Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R||IH(P), IP, WB||M||Ascites||Monoclonal Antibody|
|Presentation||Mouse monoclonal IgG1κ in buffer containing containing 10 mM sodium azide.|
|Safety Information according to GHS|
|Material Size||100 µL|
References | 99 Available | See All References
|Reference overview||Application||Pub Med ID|
|Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions. |
Farrawell, NE; Lambert-Smith, IA; Warraich, ST; Blair, IP; Saunders, DN; Hatters, DM; Yerbury, JJ
Scientific reports 5 13416 2015
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.
|BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. |
Qin, J; Zhou, Z; Chen, W; Wang, C; Zhang, H; Ge, G; Shao, M; You, D; Fan, Z; Xia, H; Liu, R; Chen, C
Nature communications 6 8471 2015
The transcription factor KLF5 is highly expressed in basal-like breast cancer and promotes breast cancer cell proliferation, survival, migration and tumour growth. Here we show that, in breast cancer cells, KLF5 is stabilized by the deubiquitinase (DUB) BAP1. With a genome-wide siRNA library screen of DUBs, we identify BAP1 as a bona fide KLF5 DUB. BAP1 interacts directly with KLF5 and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be rescued partially by ectopic expression of KLF5. Collectively, our findings not only identify BAP1 as the DUB for KLF5, but also reveal a critical mechanism that regulates KLF5 expression in breast cancer. Our findings indicate that BAP1 could be a potential therapeutic target for breast and other cancers.
|Apelin promotes diabetic nephropathy by inducing podocyte dysfunction via inhibiting proteasome activities. |
Guo, C; Liu, Y; Zhao, W; Wei, S; Zhang, X; Wang, W; Zeng, X
Journal of cellular and molecular medicine 19 2273-85 2015
Podocyte injuries are associated with progression of diabetic nephropathy (DN). Apelin, an adipocyte-derived peptide, has been reported to be a promoting factor for DN. In this study, we aim to determine whether apelin promotes progression of DN by inducing podocyte dysfunction. kk-Ay mice were used as models for DN. Apelin and its antagonist, F13A were intraperitoneally administered for 4 weeks, respectively. Renal function and foot process proteins were analysed to evaluate the effects of apelin on kk-Ay mice and podocytes. Apelin increased albuminuria and decreased podocyte foot process proteins expression in kk-Ay mice, which is consistent with the results that apelin receptor (APLNR) levels increased in glomeruli of patients or mice with DN. In cultured podocytes, high glucose increased APLNR expression and apelin administration was associated with increased permeability and decreased foot process proteins levels. All these dysfunctions were associated with decreased 26S proteasome activities and increased polyubiquitinated proteins in both kk-Ay mice and cultured podocytes, as demonstrated by 26S proteasome activation with cyclic adenosine monophosphate (cAMP) or oleuropein. These effects seemed to be related to endoplasmic reticulum (ER) stress, as apelin increased C/EBP homologous protein (CHOP) and peiFα levels while cAMP or oleuropein reduced it in high glucose and apelin treated podocytes. These results suggest that apelin induces podocyte dysfunction in DN through ER stress which was induced by decreased proteasome activities in podocytes.
|Trehalose reverses cell malfunction in fibroblasts from normal and Huntington's disease patients caused by proteosome inhibition. |
Fernandez-Estevez, MA; Casarejos, MJ; López Sendon, J; Garcia Caldentey, J; Ruiz, C; Gomez, A; Perucho, J; de Yebenes, JG; Mena, MA
PloS one 9 e90202 2014
Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and is caused by polyglutamine expansion in the huntingtin protein. Mutant huntingtin protein and its fragments are resistant to protein degradation and produce a blockade of the ubiquitin proteasome system (UPS). In HD models, the proteasome inhibitor epoxomicin aggravates protein accumulation and the inductor of autophagy, trehalose, diminishes it. We have investigated the effects of epoxomicin and trehalose in skin fibroblasts of control and HD patients. Untreated HD fibroblasts have increased the levels of ubiquitinized proteins and higher levels of reactive oxygen species (ROS), huntingtin and the autophagy marker LAMP2A. Baseline replication rates were higher in HD than in controls fibroblasts but that was reverted after 12 passages. Epoxomicin increases the activated caspase-3, HSP70, huntingtin, ubiquitinated proteins and ROS levels in both HD and controls. Treatment with trehalose counteracts the increase in ROS, ubiquitinated proteins, huntingtin and activated caspase-3 levels induced by epoxomicin, and also increases the LC3 levels more in HD fibroblast than controls. These results suggest that trehalose could revert protein processing abnormalities in patients with Huntington's Disease.
|A DERL3-associated defect in the degradation of SLC2A1 mediates the Warburg effect. |
Lopez-Serra, P; Marcilla, M; Villanueva, A; Ramos-Fernandez, A; Palau, A; Leal, L; Wahi, JE; Setien-Baranda, F; Szczesna, K; Moutinho, C; Martinez-Cardus, A; Heyn, H; Sandoval, J; Puertas, S; Vidal, A; Sanjuan, X; Martinez-Balibrea, E; Viñals, F; Perales, JC; Bramsem, JB; Ørntoft, TF; Andersen, CL; Tabernero, J; McDermott, U; Boxer, MB; Vander Heiden, MG; Albar, JP; Esteller, M
Nature communications 5 3608 2014
Cancer cells possess aberrant proteomes that can arise by the disruption of genes involved in physiological protein degradation. Here we demonstrate the presence of promoter CpG island hypermethylation-linked inactivation of DERL3 (Derlin-3), a key gene in the endoplasmic reticulum-associated protein degradation pathway, in human tumours. The restoration of in vitro and in vivo DERL3 activity highlights the tumour suppressor features of the gene. Using the stable isotopic labelling of amino acids in cell culture workflow for differential proteome analysis, we identify SLC2A1 (glucose transporter 1, GLUT1) as a downstream target of DERL3. Most importantly, SLC2A1 overexpression mediated by DERL3 epigenetic loss contributes to the Warburg effect in the studied cells and pinpoints a subset of human tumours with greater vulnerability to drugs targeting glycolysis.
|PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity. |
Kane, LA; Lazarou, M; Fogel, AI; Li, Y; Yamano, K; Sarraf, SA; Banerjee, S; Youle, RJ
The Journal of cell biology 205 143-53 2014
PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.
|Astrocytic TDP-43 pathology in Alexander disease. |
Walker, AK; Daniels, CM; Goldman, JE; Trojanowski, JQ; Lee, VM; Messing, A
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 6448-58 2014
Alexander disease (AxD) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic inclusions known as Rosenthal fibers (RFs) within astrocytes, and is caused by dominant mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP). GFAP is the major astrocytic intermediate filament, and in AxD patient brain tissue GFAP is a major component of RFs. TAR DNA binding protein of 43 kDa (TDP-43) is the major pathological protein in almost all cases of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and ∼50% of frontotemporal lobar degeneration (FTLD), designated as FTLD-TDP. In ALS and FTLD-TDP, TDP-43 becomes insoluble, ubiquitinated, and pathologically phosphorylated and accumulates in cytoplasmic inclusions in both neurons and glia of affected brain and spinal cord regions. Previously, TDP-43 was detected in RFs of human pilocytic astrocytomas; however, involvement of TDP-43 in AxD has not been determined. Here we show that TDP-43 is present in RFs in AxD patient brains, and that insoluble phosphorylated full-length and high molecular weight TDP-43 accumulates in white matter of such brains. Phosphorylated TDP-43 also accumulates in the detergent-insoluble fraction from affected brain regions of Gfap(R236H/+) knock-in mice, which harbor a GFAP mutation homologous to one that causes AxD in humans, and TDP-43 colocalizes with astrocytic RF pathology in Gfap(R236H/+) mice and transgenic mice overexpressing human wild-type GFAP. These findings suggest common pathogenic mechanisms in ALS, FTLD, and AxD, and this is the first report of TDP-43 involvement in a neurological disorder primarily affecting astrocytes.
|Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice. |
Tanaka, Y; Chambers, JK; Matsuwaki, T; Yamanouchi, K; Nishihara, M
Acta neuropathologica communications 2 78 2014
It has been shown that progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Previous studies also suggested that PGRN is involved in modulating lysosomal function. To elucidate the pathophysiological role of PGRN in the aged brain, in the present study, lysosomal function and pathological changes of the brain were investigated using 10- and 90-week-old wild-type and PGRN-deficient mice.We showed that PGRN deficiency caused enhanced CD68 expression in activated microglia and astrogliosis in the cortex and thalamus, especially in the ventral posteromedial nucleus/ventral posterolateral nucleus (VPM/VPL), in the aged brain. Immunoreactivity for Lamp1 (lysosome marker) in the VPM/VPL and expression of lysosome-related genes, i.e. cathepsin D, V-type proton ATPase subunit d2, and transcription factor EB genes, were also increased by PGRN deficiency. Aggregates of p62, which is selectively degraded by the autophagy-lysosomal system, were observed in neuronal and glial cells in the VPM/VPL of aged PGRN-deficient mice. TAR DNA binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons were also observed in aged PGRN-deficient mice. PGRN deficiency caused enhanced expression of glial cell-derived cytotoxic factors such as macrophage expressed gene 1, cytochrome b-245 light chain, cytochrome b-245 heavy chain, complement C4, tumor necrosis factor-α and lipocalin 2. In addition, neuronal loss and lipofuscinosis in the VPM/VPL and disrupted myelination in the cerebral cortex were observed in aged PGRN-deficient mice.The present study shows that aged PGRN-deficient mice present with NCL-like pathology as well as TDP-43 aggregates in the VPM/VPL, where a particular vulnerability has been reported in NCL model mice. The present results also suggest that these pathological changes in the VPM/VPL are likely a result of lysosomal dysfunction. How PGRN prevents lysosomal dysfunction with aging remains to be elucidated.
|MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis. |
Zhang, X; Chen, S; Song, L; Tang, Y; Shen, Y; Jia, L; Le, W
Autophagy 10 588-602 2014
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1(G93A) mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1(G93A) mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1(G93A) mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1(G93A) mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1(G93A) mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.
|Optineurin immunoreactivity in neuronal and glial intranuclear inclusions in adult-onset neuronal intranuclear inclusion disease. |
Nakamura, M; Murray, ME; Lin, WL; Kusaka, H; Dickson, DW
American journal of neurodegenerative disease 3 93-102 2014
Optineurin (OPTN) is a multifunctional protein involved in cellular morphogenesis, vesicle trafficking, maintenance of the Golgi complex, and transcription activation through its interactions with the Rab8, myosin 6 (MYO6), huntingtin. Recently, OPTN immunoreactivity has been reported in intranuclear inclusions in patients with neuronal intranuclear inclusions disease (NIID). Other studies have shown that the RNA-binding protein, fused in sarcoma (FUS), is a component of intranuclear inclusions in NIID. We aimed to investigate the relationship between OPTN, its binding protein MYO6 and FUS in this study. In control subjects, OPTN (C-terminal) (OPTN-C) and MYO6 immunoreactivity was mainly demonstrated in the cytoplasm of neurons. In NIID patients, both neuronal intranuclear inclusions (NII) and glial intranuclear inclusions (GII) were immunopositive for MYO6 as well as OPTN-C. However, the intensity of OPTN-C immunostaining of the neuronal cytoplasm with and without NII was less than that of the control subjects. Double immunofluorescence staining for OPTN-C, ubiquitin (Ub), p62 and FUS revealed co-localization of these proteins within NII. Moreover, Ub positive inclusions were co-localized with MYO6. The percentage of co-localization of Ub with OPTN-C, FUS or MYO6 in NII was 100%, 52% and 92%, respectively. Ultrastructurally, the inclusions consisted of thin and thick filaments. Both filaments were immunopositive for Ub and OPTN-C. These findings suggest that OPTN plays a central role in the disease pathogenesis, and that OPTN may be a major component of NII.
|CSF1R mutations link POLD and HDLS as a single disease entity. |
Nicholson, AM; Baker, MC; Finch, NA; Rutherford, NJ; Wider, C; Graff-Radford, NR; Nelson, PT; Clark, HB; Wszolek, ZK; Dickson, DW; Knopman, DS; Rademakers, R
Neurology 80 1033-40 2013
Pigmented orthochromatic leukodystrophy (POLD) and hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS) are rare neurodegenerative disorders characterized by cerebral white matter abnormalities, myelin loss, and axonal swellings. The striking overlap of clinical and pathologic features of these disorders suggested a common pathogenesis; however, no genetic or mechanistic link between POLD and HDLS has been established. Recently, we reported that mutations in the colony-stimulating factor 1 receptor (CSF1R) gene cause HDLS. In this study, we determined whether CSF1R mutations are also a cause of POLD.We performed sequencing of CSF1R in 2 pathologically confirmed POLD families. For the largest family (FTD368), a detailed case report was provided and brain samples from 2 affected family members previously diagnosed with POLD were re-evaluated to determine whether they had HDLS features. In vitro functional characterization of wild-type and mutant CSF1R was also performed.We identified CSF1R mutations in both POLD families: in family 5901, we found c.2297Tgreater than C (p.M766T), previously reported by us in HDLS family CA1, and in family FTD368, we identified c.2345Ggreater than A (p.R782H), recently reported in a biopsy-proven HDLS case. Immunohistochemical examination in family FTD368 showed the typical neuronal and glial findings of HDLS. Functional analyses of CSF1R mutant p.R782H (identified in this study) and p.M875T (previously observed in HDLS), showed a similar loss of CSF1R autophosphorylation of selected tyrosine residues in the kinase domain for both mutations when compared with wild-type CSF1R.We provide the first genetic and mechanistic evidence that POLD and HDLS are a single clinicopathologic entity.
|Unbiased screen reveals ubiquilin-1 and -2 highly associated with huntingtin inclusions. |
Rutherford, NJ; Lewis, J; Clippinger, AK; Thomas, MA; Adamson, J; Cruz, PE; Cannon, A; Xu, G; Golde, TE; Shaw, G; Borchelt, DR; Giasson, BI
Brain research 1524 62-73 2013
Recently mutations in ubiquilin-2 were identified in patients with amyotrophic lateral sclerosis (ALS) and ALS/dementia providing direct evidence for the importance of this protein in neurodegenerative diseases. Histological studies have suggested that ubiquilin-1/-2 are associated with various pathological inclusions including Lewy bodies in Parkinson's disease, neurofibrillary tangles in Alzheimer's disease, polyQ inclusions in expansion repeat diseases and various proteinopathies associated with ALS and frontotemporal dementia. Using specific ubiquilin-2 antibodies and a series of transgenic mouse models of proteinopathies associated with neurodegenerative disease, we show that ubiquilin-2 preferentially associates with huntingtin polyQ expansion aggregates compared to α-synuclein, tau and several other types of protein inclusions. These results were confirmed by similar findings for ubiquilin-1 and -2 in human brain tissue sections, where accumulation was observed in huntingtin inclusions, but only infrequently in other types of protein inclusions. In cultured cells, ubiquilin-2 associates with huntingtin/polyQ aggregates, but this is not compromised by disease-causing mutations. Although ubiquilin proteins can function as chaperones to shuttle proteins for degradation, there is persistent co-localization between ubiquilin-2 and polyQ aggregated proteins during disease progression in the N586-82Q-C63 Huntington's disease mouse model. Thus, the co-localization of ubiquilin-2 with the huntingtin aggregates does not appear to facilitate aggregate removal.
