortex and subcortical white matter. On histology, mature-looking neurons with large cytoplasmic vacuoles were distributed in a fibrillary background, where vacuoles were also noted. In the resected tumour scattered oligodendroglia-like cells were present. No ganglion cells were seen. The vacuolated cells exhibited immunopositivity for synaptophysin, HuC/HuD and p62 but were negative for NeuN, neurofilament, GFAP, IDH1, nestin and CD34. Electron microscopy showed non-membrane bound cytoplasmic vacuoles in the neurons and in some neuronal processes. The seizures recurred in Case 1.Some clinicopathological features of this lesion suggest a possible relationship with dysembryoplastic neuroepithelial tumour (DNT) although the morphological features are not typical of DNT. Case 2 demonstrates that MVNT may remain asymptomatic.
|Lipolysaccharide-Induced Neuroinflammation Is Associated with Alzheimer-Like Amyloidogenic Axonal Pathology and Dendritic Degeneration in Rats. |
Deng, X; Li, M; Ai, W; He, L; Lu, D; Patrylo, PR; Cai, H; Luo, X; Li, Z; Yan, X
Advances in Alzheimer's disease 3 78-93 2014
Chronic neuroinflammation is thought to play an etiological role in Alzheimer's disease (AD), which is characterized pathologically by amyloid and tau formation, as well as neuritic dystrophy and synaptic degeneration. The causal relationship between these pathological events is a topic of ongoing research and discussion. Recent data from transgenic AD models point to a tight spatiotemporal link between neuritic and amyloid pathology, with the obligatory enzyme for β-amyloid (Aβ) production, namely β-secretase-1 (BACE1), is overexpressed in axon terminals undergoing dystrophic change. However, the axonal pathology inherent with BACE1 elevation seen in transgenic AD mice may be secondary to increased soluble Aβ in these genetically modified animals. Here we explored the occurrence of the AD-like axonal and dendritic pathology in adult rat brain affected by LPS-induced chronic neuroinflammation. Unilateral intracerebral LPS injection induced prominent inflammatory response in glial cells in the ipsilateral cortex and hippocampal formation. BACE1 protein levels were elevated the ipsilateral hippocampal lysates in the LPS treated animals relative to controls. BACE1 immunoreactive dystrophic axons appeared in the LPS-treated ipsilateral cortex and hippocampal formation, colocalizing with increased β-amyloid precursor protein and Aβ antibody (4G8) immunolabeling. Quantitative Golgi studies revealed reduction of dendritic branching points and spine density on cortical layer III and hippocampal CA3 pyramidal neurons in the LPS-treated ipsilateral cerebrum. These findings suggest that Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration occur in wildtype mammalian brain in partnership with neuroinflammation following LPS injection.
|Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models. |
Schuh, C; Wimmer, I; Hametner, S; Haider, L; Van Dam, AM; Liblau, RS; Smith, KJ; Probert, L; Binder, CJ; Bauer, J; Bradl, M; Mahad, D; Lassmann, H
Acta neuropathologica 128 247-66 2014
Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.
|mNos2 deletion and human NOS2 replacement in Alzheimer disease models. |
Colton, CA; Wilson, JG; Everhart, A; Wilcock, DM; Puoliväli, J; Heikkinen, T; Oksman, J; Jääskeläinen, O; Lehtimäki, K; Laitinen, T; Vartiainen, N; Vitek, MP
Journal of neuropathology and experimental neurology 73 752-69 2014
Understanding the pathophysiologic mechanisms underlying Alzheimer disease relies on knowledge of disease onset and the sequence of development of brain pathologies. We present a comprehensive analysis of early and progressive changes in a mouse model that demonstrates a full spectrum of characteristic Alzheimer disease-like pathologies. This model demonstrates an altered immune redox state reminiscent of the human disease and capitalizes on data indicating critical differences between human and mouse immune responses, particularly in nitric oxide levels produced by immune activation of the NOS2 gene. Using the APPSwDI(+)/(+)mNos2(-/-) (CVN-AD) mouse strain, we show a sequence of pathologic events leading to neurodegeneration,which include pathologically hyperphosphorylated tau in the perforant pathway at 6 weeks of age progressing to insoluble tau, early appearance of β-amyloid peptides in perivascular deposits around blood vessels in brain regions known to be vulnerable to Alzheimer disease, and progression to damage and overt loss in select vulnerable neuronal populations in these regions. The role of species differences between hNOS2 and mNos2 was supported by generating mice in which the human NOS2 gene replaced mNos2. When crossed with CVN-AD mice, pathologic characteristics of this new strain (APPSwDI(+)/(-)/HuNOS2(tg+)/(+)/mNos2(-/-)) mimicked the pathologic phenotypes found in the CVN-AD strain.
|Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research. |
Ray, B; Chopra, N; Long, JM; Lahiri, DK
Molecular brain 7 63 2014
Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications.This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons.We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.
|Multiple sclerosis deep grey matter: the relation between demyelination, neurodegeneration, inflammation and iron. |
Haider, L; Simeonidou, C; Steinberger, G; Hametner, S; Grigoriadis, N; Deretzi, G; Kovacs, GG; Kutzelnigg, A; Lassmann, H; Frischer, JM
Journal of neurology, neurosurgery, and psychiatry 85 1386-95 2014
In multiple sclerosis (MS), diffuse degenerative processes in the deep grey matter have been associated with clinical disabilities. We performed a systematic study in MS deep grey matter with a focus on the incidence and topographical distribution of lesions in relation to white matter and cortex in a total sample of 75 MS autopsy patients and 12 controls. In addition, detailed analyses of inflammation, acute axonal injury, iron deposition and oxidative stress were performed. MS deep grey matter was affected by two different processes: the formation of focal demyelinating lesions and diffuse neurodegeneration. Deep grey matter demyelination was most prominent in the caudate nucleus and hypothalamus and could already be seen in early MS stages. Lesions developed on the background of inflammation. Deep grey matter inflammation was intermediate between low inflammatory cortical lesions and active white matter lesions. Demyelination and neurodegeneration were associated with oxidative injury. Iron was stored primarily within oligodendrocytes and myelin fibres and released upon demyelination. In addition to focal demyelinated plaques, the MS deep grey matter also showed diffuse and global neurodegeneration. This was reflected by a global reduction of neuronal density, the presence of acutely injured axons, and the accumulation of oxidised phospholipids and DNA in neurons, oligodendrocytes and axons. Neurodegeneration was associated with T cell infiltration, expression of inducible nitric oxide synthase in microglia and profound accumulation of iron. Thus, both focal lesions as well as diffuse neurodegeneration in the deep grey matter appeared to contribute to the neurological disabilities of MS patients.
|Mint proteins are required for synaptic activity-dependent amyloid precursor protein (APP) trafficking and amyloid β generation. |
Sullivan, SE; Dillon, GM; Sullivan, JM; Ho, A
The Journal of biological chemistry 289 15374-83 2014
Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production.
|Role of recurrent hypoxia-ischemia in preterm white matter injury severity. |
Hagen, MW; Riddle, A; McClendon, E; Gong, X; Shaver, D; Srivastava, T; Dean, JM; Bai, JZ; Fowke, TM; Gunn, AJ; Jones, DF; Sherman, LS; Grafe, MR; Hohimer, AR; Back, SA
PloS one 9 e112800 2014
Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI.We employed a preterm fetal sheep model of in utero moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI.rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing.rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.
|The problem of axonal injury in the brains of veterans with histories of blast exposure. |
Ryu, J; Horkayne-Szakaly, I; Xu, L; Pletnikova, O; Leri, F; Eberhart, C; Troncoso, JC; Koliatsos, VE
Acta neuropathologica communications 2 153 2014
Blast injury to brain, a hundred-year old problem with poorly characterized neuropathology, has resurfaced as health concern in recent deployments in Iraq and Afghanistan. To characterize the neuropathology of blast injury, we examined the brains of veterans for the presence of amyloid precursor protein (APP)-positive axonal swellings typical of diffuse axonal injury (DAI) and compared them to healthy controls as well as controls with opiate overdose, anoxic-ischemic encephalopathy, and non-blast TBI (falls and motor vehicle crashes).In cases with blast history, we found APP (+) axonal abnormalities in several brain sites, especially the medial dorsal frontal white matter. In white matter, these abnormalities were featured primarily by clusters of axonal spheroids or varicosities in a honeycomb pattern with perivascular distribution. Axonal abnormalities colocalized with IBA1 (+) reactive microglia and had an appearance that was distinct from classical DAI encountered in TBI due to motor vehicle crashes. Opiate overdose cases also showed APP (+) axonal abnormalities, but the intensity of these lesions was lower compared to cases with blast histories and there was no clear association of such lesions with microglial activation.Our findings demonstrate that many cases with history of blast exposure are featured by APP (+) axonopathy that may be related to blast exposure, but an important role for opiate overdose, antemortem anoxia, and concurrent blunt TBI events in war theater or elsewhere cannot be discounted.
|HIV-1 Tat interacts with and regulates the localization and processing of amyloid precursor protein. |
Kim, J; Yoon, JH; Kim, YS
PloS one 8 e77972 2013
HIV-1 Tat protein plays various roles in virus proliferation and in the regulation of numerous host cell functions. Accumulating evidence suggests that HIV-1 Tat also plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting intracellular communication. Amyloid beta (Aβ) is generated from amyloid precursor protein (APP) and accumulates in the senile plaques of Alzheimer's disease patients. This study demonstrates that Tat interacts with APP both in vitro and in vivo, and increases the level of Aβ42 by recruiting APP into lipid rafts. Co-localization of Tat with APP in the cytosol was observed in U-87 MG cells that expressed high levels of Tat, and redistribution of APP into lipid rafts, a site of increased β- and γ-secretase activity, was demonstrated by discontinuous sucrose density gradient ultracentrifugation in the presence of Tat. Furthermore, Tat enhanced the cleavage of APP by β-secretase in vitro, resulting in 5.5-fold higher levels of Aβ42. This was consistent with increased levels of β-C-terminal fragment (β-CTF) and reduced levels of α-CTF. Moreover, stereotaxic injection of a lentiviral Tat expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased Tat-mediated production and processing of Aβ in vivo. Increased levels of Aβ42, as well as an increase in the number and size of Aβ plaques, were observed in the hippocampus following injection of Tat virus compared with mock virus. These results suggest that HIV-1 Tat may contribute to HAND by interacting with and modifying APP processing, thereby increasing Aβ production.
|Reelin immunoreactivity in neuritic varicosities in the human hippocampal formation of non-demented subjects and Alzheimer's disease patients. |
Notter, T; Knuesel, I
Acta neuropathologica communications 1 27 2013
Reelin and its downstream signaling members are important modulators of actin and microtubule cytoskeleton dynamics, a fundamental prerequisite for proper neurodevelopment and adult neuronal functions. Reductions in Reelin levels have been suggested to contribute to Alzheimer's disease (AD) pathophysiology. We have previously reported an age-related reduction in Reelin levels and its accumulation in neuritic varicosities along the olfactory-limbic tracts, which correlated with cognitive impairments in aged mice. Here, we aimed to investigate whether a similar Reelin-associated neuropathology is observed in the aged human hippocampus and whether it correlated with dementia status.Our immunohistochemical stainings revealed the presence of N- and C-terminus-containing Reelin fragments in corpora amylacea (CAm), aging-associated spherical deposits. The density of these deposits was increased in the molecular layer of the subiculum of AD compared to non-demented individuals. Despite the limitation of a small sample size, our evaluation of several neuronal and glial markers indicates that the presence of Reelin in CAm might be related to aging-associated impairments in neuronal transport leading to accumulation of organelles and protein metabolites in neuritic varicosities, as previously suggested by the findings and discussions in rodents and primates.Our results indicate that aging- and disease-associated changes in Reelin levels and proteolytic processing might play a role in the formation of CAm by altering cytoskeletal dynamics. However, its presence may also be an indicator of a degenerative state of neuritic compartments.
|Endogenously regulated Dab2 worsens inflammatory injury in experimental autoimmune encephalomyelitis. |
Jokubaitis, VG; Gresle, MM; Kemper, DA; Doherty, W; Perreau, VM; Cipriani, TL; Jonas, A; Shaw, G; Kuhlmann, T; Kilpatrick, TJ; Butzkueven, H
Acta neuropathologica communications 1 32 2013
Neuroinflammation regulates both disease pathogenesis and repair in multiple sclerosis. In early multiple sclerosis lesion development, neuroinflammation causes demyelination and axonal injury, the likely final common determinant of disability. Here we report the identification of a novel neuroinflammatory mediator, Disabled-2 (Dab2). Dab2 is an intracellular adaptor protein with previously unknown function in the central nervous system.We report that Dab2 is up-regulated in lesional macrophages/microglia in the spinal cord in murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. We demonstrate that dab2 expression is positively correlated with experimental autoimmune encephalomyelitis disease severity during the acute disease phase. Furthermore, dab2-deficient mice have a less severe experimental autoimmune encephalomyelitis disease course and suffer less neuroinflammation and less axonal injury than their wild-type littermates. We demonstrate that dab2 expression is strongly associated with the expression of inducible nitric oxide synthase. We further demonstrate that Dab2 is expressed at the protein level by macrophages in early acute human multiple sclerosis lesions and that this correlates with axonal injury.Together, these results suggest that endogenous Dab2 exacerbates central nervous system inflammation, potentially acting to up-regulate reactive oxygen species expression in macrophages and microglia, and that it is of potential pathogenic relevance in Multiple Sclerosis.
|The response of cerebral cortex to haemorrhagic damage: experimental evidence from a penetrating injury model. |
Purushothuman, S; Marotte, L; Stowe, S; Johnstone, DM; Stone, J
PloS one 8 e59740 2013
Understanding the response of the brain to haemorrhagic damage is important in haemorrhagic stroke and increasingly in the understanding the cerebral degeneration and dementia that follow head trauma and head-impact sports. In addition, there is growing evidence that haemorrhage from small cerebral vessels is important in the pathogenesis of age-related dementia (Alzheimer's disease). In a penetration injury model of rat cerebral cortex, we have examined the neuropathology induced by a needlestick injury, with emphasis on features prominent in the ageing and dementing human brain, particularly plaque-like depositions and the expression of related proteins. Needlestick lesions were made in neo- and hippocampal cortex in Sprague Dawley rats aged 3-5 months. Brains were examined after 1-30 d survival, for haemorrhage, for the expression of hyperphosphorylated tau, Aβ, amyloid precursor protein (APP), for gliosis and for neuronal death. Temporal cortex from humans diagnosed with Alzheimer's disease was examined with the same techniques. Needlestick injury induced long-lasting changes-haem deposition, cell death, plaque-like deposits and glial invasion-along the needle track. Around the track, the lesion induced more transient changes, particularly upregulation of Aβ, APP and hyperphosporylated tau in neurons and astrocytes. Reactions were similar in hippocampus and neocortex, except that neuronal death was more widespread in the hippocampus. In summary, experimental haemorrhagic injury to rat cerebral cortex induced both permanent and transient changes. The more permanent changes reproduced features of human senile plaques, including the formation of extracellular deposits in which haem and Aβ-related proteins co-localised, neuronal loss and gliosis. The transient changes, observed in tissue around the direct lesion, included the upregulation of Aβ, APP and hyperphosphorylated tau, not associated with cell death. The findings support the possibility that haemorrhagic damage to the brain can lead to plaque-like pathology.
|A lipoprotein receptor cluster IV mutant preferentially binds amyloid-β and regulates its clearance from the mouse brain. |
Sagare, AP; Bell, RD; Srivastava, A; Sengillo, JD; Singh, I; Nishida, Y; Chow, N; Zlokovic, BV
The Journal of biological chemistry 288 15154-66 2013
Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ~70% of amyloid β-peptide (Aβ) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aβ peripheral binding and higher levels of free Aβ in plasma. Experimental studies have shown that free circulating Aβ re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aβ from entering the brain. Treatment of Alzheimer APPsw(+/0) mice with WT-LRPIV has been shown to reduce brain Aβ pathology. In addition to Aβ, LRPIV binds multiple ligands. To enhance LRPIV binding for Aβ relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aβ40 and Aβ42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3-1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aβ40 and Aβ42 25-27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw(+/0) mice with LRPIV-D3674G (40 μg/kg/day) reduced Aβ40 and Αβ42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60-80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aβ clearance therapy.
|The amyloid precursor protein (APP) triplicated gene impairs neuronal precursor differentiation and neurite development through two different domains in the Ts65Dn mouse model for Down syndrome. |
Trazzi, S; Fuchs, C; Valli, E; Perini, G; Bartesaghi, R; Ciani, E
The Journal of biological chemistry 288 20817-29 2013
Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS.
|MicroRNA-195 protects against dementia induced by chronic brain hypoperfusion via its anti-amyloidogenic effect in rats. |
Ai, J; Sun, LH; Che, H; Zhang, R; Zhang, TZ; Wu, WC; Su, XL; Chen, X; Yang, G; Li, K; Wang, N; Ban, T; Bao, YN; Guo, F; Niu, HF; Zhu, YL; Zhu, XY; Zhao, SG; Yang, BF
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 3989-4001 2013
Previous studies have demonstrated that chronic brain hypoperfusion (CBH) causes Aβ aggregation by upregulating expression of amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1) protein, which is accompanied by cognitive impairment, but the mechanisms are not fully understood. In this study, we evaluated the effect of microRNA on memory impairment in rats induced by CBH. We show here that CBH generated by bilateral common carotid artery occlusion (2VO) significantly decreased the learning and memory ability in rats, as assessed by Morris water maze, and upregulated expression of APP and BACE1 proteins in the hippocampus and cortex of rats, as evaluated by Western blot and immunofluorescence. In reciprocal, qRT-PCR analysis showed that microRNA-195 (miR-195) was downregulated in both the hippocampus and cortex of rats following CBH, and in the plasma of dementia patients. APP and BACE1 proteins were downregulated by miR-195 overexpression, upregulated by miR-195 inhibition, and unchanged by binding-site mutation or miR-masks, indicating that APP and BACE1 are two potential targets for miR-195. Knockdown of endogenous miR-195 by lentiviral vector-mediated overexpression of its antisense molecule (lenti-pre-AMO-miR-195) elicited dementia in rats, whereas overexpression of miR-195 using lenti-pre-miR-195 reduced dementia vulnerability triggered by 2VO. Additionally, chromatin immunoprecipitation analysis showed that NFκB was bound to the promoter region of miR-195 and inhibited its expression. We conclude that miR-195 may play a key role in determining dementia susceptibility in 2VO rats by regulating APP and BACE1 expression at the post-transcriptional level, and exogenous complement of miR-195 may be a potentially valuable anti-dementia approach.
|Amyloid precursor proteins interact with the heterotrimeric G protein Go in the control of neuronal migration. |
Ramaker, JM; Swanson, TL; Copenhaver, PF
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 10165-81 2013
Amyloid precursor protein (APP) belongs to a family of evolutionarily conserved transmembrane glycoproteins that has been proposed to regulate multiple aspects of cell motility in the nervous system. Although APP is best known as the source of β-amyloid fragments (Aβ) that accumulate in Alzheimer's disease, perturbations affecting normal APP signaling events may also contribute to disease progression. Previous in vitro studies showed that interactions between APP and the heterotrimeric G protein Goα-regulated Goα activity and Go-dependent apoptotic responses, independent of Aβ. However, evidence for authentic APP-Go interactions within the healthy nervous system has been lacking. To address this issue, we have used a combination of in vitro and in vivo strategies to show that endogenously expressed APP family proteins colocalize with Goα in both insect and mammalian nervous systems, including human brain. Using biochemical, pharmacological, and Bimolecular Fluorescence Complementation assays, we have shown that insect APP (APPL) directly interacts with Goα in cell culture and at synaptic terminals within the insect brain, and that this interaction is regulated by Goα activity. We have also adapted a well characterized assay of neuronal migration in the hawkmoth Manduca to show that perturbations affecting APPL and Goα signaling induce the same unique pattern of ectopic, inappropriate growth and migration, analogous to defective migration patterns seen in mice lacking all APP family proteins. These results support the model that APP and its orthologs regulate conserved aspects of neuronal migration and outgrowth in the nervous system by functioning as unconventional Goα-coupled receptors.
|Soy-based diet exacerbates seizures in mouse models of neurological disease. |
Westmark, CJ; Westmark, PR; Malter, JS
Journal of Alzheimer's disease : JAD 33 797-805 2013
Seizures are a common phenotype in many neurological disorders including Alzheimer's disease, Down syndrome, and fragile X syndrome. Mouse models of these disorders overexpress amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) and are highly susceptible to audiogenic-induced seizures (AGS). We observed decreased AGS in these mice fed a casein-based, purified diet (D07030301) as opposed to a standard soy protein-containing, non-purified diet (Purina 5015). Our objective in this manuscript was to determine if soy protein, and in particular soy isoflavones, in the Purina 5015 were contributing to the seizure phenotype. Wild running, AGS, and death rates were assessed in juvenile mice fed Purina 5015, D07030301, D07030301 containing soy protein, or D07030301 supplemented with individual isoflavones (750 mg/kg daidzein or genistein). A short treatment (3 days) with Purina 5015 induced wild running and AGS in Alzheimer's disease mice. A 3-day treatment with daidzein-supplemented diet, but not genistein, induced wild running in wild type mice. To understand the mechanism underlying daidzein activity, we assessed dendritic AβPP expression in primary, cultured, wild type neurons treated with daidzein or genistein. In vitro, daidzein significantly increased dendritic AβPP. Thus, the soy isoflavone daidzein recapitulated seizure induction in vivo and altered AβPP expression in vitro. These results have important implications for individuals on soy-based diets as well as for rodent model research.