|SIRT1 is a direct coactivator of thyroid hormone receptor β1 with gene-specific actions. |
Suh, JH; Sieglaff, DH; Zhang, A; Xia, X; Cvoro, A; Winnier, GE; Webb, P
PloS one 8 e70097 2013
Sirtuin 1 (SIRT1) NAD(+)-dependent deacetylase regulates energy metabolism by modulating expression of genes involved in gluconeogenesis and other liver fasting responses. While many effects of SIRT1 on gene expression are mediated by deacetylation and activation of peroxisome proliferator activated receptor coactivator α (PGC-1α), SIRT1 also binds directly to DNA bound transcription factors, including nuclear receptors (NRs), to modulate their activity. Since thyroid hormone receptor β1 (TRβ1) regulates several SIRT1 target genes in liver and interacts with PGC-1α, we hypothesized that SIRT1 may influence TRβ1. Here, we confirm that SIRT1 cooperates with PGC-1α to enhance response to triiodothyronine, T3. We also find, however, that SIRT1 stimulates TRβ1 activity in a manner that is independent of PGC-1α but requires SIRT1 deacetylase activity. SIRT1 interacts with TRβ1 in vitro, promotes TRβ1 deacetylation in the presence of T3 and enhances ubiquitin-dependent TRβ1 turnover; a common response of NRs to activating ligands. More surprisingly, SIRT1 knockdown only strongly inhibits T3 response of a subset of TRβ1 target genes, including glucose 6 phosphatase (G-6-Pc), and this is associated with blockade of TRβ1 binding to the G-6-Pc promoter. Drugs that target the SIRT1 pathway, resveratrol and nicotinamide, modulate T3 response at dual TRβ1/SIRT1 target genes. We propose that SIRT1 is a gene-specific TRβ1 co-regulator and TRβ1/SIRT1 interactions could play important roles in regulation of liver metabolic response. Our results open possibilities for modulation of subsets of TR target genes with drugs that influence the SIRT1 pathway.
|The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis. |
Sosna, J; Voigt, S; Mathieu, S; Kabelitz, D; Trad, A; Janssen, O; Meyer-Schwesinger, C; Schütze, S; Adam, D
Cell communication and signaling : CCS 11 76 2013
In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis.The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis.The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.
|TDP-43, an ALS linked protein, regulates fat deposition and glucose homeostasis. |
Stallings, NR; Puttaparthi, K; Dowling, KJ; Luther, CM; Burns, DK; Davis, K; Elliott, JL
PloS one 8 e71793 2013
The identification of proteins which determine fat and lean body mass composition is critical to better understanding and treating human obesity. TDP-43 is a well-conserved RNA-binding protein known to regulate alternative splicing and recently implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). While TDP-43 knockout mice show early embryonic lethality, post-natal conditional knockout mice show weight loss, fat depletion, and rapid death, suggesting an important role for TDP-43 in regulating energy metabolism. Here we report, that over-expression of TDP-43 in transgenic mice can result in a phenotype characterized by increased fat deposition and adipocyte hypertrophy. In addition, TDP-43 over-expression in skeletal muscle results in increased steady state levels of Tbc1d1, a RAB-GTPase activating protein involved in Glucose 4 transporter (Glut4) translocation. Skeletal muscle fibers isolated from TDP-43 transgenic mice show altered Glut4 translocation in response to insulin and impaired insulin mediated glucose uptake. These results indicate that levels of TDP-43 regulate body fat composition and glucose homeostasis in vivo.
|A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein. |
Baldo, B; Weiss, A; Parker, CN; Bibel, M; Paganetti, P; Kaupmann, K
The Journal of biological chemistry 287 1406-14 2012
Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.
|Right temporal variant frontotemporal dementia with motor neuron disease. |
Coon, EA; Whitwell, JL; Parisi, JE; Dickson, DW; Josephs, KA
Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia 19 85-91 2012
Patterns of atrophy in frontotemporal dementia (FTD) correlate with the clinical subtypes of behavioral variant FTD (bvFTD), semantic dementia, progressive non-fluent aphasia (PNFA) and FTD with motor neuron disease (FTD-MND). Right temporal variant FTD is associated with behavioral dyscontrol and semantic impairment, with tau abnormalities more common in right temporal bvFTD and TDP-43 accumulation in right temporal semantic dementia. However, no clinical and anatomical correlation has been described for patients with predominant right temporal atrophy and FTD-MND. Therefore, we performed a database screen for all patients diagnosed with FTD-MND at Mayo Clinic and reviewed their MRI scans to identify those with striking, dominant, right temporal lobe atrophy. For cases with volumetric MRI we performed voxel based morphometry and for those with brain tissue we performed pathological examination. Of three such patients identified, each patient had different presenting behavioral and/or aphasic characteristics. MRI, including diffusion tensor imaging in one patient, and FDG positron emission tomography revealed striking and dominant right temporal lobe atrophy, right corticospinal tract degeneration, and right temporal hypometabolism. Archived brain tissue was available in two patients; both demonstrating TDP-43 type 3 pathology (Mackenzie scheme) with predominant neuronal cytoplasmic inclusions. In one case, neurofibrillary tangles (Braak V) and neuritic plaques were also present in keeping with a diagnosis of Alzheimer's disease. There appears to be an association between FTD-MND and severe right temporal lobe atrophy. Until further characterization of such cases are determined, they may be best classified as right temporal variant FTD-MND.
|Cyclin-dependent kinase 5 immunoreactivity for granulovacuolar degeneration. |
Masahiro Nakamori,Tetsuya Takahashi,Yuu Yamazaki,Takashi Kurashige,Takemori Yamawaki,Masayasu Matsumoto
Neuroreport 23 2012
In addition to senile plaque and neurofibrillary tangles, granulovacuolar degeneration is a hallmark of Alzheimer's disease. A number of tau kinases, such as c-jun N-terminal kinase (JNK), glycogen-synthase kinase-3β (GSK3β), and casein kinase 1 (CK1), have been reported to be markers of granulovacuolar degeneration. In addition, cyclin-dependent kinase 5 (CDK5), which phosphorylates tau, has been shown to be abundantly expressed in neurofibrillary tangles in the hippocampus. CDK5 has a unique staining pattern, and therefore, has the potential to be a novel marker for granulovacuolar degeneration. In this study, we investigated the ability of CDK5 to be a marker for granulovacuolar degeneration using immunohistochemical analysis. Four Alzheimer's disease cases, three myotonic dystrophy (MyD) cases, and three control cases were subjected to immunohistochemistry and immunofluorescent techniques using anti-CDK5, anti-charged multivesicular body protein 2B (CHMP2B), anti-pSmad2/3, anti-ubiquitin (Ub), anti-phospho-TDP-43 and AT8 antibodies. Some CDK5-positive granules were morphologically similar to granulovacuolar degeneration intraluminal granules, and these granules overlapped with those immunopositive for pSmad2/3, Ub and phospho-TDP-43 established granulovacuolar degeneration markers. Moreover, CDK5-positive granulovacuolar degeneration and phosphorylated tau colocalized in pyramidal neurons in Alzheimer's disease and MyD cases. The numbers of CDK5-positive granules showed an inverse relationship with the degree of mature neurofibrillary tangles in each cell, as was the case with CHMP2B-positive granulovacuolar degeneration granules and neurofibrillary tangles. The presence of tau kinases including CDK5 in granulovacuolar degeneration might implicate that granulovacuolar degeneration is structurally involved in tau modification.
|Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes. |
Hjerpe, R; Thomas, Y; Chen, J; Zemla, A; Curran, S; Shpiro, N; Dick, LR; Kurz, T
The Biochemical journal 441 927-36 2012
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
|Increased hippocampal accumulation of autophagosomes predicts short-term recognition memory impairment in aged mice. |
Soontornniyomkij, V; Risbrough, VB; Young, JW; Soontornniyomkij, B; Jeste, DV; Achim, CL
Age (Dordrecht, Netherlands) 34 305-16 2012
Constitutive macroautophagy involved in the turnover of defective long-lived proteins and organelles is crucial for neuronal homeostasis. We hypothesized that macroautophagic dysregulation in selective brain regions was associated with memory impairment in aged mice. We used the single-trial object recognition test to measure short-term memory in 18 aged mice compared to 22 young mice and employed immunohistochemistry to assess cellular distribution of proteins involved in the selective degradation of ubiquitinated proteins via macroautophagy. Values of the discrimination ratio (DR, a measure of short-term recognition memory performance) in aged mice were significantly lower than those in young mice (median, 0.54 vs. 0.67; p = 0.005, U test). Almost exclusively in aged mice, there were clusters of puncta immunoreactive for microtubule-associated protein 1 light chain 3 (LC3), ubiquitin- and LC3-binding protein p62, and ubiquitin in neuronal processes predominantly in the hippocampal formation, olfactory bulb/tubercle, and cerebellar cortex. The hippocampal burden of clustered puncta immunoreactive for LC3 and p62 exhibited inverse linear correlations with DR in aged mice (ρ = -0.48 and -0.55, p = 0.044 and 0.018, respectively, Spearman's rank correlation). These findings suggest that increased accumulation of autophagosomes within neuronal processes in selective brain regions is characteristic of aging. The dysregulation of macroautophagy can adversely affect the turnover of aggregate-prone proteins and defective organelles, which may contribute to memory impairment in aged mice.
|Locus coeruleus neurons are resistant to dysfunction and degeneration by maintaining free ubiquitin levels although total ubiquitin levels decrease upon disruption of polyubiquitin gene Ubb. |
Chul-Woo Park,Han-Wook Ryu,Kwon-Yul Ryu
Biochemical and biophysical research communications 418 2012
Previously, we demonstrated that disruption of polyubiquitin gene Ubb leads to hypothalamic neurodegeneration and metabolic abnormalities associated with hypothalamic dysfunction. However, we cannot exclude the possibility that defects in other brain regions where Ubb is highly expressed may also contribute to the phenotypes exhibited by Ubb(-/-) mice. Upon searching for such brain regions, we identified a region in the brainstem called the locus coeruleus where both polyubiquitin genes Ubb and Ubc were highly expressed. In contrast to other brain regions, Ubc was significantly upregulated in the locus coeruleus of Ubb(-/-) mice presumably to compensate for loss of Ubb, and this upregulation was sufficient to maintain levels of free Ub, but not total Ub, in the locus coeruleus. However, in the hypothalamus of Ubb(-/-) mice, both free and total Ub levels significantly decreased. This discrepancy resulted in completely different phenotypic outcomes between the two different brain regions. While we have reported dysfunction and degeneration of hypothalamic neurons in adult Ubb(-/-) mice, there were no signs of functional impairment or degeneration in the locus coeruleus neurons, suggesting that the maintenance of free Ub above threshold levels could be an important mechanism for neuronal protection. Accordingly, we propose that, upon stress induced by disruption of Ubb, neuronal vulnerability may be determined based on the ability of neurons or neighboring cells to maintain free Ub levels for the protection of neuronal function and survival.
|Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. |
Boeve, BF; Boylan, KB; Graff-Radford, NR; DeJesus-Hernandez, M; Knopman, DS; Pedraza, O; Vemuri, P; Jones, D; Lowe, V; Murray, ME; Dickson, DW; Josephs, KA; Rush, BK; Machulda, MM; Fields, JA; Ferman, TJ; Baker, M; Rutherford, NJ; Adamson, J; Wszolek, ZK; Adeli, A; Savica, R; Boot, B; Kuntz, KM; Gavrilova, R; Reeves, A; Whitwell, J; Kantarci, K; Jack, CR; Parisi, JE; Lucas, JA; Petersen, RC; Rademakers, R
Brain : a journal of neurology 135 765-83 2012
Numerous kindreds with familial frontotemporal dementia and/or amyotrophic lateral sclerosis have been linked to chromosome 9, and an expansion of the GGGGCC hexanucleotide repeat in the non-coding region of chromosome 9 open reading frame 72 has recently been identified as the pathogenic mechanism. We describe the key characteristics in the probands and their affected relatives who have been evaluated at Mayo Clinic Rochester or Mayo Clinic Florida in whom the hexanucleotide repeat expansion were found. Forty-three probands and 10 of their affected relatives with DNA available (total 53 subjects) were shown to carry the hexanucleotide repeat expansion. Thirty-six (84%) of the 43 probands had a familial disorder, whereas seven (16%) appeared to be sporadic. Among examined subjects from the 43 families (n = 63), the age of onset ranged from 33 to 72 years (median 52 years) and survival ranged from 1 to 17 years, with the age of onset less than 40 years in six (10%) and greater than 60 in 19 (30%). Clinical diagnoses among examined subjects included behavioural variant frontotemporal dementia with or without parkinsonism (n = 30), amyotrophic lateral sclerosis (n = 18), frontotemporal dementia/amyotrophic lateral sclerosis with or without parkinsonism (n = 12), and other various syndromes (n = 3). Parkinsonism was present in 35% of examined subjects, all of whom had behavioural variant frontotemporal dementia or frontotemporal dementia/amyotrophic lateral sclerosis as the dominant clinical phenotype. No subject with a diagnosis of primary progressive aphasia was identified with this mutation. Incomplete penetrance was suggested in two kindreds, and the youngest generation had significantly earlier age of onset (greater than 10 years) compared with the next oldest generation in 11 kindreds. Neuropsychological testing showed a profile of slowed processing speed, complex attention/executive dysfunction, and impairment in rapid word retrieval. Neuroimaging studies showed bilateral frontal abnormalities most consistently, with more variable degrees of parietal with or without temporal changes; no case had strikingly focal or asymmetric findings. Neuropathological examination of 14 patients revealed a range of transactive response DNA binding protein molecular weight 43 pathology (10 type A and four type B), as well as ubiquitin-positive cerebellar granular neuron inclusions in all but one case. Motor neuron degeneration was detected in nine patients, including five patients without ante-mortem signs of motor neuron disease. While variability exists, most cases with this mutation have a characteristic spectrum of demographic, clinical, neuropsychological, neuroimaging and especially neuropathological findings.