|Recruitment of the Mint3 adaptor is necessary for export of the amyloid precursor protein (APP) from the Golgi complex. |
Caster, AH; Kahn, RA
The Journal of biological chemistry 288 28567-80 2013
The amyloid precursor protein (APP) is a ubiquitously expressed single-pass transmembrane protein that undergoes proteolytic processing by secretases to generate the pathogenic amyloid-β peptide, the major component in Alzheimer plaques. The traffic of APP through the cell determines its exposure to secretases and consequently the cleavages that generate the pathogenic or nonpathogenic peptide fragments. Despite the likely importance of APP traffic to Alzheimer disease, we still lack clear models for the routing and regulation of APP in cells. Like the traffic of most transmembrane proteins, the binding of adaptors to its cytoplasmic tail, which is 47 residues long and contains at least four distinct sorting motifs, regulates that of APP. We tested each of these for effects on the traffic of APP from the Golgi by mutating key residues within them and examining adaptor recruitment at the Golgi and traffic to post-Golgi site(s). We demonstrate strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi, a critical role for Tyr-682 (within the YENPTY motif) in Mint3 recruitment and export of APP from the Golgi, and we identify LAMP1(+) structures as the proximal destination of APP after leaving the Golgi. Together, these data provide a detailed view of the first sorting step in its route to the cell surface and processing by secretases and further highlight the critical role played by Mint3.
|Disease-specific molecular events in cortical multiple sclerosis lesions. |
Fischer, MT; Wimmer, I; Höftberger, R; Gerlach, S; Haider, L; Zrzavy, T; Hametner, S; Mahad, D; Binder, CJ; Krumbholz, M; Bauer, J; Bradl, M; Lassmann, H
Brain : a journal of neurology 136 1799-815 2013
Cortical lesions constitute an important part of multiple sclerosis pathology. Although inflammation appears to play a role in their formation, the mechanisms leading to demyelination and neurodegeneration are poorly understood. We aimed to identify some of these mechanisms by combining gene expression studies with neuropathological analysis. In our study, we showed that the combination of inflammation, plaque-like primary demyelination and neurodegeneration in the cortex is specific for multiple sclerosis and is not seen in other chronic inflammatory diseases mediated by CD8-positive T cells (Rasmussen's encephalitis), B cells (B cell lymphoma) or complex chronic inflammation (tuberculous meningitis, luetic meningitis or chronic purulent meningitis). In addition, we performed genome-wide microarray analysis comparing micro-dissected active cortical multiple sclerosis lesions with those of tuberculous meningitis (inflammatory control), Alzheimer's disease (neurodegenerative control) and with cortices of age-matched controls. More than 80% of the identified multiple sclerosis-specific genes were related to T cell-mediated inflammation, microglia activation, oxidative injury, DNA damage and repair, remyelination and regenerative processes. Finally, we confirmed by immunohistochemistry that oxidative damage in cortical multiple sclerosis lesions is associated with oligodendrocyte and neuronal injury, the latter also affecting axons and dendrites. Our study provides new insights into the complex mechanisms of neurodegeneration and regeneration in the cortex of patients with multiple sclerosis.
|Interleukin-10 expression during the acute phase is a putative prerequisite for delayed viral elimination in a murine model for multiple sclerosis. |
Vanessa Herder,Ingo Gerhauser,Stephanie Kristin Klein,Pedro Almeida,Maren Kummerfeld,Reiner Ulrich,Frauke Seehusen,Karl Rohn,Dirk Schaudien,Wolfgang Baumgärtner,Jochen Huehn,Andreas Beineke,Wolfgang Baumg
Journal of neuroimmunology 249 2012
Reduced protective immunity leads to viral persistence and demyelination in Theiler's murine encephalomyelitis. The aim of the present study was to compare the phenotype of brain-infiltrating leukocytes and cytokine expression in susceptible SJL and resistant C57BL/6 mice during Theilervirus-induced acute polioencephalitis. In contrast to C57/BL6 mice, SJL mice show an increased number of Foxp3(+) regulatory T cells and CD45R(+) B cells associated with delayed viral elimination and elevated IL-10 mRNA transcripts in the brain. Results substantiate the hypothesis that an imbalanced cytokine milieu during the early infection phase contributes to ineffective antiviral immunity in animals with a susceptible genetic background.
|Intracellular amyloid precursor protein sorting and amyloid-β secretion are regulated by Src-mediated phosphorylation of Mint2. |
Chaufty, J; Sullivan, SE; Ho, A
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 9613-25 2012
Mint adaptor proteins bind to the membrane-bound amyloid precursor protein (APP) and affect the production of pathogenic amyloid-β (Aβ) peptides related to Alzheimer's disease (AD). Previous studies have shown that loss of each of the three Mint proteins delays the age-dependent production of amyloid plaques in transgenic mouse models of AD. However, the cellular and molecular mechanisms underlying Mints effect on amyloid production are unclear. Because Aβ generation involves the internalization of membrane-bound APP via endosomes and Mints bind directly to the endocytic motif of APP, we proposed that Mints are involved in APP intracellular trafficking, which in turn, affects Aβ generation. Here, we show that APP endocytosis was attenuated in Mint knock-out neurons, revealing a role for Mints in APP trafficking. We also show that the endocytic APP sorting processes are regulated by Src-mediated phosphorylation of Mint2 and that internalized APP is differentially sorted between autophagic and recycling trafficking pathways. A Mint2 phosphomimetic mutant favored endocytosis of APP along the autophagic sorting pathway leading to increased intracellular Aβ accumulation. Conversely, the Mint2 phospho-resistant mutant increased APP localization to the recycling pathway and back to the cell surface thereby enhancing Aβ42 secretion. These results demonstrate that Src-mediated phosphorylation of Mint2 regulates the APP endocytic sorting pathway, providing a mechanism for regulating Aβ secretion.
|Inhibition of amyloid-beta peptide aggregation rescues the autophagic deficits in the TgCRND8 mouse model of Alzheimer disease. |
Lai, Aaron Y and McLaurin, Joanne
Biochim. Biophys. Acta, 1822: 1629-37 (2012) 2012
scyllo-Inositol (SI) is an endogenous inositol stereoisomer known to inhibit aggregation and fibril formation of the amyloid-beta peptide (Aβ). Human clinical trials using SI to treat Alzheimer disease (AD) patients have shown potential benefits. In light of the growing therapeutic potential of SI, the objective of our study was to gain a more thorough understanding of the mechanism of action. In addition to Aβ plaques, a prominent pathological feature of AD is the extensive accumulation of autophagic vacuoles (AVs) suggesting dysfunction in this degradation pathway. Using the TgCRND8 mouse model for AD, we examined SI treatment effects on various components of the autophagic pathway. Autophagy impairment in TgCRND8 mice occurs in the latter stages of the pathway where AV-lysosome fusion and lysosomal degradation take place. SI treatment attenuated this impairment with a decrease in the size and the number of accumulated AVs. We propose that the beneficial effects of SI-Aβ interactions may resolve autophagic deficiencies in the AD brains.
|Bepridil decreases Aβ and calcium levels in the thalamus after middle cerebral artery occlusion in rats. |
Timo Saraj,Anu Lipsanen,Petra M,Sirpa Per,Hilkka Soininen,Annakaisa Haapasalo,Jukka Jolkkonen,Mikko Hiltunen
Journal of cellular and molecular medicine 16 2012
Alzheimer's disease (AD) and cerebral ischaemia share similar features in terms of altered amyloid precursor protein (APP) processing and β-amyloid (Aβ) accumulation. We have previously shown that Aβ and calcium deposition, and β-secretase activity, are robustly increased in the ipsilateral thalamus after transient middle cerebral artery occlusion (MCAO) in rats. Here, we investigated whether the non-selective calcium channel blocker bepridil, which also inhibits β-secretase cleavage of APP, affects thalamic accumulation of Aβ and calcium and in turn influences functional recovery in rats subjected to MCAO. A 27-day bepridil treatment (50
|Memory deficits of British dementia knock-in mice are prevented by Aβ-precursor protein haploinsufficiency. |
Tamayev, R; D'Adamio, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 5481-5 2012
Familial British Dementia (FBD) is caused by an autosomal dominant mutation in the BRI2/ITM2B gene (Vidal et al., 1999). FBD(KI) mice are a model of FBD that is genetically congruous to the human disease, because they carry one mutant and one wild-type Bri2/Itm2b allele. Analysis of these mice has shown that the British mutation causes memory impairments due to loss of Bri2 function (Tamayev et al., 2010b). BRI2 is a physiologic inhibitor of processing of the Aβ-precursor protein (APP; Matsuda et al., 2008), a gene associated with Alzheimer's disease (Bertram et al., 2010). Here we show that APP haploinsufficiency prevents memory dysfunctions seen in FBD(KI) mice. This genetic suppression is consistent with a role for APP in the pathogenesis of memory deficits. Moreover, it provides compelling evidence that the memory dysfunctions caused by the British BRI2 mutant are dependent on endogenous APP and that BRI2 and APP functionally interact. This evidence establishes a mechanistic connection between Familial British and Alzheimer's dementias.
|Immunoreactivity of the amino-terminal portion of the amyloid-beta precursor protein in the nucleolus. |
Neuroscience letters 521 2012
The functioning and metabolic pathway of the amyloid β-precursor protein (APP) have not been fully elucidated. To fill this research gap, this study immunocytochemically investigated the intracellular localization of APP in the neuroblastoma cell line SK-N-SH and in normal primary cells. Using antibodies against the amino-terminal portion of the APP molecule, immunoreactivity was detected not only in the cytoplasm but also in the nucleus and nucleolus. Further analysis revealed the co-localization of amino acids 44-63 of the APP molecule with fibrillarin, a nucleolus marker. These findings indicate that a fraction of APP, including its amino-terminal portion, may be localized in the nucleus as well as in the nucleolus, suggesting an important role of APP in RNA metabolism and other intra-nucleolus functions.
|New highly sensitive rodent and human tests for soluble amyloid precursor protein alpha quantification: preclinical and clinical applications in Alzheimer's disease. |
Christiane Rose,Katell Peoc'h,Stéphanie Chasseigneaux,Claire Paquet,Julien Dumurgier,Fanchon Bourasset,Frédéric Calon,Jean-Louis Laplanche,Jacques Hugon,Bernadette Allinquant
BMC neuroscience 13 2012
|TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death. |
Chang, JY; Chiang, MF; Lin, SR; Lee, MH; He, H; Chou, PY; Chen, SJ; Chen, YA; Yang, LY; Lai, FJ; Hsieh, CC; Hsieh, TH; Sheu, HM; Sze, CI; Chang, NS
Cell death & disease 3 e302 2012
Self-aggregation of transforming growth factor β (TGF-β)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid β (Aβ) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aβ in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aβ aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aβ. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aβ formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.
|Periventricular demyelination and axonal pathology is associated with subependymal virus spread in a murine model for multiple sclerosis. |
Maren Kummerfeld,Frauke Seehusen,Stephanie Klein,Reiner Ulrich,Robert Kreutzer,Ingo Gerhauser,Vanessa Herder,Wolfgang Baumgärtner,Andreas Beineke
Intervirology 55 2012
Objectives: Theiler's murine encephalomyelitis virus (TMEV) infection of mice is a widely used animal model for demyelinating disorders, such as multiple sclerosis (MS). The aim of the present study was to identify topographical differences of TMEV spread and demyelination in the brain of experimentally infected susceptible SJL/J mice and resistant C57BL/6 mice. Methods: Demyelination was confirmed by Luxol fast blue and cresyl violet staining and axonal damage by neurofilament-specific and β-amyloid precursor protein-specific immunohistochemistry. Viral dissemination within the central nervous system (CNS) was quantified by immunohistochemistry and in situ hybridization. Further, the phenotype of infected cells was determined by confocal laser scanning microscopy. Results: An early transient infection of periventricular cells followed by demyelination and axonopathies around the fourth ventricle in SJL/J mice was noticed. Periventricular and brain stem demyelination was associated with a predominant infection of microglia/macrophages and oligodendrocytes. Conclusions: Summarized, the demonstration of ependymal infection and subjacent spread into the brain parenchyma as well as regional virus clearance despite ongoing demyelination and axonal damage in other CNS compartments allows new insights into TME pathogenesis. This novel aspect of TMEV CNS interaction will enhance the understanding of region-specific susceptibilities to injury and regenerative capacities of the brain in this MS model.
|Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons. |
Gui-Zhi Yang,Miao Yang,Yoon Lim,Jian-Jun Lu,Ting-Hua Wang,Jian-Guo Qi,Jin-Hua Zhong,Xin-Fu Zhou
Journal of neurochemistry 122 2012
J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.
|Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice. |
Krstic, D; Madhusudan, A; Doehner, J; Vogel, P; Notter, T; Imhof, C; Manalastas, A; Hilfiker, M; Pfister, S; Schwerdel, C; Riether, C; Meyer, U; Knuesel, I
Journal of neuroinflammation 9 151 2012
Alzheimer's disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain's innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease.The viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging.We found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aβ peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD.Chronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aβ plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD.
|Western Blotting, Immunohistochemistry||Mouse||22747753|
|Amyloid precursor protein is a biomarker for transformed human pluripotent stem cells. |
Venkataramani, Vivek, et al.
Am. J. Pathol., 180: 1636-52 (2012) 2012
Increasing evidence suggests an important function of the β-amyloid precursor protein (APP) in malignant disease in humans; however, the biological basis for this evidence is not well understood at present. To understand the role of APP in transformed pluripotent stem cells, we studied its expression levels in human testicular germ cell tumors using patient tissues, model cell lines, and an established xenograft mouse model. In the present study, we demonstrate the cooperative expression of APP with prominent pluripotency-related genes such as Sox2, NANOG, and POU5F1 (Oct3/4). The closest homologue family member, APLP2, showed no correlation to these stem cell factors. In addition, treatment with histone deacetylase (HDAC) inhibitors suppressed the levels of APP and stem cell markers. Loss of pluripotency, either spontaneously or as a consequence of treatment with an HDAC inhibitor, was accompanied by decreased APP protein levels both in vitro and in vivo. These observations suggest that APP represents a novel and specific biomarker in human transformed pluripotent stem cells that can be selectively modulated by HDAC inhibitors.
|Central nervous system rather than immune cell-derived BDNF mediates axonal protective effects early in autoimmune demyelination. |
Lee, DH; Geyer, E; Flach, AC; Jung, K; Gold, R; Flügel, A; Linker, RA; Lühder, F
Acta neuropathologica 123 247-58 2012
Brain-derived neurotrophic factor (BDNF) is involved in neuronal and glial development and survival. While neurons and astrocytes are its main cellular source in the central nervous system (CNS), bioactive BDNF is also expressed in immune cells and in lesions of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Previous data revealed that BDNF exerts neuroprotective effects in myelin oligodendrocyte glycoprotein-induced EAE. Using a conditional knock-out model with inducible deletion of BDNF, we here show that clinical symptoms and structural damage are increased when BDNF is absent during the initiation phase of clinical EAE. In contrast, deletion of BDNF later in the disease course of EAE did not result in significant changes, either in the disease course or in axonal integrity. Bone marrow chimeras revealed that the deletion of BDNF in the CNS alone, with no deletion of BDNF in the infiltrating immune cells, was sufficient for the observed effects. Finally, the therapeutic effect of glatiramer acetate, a well-characterized disease-modifying drug with the potential to modulate BDNF expression, was partially reversed in mice in which BDNF was deleted shortly before the onset of disease. In summary, our data argue for an early window of therapeutic opportunity where modulation of BDNF may exert neuroprotective effects in experimental autoimmune demyelination.
|BACE1 elevation is involved in amyloid plaque development in the triple transgenic model of Alzheimer's disease: differential Aβ antibody labeling of early-onset axon terminal pathology. |
Cai, Y; Zhang, XM; Macklin, LN; Cai, H; Luo, XG; Oddo, S; Laferla, FM; Struble, RG; Rose, GM; Patrylo, PR; Yan, XX
Neurotoxicity research 21 160-74 2012
β-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer's disease (FAD). Some FAD-based mouse models produce amyloid plaques, others do not. β-Amyloid (Aβ) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aβ molecules aggregate in different patterns. Is there a basic cellular process governing Aβ plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of β-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aβ antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aβ antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aβ domain and the C-terminal of APP, but not co-labeled by antibodies against the Aβ C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aβ antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments.
|Progressive neuronal inclusion formation and axonal degeneration in CHMP2B mutant transgenic mice. |
Ghazi-Noori, S; Froud, KE; Mizielinska, S; Powell, C; Smidak, M; Fernandez de Marco, M; O'Malley, C; Farmer, M; Parkinson, N; Fisher, EM; Asante, EA; Brandner, S; Collinge, J; Isaacs, AM
Brain : a journal of neurology 135 819-32 2012
Mutations in the charged multivesicular body protein 2B (CHMP2B) gene cause frontotemporal lobar degeneration. The mutations lead to C-terminal truncation of the CHMP2B protein. We generated Chmp2b knockout mice and transgenic mice expressing either wild-type or C-terminally truncated mutant CHMP2B. The transgenic CHMP2B mutant mice have decreased survival and show progressive neurodegenerative changes including gliosis and increasing accumulation of p62- and ubiquitin-positive inclusions. The inclusions are negative for the TAR DNA binding protein 43 and fused in sarcoma proteins, mimicking the inclusions observed in patients with CHMP2B mutation. Mice transgenic for mutant CHMP2B also develop an early and progressive axonopathy characterized by numerous amyloid precursor protein-positive axonal swellings, implicating altered axonal function in disease pathogenesis. These findings were not observed in Chmp2b knockout mice or in transgenic mice expressing wild-type CHMP2B, indicating that CHMP2B mutations induce degenerative changes through a gain of function mechanism. These data describe the first mouse model of dementia caused by CHMP2B mutation and provide new insights into the mechanisms of CHMP2B-induced neurodegeneration.
|Mitochondrial dysfunction and accumulation of the β-secretase-cleaved C-terminal fragment of APP in Alzheimer's disease transgenic mice. |
Devi, L; Ohno, M
Neurobiology of disease 45 417-24 2012
Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-β (Aβ) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the β-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the β-site APP-cleaving enzyme 1 (BACE1(+/-)) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1(+/-)·5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1(+/-) deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1(+/-) deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.
|Arrested preoligodendrocyte maturation contributes to myelination failure in premature infants. |
Buser, JR; Maire, J; Riddle, A; Gong, X; Nguyen, T; Nelson, K; Luo, NL; Ren, J; Struve, J; Sherman, LS; Miller, SP; Chau, V; Hendson, G; Ballabh, P; Grafe, MR; Back, SA
Annals of neurology 71 93-109 2012
The major form of magnetic resonance imaging-defined white matter injury (WMI) comprises diffuse lesions where the burden of small necrotic foci (microscopic necrosis) is poorly defined. We hypothesized that myelination failure associated with diffuse WMI involves an aberrant injury response linked to arrested preoligodendrocyte (preOL) maturation in reactive astrocyte-rich lesions.A retrospective autopsy series (1983-2000) was selected for cases with diffuse WMI and analyzed relative to prospectively collected contemporary cases (2003-2010). Controls were age- and region-matched to address regional variation in preOL maturation. Successive oligodendrocyte stages were analyzed with lineage-specific markers. Microscopic necrosis was quantified with microglial markers. Axon injury markers defined the burden of axonopathy. Extracellular matrix remodeling was defined by detection of hyaluronic acid (HA), an inhibitor of preOL maturation, and the HA receptor, CD44.In the contemporary case series, diffuse WMI was accompanied by a significant reduction in the burden of microscopic necrosis and axonopathy. Diffuse astrogliosis extended into the lesion surround with elevated HA and astrocyte-expressed CD44. The total population of OL lineage stages was significantly increased in lesions. This increase coincided with significant expansion of the preOL pool.Although these data confirm that microscopic necrosis occurs in contemporary cases, the markedly decreased burden supports that it does not contribute substantially to myelination failure. The primary mechanism of myelination failure involves a disrupted cellular response whereby preOLs fail to differentiate in diffuse astrogliotic lesions. PreOL maturation arrest converts chronic WMI to a more immature state related to the burden of astrogliosis.
|Acute blast injury reduces brain abeta in two rodent species. |
De Gasperi, R; Gama Sosa, MA; Kim, SH; Steele, JW; Shaughness, MC; Maudlin-Jeronimo, E; Hall, AA; Dekosky, ST; McCarron, RM; Nambiar, MP; Gandy, S; Ahlers, ST; Elder, GA
Frontiers in neurology 3 177 2012
Blast-induced traumatic brain injury (TBI) has been a major cause of morbidity and mortality in the conflicts in Iraq and Afghanistan. How the primary blast wave affects the brain is not well understood. In particular, it is unclear whether blast injures the brain through mechanisms similar to those found in non-blast closed impact injuries (nbTBI). The β-amyloid (Aβ) peptide associated with the development of Alzheimer's disease is elevated acutely following TBI in humans as well as in experimental animal models of nbTBI. We examined levels of brain Aβ following experimental blast injury using enzyme-linked immunosorbent assays for Aβ 40 and 42. In both rat and mouse models of blast injury, rather than being increased, endogenous rodent brain Aβ levels were decreased acutely following injury. Levels of the amyloid precursor protein (APP) were increased following blast exposure although there was no evidence of axonal pathology based on APP immunohistochemical staining. Unlike the findings in nbTBI animal models, levels of the β-secretase, β-site APP cleaving enzyme 1, and the γ-secretase component presenilin-1 were unchanged following blast exposure. These studies have implications for understanding the nature of blast injury to the brain. They also suggest that strategies aimed at lowering Aβ production may not be effective for treating acute blast injury to the brain.
|Amyloid-beta (Aβ) D7H mutation increases oligomeric Aβ42 and alters properties of Aβ-zinc/copper assemblies. |
Chen, WT; Hong, CJ; Lin, YT; Chang, WH; Huang, HT; Liao, JY; Chang, YJ; Hsieh, YF; Cheng, CY; Liu, HC; Chen, YR; Cheng, IH
PloS one 7 e35807 2012
Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (Aβ(D7H)), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn(2+) or Cu(2+), Aβ(D7H) aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn(2+) and Cu(2+) in the etiology of AD.