|Transformation by E1A oncoprotein involves ubiquitin-mediated proteolysis of the neuronal and tumor repressor REST in the nucleus. |
Guan, H; Ricciardi, RP
Journal of virology 86 5594-602 2012
The adenovirus early region 1A (E1A) protein promotes cell immortalization and transformation by mediating the activities of key cellular regulators. The repressor element 1-silencing transcription factor (REST), which is a major neuronal and tumor suppressor, was previously found mainly in the cytoplasm rather than in the nuclei of adenovirus-transformed rodent cells (22). We now demonstrate that the loss of REST in the nucleus is due to its rapid degradation by the ubiquitin-proteasome system. Only nuclear REST, but not its cytoplasmic counterpart, was ubiquitinated and degraded. REST degradation was blocked by the ubiquitination inhibitor PYR-41 and the proteasome inhibitor MG-132 but not by the nuclear export inhibitor leptomycin B. REST degradation required both of its two C-terminal degrons that are recognized by the ubiquitin ligase SCF(β-TrCP), since deletion or mutation of either degron eliminated degradation. Importantly, E1A was shown to mediate REST ubiquitination and degradation by upregulating β-TrCP. Knockdown of E1A in virus-transformed cells reduced both β-TrCP and ubiquitination of nuclear REST. In contrast, when expressed in HeLa cells, E1A enhanced the degradation of nuclear REST. Reconstitution of REST in virus-transformed cells negatively affected E1A-mediated cell proliferation and anchorage-independent growth. These data strongly indicate that E1A stimulates ubiquitination and proteolysis of REST in the nucleus, thereby abolishing the tumor suppressor functions of REST.
|Tunicamycin produces TDP-43 cytoplasmic inclusions in cultured brain organotypic slices. |
Leggett, C; McGehee, DS; Mastrianni, J; Yang, W; Bai, T; Brorson, JR
Journal of the neurological sciences 317 66-73 2012
The cellular distribution of TAR DNA binding protein (TDP-43) is disrupted in several neurodegenerative disorders, including frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U subtype) and amyotrophic lateral sclerosis (ALS). In these conditions, TDP-43 is found in neuronal cytoplasmic inclusions, with loss of the normal nuclear expression. The mechanisms leading to TDP-43 redistribution and its role in disease pathophysiology remain unknown. We describe an in vitro neural tissue model that reproduces TDP-43 relocalization and inclusion formation. Two week-old coronal organotypic mouse brain slice cultures were treated with tunicamycin for 7 days. In cortical regions of treated slice cultures, cytoplasmic inclusions of TDP-43 immunoreactivity were observed, with loss of nuclear TDP-43 immunoreactivity. These inclusions were found in both astrocytes and neurons, and were of both skein-like and round morphologies. In contrast, TDP-43 cytoplasmic inclusions were not found in slices treated with staurosporine to induce apoptosis, or with trans-4-carboxy-l-proline (PDC) to induce chronic glutamate excitotoxicity. Furthermore, TDP-43 cytoplasmic inclusions did not co-localize with cleaved caspase-3, suggesting that TDP-43 mislocalization does not generally accompany caspase activation or apoptosis. The induction of TDP-43 cytoplasmic translocation in cerebrocortical slice cultures by tunicamycin provides a platform for further mechanistic investigations of pathological processing of TDP-43.
|H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling. |
Woolley, JF; Naughton, R; Stanicka, J; Gough, DR; Bhatt, L; Dickinson, BC; Chang, CJ; Cotter, TG
PloS one 7 e34050 2012
The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
|Ubiquitin homeostasis is critical for synaptic development and function. |
Chen, PC; Bhattacharyya, BJ; Hanna, J; Minkel, H; Wilson, JA; Finley, D; Miller, RJ; Wilson, SM
The Journal of neuroscience : the official journal of the Society for Neuroscience 31 17505-13 2011
The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (ax(J)) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of ax(J) mice with neuronally expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resulting from the loss of Usp14, demonstrating that ubiquitin deficiency is a major cause of the neurological defects observed in the ax(J) mice. We also show that proteasome components are normally induced during the first 2 weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.
|Mutations of the SLX4 gene in Fanconi anemia. |
Kim, Y; Lach, FP; Desetty, R; Hanenberg, H; Auerbach, AD; Smogorzewska, A
Nature genetics 43 142-6 2011
Fanconi anemia is a rare recessive disorder characterized by genome instability, congenital malformations, progressive bone marrow failure and predisposition to hematologic malignancies and solid tumors. At the cellular level, hypersensitivity to DNA interstrand crosslinks is the defining feature in Fanconi anemia. Mutations in thirteen distinct Fanconi anemia genes have been shown to interfere with the DNA-replication-dependent repair of lesions involving crosslinked DNA at stalled replication forks. Depletion of SLX4, which interacts with multiple nucleases and has been recently identified as a Holliday junction resolvase, results in increased sensitivity of the cells to DNA crosslinking agents. Here we report the identification of biallelic SLX4 mutations in two individuals with typical clinical features of Fanconi anemia and show that the cellular defects in these individuals' cells are complemented by wildtype SLX4, demonstrating that biallelic mutations in SLX4 (renamed here as FANCP) cause a new subtype of Fanconi anemia, Fanconi anemia-P.
|The von Hippel-Lindau tumor suppressor protein promotes c-Cbl-independent poly-ubiquitylation and degradation of the activated EGFR. |
Zhou, L; Yang, H
PloS one 6 e23936 2011
Somatic mutations or reduced expression of the von Hippel-Lindau (VHL) tumor suppressor occurs in the majority of the clear cell renal cell carcinoma (ccRCC) and is a causal factor for the pathogenesis of ccRCC. pVHL was reported to suppress the oncogenic activity of Epidermal Growth Factor Receptor (EGFR) by reducing the expression of the EGFR agonist TGF-α and by reducing the translation efficiency of EGFR itself. Furthermore, it was reported that pVHL down-regulates activated EGFR by promoting efficient lysosomal degradation of the receptor. These modes of negative regulation of EGFR by pVHL were dependent on Hypoxia Inducible Factor (HIF). In this study, we report that HIF was not the only factor stabilizing the activated EGFR in VHL-deficient ccRCC cells. Down-regulation of endogenous HIF in these cells had little effect on the turnover rates of the activated EGFR. Furthermore, neither pretreatment with lysosomal inhibitors pretreatment nor down-regulation of c-Cbl, a major E3 ubiquitin ligase that targets the activated EGFR for lysosomal degradation, significantly increased the stabilities of EGFR in VHL-expressing ccRCC cells. In contrast, pretreatment with proteasomal inhibitors extended EGFR lifetime and led to similar EGFR half-lives in VHL-expressing and VHL-deficient ccRCC cells. Down-regulation of c-Cbl in VHL-deficient ccRCC cells revealed that the c-Cbl and pVHL collaborated to down-regulate the activated EGFR. Finally, we found that pVHL promoted the poly-ubiquitylation of the activated EGFR, and this function was c-Cbl-independent. Thus these results indicate that pVHL limits EGFR signaling by promoting c-Cbl-independent poly-ubiquitylation of the activated receptor, which likely results in its degradation by proteasome.