|Angiotensin II-inhibition: effect on Alzheimer's pathology in the aged triple transgenic mouse. |
Ferrington, L; Palmer, LE; Love, S; Horsburgh, KJ; Kelly, PA; Kehoe, PG
American journal of translational research 4 151-64 2012
Reducing excessive accumulation of amyloid-β (Aβ) in Alzheimer's disease (AD) is a key objective of most AD therapies, and inhibition of angiotensin-converting enzyme (ACE) may delay onset or progression of AD. The effects of an ACE-inhibitor (ACE-I) and an angiotensin II receptor blocker (ARB) on Aβ and tau pathology in a triple transgenic (3xTGAD) mouse model of AD were investigated. 9-10month 3xTGAD mice were treated with ARB, ACE-I or vehicle for 6 months. Mean arterial blood pressure (MABP) was measured periodically and mice were assessed behaviourally. Aβ, phospho-tau, amyloid precursor protein (APP) and ACE activity were analysed. MABP was significantly reduced at 2 weeks and 3 months in the ACE-I group and at 3 months in the ARB group, compared to vehicle. Neither drug altered performance of 3xTGAD mice in Morris Water Maze or T-maze, nor were Aβ, tau immunolabelling or APP levels altered. ACE-I significantly reduced ACE activity in kidney. Prolonged treatment with ACE-I or ARB does not affect Aβ or phospho-tau accumulation in brains of aged 3xTGAD mice.
|Induction of amyloid-β(1-42) in the retina and optic nerve head of chronic ocular hypertensive monkeys. |
Ito, Y; Shimazawa, M; Tsuruma, K; Mayama, C; Ishii, K; Onoe, H; Aihara, M; Araie, M; Hara, H
Molecular vision 18 2647-57 2012
Recent studies have indicated that accumulation of amyloid β(1-42) (Aβ(1-42)), which is associated with the progression of Alzheimer disease, may also be responsible for retinal ganglion cell death in glaucoma. The purpose of this study was to investigate the expression and localization of Aβ(1-42) in the retina and the optic nerve head (ONH) of monkeys with experimental glaucoma.Five cynomolgus monkeys with a glaucomatous left eye at 4, 9, 11, 15, and 24 weeks after laser photocoagulation treatment were studied by immunohistochemical methods. Another two cynomolgus monkeys with a glaucomatous left eye at 133 weeks after laser photocoagulation treatment were used to measure Aβ(1-42) concentrations in the retina by enzyme-linked immunosorbent assay.At 11 to 24 weeks after the laser photocoagulation treatment, Aβ(1-42) was upregulated in the nerve fiber layer (NFL) and the ganglion cell layer (GCL) of the retina and the ONH, but the expression of amyloid precursor protein decreased in the NFL and ONH from levels at 9 weeks. The localizations of Aβ(1-42) were merged in glial fibrillary acidic protein-positive astroglial cells but not phosphorylated neurofilament heavy- or nonphosphorylated neurofilament heavy-positive axons in the retina and the ONH. Likewise, Aβ(1-42) concentrations in the retina of monkeys increased in the chronic stage of glaucoma.These findings indicate that the upregulation of Aβ(1-42) after an intraocular pressure elevation could apply to monkeys since the structure of the ONH is more similar to humans than that of rodents.
|Dynamin 1 regulates amyloid generation through modulation of BACE-1. |
Zhu, L; Su, M; Lucast, L; Liu, L; Netzer, WJ; Gandy, SE; Cai, D
PloS one 7 e45033 2012
Several lines of investigation support the notion that endocytosis is crucial for Alzheimer's disease (AD) pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP) traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1) distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1) in Aβ biogenesis.We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF). Moreover, dyn1 knockdown (KD) leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α-secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT) or PS1 N-terminal fragment (NTF) at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1(-/-) primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts.In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its enzymatic activities.
|Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport. |
Steuble, M; Diep, TM; Schätzle, P; Ludwig, A; Tagaya, M; Kunz, B; Sonderegger, P
Biology open 1 761-74 2012
Endocytosis of amyloid-β precursor protein (APP) is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.
|Somatostatin receptor subtype-4 agonist NNC 26-9100 decreases extracellular and intracellular Aβ₁₋₄₂ trimers. |
Sandoval, KE; Farr, SA; Banks, WA; Crider, AM; Morley, JE; Witt, KA
European journal of pharmacology 683 116-24 2012
Soluble amyloid β-protein (Aβ) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimer's disease. Such Aβ oligomers exist dependent on their rates of aggregation and metabolism. Use of selective somatostatin receptor-subtype agonists have been identified as a potential means to mitigate Aβ accumulation in the brain, via regulation of the enzyme neprilysin. Herein, we first evaluated the impact of the somatostatin receptor subtype-4 agonist 1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(1H-imidazol-4-yl)propyl]thiourea (NNC 26-9100) on learning and memory in 12-month SAMP8 mice (i.c.v. injection). NNC 26-9100 (0.2 μg-dose) was shown to enhance both learning (T-maze) and memory (object recognition) compared to vehicle controls. Cortical and hippocampal tissues were evaluated subsequent to NNC 26-9100 (0.2 μg) or vehicle administration for changes in neprilysin activity, along with protein expression of amyloid-precursor protein (APP), neprilysin, and Aβ₁₋₄₂ oligomers within respective cellular fractions (extracellular, intracellular and membrane). NNC 26-9100 increased neprilysin activity in cortical tissue, with an associated protein expression increase in the extracellular fraction and decreased in the intracellular fraction. A decrease in intracellular APP expression was found with treatment in both cortical and hippocampal tissues. NNC 26-9100 also significantly decreased expression of Aβ₁₋₄₂ trimers within both the extracellular and intracellular cortical fractions. No expression changes were found in membrane fractions for any protein. These finding suggest the potential use of selective SSTR4 agonists to mitigate toxic oligomeric forms of Aβ₁₋₄₂ in critical regions of the brain identified with learning and memory decline.
|Hyperglycaemia and apoptosis of microglial cells in human septic shock. |
Polito, A; Brouland, JP; Porcher, R; Sonneville, R; Siami, S; Stevens, RD; Guidoux, C; Maxime, V; de la Grandmaison, GL; Chrétien, FC; Gray, F; Annane, D; Sharshar, T
Critical care (London, England) 15 R131 2011
The effect of hyperglycaemia on the brain cells of septic shock patients is unknown. The objective of this study was to evaluate the relationship between hyperglycaemia and apoptosis in the brains of septic shock patients.In a prospective study of 17 patients who died from septic shock, hippocampal tissue was assessed for neuronal ischaemia, neuronal and microglial apoptosis, neuronal Glucose Transporter (GLUT) 4, endothelial inducible Nitric Oxide Synthase (iNOS), microglial GLUT5 expression, microglial and astrocyte activation. Blood glucose (BG) was recorded five times a day from ICU admission to death. Hyperglycaemia was defined as a BG 200 mg/dL g/l and the area under the BG curve (AUBGC) greater than 2 g/l was assessed.Median BG over ICU stay was 2.2 g/l. Neuronal apoptosis was correlated with endothelial iNOS expression (rho = 0.68, P = 0.04), while microglial apoptosis was associated with AUBGC greater than 2 g/l (rho = 0.70; P = 0.002). Neuronal and microglial apoptosis correlated with each other (rho = 0.69, P = 0.006), but neither correlated with the duration of septic shock, nor with GLUT4 and 5 expression. Neuronal apoptosis and ischaemia tended to correlate with duration of hypotension.In patients with septic shock, neuronal apoptosis is rather associated with iNOS expression and microglial apoptosis with hyperglycaemia, possibly because GLUT5 is not downregulated. These data provide a mechanistic basis for understanding the neuroprotective effects of glycemic control.
|Retromer disruption promotes amyloidogenic APP processing. |
Sullivan, CP; Jay, AG; Stack, EC; Pakaluk, M; Wadlinger, E; Fine, RE; Wells, JM; Morin, PJ
Neurobiology of disease 43 338-45 2011
Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aβ is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aβ42:Aβ40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aβ40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aβ. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.
|Axon-glial interaction in the CNS: what we have learned from mouse models of Pelizaeus-Merzbacher disease. |
Gruenenfelder FI, Thomson G, Penderis J, Edgar JM
Journal of anatomy 219 33-43. doi 2011
In the central nervous system (CNS) the majority of axons are surrounded by a myelin sheath, which is produced by oligodendrocytes. Myelin is a lipid-rich insulating material that facilitates the rapid conduction of electrical impulses along the myelinated nerve fibre. Proteolipid protein and its isoform DM20 constitute the most abundant protein component of CNS myelin. Mutations in the PLP1 gene encoding these myelin proteins cause Pelizaeus-Merzbacher disease and the related allelic disorder, spastic paraplegia type 2. Animal models of these diseases, particularly models lacking or overexpressing Plp1, have shed light on the interplay between axons and oligodendrocytes, and how one component influences the other.© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society of Great Britain and Ireland.
|Retrograde study of projections from the tuberomammillary nucleus to the mesopontine cholinergic complex in the rat. |
Hong EY, Lee HS
Brain Res 1383 169-78. Epub 2011 Jan 31. 2011
The mesopontine cholinergic complex comprising the pedunculopontine (PPTg) and laterodorsal (LDTg) tegmental nuclei is a key component of ascending reticular activating system. The brainstem cholinergic nuclei are yet under the control of descending projections from hypothalamic arousal centers such as the hypocretinergic, lateral hypothalamus (LH), and the histaminergic, tuberomammillary nucleus (TMN). The present study was designed to determine the differential projection pattern as well as the laterality of descending TMN projections to the PPTg and LDTg. Our results showed that each TMN subdivision provided differential projections to the mesopontine complex. The majority of PPTg-projecting neurons were located in ventrolateral TMN mainly at its subpial border, whereas LDTg-projecting cells were in dorsomedial TMN with a few in the medial border of the ventrolateral subdivision. For both PPTg and LDTg cases, retrogradely labeled neurons were more pronounced in each TMN subdivision ipsilateral to the injection site. The light microscopic observation also indicated that hypocretinergic, terminal-like boutons formed close appositions to somata as well as proximal dendrites of PPTg- or LDTg-projecting TMN neurons. Taken together, the present observations suggested that each TMN subdivision provide differential projections to the mesopontine cholinergic complex and that the LH, via the TMN, provides indirect, descending projections to the complex as well.Copyright © 2011 Elsevier B.V. All rights reserved.
|Angiotensin II-inhibiting drugs have no effect on intraneuronal Aβ or oligomeric Aβ levels in a triple transgenic mouse model of Alzheimer's disease. |
Ferrington, L; Miners, JS; Palmer, LE; Bond, SM; Povey, JE; Kelly, PA; Love, S; Horsburgh, KJ; Kehoe, PG
American journal of translational research 3 197-208 2011
Reducing the excessive accumulation of amyloid β-protein (Aβ) in Alzheimer's disease (AD) is a key objective of most AD therapies. Several studies suggest that pharmacological inhibition of angiotensin-converting enzyme (ACE) or its by-product angiotensin II may delay onset or progression of dementia and it has been suggested that this occurs via regulation of Aβ. Intraneuronal oligomeric accumulation of Aβ is postulated to be one of the earliest pathological events. Thus this study investigated the effect of an ACE-inhibitor, captopril, and two angiotensin II receptor blockers (ARBs), eprosartan and valsartan, on intraneuronal Aβ pathology and oligomeric Aβ levels in a triple transgenic (3xTGAD) mouse model of AD.Male, adult (3-4 month old) 3xTgAD mice (n=39) were randomly assigned to 4 treatment groups: valsartan (0.17g/l), eprosartan (0.8g/l), captopril (5g/l) or normal drinking water and the drugs given ad libitum for 2 months. Mean arterial blood pressure (MABP) was measured at baseline, at 2 weeks and at 2 months when the mice were sacrificed and the brains hemisected for analysis. One hemisphere was processed for Aβ and amyloid precursor protein (APP) immunohistochemistry and the other for biochemical measurement of oligomeric Aβ and APP. ACE activity was measured in the brain and kidney.MABP was significantly reduced at 2 weeks and 2 months in the ACE-I group (p=0.0006) but was unaltered in the ARB groups compared to vehicle. Neither ACE-I nor ARB treatment altered Aβ and APP immunolabelling or the level of Aβ or APP in brain tissue homogenates. Similarly neither ACE-I nor ARB treatment altered ACE activity in either brain or kidney compared to control tissue.ACE-I or ARB administration over 2 months did not affect APP levels or either intraneuronal Aβ or oligomeric Aβ levels in 3xTGAD mice. While ARBs did not alter MABP, captopril did mediate reductions in MABP in the 3xTGAD mice which appeared to be independent of ACE activity. Further studies are needed to examine the effects of these drugs over a longer term and in older mice (i.e. when AD-like changes are more pronounced).Full Text Article
|BACE1 activity is modulated by cell-associated sphingosine-1-phosphate. |
Takasugi, N; Sasaki, T; Suzuki, K; Osawa, S; Isshiki, H; Hori, Y; Shimada, N; Higo, T; Yokoshima, S; Fukuyama, T; Lee, VM; Trojanowski, JQ; Tomita, T; Iwatsubo, T
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 6850-7 2011
Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to generate sphingosine-1-phosphate (S1P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of β-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for amyloid-β peptide (Aβ) production, is modulated by S1P in mouse neurons. Treatment by SphK inhibitor, RNA interference knockdown of SphK, or overexpression of S1P degrading enzymes decreased BACE1 activity, which reduced Aβ production. S1P specifically bound to full-length BACE1 and increased its proteolytic activity, suggesting that cellular S1P directly modulates BACE1 activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimer's disease. The unique modulatory effect of cellular S1P on BACE1 activity is a novel potential therapeutic target for Alzheimer's disease.
|In vitro Pb exposure disturbs the balance between Aβ production and elimination: the role of AβPP and neprilysin. |
Huang, H; Bihaqi, SW; Cui, L; Zawia, NH
Neurotoxicology 32 300-6 2011
Metabolism of β-amyloid peptide (Aβ) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Our previous studies on aging primates and rodents have revealed that early life lead exposure increases the expression of the β-amyloid precursor protein (AβPP), elevates Aβ levels, and promotes neurodegeneration in old age. These effects were attributed to de novo synthetic pathways; however, the impact on Aβ degradation was not explored. Neprilysin (NEP), a rate-limiting catabolic peptidase is involved in Aβ metabolism in vivo. In the present study we sought to investigate whether accumulation of Aβ induced by Pb exposure is partially due to its ability to subdue NEP expression and consequently NEP activity. SH-SY5Y cells were exposed to Pb concentrations of 0, 5, 10, 20, and 50 μM for 48 h and AβPP, NEP protein and mRNA levels were measured. Additionally, NEP enzymatic activity and Aβ levels were also assessed. Western blot and RT-PCR analysis indicated significant increases in the protein and mRNA expression of AβPP, which appeared to be concentration and time-dependent, while the protein and mRNA expression of NEP as well as NEP activity declined. These actions of Pb were specific and were not observed when substituted by another metal. These results suggest that Pb causes both the overexpression of AβPP and repression of NEP resulting in the buildup of Aβ.
|Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits. |
Yang, DS; Stavrides, P; Mohan, PS; Kaushik, S; Kumar, A; Ohno, M; Schmidt, SD; Wesson, D; Bandyopadhyay, U; Jiang, Y; Pawlik, M; Peterhoff, CM; Yang, AJ; Wilson, DA; St George-Hyslop, P; Westaway, D; Mathews, PM; Levy, E; Cuervo, AM; Nixon, RA
Brain : a journal of neurology 134 258-77 2011
Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.Full Text Article
|Cysteine 27 variant of the delta-opioid receptor affects amyloid precursor protein processing through altered endocytic trafficking. |
Sarajärvi, T; Tuusa, JT; Haapasalo, A; Lackman, JJ; Sormunen, R; Helisalmi, S; Roehr, JT; Parrado, AR; Mäkinen, P; Bertram, L; Soininen, H; Tanzi, RE; Petäjä-Repo, UE; Hiltunen, M
Molecular and cellular biology 31 2326-40 2011
Agonist-induced activation of the δ-opioid receptor (δOR) was recently shown to augment β- and γ-secretase activities, which increased the production of β-amyloid peptide (Aβ), known to accumulate in the brain tissues of Alzheimer's disease (AD) patients. Previously, the δOR variant with a phenylalanine at position 27 (δOR-Phe27) exhibited more efficient receptor maturation and higher stability at the cell surface than did the less common cysteine (δOR-Cys27) variant. For this study, we expressed these variants in human SH-SY5Y and HEK293 cells expressing exogenous or endogenous amyloid precursor protein (APP) and assessed the effects on APP processing. Expression of δOR-Cys27, but not δOR-Phe27, resulted in a robust accumulation of the APP C83 C-terminal fragment and the APP intracellular domain, while the total soluble APP and, particularly, the β-amyloid 40 levels were decreased. These changes upon δOR-Cys27 expression coincided with decreased localization of APP C-terminal fragments in late endosomes and lysosomes. Importantly, a long-term treatment with a subset of δOR-specific ligands or a c-Src tyrosine kinase inhibitor suppressed the δOR-Cys27-induced APP phenotype. These data suggest that an increased constitutive internalization and/or concurrent signaling of the δOR-Cys27 variant affects APP processing through altered endocytic trafficking of APP.
|Biochemical studies in Normal Pressure Hydrocephalus (NPH) patients: change in CSF levels of amyloid precursor protein (APP), amyloid-beta (Aβ) peptide and phospho-tau. |
Ray, Balmiki, et al.
J Psychiatr Res, 45: 539-47 (2011) 2011
Normal Pressure Hydrocephalus (NPH) is one of the causes of dementia of the elderly characterized by impaired mental function, gait difficulties and urinary incontinence. Previously, it was proposed that some of the NPH patients may develop Alzheimer's disease (AD) like pathology. Aim of this study was to compare levels of different CSF biomarkers, including total secreted β-amyloid precursor protein (sAPP), sAPP-alpha form (sAPPα), amyloid-beta (Aβ) peptide, total-tau protein and hyperphosphorylated-tau protein in subjects from NPH and Non-NPH Control (NNC). CSF was collected from 23 NPH patients and 13 Non-NPH controls by lumber puncture. Western blot analysis was performed to measure levels of sAPP-total. ELISA was used separately to determine levels of sAPPα, Aβ peptide, total-tau and phospho-tau proteins. We found a significant decrease in levels of total secreted APP, sAPPα and Aβ (1-42) in the CSF sample of NPH patients vs. NNC. We did not observe any change in levels of total-tau or phospho-tau in NPH vs. NNC subjects. Notably, phospho-tau level was significantly increased in the NPH patients, who were suffering from the disease for more than one year, vs. NNC. Among five biomarkers studied, decreased sAPP, sAPPα and Aβ (1-42) levels in CSF can be molecular markers to distinguish NPH cases from NNC. Disease severity can also be assessed by increased levels of CSF phospho-tau protein and the ratio of phospho-tau to Aβ (1-42), which might be a useful tool for predicting conversion of NPH individuals to other neurodegenerative disorders including Alzheimer's disease (AD).
|Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein. |
Pavlov, PF; Wiehager, B; Sakai, J; Frykman, S; Behbahani, H; Winblad, B; Ankarcrona, M
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 25 78-88 2011
Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.
|Restraint stress and repeated corticotrophin-releasing factor receptor activation in the amygdala both increase amyloid-β precursor protein and amyloid-β peptide but have divergent effects on brain-derived neurotrophic factor and pre-synaptic proteins in the prefrontal cortex of rats. |
Ray, B; Gaskins, DL; Sajdyk, TJ; Spence, JP; Fitz, SD; Shekhar, A; Lahiri, DK
Neuroscience 184 139-50 2011
Both environmental stress and anxiety may represent important risk factors for Alzheimer's disease (AD) pathogenesis. Previous studies demonstrate that restraint stress is associated with increased amyloid beta (Aβ) and decreased brain-derived neurotrophic factor (BDNF) levels in the brain. Aβ deposition, synaptic loss, and neurodegeneration define major hallmarks of AD, and BDNF is responsible for the maintenance of neurons. In contrast to restraint stress, repeated injections of sub-anxiogenic doses of the corticotrophin releasing factor receptor agonist urocortin1 (Ucn1) administered in the basolateral amygdala (BLA) of rats elicits persistent anxiety-like responses. We hypothesized that both restraint stress and Ucn1-induced anxiety would contribute to a neurobiological abnormality that would change the levels of Aβ precursor protein (APP) and Aβ as well as BDNF and pre-synaptic markers. In the first experiment, adult male Wister rats (n=5) were subjected to 3-h restraint, as compared to unstressed controls. In the second experiment, adult male Wistar rats (n=6) were subjected to sub-anxiogenic doses of Ucn1 (6 fmol/100 nl) administered in the BLA for 5 consecutive days, as compared to controls. Following each respective treatment, the social interaction (SI) test was performed to measure anxiety-like behavior. Protein studies were then conducted to quantify levels of APP, Aβ, BDNF and presynaptic proteins in the prefrontal cortex (PFC). In both experiments, we detected differences in either corticosterone levels or the SI test associated with a stress response. Furthermore, our findings indicate that both restraint stress and Ucn1 administration in the BLA lead to increased APP and Aβ deposition. However, restraint-induced stress leads to reductions in the levels of BDNF and presynaptic markers, while Ucn1-induced anxiety is associated with increases in the levels of each respective protein. This demonstrates a convergent role for stress response and Ucn1-induced anxiety in the regulation of APP and Aβ, but opposing roles for each respective treatment in the regulation of BDNF and presynaptic markers.
|Acid-sensing ion channel 1 is involved in both axonal injury and demyelination in multiple sclerosis and its animal model. |
Vergo, S; Craner, MJ; Etzensperger, R; Attfield, K; Friese, MA; Newcombe, J; Esiri, M; Fugger, L
Brain : a journal of neurology 134 571-84 2011
Although there is growing evidence for a role of excess intracellular cations, particularly calcium ions, in neuronal and glial cell injury in multiple sclerosis, as well as in non-inflammatory neurological conditions, the molecular mechanisms involved are not fully determined. We previously showed that the acid-sensing ion channel 1 which, when activated under the acidotic tissue conditions found in inflammatory lesions opens to allow influx of sodium and calcium ions, contributes to axonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, the extent and cellular distribution of acid-sensing ion channel 1 expression in neurons and glia in inflammatory lesions is unknown and, crucially, acid-sensing ion channel 1 expression has not been determined in multiple sclerosis lesions. Here we studied acute and chronic experimental autoimmune encephalomyelitis and multiple sclerosis spinal cord and optic nerve tissues to describe in detail the distribution of acid-sensing ion channel 1 and its relationship with neuronal and glial damage. We also tested the effects of amiloride treatment on tissue damage in the mouse models. We found that acid-sensing ion channel 1 was upregulated in axons and oligodendrocytes within lesions from mice with acute experimental autoimmune encephalomyelitis and from patients with active multiple sclerosis. The expression of acid-sensing ion channel 1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking acid-sensing ion channel 1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing experimental autoimmune encephalomyelitis. Together these findings suggest that blockade of acid-sensing ion channel 1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis.
|Increased p75 neurotrophin receptor expression in the canine distemper virus model of multiple sclerosis identifies aldynoglial schwann cells that emerge in response to axonal damage. |
Imbschweiler I, Seehusen F, Peck CT, Omar M, Baumgärtner W, Wewetzer K
Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide regeneration-promoting cells recruitable for therapeutic purposes. There is accumulating evidence that aldynoglial cells with Schwann cell-like growth-promoting properties emerge in the lesioned CNS. However, the characterization of these cells and the signals triggering their in situ generation have remained enigmatic. In the present study, we used the p75 neurotrophin receptor (p75(NTR) ) as a marker for Schwann cells to study gliogenesis in the well-defined canine distemper virus (CDV)-induced demyelination model. White matter lesions of CDV-infected dogs contained bi- to multipolar, p75(NTR) -expressing cells that neither expressed MBP, GFAP, BS-1, or P0 identifying oligodendroglia, astrocytes, microglia, and myelinating Schwann cells nor CDV antigen. Interestingly, p75(NTR) -expression became apparent prior to the onset of demyelination in parallel to the expression of β-amyloid precursor protein (β-APP), nonphosphorylated neurofilament (n-NF), BS-1, and CD3, and peaked in subacute lesions with inflammation. To study the role of infiltrating immune cells during differentiation of Schwann cell-like glia, organotypic slice cultures from the normal olfactory bulb were established. Despite the absence of infiltrating lymphocytes and macrophages, a massive appearance of p75(NTR) -positive Schwann-like cells and BS-1-positive microglia was noticed at 10 days in vitro. It is concluded that axonal damage as an early signal triggers the differentiation of tissue-resident precursor cells into p75(NTR) -expressing aldynoglial Schwann cells that retain an immature pre-myelin state. Further studies have to address the role of microglia during this process and the regenerative potential of aldynoglial cells in CDV infection and other demyelinating diseases. © 2011 Wiley-Liss, Inc.Copyright © 2011 Wiley-Liss, Inc.
|Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse. |
Magdesian, MH; Gralle, M; Guerreiro, LH; Beltrão, PJ; Carvalho, MM; Santos, LE; de Mello, FG; Reis, RA; Ferreira, ST
PloS one 6 e22857 2011
The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.
|Reversal of fragile X phenotypes by manipulation of AβPP/Aβ levels in Fmr1KO mice. |
Westmark, CJ; Westmark, PR; O'Riordan, KJ; Ray, BC; Hervey, CM; Salamat, MS; Abozeid, SH; Stein, KM; Stodola, LA; Tranfaglia, M; Burger, C; Berry-Kravis, EM; Malter, JS
PloS one 6 e26549 2011
Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading known genetic cause of autism. Fragile X mental retardation protein (FMRP), which is absent or expressed at substantially reduced levels in FXS, binds to and controls the postsynaptic translation of amyloid β-protein precursor (AβPP) mRNA. Cleavage of AβPP can produce β-amyloid (Aβ), a 39-43 amino acid peptide mis-expressed in Alzheimer's disease (AD) and Down syndrome (DS). Aβ is over-expressed in the brain of Fmr1(KO) mice, suggesting a pathogenic role in FXS. To determine if genetic reduction of AβPP/Aβ rescues characteristic FXS phenotypes, we assessed audiogenic seizures (AGS), anxiety, the ratio of mature versus immature dendritic spines and metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in Fmr1(KO) mice after removal of one App allele. All of these phenotypes were partially or completely reverted to normal. Plasma Aβ(1-42) was significantly reduced in full-mutation FXS males compared to age-matched controls while cortical and hippocampal levels were somewhat increased, suggesting that Aβ is sequestered in the brain. Evolving therapies directed at reducing Aβ in AD may be applicable to FXS and Aβ may serve as a plasma-based biomarker to facilitate disease diagnosis or assess therapeutic efficacy.
|Alzheimer's disease and non-demented high pathology control nonagenarians: comparing and contrasting the biochemistry of cognitively successful aging. |
Maarouf, CL; Daugs, ID; Kokjohn, TA; Walker, DG; Hunter, JM; Kruchowsky, JC; Woltjer, R; Kaye, J; Castaño, EM; Sabbagh, MN; Beach, TG; Roher, AE
PloS one 6 e27291 2011
The amyloid cascade hypothesis provides an economical mechanistic explanation for Alzheimer's disease (AD) dementia and correlated neuropathology. However, some nonagenarian individuals (high pathology controls, HPC) remain cognitively intact while enduring high amyloid plaque loads for decades. If amyloid accumulation is the prime instigator of neurotoxicity and dementia, specific protective mechanisms must enable these HPC to evade cognitive decline. We evaluated the neuropathological and biochemical differences existing between non-demented (ND)-HPC and an age-matched cohort with AD dementia. The ND-HPC selected for our study were clinically assessed as ND and possessed high amyloid plaque burdens. ELISA and Western blot analyses were used to quantify a group of proteins related to APP/Aβ/tau metabolism and other neurotrophic and inflammation-related molecules that have been found to be altered in neurodegenerative disorders and are pivotal to brain homeostasis and mental health. The molecules assumed to be critical in AD dementia, such as soluble or insoluble Aβ40, Aβ42 and tau were quantified by ELISA. Interestingly, only Aβ42 demonstrated a significant increase in ND-HPC when compared to the AD group. The vascular amyloid load which was not used in the selection of cases, was on the average almost 2-fold greater in AD than the ND-HPC, suggesting that a higher degree of microvascular dysfunction and perfusion compromise was present in the demented cohort. Neurofibrillary tangles were less frequent in the frontal cortices of ND-HPC. Biochemical findings included elevated vascular endothelial growth factor, apolipoprotein E and the neuroprotective factor S100B in ND-HPC, while anti-angiogenic pigment epithelium derived factor levels were lower. The lack of clear Aβ-related pathological/biochemical demarcation between AD and ND-HPC suggests that in addition to amyloid plaques other factors, such as neurofibrillary tangle density and vascular integrity, must play important roles in cognitive failure.
|Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis. |
Rau, CR; Hein, K; Sättler, MB; Kretzschmar, B; Hillgruber, C; McRae, BL; Diem, R; Bähr, M
The American journal of pathology 178 1770-81 2011
In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.
|Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles. |
Matsuda, S; Matsuda, Y; Snapp, EL; D'Adamio, L
Neurobiology of aging 32 1400-8 2011
Processing of the amyloid-β (Aβ) precursor protein (APP) has been extensively studied since it leads to production of Aβ peptides. Toxic forms of Aβ aggregates are considered the cause of Alzheimer's disease (AD). On the other end, BRI2 is implicated in APP processing and Aβ production. We have investigated the precise mechanism by which BRI2 modulates APP cleavages and have found that BRI2 forms a mature BRI2 polypeptide that is transported to the plasma membrane and endosomes where it interacts with mature APP. Notably, immature forms of APP and BRI2 fail to interact. Mature BRI2 inhibits APP processing by α-, β- and γ-secretases on the plasma membrane and in endocytic compartments. Thus, BRI2 is a specific inhibitor that reduces secretases' access to APP in the intracellular compartments where APP is normally processed.
|Tolfenamic acid interrupts the de novo synthesis of the β- amyloid precursor protein and lowers amyloid beta via a transcriptional pathway. |
Adwan LI, Basha R, Abdelrahim M, Subaiea GM, Zawia NH
Current Alzheimer research 8 385-92. 2011
Amyloid beta (Aβ) peptides are related to the pathogenesis of Alzheimer\'s disease (AD). The search for therapeutic strategies that lower these peptides has mainly focused on the proteolytic processing of the β-amyloid precursor protein (APP), and other post-transcriptional pathways. The transcription factor specificity protein 1 (Sp1) is vital for the regulation of several genes involved in AD including APP and the beta site APP cleaving enzyme 1 (BACE1). We have previously reported that tolfenamic acid promotes the degradation of Sp1 protein (SP1) in pancreatic human cancer cells and mice tumors. This study examines the ability of tolfenamic acid to reduce SP1 levels, and thereby decrease APP transcription and Aβ levels in rodent brains. Tolfenamic acid was administered by oral gavage to C57BL/6 mice at variable dosages and for different time periods. Results have shown that tolfenamic acid was able to down regulate brain protein levels of SP1, APP, and Aβ. These findings demonstrate that interference with upstream transcriptional pathways can lower pathogenic intermediates associated with AD, and thus tolfenamic acid represents a novel approach for the development of a therapeutic intervention for AD.
|Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production. |
Bordji, K; Becerril-Ortega, J; Nicole, O; Buisson, A
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 15927-42 2010
Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.
|A NH2 tau fragment targets neuronal mitochondria at AD synapses: possible implications for neurodegeneration. |
Amadoro G, Corsetti V, Stringaro A, Colone M, D'Aguanno S, Meli G, Ciotti M, Sancesario G, Cattaneo A, Bussani R, Mercanti D, Calissano P
J Alzheimers Dis 21 445-70. 2010
Synapses are ultrastructural sites for memory storage in brain, and synaptic damage is the best pathologic correlate of cognitive decline in Alzheimer's disease (AD). Post-translational hyperphosphorylation, enzyme-mediated truncation, conformational modifications, and aggregation of tau protein into neurofibrillary tangles (NFTs) are hallmarks for a heterogeneous group of neurodegenerative disorders, so-called tauopathies. AD is a secondary tauopathy since it is pathologically distinguished by the presence of amyloid-beta (Abeta)-containing senile plaques and the presence of tau-positive NFTs in the neocortex and hippocampus. Here, we report that a 20-22 kDa NH2-truncated tau fragment is largely enriched in human mitochondria from cryopreserved synaptosomes of AD brains and that its amount in terminal fields correlates with the pathological synaptic changes and with the organelle functional impairment. This NH2-truncated tau form is also found in other human, not AD-tauopathies, while its presence in AD patients is linked to Abeta multimeric species and likely to pathology severity. Finally native, patient-derived, Abeta oligomers-enriched extracts likely impair the mitochondrial function by the in vitro production of 20-22 kDa NH2-tau fragments in mature human SY5Y and in rat hippocampal neurons. Thus our findings suggest that the mitochondrial NH2-derived tau peptide distribution may exacerbate the synapse degeneration occurring in tauopathies, including AD, and sustain the in vivo NH-2 tau cleavage inhibitors as an alternative drug discovery strategies for AD therapy.
|Syndecan-3 and Notch cooperate in regulating adult myogenesis. |
Pisconti A, Cornelison DD, Olguín HC, Antwine TL, Olwin BB
J Cell Biol 190 427-41. 2010
Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7(+) satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3-null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-alpha-converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.
|Memory impairment in transgenic Alzheimer mice requires cellular prion protein. |
David A Gimbel,Haakon B Nygaard,Erin E Coffey,Erik C Gunther,Juha Laurén,Zachary A Gimbel,Stephen M Strittmatter
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 2010
Soluble oligomers of the amyloid-beta (Abeta) peptide are thought to play a key role in the pathophysiology of Alzheimer's disease (AD). Recently, we reported that synthetic Abeta oligomers bind to cellular prion protein (PrP(C)) and that this interaction is required for suppression of synaptic plasticity in hippocampal slices by oligomeric Abeta peptide. We hypothesized that PrP(C) is essential for the ability of brain-derived Abeta to suppress cognitive function. Here, we crossed familial AD transgenes encoding APPswe and PSen1DeltaE9 into Prnp-/- mice to examine the necessity of PrP(C) for AD-related phenotypes. Neither APP expression nor Abeta level is altered by PrP(C) absence in this transgenic AD model, and astrogliosis is unchanged. However, deletion of PrP(C) expression rescues 5-HT axonal degeneration, loss of synaptic markers, and early death in APPswe/PSen1DeltaE9 transgenic mice. The AD transgenic mice with intact PrP(C) expression exhibit deficits in spatial learning and memory. Mice lacking PrP(C), but containing Abeta plaque derived from APPswe/PSen1DeltaE9 transgenes, show no detectable impairment of spatial learning and memory. Thus, deletion of PrP(C) expression dissociates Abeta accumulation from behavioral impairment in these AD mice, with the cognitive deficits selectively requiring PrP(C).
|Amyloid precursor protein cleavage-dependent and -independent axonal degeneration programs share a common nicotinamide mononucleotide adenylyltransferase 1-sensitive pathway. |
Vohra, BP; Sasaki, Y; Miller, BR; Chang, J; DiAntonio, A; Milbrandt, J
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 13729-38 2010
Axonal degeneration is a hallmark of many debilitating neurological disorders and is thought to be regulated by mechanisms distinct from those governing cell body death. Recently, caspase 6 activation via amyloid precursor protein (APP) cleavage and activation of DR6 was discovered to induce axon degeneration after NGF withdrawal. We tested whether this pathway is involved in axonal degeneration caused by withdrawal of other trophic support, axotomy or vincristine exposure. Neurturin deprivation, like NGF withdrawal activated this APP/DR6/caspase 6 pathway and resulted in axonal degeneration, however, APP cleavage and caspase 6 activation were not involved in axonal degeneration induced by mechanical or toxic insults. However, loss of surface APP (sAPP) and caspase 6 activation were observed during axonal degeneration induced by dynactin 1(Dctn1) dysfunction, which disrupts axonal transport. Mutations in Dctn1 are associated with motor neuron disease and frontal temporal dementia, thus suggesting that the APP/caspase 6 pathway could be important in specific types of disease-associated axonal degeneration. The NGF deprivation paradigm, with its defined molecular pathway, was used to examine the context of Nmnat-mediated axonal protection. We found that although Nmnat blocks axonal degeneration after trophic factor withdrawal, it did not prevent loss of axon sAPP or caspase 6 activation within the axon, suggesting it acts downstream of caspase 6. These results indicate that diverse insults induce axonal degeneration via multiple pathways and that these degeneration signals converge on a common, Nmnat-sensitive program that is uniquely involved in axonal, but not cell body, degeneration.
|Memantine treatment decreases levels of secreted Alzheimer's amyloid precursor protein (APP) and amyloid beta (A beta) peptide in the human neuroblastoma cells. |
Ray, Balmiki, et al.
Neurosci. Lett., 470: 1-5 (2010) 2010
Memantine, an uncompetitive NMDA receptor antagonist, is a FDA-approved drug used for the treatment of moderate-to-severe Alzheimer's disease (AD). Several studies have documented protective roles of memantine against amyloid beta (A beta) peptide-mediated damage to neurons in both in vitro and in vivo models. Memantine is also effective in reducing amyloid burden in the brain of APP transgenic mice. However, the exact mechanism by which memantine provides protection against A beta-mediated neurodegenerative cascade, including APP metabolism, remains to be elucidated. Herein, we investigated the effect of memantine on levels of the secreted form of A beta precursor protein (APP), secreted A beta and cell viability markers under short/acute conditions. We treated neuronal SK-N-SH cells with 10 microM memantine and measured levels of secreted total APP (sAPP), APP alpha isoform and A beta((1-40)) in a time dependent manner for up to 24h. Memantine significantly decreased the levels of the secreted form of sAPP, sAPP alpha and A beta((1-40)) compared to vehicle treated cells. This change started as early as 8h and continued for up to 24h of drug treatment. Unlike sAPP, a slight non-significant increase in total intracellular APP level was observed in 24-h treated memantine cells. Taken together, these results suggest a role for memantine in the transport or trafficking of APP molecules away from the site of their proteolytic cleavage by the secretase enzymes. Such a novel property of memantine warrants further study to define its therapeutic utility.
|GLP-1 receptor stimulation reduces amyloid-beta peptide accumulation and cytotoxicity in cellular and animal models of Alzheimer's disease. |
Li, Yazhou, et al.
J. Alzheimers Dis., 19: 1205-19 (2010) 2010
Type 2 (T2) diabetes mellitus (DM) has been associated with an increased incidence of neurodegenerative disorders, including Alzheimer's disease (AD). Several pathological features are shared between diabetes and AD, including dysfunctional insulin signaling and a dysregulation of glucose metabolism. It has therefore been suggested that not only may the two conditions share specific molecular mechanisms but also that agents with proven efficacy in one may be useful against the other. Hence, the present study characterized the effects of a clinically approved long-acting analogue, exendin-4 (Ex-4), of the endogenous insulin releasing incretin, glucagon-like peptide-1 (GLP-1), on stress-induced toxicity in neuronal cultures and on amyloid-beta protein (Abeta) and tau levels in triple transgenic AD (3xTg-AD) mice with and without streptozocin (STZ)-induced diabetes. Ex-4 ameliorated the toxicity of Abeta and oxidative challenge in primary neuronal cultures and human SH-SY5Y cells in a concentration-dependent manner. When 11 to 12.5 month old female 3xTg AD mice were challenged with STZ or saline, and thereafter treated with a continuous subcutaneous infusion of Ex-4 or vehicle, Ex-4 ameliorated the diabetic effects of STZ in 3xTg-AD mice, elevating plasma insulin and lowering both plasma glucose and hemoglobin A1c (HbA1c) levels. Furthermore, brain levels of Abeta protein precursor and Abeta, which were elevated in STZ 3xTg-AD mice, were significantly reduced in Ex-4 treated mice. Brain tau levels were unaffected following STZ challenge, but showed a trend toward elevation that was absent following Ex-4 treatment. Together, these results suggest a potential value of Ex-4 in AD, particularly when associated with T2DM or glucose intolerance.
|β-Secretase-1 elevation in aged monkey and Alzheimer's disease human cerebral cortex occurs around the vasculature in partnership with multisystem axon terminal pathogenesis and β-amyloid accumulation. |
Cai, Y; Xiong, K; Zhang, XM; Cai, H; Luo, XG; Feng, JC; Clough, RW; Struble, RG; Patrylo, PR; Chu, Y; Kordower, JH; Yan, XX
The European journal of neuroscience 32 1223-38 2010
Alzheimer's disease (AD) is the most common dementia-causing disorder in the elderly; it may be related to multiple risk factors, and is characterized pathologically by cerebral hypometabolism, paravascular β-amyloid peptide (Aβ) plaques, neuritic dystrophy, and intra-neuronal aggregation of phosphorylated tau. To explore potential pathogenic links among some of these lesions, we examined β-secretase-1 (BACE1) alterations relative to Aβ deposition, neuritic pathology and vascular organization in aged monkey and AD human cerebral cortex. Western blot analyses detected increased levels of BACE1 protein and β-site-cleavage amyloid precursor protein C-terminal fragments in plaque-bearing human and monkey cortex relative to controls. In immunohistochemistry, locally elevated BACE1 immunoreactivity (IR) occurred in AD but not in control human cortex, with a trend for increased overall density among cases with greater plaque pathology. In double-labeling preparations, BACE1 IR colocalized with immunolabeling for Aβ but not for phosphorylated tau. In perfusion-fixed monkey cortex, locally increased BACE1 IR co-existed with intra-axonal and extracellular Aβ IR among virtually all neuritic plaques, ranging from primitive to typical cored forms. This BACE1 labeling localized to swollen/sprouting axon terminals that might co-express one or another neuronal phenotype markers (GABAergic, glutamatergic, cholinergic, or catecholaminergic). Importantly, these BACE1-labeled dystrophic axons resided near to or in direct contact with blood vessels. These findings suggest that plaque formation in AD or normal aged primates relates to a multisystem axonal pathogenesis that occurs in partnership with a potential vascular or metabolic deficit. The data provide a mechanistic explanation for why senile plaques are present preferentially near the cerebral vasculature.Full Text Article
|The histone deacetylase inhibitor valproic acid inhibits cancer cell proliferation via down-regulation of the Alzheimer amyloid precursor protein. |
Venkataramani, Vivek, et al.