|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
|Proteome studies on liver tissue in a phenobarbital-induced rat model. |
Wu Q, Zhang YH, Zhao X, Shi WL, Pu XP
European journal of pharmacology 670 333-40. Epub 2011 Sep 24. 2011
Many drugs may affect the activity of cytochrome P450 (CYP), which is a major source of adverse drug interactions (ADR). Phenobarbital (PB) is the typical inducer of cytochrome P450. The aim of our study was to determine the changes in the cytosolic proteins expression in rat liver at a protein level following induction of cytochrome P450. Firstly, we made a phenobarbital-induced cytochrome P450 rat model. The total cytosolic proteins were then extracted from rat liver tissue and separated by 2-D gel electrophoresis (2-DE). Differentially expressed spots were identified by MALDI-TOF/TOF tandem mass spectrometry followed by database searching. Eight differentially expressed proteins were identified and these proteins were found to be involved in protein degradation, oxidative stress, energy metabolism, biotransformation, and the synthesis of quinolinic acid (QUIN). These findings should provide useful information for research into the regulation of cytochrome P450 gene expression, drug metabolism and drug interaction.Copyright Â© 2011 Elsevier B.V. All rights reserved.
|Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease. |
Dammer, EB; Na, CH; Xu, P; Seyfried, NT; Duong, DM; Cheng, D; Gearing, M; Rees, H; Lah, JJ; Levey, AI; Rush, J; Peng, J
The Journal of biological chemistry 286 10457-65 2011
Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys(48)), 38% (Lys(63)), 8% (Lys(29)), 2% (Lys(11)), and 0.5% or less for linear, Lys(6), Lys(27), and Lys(33) linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys(6), Lys(11), Lys(27), Lys(29), and Lys(48) mediate proteasomal degradation, whereas Lys(63) chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys(48), may also contribute to lysosomal targeting, as both Lys(63) and Lys(48) linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys(11), Lys(48), and Lys(63) but not Lys(29) linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.
|Granulovacuolar degenerations appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders. |
Yamazaki, Y; Matsubara, T; Takahashi, T; Kurashige, T; Dohi, E; Hiji, M; Nagano, Y; Yamawaki, T; Matsumoto, M
PloS one 6 e26996 2011
Granulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimer's disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders.An autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized.The number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs.Our data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation.Full Text Article
|Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. |
Hartleben, B; Gödel, M; Meyer-Schwesinger, C; Liu, S; Ulrich, T; Köbler, S; Wiech, T; Grahammer, F; Arnold, SJ; Lindenmeyer, MT; Cohen, CD; Pavenstädt, H; Kerjaschki, D; Mizushima, N; Shaw, AS; Walz, G; Huber, TB
The Journal of clinical investigation 120 1084-96 2010
Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.
|Transgenic rat model of neurodegeneration caused by mutation in the TDP gene. |
Zhou, H; Huang, C; Chen, H; Wang, D; Landel, CP; Xia, PY; Bowser, R; Liu, YJ; Xia, XG
PLoS genetics 6 e1000887 2010
TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons and denervation atrophy of skeletal muscles. This robust rat model also recapitulated features of TDP-43 proteinopathies including the formation of TDP-43 inclusions, cytoplasmic localization of phosphorylated TDP-43, and fragmentation of TDP-43 protein. TDP transgenic rats will be useful for deciphering the mechanisms underlying TDP-43-related neurodegenerative diseases.
|Combined chemical and genetic approach to inhibit proteolysis by the proteasome. |
Collins, GA; Gomez, TA; Deshaies, RJ; Tansey, WP
Yeast (Chichester, England) 27 965-74 2010
Regulated protein destruction by the proteasome is crucial for the maintenance of normal cellular homeostasis. Much of our understanding of proteasome function stems from the use of drugs that inhibit its activity. Curiously, despite the importance of proteasomal proteolysis, previous studies have found that proliferation of the yeast Saccharomyces cerevisiae is relatively resistant to the effects of proteasome inhibitors such as MG132, even in the presence of mutations that increase inhibitor levels in cells. We reasoned that part of the resistance of S. cerevisiae to proteasome inhibitors stems from the fact that most proteasome inhibitors preferentially target the chymotryptic activity of the proteasome, and that the caspase-like and tryptic sites within the 20S core could compensate for proteasome function under these conditions. To test this hypothesis, we generated a strain of yeast in which the gene encoding the drug efflux pump Pdr5 is deleted, and the tryptic and caspase-like proteasome activities are inactivated by mutation. We find that this strain has dramatically increased sensitivity to the proteasome inhibitor MG132. Under these conditions, treatment of yeast with MG132 blocks progression through the cell cycle, increases the accumulation of polyubiquitylated proteins and decreases the ability to induce transcription of certain genes. These results highlight the contribution of the caspase-like and tryptic activities of the proteasome to its function, and provide a strategy to potently block proteasomal proteolysis in yeast that has practical applications.
|Characterization of the Brain 26S Proteasome and its Interacting Proteins. |
Tai, HC; Besche, H; Goldberg, AL; Schuman, EM
Frontiers in molecular neuroscience 3 2010
Proteasome-mediated proteolysis is important for synaptic plasticity, neuronal development, protein quality control, and many other processes in neurons. To define proteasome composition in brain, we affinity purified 26S proteasomes from cytosolic and synaptic compartments of the rat cortex. Using tandem mass spectrometry, we identified the standard 26S subunits and a set of 28 proteasome-interacting proteins that associated substoichiometrically and may serve as regulators or cofactors. This set differed from those in other tissues and we also found several proteins that associated only with either the cytosolic or the synaptic proteasome. The latter included the ubiquitin-binding factor TAX1BP1 and synaptic vesicle protein SNAP-25. Native gel electrophoresis revealed a higher proportion of doubly-capped 26S proteasome (19S-20S-19S) in the cortex than in the liver or kidney. To investigate the interplay between proteasome regulation and synaptic plasticity, we exposed cultured neurons to glutamate receptor agonist NMDA. Within 4 h, this agent caused a prolonged decrease in the activity of the ubiquitin-proteasome system as shown by disassembly of 26S proteasomes, decrease in ubiquitin-protein conjugates, and dissociation of the ubiquitin ligases UBE3A (E6-AP) and HUWE1 from the proteasome. Surprisingly, the regulatory 19S particles were rapidly degraded by proteasomal, not lysosomal degradation, and the dissociated E3 enzymes also degraded. Thus the content of proteasomes and their set of associated proteins can be altered by neuronal activity, in a manner likely to influence synaptic plasticity and learning.Full Text Article
|The RNA-binding protein FUS/TLS is a common aggregate-interacting protein in polyglutamine diseases. |
Hiroshi Doi,Shigeru Koyano,Yume Suzuki,Nobuyuki Nukina,Yoshiyuki Kuroiwa
Neuroscience research 66 2010
Neuronal intranuclear inclusions (NIIs) are the pathological hallmark of polyglutamine (polyQ) diseases. We previously found that the RNA-binding protein FUS/TLS is the major component of nuclear polyQ aggregates of a cellular model of Huntington disease. In this study, we revealed that FUS/TLS binds to NIIs in the human brains from patients with spinocerebellar ataxia type 1, 2, 3, and dentatorubral-pallidoluysian atrophy. Recent reports have revealed that mutations in FUS/TLS gene are responsible for familial amyotrophic lateral sclerosis 6 (ALS6). Our results indicated that changing FUS/TLS to an insoluble form may be a common process in polyQ diseases and ALS6.
|Lithium treatment arrests the development of neurofibrillary tangles in mutant tau transgenic mice with advanced neurofibrillary pathology. |
Leroy, Karelle, et al.
J. Alzheimers Dis., 19: 705-19 (2010) 2010
Neurofibrillary tangles (NFTs) made of phosphorylated tau proteins are a key lesion of Alzheimer's disease and other neurodegenerative diseases, and previous studies have indicated that lithium can decrease tau phosphorylation in tau transgenic models. In this study, we have reassessed the effectiveness of treatment per os with lithium on the prevention, the arrest, or the reversal of NFT development in a tau transgenic line (Tg30tau) developing severe neurofibrillary pathology in the brain and the spinal cord. Wild-type and Tgtau30 mice were treated per os with lithium carbonate or with natrium carbonate by chronic chow feeding for 8 months starting at the age of 3 months (to test for a preventive effect on NFT formation) or by oral gavage for 1 month starting at the age of 9 months (after development of NFTs). In mice treated by oral gavage, a decrease of tau phosphorylation and of Sarkosyl-insoluble aggregated tau was observed in the brain and in the spinal cord. The density of NFTs identified by Gallyas staining in the hippocampus and in the spinal cord was also significantly reduced and was similar to that observed at the beginning of the lithium treatment. In these animals, the level of brain beta-catenin was increased probably as a result of its stabilization by glycogen synthase kinase-3beta inhibition. Despite this inhibitory effect of lithium on NFT development, the motor and working memory deficits were not significantly rescued in these aged animals. Chronic chow feeding with lithium did not alter the development of NFT. Nevertheless, this study indicates that even a relatively short-term per os treatment leading to high blood concentration of lithium is effective in arresting the formation of NFTs in the hippocampus and the spinal cord of a tau transgenic model.
|Accelerated lipofuscinosis and ubiquitination in granulin knockout mice suggest a role for progranulin in successful aging. |
Ahmed Z, Sheng H, Xu YF, Lin WL, Innes AE, Gass J, Yu X, Hou H, Chiba S, Yamanouchi K, Leissring M, Petrucelli L, Nishihara M, Hutton ML, McGowan E, Dickson DW, Lewis J
Am J Pathol 177 311-24. Epub 2010 Jun 3. 2010
Progranulin (PGRN) is involved in wound repair, inflammation, and tumor formation, but its function in the central nervous system is unknown. Roles in development, sexual differentiation, and long-term neuronal survival have been suggested. Mutations in the GRN gene resulting in partial loss of the encoded PGRN protein cause frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions. We sought to understand the neuropathological consequences of loss of PGRN function throughout the lifespan of GRN-deficient ((-/+) and (-/-)) mice. An aged series of GRN-deficient and wild-type mice were compared by histology, immunohistochemistry, and electron microscopy. Although GRN-deficient mice were viable, GRN(-/-) mice were produced at lower than predicted frequency. Neuropathologically, GRN(-/+) were indistinguishable from controls; however, GRN(-/-) mice developed age-associated, abnormal intraneuronal ubiquitin-positive autofluorescent lipofuscin. Lipofuscin was noted in aged GRN(+/+) mice at levels comparable with those of young GRN(-/-) mice. GRN(-/-) mice developed microgliosis, astrogliosis, and tissue vacuolation, with focal neuronal loss and severe gliosis apparent in the oldest GRN(-/-) mice. Although no overt frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions type- or TAR DNA binding protein-43-positive lesions were observed, robust lipofuscinosis and ubiquitination in GRN(-/-) mice is strikingly similar to changes associated with aging and cellular decline in humans and animal models. Our data suggests that PGRN plays a key role in maintaining neuronal function during aging and supports the notion that PGRN is a trophic factor essential for long-term neuronal survival.Full Text Article
|Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction. |
Yamanaka T, Tosaki A, Miyazaki H, Kurosawa M, Furukawa Y, Yamada M, Nukina N
Human molecular genetics 2010
In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.
|Engineering of stable bispecific antibodies targeting IL-17A and IL-23. |
Mabry R, Lewis KE, Moore M, McKernan PA, Bukowski TR, Bontadelli K, Brender T, Okada S, Lum K, West J, Kuijper JL, Ardourel D, Franke S, Lockwood L, Vu T, Frank A, Appleby MW, Wolf A, Reardon B, Hamacher NB, Stevens B, Lewis P, Lewis KB, Gilbertson DG, Lantry M, Julien SH, Ostrander C, Chan C, Byrnes-Blake K, Brody J, Presnell S, Meengs B, Levin SD, Snavely M
Protein engineering, design & selection : PEDS 23 115-27 2010
Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
|An inhibitor of a deubiquitinating enzyme regulates ubiquitin homeostasis. |
Kimura, Y; Yashiroda, H; Kudo, T; Koitabashi, S; Murata, S; Kakizuka, A; Tanaka, K
Cell 137 549-59 2009
The dynamic and reversible process of ubiquitin modification controls various cellular activities. Ubiquitin exists as monomers, unanchored chains, or protein-conjugated forms, but the regulation of these interconversions remains largely unknown. Here, we identified a protein designated Rfu1 (regulator of free ubiquitin chains 1), which regulates intracellular concentrations of monomeric ubiquitins and free ubiquitin chains in Saccharomyces cerevisiae. Rfu1 functions as an inhibitor of Doa4, a deubiquitinating enzyme. Rapid loss of free ubiquitin chains upon heat shock, a condition in which more proteins require ubiquitin conjugation, was mediated in part by Doa4 and Rfu1. Thus, regulation of ubiquitin homeostasis is controlled by a balance between a deubiquitinating enzyme and its inhibitor. We propose that free ubiquitin chains function as a ubiquitin reservoir that allows maintenance of monomeric ubiquitins at adequate levels under normal conditions and rapid supply for substrate conjugation under stress conditions.
|TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. |
Iga Wegorzewska,Shaughn Bell,Nigel J Cairns,Timothy M Miller,Robert H Baloh
Proceedings of the National Academy of Sciences of the United States of America 106 2009
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that show considerable clinical and pathologic overlap, with no effective treatments available. Mutations in the RNA binding protein TDP-43 were recently identified in patients with familial amyotrophic lateral sclerosis (ALS), and TDP-43 aggregates are found in both ALS and FTLD-U (FTLD with ubiquitin aggregates), suggesting a common underlying mechanism. We report that mice expressing a mutant form of human TDP-43 develop a progressive and fatal neurodegenerative disease reminiscent of both ALS and FTLD-U. Despite universal transgene expression throughout the nervous system, pathologic aggregates of ubiquitinated proteins accumulate only in specific neuronal populations, including layer 5 pyramidal neurons in frontal cortex, as well as spinal motor neurons, recapitulating the phenomenon of selective vulnerability seen in patients with FTLD-U and ALS. Surprisingly, cytoplasmic TDP-43 aggregates are not present, and hence are not required for TDP-43-induced neurodegeneration. These results indicate that the cellular and molecular substrates for selective vulnerability in FTLD-U and ALS are shared between mice and humans, and suggest that altered DNA/RNA-binding protein function, rather than toxic aggregation, is central to TDP-43-related neurodegeneration.