The Journal of biological chemistry, (2010) 2010
The beta-amyloid precursor protein (APP) represents a type I transmembrane glycoprotein that is ubiquitously expressed. In brain it is a key player in the molecular pathogenesis of Alzheimer's disease. Its physiological function is however less well understood. Previous studies showed that APP is up-regulated in prostate, colon, pancreatic tumor and oral squamous cell carcinoma. In the present study, we show that APP has an essential role in growth control of pancreatic and colon cancer. Abundant APP staining was found in human pancreatic adenocarcinoma and colon cancer tissue. Interestingly, treating pancreatic and colon cancer cells with valproic acid (VPA, 2-propylpentanoic acid), a known histone deacetylase inhibitor (HDACi), leads to up-regulation of GRP78, an ER chaperone immunoglobulin binding protein. GRP78 is involved in APP maturation, and inhibition of tumor cell growth by down-regulation of APP and secreted sAPPalpha. Trichostatin A (TSA), a pan-HDAC inhibitor also lowered APP and increased GRP78 levels. In contrast, treating cells with valpromide, a VPA derivative lacking HDAC inhibitory properties had no effect on APP levels. VPA did not modify the level of epidermal growth factor receptor, another type I transmembrane protein, and APLP2, a member of the APP-family, demonstrating the specificity of the VPA effect on APP. Small interfering RNA (siRNA)-mediated knock-down of APP also resulted in significantly decreased cell growth. Based on these observations, the data suggest that APP down-regulation via HDAC inhibition provides a novel mechanism for pancreatic and colon cancer therapy.
|Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis. |
Linker, RA; Lee, DH; Demir, S; Wiese, S; Kruse, N; Siglienti, I; Gerhardt, E; Neumann, H; Sendtner, M; Lühder, F; Gold, R
Brain : a journal of neurology 133 2248-63 2010
Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects. We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease. In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed. In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection. Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.
|Functional deprivation promotes amyloid plaque pathogenesis in Tg2576 mouse olfactory bulb and piriform cortex. |
Zhang, XM; Xiong, K; Cai, Y; Cai, H; Luo, XG; Feng, JC; Clough, RW; Patrylo, PR; Struble, RG; Yan, XX
The European journal of neuroscience 31 710-21 2010
Cerebral hypometabolism and amyloid accumulation are principal neuropathological manifestations of Alzheimer's disease (AD). Whether and how brain/neuronal activity might modulate certain pathological processes of AD are interesting topics of recent clinical and basic research in the field, and may be of potential medical relevance in regard to both the disease etiology and intervention. Using the Tg2576 transgenic mouse model of AD, this study characterized a promotive effect of neuronal hypoactivity associated with functional deprivation on amyloid plaque pathogenesis in the olfactory pathway. Unilateral naris-occlusion caused beta-secretase-1 (BACE1) elevation in neuronal terminals in the deprived relative to the non-deprived bulb and piriform cortex in young adult mice. In parallel with the overall age-related plaque development in the forebrain, locally increased BACE1 immunoreactivity co-occurred with amyloid deposition first in the piriform cortex then within the bulb, more prominent on the deprived relative to the non-deprived side. Biochemical analyses confirmed elevated BACE1 protein levels, enzymatic activity and products in the deprived relative to non-deprived bulbs. Plaque-associated BACE1 immunoreactivity in the bulb and piriform cortex was localized preferentially to swollen/sprouting glutamatergic axonal terminals, with Abeta immunoreactivity occurring inside as well as around these terminals. Together, these findings suggest that functional deprivation or neuronal hypoactivity facilitates amyloid plaque formation in the forebrain in a transgenic model of AD, which operates synergistically with age effect. The data also implicate an intrinsic association of amyloid accumulation and plaque formation with progressive axonal pathology.Full Text Article
|APP processing induced by herpes simplex virus type 1 (HSV-1) yields several APP fragments in human and rat neuronal cells. |
De Chiara, G; Marcocci, ME; Civitelli, L; Argnani, R; Piacentini, R; Ripoli, C; Manservigi, R; Grassi, C; Garaci, E; Palamara, AT
PloS one 5 e13989 2010
Lifelong latent infections of the trigeminal ganglion by the neurotropic herpes simplex virus type 1 (HSV-1) are characterized by periodic reactivation. During these episodes, newly produced virions may also reach the central nervous system (CNS), causing productive but generally asymptomatic infections. Epidemiological and experimental findings suggest that HSV-1 might contribute to the pathogenesis of Alzheimer's disease (AD). This multifactorial neurodegenerative disorder is related to an overproduction of amyloid beta (Aβ) and other neurotoxic peptides, which occurs during amyloidogenic endoproteolytic processing of the transmembrane amyloid precursor protein (APP). The aim of our study was to identify the effects of productive HSV-1 infection on APP processing in neuronal cells. We found that infection of SH-SY5Y human neuroblastoma cells and rat cortical neurons is followed by multiple cleavages of APP, which result in the intra- and/or extra-cellular accumulation of various neurotoxic species. These include: i) APP fragments (APP-Fs) of 35 and 45 kDa (APP-F35 and APP-F45) that comprise portions of Aβ; ii) N-terminal APP-Fs that are secreted; iii) intracellular C-terminal APP-Fs; and iv) Aβ(1-40) and Aβ(1-42). Western blot analysis of infected-cell lysates treated with formic acid suggests that APP-F35 may be an Aβ oligomer. The multiple cleavages of APP that occur in infected cells are produced in part by known components of the amyloidogenic APP processing pathway, i.e., host-cell β-secretase, γ-secretase, and caspase-3-like enzymes. These findings demonstrate that HSV-1 infection of neuronal cells can generate multiple APP fragments with well-documented neurotoxic potentials. It is tempting to speculate that intra- and extracellular accumulation of these species in the CNS resulting from repeated HSV-1 reactivation could, in the presence of other risk factors, play a co-factorial role in the development of AD.Full Text Article
|International Workshop on Inclusion Body Myositis held at the Institute of Myology, Paris, on 29 May 2009. |
Olivier Benveniste,David Hilton-Jones
Neuromuscular disorders : NMD 20 2010
|The amyloid precursor protein/protease nexin 2 Kunitz inhibitor domain is a highly specific substrate of mesotrypsin. |
Salameh, MA; Robinson, JL; Navaneetham, D; Sinha, D; Madden, BJ; Walsh, PN; Radisky, ES
The Journal of biological chemistry 285 1939-49 2010
The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-beta peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg(15)-Ala(16) reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.
|Predicting memapsin 2 (β-secretase) hydrolytic activity. |
Li, X; Bo, H; Zhang, XC; Hartsuck, JA; Tang, J
Protein science : a publication of the Protein Society 19 2175-85 2010
Memapsin 2 (BACE1, β-secretase), a membrane aspartic protease, functions in the cleavage of brain β-amyloid precursor protein (APP) leading to the production of β-amyloid. Because the excess level of β-amyloid in the brain is a leading factor in Alzheimer's disease (AD), memapsin 2 is a major therapeutic target for inhibitor drugs. The substrate-binding cleft of memapsin 2 accommodates 12 subsite residues, from P(8) to P(4)'. We have determined the hydrolytic preference as relative k(cat)/K(M) (preference constant) in all 12 subsites and used these data to establish a predictive algorithm for substrate hydrolytic efficiency. Using the sequences from 12 reported memapsin 2 protein substrates, the predicted and experimentally determined preference constants have an excellent correlation coefficient of 0.97. The predictive model indicates that the hydrolytic preference of memapsin 2 is determined mainly by the interaction with six subsites (from P(4) to P(2)'), a conclusion supported by the crystal structure B-factors calculated for the various residues of transition-state analogs bound to different memapsin 2 subsites. The algorithm also predicted that the replacement of the P(3), P(2), and P(1) subsites of APP from Val, Lys, and Met, respectively, to Ile, Asp, and Phe, respectively, (APP(IDF)) would result in a highest hydrolytic rate for β-amyloid-generating APP variants. Because more β-amyloid was produced from cells expressing APP(IDF) than those expressing APP with Swedish mutations, this designed APP variant may be useful in new memapsin 2 substrates or transgenic mice for AD studies.
|Oligodendrocyte PTEN is required for myelin and axonal integrity, not remyelination. |
Harrington, EP; Zhao, C; Fancy, SP; Kaing, S; Franklin, RJ; Rowitch, DH
Annals of neurology 68 703-16 2010
Repair of myelin injury in multiple sclerosis may fail, resulting in chronic demyelination, axonal loss, and disease progression. As cellular pathways regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN; eg, phosphatidylinositol-3-kinase [PI-3K]) have been reported to enhance axon regeneration and oligodendrocyte maturation, we investigated potentially beneficial effects of Pten loss of function in the oligodendrocyte lineage on remyelination.We characterized oligodendrocyte numbers and myelin sheath thickness in mice with conditional inactivation of Pten in oligodendrocytes, Olig2-cre, Pten(fl/fl) mice. Using a model of central nervous system demyelination, lysolecithin injection into the spinal cord white matter, we performed short- and long-term lesioning experiments and quantified oligodendrocyte maturation and myelin sheath thickness in remyelinating lesions.During development, we observed dramatic hypermyelination in the corpus callosum and spinal cord. Following white matter injury, however, there was no detectable improvement in remyelination. Moreover, we observed progressive myelin sheath abnormalities and massive axon degeneration in the fasciculus gracilis of mutant animals, as indicated by ultrastructure and expression of SMI-32, amyloid precursor protein, and caspase 6.These studies indicate adverse effects of chronic Pten inactivation (and by extension, activation PI-3K signaling) on myelinating oligodendrocytes and their axonal targets. We conclude that PTEN function in oligodendrocytes is required to regulate myelin thickness and preserve axon integrity. In contrast, PTEN is dispensable during myelin repair, and its inactivation confers no detectable benefit.
|Phospho-eIF2α level is important for determining abilities of BACE1 reduction to rescue cholinergic neurodegeneration and memory defects in 5XFAD mice. |
Devi, L; Ohno, M
PloS one 5 e12974 2010
β-Site APP-cleaving enzyme 1 (BACE1) initiates amyloid-β (Aβ) generation and thus represents a prime therapeutic target in treating Alzheimer's disease (AD). Notably, increasing evidence indicates that BACE1 levels become elevated in AD brains as disease progresses; however, it remains unclear how the BACE1 upregulation may affect efficacies of therapeutic interventions including BACE1-inhibiting approaches. Here, we crossed heterozygous BACE1 knockout mice with AD transgenic mice (5XFAD model) and compared the abilities of partial BACE1 reduction to rescue AD-like phenotypes at earlier (6-month-old) and advanced (15-18-month-old) stages of disease, which expressed normal (∼100%) and elevated (∼200%) levels of BACE1, respectively. BACE1(+/-) deletion rescued memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at the earlier stage and prevented their septohippocampal cholinergic deficits associated with significant neuronal loss. Importantly, BACE1(+/-) deletion was no longer able to rescue memory deficits or cholinergic neurodegeneration in 5XFAD mice at the advanced stage. Moreover, BACE1(+/-) deletion significantly reduced levels of Aβ42 and the β-secretase-cleaved C-terminal fragment (C99) in 6-month-old 5XFAD mouse brains, while these neurotoxic β-cleavage products dramatically elevated with age and were not affected by BACE1(+/-) deletion in 15-18-month-old 5XFAD brains. Interestingly, although BACE1(+/-) deletion lowered BACE1 expression by ∼50% in 5XFAD mice irrespective of age in concordance with the reduction in gene copy number, BACE1 equivalent to wild-type controls remained in BACE1(+/-)·5XFAD mice at the advanced age. In accord, phosphorylation of the translation initiation factor eIF2α, an important mediator of BACE1 elevation, was dramatically increased (∼9-fold) in 15-18-month-old 5XFAD mice and remained highly upregulated (∼6-fold) in age-matched BACE1(+/-)·5XFAD mice. Together, our results indicate that partial reduction of BACE1 is not sufficient to block the phospho-eIF2α-dependent BACE1 elevation during the progression of AD, thus limiting its abilities to reduce cerebral Aβ/C99 levels and rescue memory deficits and cholinergic neurodegeneration.
|TGF-β induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid β plaques in Alzheimer's disease. |
Lee, MH; Lin, SR; Chang, JY; Schultz, L; Heath, J; Hsu, LJ; Kuo, YM; Hong, Q; Chiang, MF; Gong, CX; Sze, CI; Chang, NS
Cell death & disease 1 e110 2010
The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.
|Beta amyloid and hyperphosphorylated tau deposits in the pancreas in type 2 diabetes. |
Miklossy, J; Qing, H; Radenovic, A; Kis, A; Vileno, B; Làszló, F; Miller, L; Martins, RN; Waeber, G; Mooser, V; Bosman, F; Khalili, K; Darbinian, N; McGeer, PL
Neurobiology of aging 31 1503-15 2010
Strong epidemiologic evidence suggests an association between Alzheimer disease (AD) and type 2 diabetes. To determine if amyloid beta (Abeta) and hyperphosphorylated tau occurs in type 2 diabetes, pancreas tissues from 21 autopsy cases (10 type 2 diabetes and 11 controls) were analyzed. APP and tau mRNAs were identified in human pancreas and in cultured insulinoma beta cells (INS-1) by RT-PCR. Prominent APP and tau bands were detected by Western blotting in pancreatic extracts. Aggregated Abeta, hyperphosphorylated tau, ubiquitin, apolipoprotein E, apolipoprotein(a), IB1/JIP-1 and JNK1 were detected in Langerhans islets in type 2 diabetic patients. Abeta was co-localized with amylin in islet amyloid deposits. In situ beta sheet formation of islet amyloid deposits was shown by infrared microspectroscopy (SIRMS). LPS increased APP in non-neuronal cells as well. We conclude that Abeta deposits and hyperphosphorylated tau are also associated with type 2 diabetes, highlighting common pathogenetic features in neurodegenerative disorders, including AD and type 2 diabetes and suggesting that Abeta deposits and hyperphosphorylated tau may also occur in other organs than the brain.
|Memory deficits due to familial British dementia BRI2 mutation are caused by loss of BRI2 function rather than amyloidosis. |
Tamayev, R; Giliberto, L; Li, W; d'Abramo, C; Arancio, O; Vidal, R; D'Adamio, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 30 14915-24 2010
Familial dementias, which include Alzheimer disease (AD), familial British dementia (FBD), and familial Danish dementia (FDD), are caused by dominantly inherited autosomal mutations and are characterized by the production of amyloidogenic peptides, neurofibrillary tangles (NFTs) and neurodegeneration (St George-Hyslop and Petit, 2005; Garringer et al., 2009). The prevailing pathogenic theory, the "amyloid cascade hypothesis" (Hardy and Selkoe, 2002), posits that the accumulation of amyloidogenic peptides triggers tauopathy, neurodegeneration, and cognitive and behavioral changes. However, this hypothesis is yet to be validated, and causes of dementia may be multifaceted and involve other mechanisms, such as loss of function due to pathogenic mutations. Mouse models of human dementia invariably use transgenic expression systems (LaFerla and Oddo, 2005; McGowan et al., 2006; Vidal et al., 2009; Coomaraswamy et al., 2010) that do not reflect the genotypes of human disease and cannot replicate loss of function. Therefore, we generated a knock-in (KI) mouse model of FBD (FBD(KI)) genetically congruous with the human disease. FBD is caused by a missense mutation at the stop codon of the BRI2 gene (Vidal et al., 1999) and, like FBD patients, FBD(KI) mice carry this mutation in one of the two murine Bri2 alleles. We report that the British mutation drastically reduces expression of mature BRI2 in both KI mice and human FBD brains. This deficit is associated with severe hippocampal memory deficits in FBD(KI) mice. Remarkably, these animals showed no cerebral amyloidosis and tauopathy. Bri2(+/-) mice present memory deficits similar to those in FBD(KI) animals. Collectively, these results indicate that the British BRI2 mutation underlies abnormal memory due to loss of BRI2 function and independently of histopathological alterations typically evident in advanced neurodegenerative disease.
|Molecular and immunocytochemical characterization of primary neuronal cultures from adult rat brain: Differential expression of neuronal and glial protein markers. |
Ray, Balmiki, et al.
J. Neurosci. Methods, 184: 294-302 (2009) 2009
Neurobiological studies using primary neuronal cultures commonly employ fetal-derived neurons, but much less often adult brain-derived neurons. Our goal is to perform morphological and molecular characterization of primary neuronal cultures from adult rat brain, including the relative expression of neuronal and glial cell markers at different time points. We tested the hypothesis that long-term neuronal viability is compatible with glial proliferation in adult neuron culture. We examined neuron culture from adult rat brain, which was maintained at steady state up to 24 days, and characterized them on the basis of cellular, molecular and biochemical properties at different time points of the culture. We identified neuronal and glial cells by both immunocytochemical and western immunoblotting techniques using NSE and Tau as neuronal markers and GFAP as glial protein marker, which revealed the presence of predominantly neuronal cells in the initial phase of the culture and a rise in glial cells from day 12 onwards. Notably, neuronal cells were preserved in the culture along with the glial cells even at day 24. Transfection of the cultured cells with a GFP expression vector and plasmids containing a luciferase reporter gene under the control of two different gene promoters demonstrated DNA transfectability. Taken together, these results suggest a differential expression of neuronal and glial cells at different time points and long-term neuronal viability in the presence of glial proliferation. Such adult neurons serve as a suitable system for the application of neurodegeneration models and for drug target discovery in various brain disorders including Alzheimer's disease.
|The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites. |
Muresan, V; Varvel, NH; Lamb, BT; Muresan, Z
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 3565-78 2009
The amyloid-beta (Abeta) precursor protein (APP), a transmembrane protein that undergoes proteolytic cleavage into defined fragments, has been implicated in axonal transport. The proposed role of APP as a vesicle receptor for the microtubule motor kinesin-1 has relevance for the pathogenesis of Alzheimer's disease. Nevertheless, this function, which relies on the transport to the cell periphery of full-length APP rather than its cleavage fragments, remains controversial. Other proposed functions of APP, such as regulating transcription, neurogenesis, cell movement, or neurite growth also rely on APP's presence as a full-length protein at the cell surface, implying that APP cleavage occurs after its transport to the cell periphery. To test this hypothesis, we mapped the localization of various APP epitopes in neurons in culture and in the mouse brain. Surprisingly, epitopes from the N-terminal, C-terminal, and central (Abeta) domains of APP each showed a distinct distribution throughout the cell and rarely colocalized. Within neurites, these epitopes were localized to distinct transport vesicles that associated with different sets of microtubules and, occasionally, actin filaments. C-terminal APP fragments were preferentially transported into neurites as phosphorylated forms, entered the lamellipodium and filopodia of growth cones, and concentrated in regions of growth cone turning and advancement (unlike the N-terminal and Abeta fragments). We conclude that, under normal conditions, the proteolytic cleavage of APP primarily occurs before its sorting into axonal transport vesicles and the cleaved fragments segregate into separate vesicle populations that reach different destinations, and thus have different functions.Full Text Article
|Neuropathy target esterase is required for adult vertebrate axon maintenance. |
Read DJ, Li Y, Chao MV, Cavanagh JB, Glynn P
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 11594-600 2009
The enzyme neuropathy target esterase (NTE) is present in neurons and deacylates the major membrane phospholipid, phosphatidylcholine (PtdCho). Mutation of the NTE gene or poisoning by neuropathic organophosphates--chemical inhibitors of NTE--causes distal degeneration of long spinal axons in humans. However, analogous neuropathological changes have not been reported in nestin-cre:NTEfl/fl mice with NTE-deficient neural tissue. Furthermore, altered PtdCho homeostasis has not been detected in NTE-deficient vertebrates. Here, we describe distal degeneration of the longest spinal axons in approximately 3-week-old nestin-cre:NTEfl/fl mice and in adult C57BL/6J mice after acute dosing with a neuropathic organophosphate: in both groups early degenerative lesions were followed by swellings comprising accumulated axoplasmic material. In mice dosed acutely with organophosphate, maximal numbers of lesions, in the longest spinal sensory axon tract, were attained within days and were preceded by a transient rise in neural PtdCho. In nestin-cre:NTEfl/fl mice, sustained elevation of PtdCho over many months was accompanied by progressive degeneration and massive swelling of axons in sensory and motor spinal tracts and by increasing hindlimb dysfunction. Axonal lesion distribution closely resembled that in hereditary spastic paraplegia (HSP). The importance of defective membrane trafficking in HSP and the association of NTE with the endoplasmic reticulum--the starting point for the constitutive secretory pathway and transport of neuronal materials into axons--prompted investigation for a role of NTE in secretion. Cultured NTE-deficient neurons displayed modestly impaired secretion, consistent with neuronal viability and damage in vivo initially restricted to distal parts of the longest axons.
|Calsyntenins Mediate TGN Exit of APP in a Kinesin-1-Dependent Manner. |
Alexander Ludwig, Jessica Blume, Tu-My Diep, Ju Yuan, José María Mateos, Kerstin Leuthäuser, Martin Steuble, Peter Streit, Peter Sonderegger
Traffic (Copenhagen, Denmark) 10 572-89 2009
Kinesin motors are required for the export of membranous cargo from the trans-Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin-1 docking protein calsyntenin-1 localizes to the TGN in vivo and directly and specifically recruits Kinesin-1 to Golgi/TGN membranes as well as to dynamic post-Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin-1-decorated tubular structures which may serve as carriers for calsyntenin-1-mediated post-TGN transport of APP. Abrogation of this pathway via virus-mediated knockdown of calsyntenin-1 expression in primary cultured neurons caused a marked elevation of APP C-terminal fragments. Together, these results indicate a role for calsyntenin-1 in Kinesin-1-dependent TGN exit and post-Golgi transport of APP-containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.
|Down-regulation of seladin-1 increases BACE1 levels and activity through enhanced GGA3 depletion during apoptosis. |
Sarajarvi T, Haapasalo A, Viswanathan J, Makinen P, Laitinen M, Soininen H, Hiltunen M
The Journal of biological chemistry 284 34433-43 2009
Seladin-1 is a neuroprotective protein selectively down-regulated in brain regions affected in Alzheimer disease (AD). Seladin-1 protects cells against beta-amyloid (Abeta) peptide 42- and oxidative stress-induced apoptosis activated by caspase-3, a key mediator of apoptosis. Here, we have employed RNA interference to assess the molecular effects of seladin-1 down-regulation on the beta-secretase (BACE1) function and beta-amyloid precursor protein (APP) processing in SH-SY5Y human neuroblastoma cells in both normal and apoptotic conditions. Our results show that approximately 60% reduction in seladin-1 protein levels, resembling the decrease observed in AD brain, did not significantly affect APP processing or Abeta secretion in normal growth conditions. However, under apoptosis, seladin-1 small interfering RNA (siRNA)-transfected cells showed increased caspase-3 activity on average by 2-fold when compared with control siRNA-transfected cells. Increased caspase-3 activity coincided with a significant depletion of the BACE1-sorting protein, GGA3 (Golgi-localized gamma-ear-containing ADP-ribosylation factor-binding protein), and subsequently augmented BACE1 protein levels and activity. Augmented BACE1 activity in turn correlated with the enhanced beta-amyloidogenic processing of APP and ultimately increased Abeta production. These adverse changes associated with decreased cell viability in seladin-1 siRNA-transfected cells under apoptosis. No changes in GGA3 or BACE1 levels were found after seladin-1 knockdown in normal growth conditions. Collectively, our results suggest that under stress conditions, reduced seladin-1 expression results in enhanced GGA3 depletion, which further leads to augmented post-translational stabilization of BACE1 and increased beta-amyloidogenic processing of APP. These mechanistic findings related to seladin-1 down-regulation are important in the context of AD as the oxidative stress-induced apoptosis plays a key role in the disease pathogenesis.
|Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells. |
V Rhein, G Baysang, S Rao, F Meier, A Bonert, F Müller-Spahn, A Eckert, V Rhein, G Baysang, S Rao, F Meier, A Bonert, F Müller-Spahn, A Eckert
Cellular and molecular neurobiology 29 1063-71 2009
Evidence suggests that amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Here we investigated the specific effects of Abeta on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Abeta protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.
|Gene expression profiles of APP and BACE1 in Tg SOD1G93A cortical cells. |
O Spadoni, A Crestini, P Piscopo, L Malvezzi-Campeggi, I Carunchio, M Pieri, C Zona, A Confaloni
Cell Mol Neurobiol. 29 635-41 Epub 2009 Feb 13 2009
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Abeta peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer's disease, as APP, beta- and gamma-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of gamma-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.
|CD74 interacts with APP and suppresses the production of Abeta. |
Matsuda, S; Matsuda, Y; D'Adamio, L
Molecular neurodegeneration 4 41 2009
Alzheimer disease (AD) is characterized by senile plaques, which are mainly composed of beta amyloid (Abeta) peptides. Abeta is cleaved off from amyloid precursor protein (APP) with consecutive proteolytic processing by beta-secretase and gamma-secretase.Here, we show that CD74, the invariant chain of class II major histocompatibility complex, interacts with APP and serves as a negative regulator of Abeta. CD74 resembles other APP interacters such as BRI2 and BRI3, since all of them reduce the level of Abeta. However, unlike BRIs, CD74 does not reduce the secretion of sAPPalpha or sAPPbeta. Interestingly, in HeLa cells, over expression of CD74 steers APP, but not Notch, to large vacuoles created by CD74.Taken together, we propose that CD74 inhibits Abeta production by interacting with and derailing normal trafficking of APP.
|Altered expression of Abeta metabolism-associated molecules from d-galactose/AlCl(3) induced mouse brain. |
Yun Luo, Fengnan Niu, Zongzheng Sun, Wangsen Cao, Xin Zhang, Dening Guan, Zhimai Lv, Bing Zhang, Yun Xu, Yun Luo, Fengnan Niu, Zongzheng Sun, Wangsen Cao, Xin Zhang, Dening Guan, Zhimai Lv, Bing Zhang, Yun Xu, Yun Luo, Fengnan Niu, Zongzheng Sun, Wangsen Cao, Xin Zhang, Dening Guan, Zhimai Lv, Bing Zhang, Yun Xu, Yun Luo, Fengnan Niu, Zongzheng Sun, Wangsen Cao, Xin Zhang, Dening Guan, Zhimai Lv, Bing Zhang, Yun Xu
Mechanisms of ageing and development 130 248-52 2009
Cerebral deposition of amyloid-beta peptide (Abeta) is a critical feature of Alzheimer's disease (AD). Either aluminium trichloride (Al) or d-galactose (d-gal) induces Abeta overproduction in rat or mouse brain and has been used to produce models of aging and AD. Here it is shown that mice treated with Al plus d-gal represent a good model of AD with altered expression of Abeta metabolism-associated molecules. The work shows that Al/d-gal causes memory impairment and high Abeta levels in the cortex (Co) and hippocampus (Hi). Then, we found that beta-site APP cleavage enzyme 1 (BACE1) was increased in mouse Co and Hi. Al or Al plus d-gal suppressed mRNA of the low-density lipoprotein receptor-related protein 1(LRP1). d-gal also decreased the LRP expression in Hi, but not in Co. However, Al/d-gal did not affect the receptor for advanced glycation end products (RAGE) expression in mouse brains. Furthermore, Al/d-gal reduced the expression of neprilysin (NEP), but not the insulin degrading enzyme (IDE). This study indicates that Al/d-gal affects the expression of Abeta metabolism-associated molecules that are responsible for Abeta deposition during AD, suggesting that this mouse model can be a useful model for studying the mechanisms and biomarkers of AD and for drug screening.
|MPEP reduces seizure severity in Fmr-1 KO mice over expressing human Abeta. |
Westmark, CJ; Westmark, PR; Malter, JS
International journal of clinical and experimental pathology 3 56-68 2009
Metabotropic glutamate receptor 5 (mGluR(5)) regulates the translation of amyloid precursor protein (APP) mRNA. Under resting conditions, mRNA is bound to and translationally repressed by the fragile X mental retardation protein (FMRP). Upon group 1 mGluR activation, FMRP dissociates from the mRNA and translation ensues. APP levels are elevated in the dendrites of primary neuronal cultures as well as in synaptoneurosomes (SN) prepared from embryonic and juvenile fmr-1 knockout (KO) mice, respectively. In order to study the effects of APP and its proteolytic product Abeta on Fragile X syndrome (FXS) phenotypes, we created a novel mouse model (FRAXAD) that over-expresses human APPSwe/Abeta in an fmr-1 KO background. Herein, we assess (1) human APP(Swe) and Abeta levels as a function of age in FRAXAD mice, and (2) seizure susceptibility to pentylenetetrazol (PTZ) after mGluR(5) blockade. PTZ-induced seizure severity is decreased in FRAXAD mice pre-treated with the mGluR(5) antagonist MPEP. These data suggest that Abeta contributes to seizure incidence and may be an appropriate therapeutic target to lessen seizure pathology in FXS, Alzheimer's disease (AD) and Down syndrome (DS) patients.
|Generation and initial characterization of FDD knock in mice. |
Giliberto, L; Matsuda, S; Vidal, R; D'Adamio, L
PloS one 4 e7900 2009
Mutations in the integral membrane protein 2B, also known as BRI(2), a type II trans-membrane domain protein cause two autosomal dominant neurodegenerative diseases, Familial British and Danish Dementia. In these conditions, accumulation of a C-terminal peptide (ABri and ADan) cleaved off from the mutated precursor protein by the pro-protein convertase furin, leads to amyloid deposition in the walls of blood vessels and parenchyma of the brain. Recent advances in the understanding of the generation of amyloid in Alzheimer's disease has lead to the finding that BRI(2) interacts with the Amyloid Precursor Protein (APP), decreasing the efficiency of APP processing to generate Abeta. The interaction between the two precursors, APP and BRI(2), and possibly between Abeta and ABri or ADan, could be important in influencing the rate of amyloid production or the tendency of these peptides to aggregate.We have generated the first BRI(2) Danish Knock-In (FDD(KI)) murine model of FDD, expressing the pathogenic decamer duplication in exon 6 of the BRI(2) gene. FDD(KI) mice do not show any evident abnormal phenotype, with normal brain histology and no detectable amyloid deposition in blood vessel walls or parenchyma.This new murine mouse model will be important to further understand the interaction between APP and BRI(2), and to provide insights into the molecular basis of FDD.
|Loss of modifier of cell adhesion reveals a pathway leading to axonal degeneration. |
Chen, Q; Peto, CA; Shelton, GD; Mizisin, A; Sawchenko, PE; Schubert, D
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 118-30 2009
Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration.Full Text Article
|BRI3 inhibits amyloid precursor protein processing in a mechanistically distinct manner from its homologue dementia gene BRI2. |
Matsuda, S; Matsuda, Y; D'Adamio, L
The Journal of biological chemistry 284 15815-25 2009
Alzheimer disease (AD) is characterized by senile plaques, which are mainly composed of beta amyloid (Abeta) peptides. Abeta is cleaved off from amyloid precursor protein (APP) with consecutive proteolytic processing: beta-secretase, followed by gamma-secretase. Here, we show that BRI3, a member of the BRI gene family that includes the familial British and Danish dementia gene BRI2, interacts with APP and serves as an endogenous negative regulator of Abeta production. BRI3 colocalizes with APP along neuritis in differentiated N2a cells; endogenous BRI3-APP complexes are readily detectable in mouse brain extract; reducing endogenous BRI3 levels by RNA interference results in increased Abeta secretion. BRI3 resembles BRI2, because BRI3 overexpression reduces both alpha- and beta-APP cleavage. We propose that BRI3 inhibits the various processing of APP by blocking the access of alpha- and beta-secretases to APP. However, unlike BRI2, the binding of BRI3 to the beta-secretase cleaved APP C-terminal fragment is negligible and BRI3 does not cause the massive accumulation of this APP fragment, suggesting that, unlike BRI2, BRI3 is a poor gamma-cleavage inhibitor. Competitive inhibition of APP processing by BRI3 may provide a new approach to AD therapy and prevention.
|Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo. |
Beirowski, B; Babetto, E; Gilley, J; Mazzola, F; Conforti, L; Janeckova, L; Magni, G; Ribchester, RR; Coleman, MP
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 653-68 2009
Axon degeneration contributes widely to neurodegenerative disease but its regulation is poorly understood. The Wallerian degeneration slow (Wld(S)) protein protects axons dose-dependently in many circumstances but is paradoxically abundant in nuclei. To test the hypothesis that Wld(S) acts within nuclei in vivo, we redistributed it from nucleus to cytoplasm in transgenic mice. Surprisingly, instead of weakening the phenotype as expected, extranuclear Wld(S) significantly enhanced structural and functional preservation of transected distal axons and their synapses. In contrast to native Wld(S) mutants, distal axon stumps remained continuous and ultrastructurally intact up to 7 weeks after injury and motor nerve terminals were robustly preserved even in older mice, remaining functional for 6 d. Moreover, we detect extranuclear Wld(S) for the first time in vivo, and higher axoplasmic levels in transgenic mice with Wld(S) redistribution. Cytoplasmic Wld(S) fractionated predominantly with mitochondria and microsomes. We conclude that Wld(S) can act in one or more non-nuclear compartments to protect axons and synapses, and that molecular changes can enhance its therapeutic potential.
|Flupirtine as neuroprotective add-on therapy in autoimmune optic neuritis. |
Sättler, MB; Williams, SK; Neusch, C; Otto, M; Pehlke, JR; Bähr, M; Diem, R
The American journal of pathology 173 1496-507 2008
Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system that results in persistent impairment in young adults. During chronic progressive disease stages, there is a strong correlation between neurodegeneration and disability. Current therapies fail to prevent progression of neurological impairment during these disease stages. Flupirtine, a drug approved for oral use in patients suffering from chronic pain, was used in a rat model of autoimmune optic neuritis and significantly increased the survival of retinal ganglion cells, the neurons that form the axons of the optic nerve. When flupirtine was combined with interferon-beta, an established immunomodulatory therapy for MS, visual functions of the animals were improved during the acute phase of optic neuritis. Furthermore, flupirtine protected retinal ganglion cells from degeneration in a noninflammatory animal model of optic nerve transection. Although flupirtine was shown previously to increase neuronal survival by Bcl-2 up-regulation, this mechanism does not appear to play a role in flupirtine-mediated protection of retinal ganglion cells either in vitro or in vivo. Instead, we showed through patch-clamp investigations that the activation of inwardly rectifying potassium channels is involved in flupirtine-mediated neuroprotection. Considering the few side effects reported in patients who receive long-term flupirtine treatment for chronic pain, our results indicate that this drug is an interesting candidate for further evaluation of its neuroprotective potential in MS.Full Text Article
|Strain-specific susceptibility for neurodegeneration in a rat model of autoimmune optic neuritis. |
Muriel B Sättler, Mauro Togni, Ivana Gadjanski, Kurt-Wolfram Sühs, Nadine Meyer, Mathias Bähr, Ricarda Diem
Journal of neuroimmunology 193 77-86 2008
Heterogeneity in clinical disease course and histopathology complicates the treatment of multiple sclerosis. We detected important differences in neurodegeneration in various subtypes of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. Dark Agouti (DA) rats showed a significantly higher survival of retinal ganglion cells in comparison to Brown Norway rats. After surgical transection of the optic nerve neuronal loss was similar in both rat strains. We identified an increased expression of interleukin 1beta and glial cell line-derived neurotrophic factor in DA rats as the possible mechanism of the observed endogenous neuroprotection in MOG-induced optic neuritis.
|Enhanced sensitivity of striatal neurons to axonal transport defects induced by mutant huntingtin. |
Her, LS; Goldstein, LS
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 13662-72 2008
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease linked to a polyQ (polyglutamine) expansion in the huntingtin protein. Although general brain atrophy is found in HD patients, the striatum is the most severely affected region. Loss or mutant forms of huntingtin were reported to disrupt fast axonal transport in Drosophila, squid, and mice. However, previous work did not resolve whether mutant huntingtin affects global axonal transport or only a subset of cargoes, nor did it resolve whether striatal neurons are preferentially sensitive to huntingtin-mediated defects. We used amyloid precursor protein (APP)-yellow fluorescent protein and brain-derived neurotrophic factor (BDNF)-mCherry fusion proteins as markers for fast axonal transport when huntingtin is altered. We found that movement of APP and BDNF is impaired in striatal and hippocampal, but not cortical, neurons from presymptomatic homozygous mutant mice carrying 150Q huntingtin knock-in mutations. In addition, loss of huntingtin disrupts APP axonal transport, whereas overexpression of wild-type, but not mutant, huntingtin enhances APP transport in all three types of neurons tested. These data suggest that a loss of wild-type huntingtin function in fast axonal transport plays important roles in the development of cell-type-specific defects in HD.
|Activity requires soluble amyloid precursor protein alpha to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway. |
Nidhi Gakhar-Koppole, Phillip Hundeshagen, Claudia Mandl, Sascha W Weyer, Bernadette Allinquant, Ulrike Müller, Francesca Ciccolini, Nidhi Gakhar-Koppole, Phillip Hundeshagen, Claudia Mandl, Sascha W Weyer, Bernadette Allinquant, Ulrike Müller, Francesca Ciccolini
The European journal of neuroscience 28 871-82 2008
It is known that activity modulates neuronal differentiation in the adult brain but the signalling mechanisms underlying this process remain to be identified. We show here that activity requires soluble amyloid precursor protein (sAPP) to enhance neurite outgrowth of young neurons differentiating from neural stem cells. Inhibition of sAPP secretion and anti-APP antibodies both abolished the effect of depolarization on neurite outgrowth, whereas exogenous sAPPalpha, similar to depolarization, induced neurite elongation. Depolarization and sAPPalpha both required active N-methyl-D-aspartic acid receptor (NMDAR) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) recruitment to induce neurite outgrowth. However, depolarization and sAPPalpha played different roles in modulating this signalling cascade. Depolarization induced ERK phosphorylation with fast kinetics via activation of NMDAR. By contrast, acute application of sAPPalpha did not lead to ERK activation. However, continuous generation of sAPPalpha was necessary for depolarization-induced ERK phosphorylation, indicating that sAPPalpha promotes MAPK/ERK recruitment by an indirect mechanism. In addition, we found that blockade of NMDAR down-regulated APP expression, whereas depolarization increased sAPPalpha, suggesting that activity may also act upstream of sAPP signalling by regulating the amount of cellular APP and extracellular sAPPalpha. Finally, we show that soluble amyloid precursor-like protein 2 (sAPLP2), but not sAPLP1, is functionally redundant to sAPP in promoting neurite outgrowth and that soluble members of the APP family require membrane-bound APP to enhance neurite outgrowth. In summary, these experiments indicate a novel role of APP family members in activity-dependent neuronal differentiation.
|BRI2 inhibits amyloid beta-peptide precursor protein processing by interfering with the docking of secretases to the substrate. |
Matsuda, S; Giliberto, L; Matsuda, Y; McGowan, EM; D'Adamio, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 8668-76 2008
Genetic alterations of amyloid beta-peptide (Abeta) production caused by mutations in the Abeta precursor protein (APP) cause familial Alzheimer's disease (AD). Mutations in BRI2, a gene of undefined function, are linked to familial British and Danish dementias, which are pathologically and clinically similar to Alzheimer's disease. We report that BRI2 is a physiological suppressor of Abeta production. BRI2 restrict docking of gamma-secretase to APP and access of alpha- and beta-secretases to their cleavage APP sequences. Alterations of BRI2 by gene targeting or transgenic expression regulate Abeta levels and AD pathology in mouse models of AD. Competitive inhibition of APP processing by BRI2 may provide a new approach to AD therapy and prevention.
|Deletion of Mint proteins decreases amyloid production in transgenic mouse models of Alzheimer's disease. |
Ho, A; Liu, X; Südhof, TC
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 14392-400 2008
Mints/X11s are neuronal adaptor proteins that bind to amyloid-beta precursor protein (APP). Previous studies suggested that Mint/X11 proteins influence APP cleavage and affect production of pathogenic amyloid-beta (Abeta) peptides in Alzheimer's disease; however, the biological significance of Mint/X11 binding to APP and their possible role in Abeta production remain unclear. Here, we crossed conditional and constitutive Mint1, Mint2, and Mint3 knock-out mice with transgenic mouse models of Alzheimer's disease overproducing human Abeta peptides. We show that deletion of all three individual Mint proteins delays the age-dependent production of amyloid plaque numbers and Abeta40 and Abeta42 levels with loss of Mint2 having the largest effect. Acute conditional deletion of all three Mints in cultured neurons suppresses the accumulation of APP C-terminal fragments and the secretion of ectodomain APP by decreasing beta-cleavage but does not impair subsequent gamma-cleavage. These results suggest that the three Mint/X11 proteins regulate Abeta production by a novel mechanism that may have implications for therapeutic approaches to altering APP cleavage in Alzheimer's disease.Full Text Article
|Shaken baby syndrome: re-examination of diffuse axonal injury as cause of death. |
Manfred Oehmichen, Daniela Schleiss, Ingo Pedal, Klaus-Steffen Saternus, Ivana Gerling, Christoph Meissner
Acta neuropathologica 116 317-29 2008
The discussion surrounding shaken baby syndrome (SBS) arose from the lack of evidence implicating diffuse axonal injury (DAI) as a cause of death. It was assumed instead that injury to the cervical cord, medulla, and nerve roots played a causal role. The present pathomorphological study examines 18 selected infants (1-year-old) whose deaths were highly suspicious for SBS, exhibiting the classical SBS triad of acute subdural hemorrhage (SDH), retinal bleeding, and encephalopathy. Gross autopsy and microscopic findings of these infants were compared with those of 19 victims of sudden infant death syndrome (SIDS; control group 1) and of 14 infants who died of disease or injuries/violence not involving the head, neck or eyes (control group 2). Symptoms of mechanical impact to the head were evident in seven of the SBS infants, but in none of the control infants. DAI was not detected in either the SBS or control cases. Localized axonal injury (AI) was regularly present in the brains of the SBS infants surviving longer than 1.5-3.0 h, but only occasionally in the craniocervical junction and within the nerve roots of the upper cervical cord; it was never present in the medulla. Epidural hemorrhage of the cervical cord was seen in four of the ten examined SBS cases, but in none of the control cases. Based on the absence of DAI in the brain and of signs of generalized cervical cord or nerve root injuries, we conclude that the cause of death in the SBS victims was a global cerebral ischemia secondary to SDH, focal vasospasm, trauma-induced transitory respiratory and/or circulatory failure.
|Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice. |
Liao, CW; Fan, CK; Kao, TC; Ji, DD; Su, KE; Lin, YH; Cho, WL
BMC infectious diseases 8 84 2008
Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT.BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR.Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain.Further studies are needed to determine whether there is an increased risk of CT progression into neurodegenerative disease because neurodegeneration-associated AbetaPP and phosphorylated tau emerged in the brain.
|Histological and functional outcomes after traumatic brain injury in mice null for the erythropoietin receptor in the central nervous system. |
Xiong, Y; Mahmood, A; Lu, D; Qu, C; Kazmi, H; Goussev, A; Zhang, ZG; Noguchi, CT; Schallert, T; Chopp, M
Brain research 1230 247-57 2008
Erythropoietin (EPO) and its receptor (EPOR), essential for erythropoiesis, are expressed in the nervous system. Recombinant human EPO treatment promotes functional outcome after traumatic brain injury (TBI) and stroke, suggesting that the endogenous EPO/EPOR system plays an important role in neuroprotection and neurorestoration. This study was designed to investigate effects of the EPOR on histological and functional outcomes after TBI. Experimental TBI was induced in adult EPOR-null and wild-type mice by controlled cortical impact. Neurological function was assessed using the modified Morris Water Maze and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemistry. As compared to the wild-type injured mice, EPOR-null mice did not exhibit higher susceptibility to TBI as exemplified by tissue loss in the cortex, cell loss in the dentate gyrus, impaired spatial learning, angiogenesis and cell proliferation. We observed that less cortical neurogenesis occurred and that sensorimotor function (i.e., footfault) was more impaired in the EPOR-null mice after TBI. Co-accumulation of amyloid precursor protein (axonal injury marker) and calcium was observed in the ipsilateral thalamus in both EPOR-null and wild-type mice after TBI with more calcium deposits present in the wild-type mice. This study demonstrates for the first time that EPOR null in the nervous system aggravates sensorimotor deficits, impairs cortical neurogenesis and reduces thalamic calcium precipitation after TBI.
|In vivo beta-secretase 1 inhibition leads to brain Abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin-1. |
Sankaranarayanan, S; Price, EA; Wu, G; Crouthamel, MC; Shi, XP; Tugusheva, K; Tyler, KX; Kahana, J; Ellis, J; Jin, L; Steele, T; Stachel, S; Coburn, C; Simon, AJ
The Journal of pharmacology and experimental therapeutics 324 957-69 2008
beta-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid beta peptide Abeta42, the putative neurotoxic species in Alzheimer's disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC(50) approximately 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Abeta40 and Abeta42. BACE1 inhibition also led to a robust brain secreted (s)APPbeta lowering that was accompanied by an increase in brain sAPPalpha levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (-/-) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/-) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (-/-) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Abeta lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major beta-site cleavage enzyme for APP and that its inhibition can lower brain Abeta and redirect APP processing via the potentially nonamyloidogenic alpha-secretase pathway, without significantly altering NRG-1 processing.
|Myelin localization of peptidylarginine deiminases 2 and 4: comparison of PAD2 and PAD4 activities. |
Wood, DD; Ackerley, CA; Brand, Bv; Zhang, L; Raijmakers, R; Mastronardi, FG; Moscarello, MA
Laboratory investigation; a journal of technical methods and pathology 88 354-64 2008
An understanding of the structure and composition of the myelin sheath is essential to understand the pathogenesis of demyelinating diseases such as multiple sclerosis (MS). The presence of citrulline in myelin proteins in particular myelin basic protein (MBP) causes an important change in myelin structure, which destabilizes myelin. The peptidylarginine deiminases (PADs) are responsible for converting arginine in proteins to citrulline. Two of these, PAD2 and PAD4, were localized to the myelin sheath by immunogold electron microscopy. Deimination of MBP by the recombinant forms of these enzymes showed that it was extensive, that is, PAD2 deiminated 18 of 19 arginyl residues in MBP, whereas PAD4 deiminated 14 of 19 residues. In the absence of PAD2 (the PAD2-knockout mouse) PAD4 remained active with limited deimination of arginyl residues. In myelin isolated from patients with MS, the amounts of both PAD2 and PAD4 enzymes were increased compared with that in normals, and the citrullinated proteins were also increased. These data support the view that an increase in citrullinated proteins resulting from increased PAD2 and 4 is an important change in the pathogenesis of MS.
|Beta-amyloid expression, release and extracellular deposition in aged rat brain slices. |
Marksteiner, J; Humpel, C
Molecular psychiatry 13 939-52 2008
Alzheimer's disease (AD) is characterized by beta-amyloid plaques, tau pathology, cholinergic cell death and inflammation. The aim of this study was to investigate whether beta-amyloid is generated, released and extracellularly deposited in organotypic brain slices. In developing slices, no amyloid-precursor protein (APP) was detectable; however, there was a strong upregulation in aging slices. In such slices, rat beta-amyloid(1-42) and -(1-40) peptides were found using four sequence-specific antibodies. APP and beta-amyloid were expressed in neurons and to a lesser extent in astrocytes. Beta-amyloid was secreted into the medium. Beta-amyloid was located extracellularly when aging slices were incubated with medium at pH 6.0 including apolipoprotein E4 (ApoE4). It is concluded that aging organotypic brain slices express beta-amyloid and that acidosis induces cell death with efflux of beta-amyloid and extracellular depositions, which is triggered by ApoE4. This novel in vitro model may enable us to investigate further the pathological cascade for AD and may be useful to explore future therapeutics.
|Histopathological and molecular heterogeneity among individuals with dementia associated with Presenilin mutations. |
Maarouf, CL; Daugs, ID; Spina, S; Vidal, R; Kokjohn, TA; Patton, RL; Kalback, WM; Luehrs, DC; Walker, DG; Castaño, EM; Beach, TG; Ghetti, B; Roher, AE
Molecular neurodegeneration 3 20 2008
Mutations in the presenilin (PSEN) genes are associated with early-onset familial Alzheimer's disease (FAD). Biochemical characterizations and comparisons have revealed that many PSEN mutations alter gamma-secretase activity to promote accumulation of toxic Abeta42 peptides. In this study, we compared the histopathologic and biochemical profiles of ten FAD cases expressing independent PSEN mutations and determined the degradation patterns of amyloid-beta precursor protein (AbetaPP), Notch, N-cadherin and Erb-B4 by gamma-secretase. In addition, the levels of Abeta40/42 peptides were quantified by ELISA.We observed a wide variation in type, number and distribution of amyloid deposits and neurofibrillary tangles. Four of the ten cases examined exhibited a substantial enrichment in the relative proportions of Abeta40 over Abeta42. The AbetaPP N-terminal and C-terminal fragments and Tau species, assessed by Western blots and scanning densitometry, also demonstrated a wide variation. The Notch-1 intracellular domain was negligible by Western blotting in seven PSEN cases. There was significant N-cadherin and Erb-B4 peptide heterogeneity among the different PSEN mutations.These observations imply that missense mutations in PSEN genes can alter a range of key gamma-secretase activities to produce an array of subtly different biochemical, neuropathological and clinical manifestations. Beyond the broad common features of dementia, plaques and tangles, the various PSEN mutations resulted in a wide heterogeneity and complexity and differed from sporadic AD.
|3,4-Methylenedioxymethamphetamine (MDMA), but not morphine, alters APP processing in the rat brain. |
Kálmán, János, et al.
Int. J. Neuropsychopharmacol., 10: 183-90 (2007) 2007
The abuse of drugs such as opioids and 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') can have detrimental effects on the cognitive functions, but the exact molecular mechanism whereby these drugs promote neurodegeneration remains to be elucidated. The major purpose of the present pilot study was to determine whether the chronic in-vivo administration of morphine (10 mg/kg) or MDMA (1 mg/kg) to rats can alter the expression and processing of amyloid precursor protein (APP), the central molecule in the proposed pathomechanism of Alzheimer's disease. MDMA treatment significantly decreased the production of APP in the cytosolic fraction of the brain cortex. A concomitant 25% increase was found both in the beta-secretase (BACE) and APP mRNA levels (108%). In contrast, in the applied single dosage chronic morphine treatment did not influence either the APP and BACE protein levels or the APP mRNA production. These results indicate that the chronic use of 'ecstasy', but not morphine, may be harmful via a novel mode of action, i.e. by altering the APP expression and processing in the brain.
|Cholesterol independent effect of LXR agonist TO-901317 on gamma-secretase. |
Christian Czech, Mark P Burns, Lilit Vardanian, Angelique Augustin, Helmut Jacobsen, Karlheinz Baumann, G William Rebeck
Journal of neurochemistry 101 929-36 2007
The balance of intracellular cholesterol has proven to be critical to the production of beta-amyloid (A beta). Reducing cholesterol in vitro leads to decreased production of A beta, whereas an increase in cellular cholesterol induces A beta production. Liver X Receptor (LXR) agonists are known to increase cholesterol efflux from cells, but there are conflicting reports as to the effects of these agonists on A beta production. We therefore examined the effects of efflux-inducing agents on A beta production in vitro. We used methyl-beta-cyclodextrin and an LXR agonist (TO-901317) to induce cholesterol efflux and studied the resulting A beta production in a stable amyloid precursor protein (APP) -transfected cell line. When cholesterol efflux was induced with methyl-beta-cyclodextrin there was a >60% decrease in A beta(40) and A beta(42) production. However, while activation of LXR using TO-901317-induced cholesterol efflux in the presence of a cholesterol acceptor, no changes in A beta levels were recorded. When cells were incubated with TO-901317 above the concentration required for maximal cholesterol efflux, there was a 150% increase in A beta(42) levels. The absence of a cholesterol acceptor from the culture media (preventing cholesterol efflux) did not blunt this increase in A beta(42), suggesting that the effects of TO-901317 on A beta(42) are efflux independent. These results were confirmed in APP stably transfected human H4 cells, which revealed in addition to a 200% increase in A beta(42) levels, a concomitant 80% reduction in A beta(38). A cell-free gamma-secretase assay confirmed that TO-901317 can directly alter gamma-secretase activity. These data demonstrate that TO-901317 can directly modulate the site of cleavage of APP by gamma-secretase in vitro.
|Nogo-A is involved in secondary axonal degeneration of thalamus in hypertensive rats with focal cortical infarction. |
Fang Wang, Zhijian Liang, Qinghua Hou, Shihui Xing, Li Ling, Meixia He, Zhong Pei, Jinsheng Zeng
Neuroscience letters 417 255-60 2007
We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid betaA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.
|Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival. |
Gross, I; Armant, O; Benosman, S; de Aguilar, JL; Freund, JN; Kedinger, M; Licht, JD; Gaiddon, C; Loeffler, JP
Cell death and differentiation 14 1802-12 2007
Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.
|The amyloid-beta precursor protein is phosphorylated via distinct pathways during differentiation, mitosis, stress, and degeneration. |
Muresan, Z; Muresan, V
Molecular biology of the cell 18 3835-44 2007
Phosphorylation of amyloid-beta precursor protein (APP) at Thr(668) is a normal process linked to neurite extension and anterograde transport of vesicular cargo. By contrast, increased phosphorylation of APP is a pathological trait of Alzheimer's disease. APP is overexpressed in Down's syndrome, a condition that occasionally leads to increased APP phosphorylation, in cultured cells. Whether phosphorylation of APP in normal versus high APP conditions occurs by similar or distinct signaling pathways is not known. Here, we addressed this problem using brainstem-derived neurons (CAD cells). CAD cells that ectopically overexpress APP frequently show features of degenerating neurons. We found that, in degenerating cells, APP is hyperphosphorylated and colocalizes with early endosomes. By contrast, in normal CAD cells, phosphorylated APP (pAPP) is excluded from endosomes, and localizes to the Golgi apparatus and to transport vesicles within the neurites. Whereas the neuritic APP is phosphorylated by c-Jun NH(2)-terminal kinase through a pathway that is modulated by glycogen synthase kinase 3beta, the endosomal pAPP in degenerated CAD cells results from activation of cyclin-dependent kinase 5. Additional signaling pathways, leading to APP phosphorylation, become active during stress and mitosis. We conclude that distinct pathways of APP phosphorylation operate in proliferating, differentiating, stressed, and degenerating neurons.Full Text Article
|Interaction of ASK1 and the beta-amyloid precursor protein in a stress-signaling complex. |
Galvan, V; Banwait, S; Spilman, P; Gorostiza, OF; Peel, A; Ataie, M; Crippen, D; Huang, W; Sidhu, G; Ichijo, H; Bredesen, DE
Neurobiology of disease 28 65-75 2007
The amyloid precursor protein (APP) is a type I transmembrane protein translocated to neuronal terminals, whose function is still unknown. The C-terminus of APP mediates its interaction with cellular adaptor and signaling proteins, some of which signal to the stress-activated protein kinase (SAPK) pathway. Here we show that ASK1, a MAPKKK that activates two SAPKs, c-Jun N-terminal-kinase (JNK) and p38, is present in a complex containing APP, phospho-MKK6, JIP1 and JNK1. In primary neurons deprived of growth factors, as well as in brains of (FAD)APP-transgenic mice, ASK1 was upregulated in neuronal projections, where it interacted with APP. In non-transgenic brains, ASK1 and APP associated mainly in the ER. Our results indicate that recruitment of ASK1 to stress-signaling complexes assembled with APP may be triggered and enhanced by cellular stress. Thus, ASK1 may be the apical MAPKKK in a signaling complex assembled with APP as a response to stress.
|FMRP mediates mGluR5-dependent translation of amyloid precursor protein. |
Westmark, CJ; Malter, JS
PLoS biology 5 e52 2007
Amyloid precursor protein (APP) facilitates synapse formation in the developing brain, while beta-amyloid (Abeta) accumulation, which is associated with Alzheimer disease, results in synaptic loss and impaired neurotransmission. Fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA binding protein whose expression is lost in fragile X syndrome. Here we show that FMRP binds to the coding region of APP mRNA at a guanine-rich, G-quartet-like sequence. Stimulation of cortical synaptoneurosomes or primary neuronal cells with the metabotropic glutamate receptor agonist DHPG increased APP translation in wild-type but not fmr-1 knockout samples. APP mRNA coimmunoprecipitated with FMRP in resting synaptoneurosomes, but the interaction was lost shortly after DHPG treatment. Soluble Abeta40 or Abeta42 levels were significantly higher in multiple strains of fmr-1 knockout mice compared to wild-type controls. Our data indicate that postsynaptic FMRP binds to and regulates the translation of APP mRNA through metabotropic glutamate receptor activation and suggests a possible link between Alzheimer disease and fragile X syndrome.
|Mechanisms of copper ion mediated Huntington's disease progression. |
Fox, JH; Kama, JA; Lieberman, G; Chopra, R; Dorsey, K; Chopra, V; Volitakis, I; Cherny, RA; Bush, AI; Hersch, S
PloS one 2 e334 2007
Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+)in vitro in a 1:1 copper:protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics.
|Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis. |
Bjelik, Annamária, et al.
Neurochem. Int., 49: 393-400 (2006) 2006
Epidemiological and biochemical data suggest a link between the cholesterol metabolism, the amyloid precursor protein (APP) processing and the increased cerebral beta-amyloid (Abeta) deposition in Alzheimer's disease (AD). The individual and combined effects of a high-cholesterol (HC) diet and the overexpression of the human apoB-100 gene were therefore examined on the cerebral expression and processing of APP in homozygous apoB-100 transgenic mice [Tg (apoB(+/+))], a validated model of atherosclerosis. When fed with 2% cholesterol for 17 weeks, only the wild-type mice exhibited significantly increased APP695 (123%) and APP770 (138%) mRNA levels in the cortex. The HC diet-induced hypercholesterolemia significantly increased the APP isoform levels in the membrane-bound fraction, not only in the wild-type animals (114%), but also in the Tg apoB(+/+) group (171%). The overexpression of human apoB-100 gene by the liver alone reduced the brain APP isoform levels in the membrane-bound fraction (78%), whereas the levels were increased by the combined effect of HC and the overexpression of the human apoB-100 gene (134%). The protein kinase C and beta-secretase protein levels were not altered by the individual or combined effects of these two factors. Our data indicate that the two atherogenic factors, the HC diet and the overexpression of the human apoB-100 gene by the liver, could exert different effects on the processing and expression of APP in the mice brain.
|Transient axonal injury in the absence of demyelination: a correlate of clinical disease in acute experimental autoimmune encephalomyelitis. |
Aboul-Enein, Fahmy, et al.
Acta Neuropathol., 111: 539-47 (2006) 2006
Axonal degeneration contributes to the transient and permanent neurological deficits seen in multiple sclerosis, an inflammatory disease of the central nervous system. To study the immunological mechanisms causing axonal degeneration, we induced experimental autoimmune encephalomyelitis (EAE) in wildtype Lewis rats and Lewis rats with a slowly progressive myelin degeneration due to proteolipid protein (PLP) overexpression. EAE was triggered either by the transfer of encephalitogenic T-cells alone or by the co-transfer of T-cells with demyelinating antibodies. Inducible nitric oxide synthase (iNOS) expression in perivascular macrophages was associated with a transient functional disturbance of axons, reflected by the focal and reversible accumulation of amyloid precursor protein. Clinical disease correlated with the numbers of APP positive axon spheroids. Demyelination was associated with a further increase of iNOS expression in macrophages and with a higher degree of axonal injury. Our studies suggest that nitric oxide and its metabolites contribute to axonal pathology and possibly also to subsequent neurological dysfunction in EAE.
|Heterogeneous hyperactivity and distribution of ischemic lesions after focal cerebral ischemia in Mongolian gerbils. |
Noriko Katsumata, Toshihiko Kuroiwa, Satoru Ishibashi, Shihong Li, Shu Endo, Kikuo Ohno
Neuropathology : official journal of the Japanese Society of Neuropathology 26 283-92 2006
Various types of poststroke hyperactivity exist in humans, but studies of each mechanism using animal models are scarce. We aimed to analyze the heterogeneity of postischemic hyperlocomotion and to identify the ischemic lesions responsible for postischemic hyperlocomotion in rodent models of focal ischemia. Mongolian gerbils underwent right common carotid artery occlusion (CCAO) for 10 or 20 min. At 24 h, 2 days, and 7 days postischemia, we performed quantitative and qualitative locomotor analysis and correlated these results with the extent of ischemic lesions. Intermittent explosive hyperlocomotion was induced transiently in a 10-min CCAO group at 24 h after ischemia and continual unexplosive hyperlocomotion persisted for 7 days in the 20-min CCAO animals. Selective neuronal death, confined to the hippocampal cornu ammonis 1 (CA1), was observed in the 10-min CCAO group and widespread cortical and basal ganglia infarction was observed in the 20-min CCAO group. Amyloid precursor protein was transiently observed in the hippocampus at 24 h postischemia in the 10-min CCAO animals, while it was widely distributed over the ischemic regions throughout the 7 days postischemia in the 20-min CCAO animals. Incidence maps and correlation analysis revealed hippocampal neuronal death of the CA1 sector and widespread hemispheric infarction, including the cortex, as the region responsible for the 10-min and 20-min CCAO-induced hyperactivity, respectively. Two distinct types of locomotor hyperactivity were observed that varied with regard to the distribution of the ischemic lesion, that is, hippocampal neuronal death and widespread infarction involving the cortex. These two types of locomotor hyperactivity appear to be models of the different types of poststroke hyperactivity seen in stroke patients.
|Polychlorinated biphenyls alter expression of alpha-synuclein, synaptophysin and parkin in the rat brain. |
Katarzyna Malkiewicz, Roma Mohammed, Ronnie Folkesson, Bengt Winblad, Miroslaw Szutowski, Eirikur Benedikz
Toxicology letters 161 152-8 2006
Polychlorinated Biphenyls (PCBs)-induced changes in synaptic transmission are one of the effects of their neurotoxicity but the mechanism remains unknown. We assessed the in vivo effects of the PCBs mixture, Aroclor 1254 on the expression of neuronal proteins that are involved in the synaptic function and/or are associated with neurodegeneration. Wistar rats were treated orally with repeated doses of Aroclor 1254 and the levels of soluble alpha-synuclein, parkin, synaptophysin and amyloid precursor protein (APP) in the brain were determined by Western blotting. The results showed that Aroclor did not cause changes in the expression and processing of APP but at a dose 100 microg/g/day repeated for 6 days caused a decrease in the expression of alpha-synuclein in the cerebellum, cortex, hippocampus and hypothalamus of the animals sacrificed 2 days after treatment. The decrease in alpha-synuclein was accompanied by a transient increase in parkin and synaptophysin levels. Interestingly, in the hypothalamus the levels of alpha-synuclein remained decreased after 21 days post treatment perhaps due to regional differences in the PCBs elimination or perhaps a more specific interaction with the dopaminergic cells that are present in the hypothalamus that needs to be investigated further.
|Effects of interferon-beta-1a on neuronal survival under autoimmune inflammatory conditions. |
Muriel B Sättler, Iris Demmer, Sarah K Williams, Katharina Maier, Doron Merkler, Ivana Gadjanski, Christine Stadelmann, Mathias Bähr, Ricarda Diem
Experimental neurology 201 172-81 2006
Interferon-beta-1a (IFN-beta-1a) is an approved treatment for multiple sclerosis (MS). It improves the disease course by reducing the relapse rate as well as the persistent neurological deficits. Recent MRI and post-mortem studies revealed that neuronal and axonal damage are most relevant for chronic disability in MS patients. We have characterized previously time course and mechanisms of neuronal apoptosis in a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis. In this animal model, application of IFN-beta-1a three times per week slightly decreases the loss of retinal ganglion cells (RGCs), the neurons that form the axons within the optic nerve. In contrast to neurotrophic factors, this cytokine does not directly protect cultured RGCs from apoptosis. We conclude that IFN-beta-1a is a suitable candidate to be combined with a directly neuroprotective agent in order to further decrease axonal and neuronal degeneration in MS patients.
|Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells. |
Muresan, Z; Muresan, V
Molecular and cellular biology 26 4982-97 2006
Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.
|EAE in beta-2 microglobulin-deficient mice: axonal damage is not dependent on MHC-I restricted immune responses. |
Ralf A Linker, Evelyn Rott, H H Hofstetter, T Hanke, Klaus V Toyka, Ralf Gold
Neurobiology of disease 19 218-28 2005
There is accumulating evidence that CD8-positive (CD8+) T-cells and MHC-I expression may also play a role in neurodegeneration associated with multiple sclerosis (MS). We investigated the role of MHC-I and CD8+ T-cells by studying experimental autoimmune encephalomyelitis (EAE) in beta-2 microglobulin knockout mice induced by myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 or whole rat myelin basic protein (rMBP). For both encephalitogens and even after reconstitution of the immune system with MHC-I-positive bone marrow and transfer of mature CD8+ T-cells (iMHC-I+ CD8+ beta2m-/- mice), the disease course in beta2m-/- mice was significantly more severe with a 10-fold increased mortality in the beta2m-/- mice as compared to wild-type C57BL/6 mice. EAE in beta2m-/- mice caused more severe demyelination after immunization with MOG than with rMBP and axonal damage was more marked with rMBP as well as MOG even in iMHC-I+ CD8+ beta2m-/- mice. Immunocytochemical analysis of spinal cord tissue revealed a significant increase in macrophage and microglia infiltration in beta2m-/- and iMHC-I+ CD8+ beta2m-/- mice. The different pattern of T-cell infiltration was underscored by a 2.5-fold increase in CD4-positive (CD4+) T-cells in beta2m-/- mice after induction of MOG 35-55 EAE. We conclude that lack of functional MHC-I molecules and CD8+ T-cells aggravates autoimmune tissue destruction in the CNS. Enhanced axonal damage speaks for pathways of tissue damage independent of CD8+ T-cells and neuronal MHC-I expression.
|Nuclear translocation of the p75 neurotrophin receptor cytoplasmic domain in response to neurotrophin binding |
Frade, J. M.
J. Neurosci., 25(6):1407-1411 (2005) 2005
|Imipramine and citalopram facilitate amyloid precursor protein secretion in vitro. |
Pákáski, Magdolna, et al.
Neurochem. Int., 47: 190-5 (2005) 2005
|Lengthening of G2/mitosis in cortical precursors from mice lacking beta-amyloid precursor protein. |
López-Sánchez, N, et al.
Neuroscience, 130: 51-60 (2005) 2005
The beta-amyloid precursor protein (APP) is expressed within the nervous system, even at the earliest stages of embryonic development when cell growth and proliferation is particularly important. In order to study the function of APP at these early developmental stages, we have studied the development of the cerebral cortex in both wild type and App-/- mutant mice. Here, we demonstrate that APP mRNA is expressed in cortical precursor cells and that APP protein is concentrated within their apical domains during interphase. However, during mitosis, APP re-localizes to the peripheral space surrounding the metaphase plate. In APP-deficient cortical precursors, the duration of mitosis is increased and a higher proportion of cortical precursor cells contained nuclei in late G2. We conclude that during cortical development APP plays a role in controlling cell cycle progression, particularly affecting G2 and mitosis. These observations may have important implications for our understanding of how APP influences the progression of Alzheimer's disease, since degenerating cortical neurons have been shown to up-regulate cell cycle markers and re-enter the mitotic cycle before dying.
|Prion disease with a 144 base pair insertion: unusual cerebellar prion protein immunoreactivity. |
Ellen Gelpi, Gabor G Kovacs, Thomas Ströbel, Oskar Koperek, Till Voigtländer, Pawel P Liberski, Herbert Budka
Acta neuropathologica 110 513-9 2005
Sporadic, acquired, and genetic human prion diseases are characterized neuropathologically by distinct deposition patterns of the abnormal, disease-associated form of the prion protein (PrP(sc)). In addition to mutations in the prion protein gene (PRNP), PrP(sc) immunostaining patterns correlate with molecular phenotypes of prion diseases defined by the PRNP polymorphism at codon 129 and with protease-resistant PrP classified by Western blotting. Some point or insertional PRNP mutations share similar clinical and neuropathological phenotypes, whereas others show great variability even within the same family. Here we report a patient who presented clinically as sporadic Creutzfeldt-Jakob disease (CJD). Histologically moderate spongiform change was seen in cerebral and cerebellar cortical areas. Neuronal loss was restricted mainly to the occipital cortex and the basal ganglia. Surprisingly, numerous eosinophilic globular structures were noted in the molecular layer and the parahippocampal gyrus. These globules showed intense PrP immunopositivity using anti-PrP antibodies against different epitopes. They were stained with PAS but lacked congophilia and birefringence in polarized light. Ultrastructurally, globules were composed of 21-nm-thick intermingled filaments without dense core. Genetic analysis revealed a PRNP 144 base pair insertion. Our case reinforces the importance of molecular genetic diagnosis, especially in those patients who lack a family history of prion disease and show unusual neuropathological changes. It also widens the phenotypic spectrum of prion diseases. The phenotypic variability within the same mutation suggests further, yet uncharacterized, genetic or epigenetic influence on phenotype in these diseases.
|The potential role of amyloid beta in the pathogenesis of age-related macular degeneration. |
Takeshi Yoshida, Kyoko Ohno-Matsui, Shizuko Ichinose, Tetsuji Sato, Nobuhisa Iwata, Takaomi C Saido, Toshio Hisatomi, Manabu Mochizuki, Ikuo Morita
The Journal of clinical investigation 115 2793-800 2005
Drusen are extracellular deposits that lie beneath the retinal pigment epithelium (RPE) and are the earliest signs of age-related macular degeneration (AMD). Recent proteome analysis demonstrated that amyloid beta (Abeta) deposition was specific to drusen from eyes with AMD. To work toward a molecular understanding of the development of AMD from drusen, we investigated the effect of Abeta on cultured human RPE cells as well as ocular findings in neprilysin gene-disrupted mice, which leads to an increased deposition Abeta. The results showed that Abeta treatment induced a marked increase in VEGF as well as a marked decrease in pigment epithelium-derived factor (PEDF). Conditioned media from Abeta-exposed RPE cells caused a dramatic increase in tubular formation by human umbilical vein endothelial cells. Light microscopy of senescent neprilysin gene-disrupted mice showed an increased number of degenerated RPE cells with vacuoles. Electron microscopy revealed basal laminar and linear deposits beneath the RPE layer, but we did not observe choroidal neovascularization (CNV). The present study demonstrates that Abeta accumulation affects the balance between VEGF and PEDF in the RPE, and an accumulation of Abeta reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation. Some other factors, such as breakdown of integrity of Bruch membrane, might be necessary to induce CNV of AMD.Full Text Article
|CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production. |
Zhou, S; Zhou, H; Walian, PJ; Jap, BK
Proceedings of the National Academy of Sciences of the United States of America 102 7499-504 2005
gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. Here, we report the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression had no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides.
|Comparison of myelin, axon, lipid, and immunopathology in the central nervous system of differentially myelin-compromised mutant mice: a morphological and biochemical study. |
Loers, Gabriele, et al.
Mol. Cell. Neurosci., 27: 175-89 (2004) 2004
|X11alpha impairs gamma- but not beta-cleavage of amyloid precursor protein. |
King, Gwendalyn D, et al.
J. Neurochem., 88: 971-82 (2004) 2004
The phosphotyrosine binding domain of the neuronal protein X11alpha/mint-1 binds to the C-terminus of amyloid precursor protein (APP) and inhibits catabolism to beta-amyloid (Abeta), but the mechanism of this effect is unclear. Coexpression of X11alpha or its PTB domain with APPswe inhibited secretion of Abeta40 but not APPsbetaswe, suggesting inhibition of gamma- but not beta-secretase. To further probe cleavage(s) inhibited by X11alpha, we coexpressed beta-secretase (BACE-1) or a component of the gamma-secretase complex (PS-1Delta9) with APP, APPswe, or C99, with and without X11alpha, in HEK293 cells. X11alpha suppressed the PS-1Delta9-induced increase in Abeta42 secretion generated from APPswe or C99. However, X11alpha did not impair BACE-1-mediated proteolysis of APP or APPswe to C99. In contrast to impaired gamma-cleavage of APPswe, X11alpha or its PTB domain did not inhibit gamma-cleavage of NotchDeltaE to NICD (the Notch intracellular domain). The X11alpha PDZ-PS.1Delta9 interaction did not affect gamma-cleavage activity. In a cell-free system, X11alpha did not inhibit the catabolism of APP C-terminal fragments. These data suggest that X11alpha may inhibit Abeta secretion from APP by impairing its trafficking to sites of active gamma-secretase complexes. By specifically targeting substrate instead of enzyme X11alpha may function as a relatively specific gamma-secretase inhibitor.
|Axonal injury, a neglected cause of CNS damage in bacterial meningitis. |
Roland Nau, Joachim Gerber, Stephanie Bunkowski, Wolfgang Brück
Neurology 62 509-11 2004
The contribution of axonal injury to CNS damage in bacterial meningitis was studied by histology and immunohistochemistry for amyloid-beta precursor protein in humans and experimental rabbits. Axonal injury in the white matter caused predominantly but not exclusively by ischemia was detected in all autopsy cases (n = 5) and in 11 of 15 brains of rabbits 18 to 24 hours after intracisternal infection with Streptococcus pneumoniae. This suggests a substantial contribution of axonal pathology to neurologic sequelae after bacterial meningitis.
|The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor. |
von Rotz, RC; Kohli, BM; Bosset, J; Meier, M; Suzuki, T; Nitsch, RM; Konietzko, U
Journal of cell science 117 4435-48 2004
The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11alpha (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. gamma-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11alpha trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3beta and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.
|Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells. |
Valentina Venezia, Claudio Russo, Emanuela Repetto, Serena Salis, Virginia Dolcini, Francesca Genova, Mario Nizzari, Ulrike Mueller, Gennaro Schettini
Journal of neurochemistry 90 1359-70 2004
The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.
|Immunohistochemical detection of beta-amyloid and beta-amyloid precursor protein in the canine brain and non-neuronal epithelial tissues. |
Ayumi Fukuoka, Hiroyuki Nakayama, Kunio Doi
Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis 11 173-8 2004
We examined the immunohistochemical localization of beta-amyloid (Abeta) and beta-amyloid precursor protein (AFPP) in the neuronal and non-neuronal tissues of 14 aged (over 10 years old) and 4 young (0-4 years old) dogs. Abeta was detected only in the senile plaque in the cerebrum of aged dogs. AbetaPP was expressed in the neuronal cell body, neuronal fiber, senile plaque and perimalacic tissue independent of age. In addition, epithelial cells in the bronchus, bronchial glands, gastric and intestinal mucosa, intrahepatic bile ducts, and pancreatic ducts and exocrine glands were immunopositive for AbetaPP. Thus, it is suggested that AbetaPP may be expressed in the non-neuronal epithelial tissues independent of age and of the presence of senile plaques, and the expression may depend on individual differences or physiological conditions.
|Increased mortality and spatial memory deficits in TNF-alpha-deficient mice in ceftriaxone-treated experimental pneumococcal meningitis. |
Joachim Gerber, Tobias Böttcher, Michael Hahn, Alexander Siemer, Stephanie Bunkowski, Roland Nau
Neurobiology of disease 16 133-8 2004
Tumor necrosis factor-alpha (TNF-alpha) is critically involved in inflammation and may participate in hippocampal injury in bacterial meningitis. In a mouse model of ceftriaxone-treated pneumococcal meningitis, spatial memory and motor performance of TNF-alpha-deficient (n = 57) and control mice (n = 55) were investigated. After infection, therapy was initiated with ceftriaxone (100 mg/kg twice daily for 5 days). Sixty-three percent TNF-alpha-deficient mice and 40% control animals died within 6 days (Fisher's exact test: P = 0.02). TNF-alpha-deficient mice surviving pneumococcal meningitis took substantially longer to reach the hidden platform than controls, and the distance of swim tracks was longer (P = 0.02). The swim speed in both groups was similar (P = 0.59). The proliferation of dentate granule cells was lower in TNF-alpha-deficient than in wild-type mice (P = 0.03). In pneumococcal meningitis, TNF-alpha deficiency caused increased mortality and stronger deficits in spatial memory possibly due to impaired neurogenesis.
|Prion protein accumulation and neuroprotection in hypoxic brain damage. |
McLennan, NF; Brennan, PM; McNeill, A; Davies, I; Fotheringham, A; Rennison, KA; Ritchie, D; Brannan, F; Head, MW; Ironside, JW; Williams, A; Bell, JE
The American journal of pathology 165 227-35 2004
The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.Full Text Article
|A phosphorylated, carboxy-terminal fragment of beta-amyloid precursor protein localizes to the splicing factor compartment. |
Muresan, Z; Muresan, V
Human molecular genetics 13 475-88 2004
Beta-amyloid precursor protein (APP) is implicated in the pathobiology of Alzheimer's disease (AD). To gain insight into its function, we have investigated the proteolytic processing and post-translational modification of APP in relation to its intracellular traffic and localization. The proteolytic processing that generates the amyloid beta-peptide (Abeta) also releases into the cytoplasm the carboxy-terminal fragment of APP, Cgamma. Using the catecholaminergic cell line, CAD, and an antibody to a form of APP that is phosphorylated at Thr668 (pAPP; numbering for APP695), we show that a phosphorylated, carboxy-terminal fragment of APP, probably Cgamma, is present in the nucleus, where it localizes to subnuclear particles. The labeling with anti-pAPP antibody co-localizes with proteins that define the splicing factor compartment (SFC) [e.g. the small nuclear ribonucleoprotein (snRNP), U2B, and serine/arginine-rich (SR) proteins], but is excluded from the coiled bodies and the gems. This distribution of pAPP epitopes was found in CAD cells independent of their state of differentiation, as well as in primary cortical neurons, epithelial cells and fibroblasts. We further show that exogenously expressed Cgamma becomes phosphorylated, and distributes throughout the cell. A fraction of this Cgamma is translocated into the nucleus, where it co-localizes with endogenous pAPP epitopes. Finally, we show that the APP binding, scaffolding protein, Fe65 co-localizes with pAPP epitopes and with expressed Cgamma at intranuclear speckles. These results suggest that phosphorylated Cgamma accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.
|Apoptotic cell death and amyloid precursor protein signaling in neuroblastoma SH-SY5Y cells. |
Valentina Venezia, Claudio Russo, Emanuela Repetto, Mario Nizzari, Elisabetta Violani, Pia Carlo, Bianca Marchetti, Gennaro Schettini
Annals of the New York Academy of Sciences 1030 339-47 2004
We have recently shown that the amyloid precursor protein (APP) and a subset of its C-terminal fragments (CTFs) are tyrosine phosphorylated in human brain and in cultured cells. Tyrosine phosphorylation generates a substrate that is sequentially bound by the adaptor proteins ShcA and Grb2, and this interaction is significantly enhanced in Alzheimer's disease brains. Here we have studied the APP/CTFs phosphorylation and ShcA activation in a human neuroblastoma cell line, SH-SY5Y, under basal and apoptotic conditions. To commit these cells to apoptosis, we used staurosporin, a well-known apoptotic inducer and protein kinase C blocker. Our data suggest the following: (1) in normally proliferating SH-SY5Y cells, full-length APP is complexed with Grb2[Q3], likely through its SH2 domain; (2) upon induction of apoptosis, APP is degraded and ShcA-Grb2 coimmunoprecipitates with CTFs recognized by anti-APP antibodies; and (3) caspase inhibitors partially block the degradation of APP and the coprecipitation of CTFs with ShcA-Grb2 adaptors. In summary, our data suggest that in SH-SY5Y cells, tyrosine-phosphorylated APP is involved in a complex with ShcA-Grb2 adaptors that is disrupted during apoptosis. The abnormal degradation of APP and consequent increased levels of CTFs (as has been observed in Alzheimer's disease and Down's syndrome) generate a complex between tyrosine-phosphorylated CTFs and intracellular adaptors. The signaling through APP and its CTFs may have significant relevance for apoptotic cell death in Alzheimer's disease.
|APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain |
Chen, Y. et al.
J. Cell Biol., 163:27-33 (2003) 2003
|Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. |
Farris, W; Mansourian, S; Chang, Y; Lindsley, L; Eckman, EA; Frosch, MP; Eckman, CB; Tanzi, RE; Selkoe, DJ; Guenette, S
Proceedings of the National Academy of Sciences of the United States of America 100 4162-7 2003
Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE --). IDE deficiency resulted in a greater than 50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE -- mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.
|Down-regulation of amyloid precursor protein by peptide nucleic acid oligomer in cultured rat primary neurons and astrocytes. |
Linda Adlerz, Ursel Soomets, Linda Holmlund, Säde Viirlaid, Ulo Langel, Kerstin Iverfeldt
Neuroscience letters 336 55-9 2003
The amyloid precursor protein (APP) and its proteolytic cleavage products, the amyloid beta peptides, have been implicated as a cause of Alzheimer's disease. Peptide nucleic acids (PNA), the DNA mimics, have been shown to block the expression of specific proteins at both transcriptional and translational levels. Generally, the cellular uptake of PNA is low. However, recent studies have indicated that the effect of unmodified antisense PNA uptake is more pronounced in nervous tissue. In this study we have shown that biotinylated PNA directed to the initiator codon region of the APP mRNA (-4 - +11) was taken up into the cytoplasm of primary rat cerebellar granule cells and cortical astrocytes, using fluorescence and confocal microscopy studies. Uptake of PNA was faster in neurons than in astrocytes. Western blotting analysis showed that APP was strongly down-regulated in both neurons and astrocytes. Thus, unmodified PNA can be used for studies on the function of APP in neurons and astrocytes.
|Accumulation of the amyloid precursor-like protein APLP2 and reduction of APLP1 in retinoic acid-differentiated human neuroblastoma cells upon curcumin-induced neurite retraction. |
Linda Adlerz, Marie Beckman, Sofia Holback, Roya Tehranian, Veronica Cortés Toro, Kerstin Iverfeldt
Brain research. Molecular brain research 119 62-72 2003
Amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. The function of these three proteins is not yet fully understood. One of the proposed roles of APP is to promote neurite outgrowth. The aim of this study was to investigate the regulation of the expression levels of APP family members during neurite outgrowth. We observed that retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y cells resulted in increased expression of APP, APLP1 and APLP2. We also examined the effect of the NFkappaB, AP-1 and c-Jun N-terminal kinase inhibitor curcumin (diferuloylmethane) on the RA-induced expression levels of these proteins. We found that treatment with curcumin counteracted the RA-induced mRNA expression of all APP family members. In addition, we observed that curcumin treatment resulted in neurite retraction without any effect on cell viability. Surprisingly, curcumin had differential effects on the APLP protein levels in RA-differentiated cells. RA-induced APLP1 protein expression was blocked by curcumin, while the APLP2 protein levels were further increased. APP protein levels were not affected by curcumin treatment. We propose that the sustained levels of APP and the elevated levels of APLP2, in spite of the reduced mRNA expression, are due to altered proteolytic processing of these proteins. Furthermore, our results suggest that APLP1 does not undergo the same type of regulated processing as APP and APLP2.
|Heme deficiency may be a factor in the mitochondrial and neuronal decay of aging. |
Atamna, H; Killilea, DW; Killilea, AN; Ames, BN
Proceedings of the National Academy of Sciences of the United States of America 99 14807-12 2002
Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.Full Text Article
|Signal transduction through tyrosine-phosphorylated C-terminal fragments of amyloid precursor protein via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer's disease brain. |
Russo, C; Dolcini, V; Salis, S; Venezia, V; Zambrano, N; Russo, T; Schettini, G
The Journal of biological chemistry 277 35282-8 2002
The proteolytic processing of amyloid precursor protein (APP) through the formation of membrane-bound C-terminal fragments (CTFs) and of soluble beta-amyloid peptides likely influences the development of Alzheimer's disease (AD). We show that in human brain a subset of CTFs are tyrosine-phosphorylated and form stable complexes with the adaptor protein ShcA. Grb2 is also part of these complexes, which are present in higher amounts in AD than in control brains. ShcA immunoreactivity is also greatly enhanced in patients with AD and occurs at reactive astrocytes surrounding cerebral vessels and amyloid plaques. A higher amount of phospho-ERK1,2, likely as result of the ShcA activation, is present in AD brains. In vitro experiments show that the ShcA-CTFs interaction is strictly confined to glial cells when treated with thrombin, which is a well known ShcA and ERK1,2 activator and a regulator of APP cleavage. In untreated cells ShcA does not interact with either APP or CTFs, although they are normally generated. Altogether these data suggest that CTFs are implicated in cell signaling via Shc transduction machinery, likely influencing MAPK activity and glial reaction in AD patients.
|Grb2-mediated alteration in the trafficking of AbetaPP: insights from Grb2-AICD interaction. |
Raychaudhuri M, Mukhopadhyay D
J Alzheimers Dis 20 275-92. 2001
The amyloid-beta protein precursor (AbetaPP) is processed by various proteases located along the endosomal lysosomal pathway and any alteration in its trafficking would be important in the pathogenesis of Alzheimer's disease (AD). Our current study is based on the clinical evidence that an AbetaPP intracellular domain (AICD) "adaptor" protein, growth factor receptor protein binding protein 2 (Grb2), gets concentrated in neuronal cell bodies in AD patients. Here we show that both endogenous and exogenously transfected Grb2 interact with AbetaPP in Neuro 2A cells. Endogenous Grb2 partially co-localizes to late endosomal compartments along with AbetaPP and AICD. Increase in the concentration of Grb2 confines it in enlarged late endosomes leading to more sequestration of AbetaPP and AICD within these compartments. This confinement of AbetaPP due to Grb2 overexpression affects its turnover by inhibiting its release via exosomal vesicles. As a consequence, the level of intracellular AbetaPP and AICD increases. The effect of Grb2 overexpression has been verified by knocking down Grb2 as well as by overexpressing Grb2 in Grb2 knocked down cells. Having established the Grb2-mediated trafficking of AICD and its impairment, the significance of its consequence has now become apparent in the downstream events of AD pathogenesis.
|Evaluation of brain damage resulting from penetrating and non-penetrating captive bolt stunning using lambs |
Finnie, J W, et al
Aust Vet J, 78:775-8 (2000) 2000
|Prion protein devoid of the octapeptide repeat region restores susceptibility to scrapie in PrP knockout mice. |
Flechsig, E, et al.
Neuron, 27: 399-408 (2000) 2000
Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice. Surprisingly, brains of terminally ill animals show no histopathology typical for scrapie. However, in the spinal cord, infectivity, gliosis, and motor neuron loss are as in scrapie-infected wild-type controls. Thus, while the region comprising the octarepeats is not essential for mediating pathogenesis and prion replication, it modulates the extent of these events and of disease presentation.
|Expression of an ubiquitous, cross-reactive homologue of the mouse beta-amyloid precursor protein (APP) |
Slunt, H. et al.
J. Biol. Chem., 269:2637-2644 (1994) 1994
|Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein. |
Weidemann, A, et al.
Cell, 57: 115-26 (1989) 1989
To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.
|Organization and morphological characteristics of cholinergic neurons: an immunocytochemical study with a monoclonal antibody to choline acetyltransferase. |
Houser, C R, et al.
Brain Res., 266: 97-119 (1983) 1983
Choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, has been localized immunocytochemically with a monoclonal antibody in light and electron microscopic preparations of rat central nervous system (CNS). The antibody was an IgG1 subclass immunoglobulin that removed ChAT activity from solution. The specificity of the antibody and immunocytochemical methods has been confirmed by the demonstration of ChAT-positive neurons in a number of well-characterized cholinergic systems. For example, ChAT-positive reaction product was present in the cell bodies of spinal and cranial nerve motoneurons, as well as in their axons and terminations as motor end-plates in skeletal muscle. In addition, the somata of preganglionic sympathetic and parasympathetic neurons were ChAT-positive. The specificity of staining was further supported by a lack of reaction product in several groups of neurons thought to use neuroactive substances other than acetylcholine. No specific staining was observed in control specimens. The findings indicated that ChAT had an extensive intraneuronal distribution in many cholinergic neurons, being present in cell bodies, dendrites, axons and axon terminals. ChAT-positive somata were found in the medial septum and diagonal band, the medial habenula, and the basal nucleus of, the forebrain, 3 regions that are sources of cholinergic afferents to the hippocampus, interpeduncular nucleus and cerebral cortex, respectively. In addition, positively stained cell bodies were present within the cerebral cortex. ChAT-positive punctate structures were observed in the ventral horn of the spinal cord, where electron microscopic studies demonstrated that some of these structures were synaptic terminals. Other regions containing numerous ChAT-positive puncta included the hippocampus, the interpeduncular nucleus and the cerebral cortex. The light microscopic appearance of these putative cholinergic terminals varied among different brain regions. Large punctate structures related to well-defined post-synaptic elements were characteristic of some regions, such as the ventral horn of the spinal cord, while smaller punctate structures and varicose fibers with a diffuse pattern of organization distinguished other regions, such as the cerebral cortex.
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