|Induction of the lysosomal apoptosis pathway by inhibitors of the ubiquitin-proteasome system. |
Maria Berndtsson, Melanie Beaujouin, Linda Rickardson, Aleksandra Mandic Havelka, Rolf Larsson, Jacob Westman, Emmanuelle Liaudet-Coopman, Stig Linder, Maria Berndtsson, Melanie Beaujouin, Linda Rickardson, Aleksandra Mandic Havelka, Rolf Larsson, Jacob Westman, Emmanuelle Liaudet-Coopman, Stig Linder
International journal of cancer. Journal international du cancer 124 1463-9 2009
The lysosomal apoptosis pathway is a potentially interesting therapeutic target. Since apoptosis involving the lysosomal pathway has been described to involve cathepsins, we screened a drug library for agents that induce cathepsin-dependent apoptosis. Using pharmacological inhibitors and siRNA, we identified 2 structurally related agents (NSC687852 and NSC638646) that induced cathepsin D-dependent caspase-cleavage activity in human breast cancer cells. Both agents were found to induce the mitochondrial apoptosis pathway. NSC687852 and NSC638646 were found to inhibit the activity of ubiquitin isopeptidases and to induce the accumulation of high-molecular-mass ubiquitins in cells. We show that 3 other inhibitors of the proteasome degradation pathway induce lysosomal membrane permeabilization (LMP) and that cathepsin-D siRNA inhibits apoptosis induced by these agents. We conclude that a screen for cathepsin-dependent apoptosis-inducing agents resulted in the identification of ubiquitin isopeptidase inhibitors and that proteasome inhibitors with different mechanisms of action induce LMP and cathepsin D-dependent apoptosis.
|Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells. |
Takashi Nonaka, Tetsuaki Arai, Emanuele Buratti, Francisco E Baralle, Haruhiko Akiyama, Masato Hasegawa, Takashi Nonaka, Tetsuaki Arai, Emanuele Buratti, Francisco E Baralle, Haruhiko Akiyama, Masato Hasegawa, Takashi Nonaka, Tetsuaki Arai, Emanuele Buratti, Francisco E Baralle, Haruhiko Akiyama, Masato Hasegawa
FEBS letters 583 394-400 2009
We report phosphorylated and ubiquitinated aggregates of TAR DNA binding protein of 43 kDa (TDP-43) in SH-SY5Y cells similar to those in brains of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). Two candidate sequences for the nuclear localization signal were examined. Deletion of residues 78-84 resulted in cytoplasmic localization of TDP-43, whereas the mutant lacking residues 187-192 localized in nuclei, forming unique dot-like structures. Proteasome inhibition caused these to assemble into phosphorylated and ubiquitinated TDP-43 aggregates. The deletion mutants lacked the exon skipping activity of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Our results suggest that intracellular localization of TDP-43 and proteasomal function may be involved in inclusion formation and neurodegeneration in TDP-43 proteinopathies.
|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
|Neuropathological investigation of regions responsible for semantic aphasia in frontotemporal lobar degeneration. |
Ryoko Yamamoto, Eizo Iseki, Shinji Higashi, Norio Murayama, Michiko Minegishi, Kiyoshi Sato, Hiroaki Hino, Koshiro Fujisawa, Kenji Kosaka, Takashi Togo, Omi Katsuse, Hirotake Uchikado, Yoshiko Furukawa, Mari Yoshida, Yoshio Hashizume, Heii Arai, Ryoko Yamamoto, Eizo Iseki, Shinji Higashi, Norio Murayama, Michiko Minegishi, Kiyoshi Sato, Hiroaki Hino, Koshiro Fujisawa, Kenji Kosaka, Takashi Togo, Omi Katsuse, Hirotake Uchikado, Yoshiko Furukawa, Mari Yoshida, Yoshio Hashizume, Heii Arai
Dementia and geriatric cognitive disorders 27 214-23 2009
BACKGROUND/AIMS: Semantic dementia is a subtype of frontotemporal lobar degeneration, of which an initial symptom is semantic aphasia. Semantic dementia pathologically corresponds to atypical Pick's disease (aPiD), showing ubiq- uitin-positive inclusions similar to those in dementia with motor neuron disease (D-MND). Previous studies have not clarified the regions responsible for semantic aphasia in aPiD, and there have been no reported neuropathological studies concerning its pathomechanism. METHODS: We neuropathologically investigated aPiD and D-MND cases with and without semantic aphasia. RESULTS: We determined that the regions involved in the early stage of the disease course of semantic dementia were more restricted to the anterior and inferior portion of the temporal lobe on the side of the dominant hemisphere. CONCLUSION: Degeneration of the temporal pole is most likely to participate in the pathomechanism of SA in semantic dementia.
|Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43. |
Takashi Nonaka, Fuyuki Kametani, Tetsuaki Arai, Haruhiko Akiyama, Masato Hasegawa
Human molecular genetics 18 3353-64 2009
TAR DNA binding protein of 43 kDa (TDP-43) is a major component of the ubiquitin-positive inclusions found in the brain of patients with frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). Here, we report that expression of TDP-43 C-terminal fragments as green fluorescent protein (GFP) fusions in SH-SY5Y cells results in the formation of abnormally phosphorylated and ubiquitinated inclusions that are similar to those found in FTLD-U and ALS. Co-expression of DsRed-tagged full-length TDP-43 with GFP-tagged C-terminal fragments of TDP-43 causes formation of cytoplasmic inclusions positive for both GFP and DsRed. Cells with GFP and DsRed positive inclusions lack normal nuclear staining for endogenous TDP-43. These results suggest that GFP-tagged C-terminal fragments of TDP-43 are bound not only to transfected DsRed-full-length TDP-43 but also to endogenous TDP-43. Endogenous TDP-43 may be recruited to cytoplasmic aggregates of TDP-43 C-terminal fragments, which results in the failure of its nuclear localization and function. Interestingly, expression of GFP-tagged TDP-43 C-terminal fragments harboring pathogenic mutations that cause ALS significantly enhances the formation of inclusions. We also identified cleavage sites of TDP-43 C-terminal fragments deposited in the FTLD-U brains using mass spectrometric analyses. We propose that generation and aggregation of phosphorylated C-terminal fragments of TDP-43 play a primary role in the formation of inclusions and resultant loss of normal TDP-43 localization, leading to neuronal degeneration in TDP-43 proteinopathy.
|Absence of ubiquitinated inclusions in hypocretin neurons of patients with narcolepsy. |
M Honda, T Arai, M Fukazawa, Y Honda, K Tsuchiya, A Salehi, H Akiyama, E Mignot
Neurology 73 511-517 2009
OBJECTIVES: The cause of hypocretin cell loss in human narcolepsy-cataplexy is unknown but has been suggested to be neurodegenerative in nature. To test this hypothesis, we evaluated the remaining hypocretin cells in human narcolepsy brains for the presence of aggregated protein inclusions, gliosis, and inflammation. METHODS: Brains were examined by routine histologic methods for potential comorbid neurodegenerative diseases and through immunohistochemical screening for protein inclusions in the hypothalamus. Hypothalamic sections of 4 subjects with narcolepsy and 5 nonneurologic controls were examined immunohistochemically with antibodies against ubiquitin (a marker of aggregated protein), allograft inflammatory factor 1 (AIF1, a microglial activation marker), glial fibrillary acidic protein (GFAP, a reactive astrocytic marker), and hypocretin. Hypothalami of subjects with narcolepsy were additionally examined for the presence of known components of protein aggregates (tau, alpha-synuclein, amyloid beta, and TDP-43). RESULTS: Hypocretin cells were markedly decreased in all 4 subjects with narcolepsy. Ubiquitinated inclusions were not observed in the total of 96 remaining hypocretin cells in these subjects. Further, we noted that even in patients with dementia neuropathology, the lateral hypothalamic hypocretin area was spared from ubiquitinated inclusions. AIF1 and GFAP staining in the perifornical area was unremarkable. CONCLUSIONS: Our findings suggest that hypocretin cell loss does not involve ubiquitinated inclusions, the hallmark of most neurodegenerative diseases. The lack of increased markers of inflammation also argues against a progressive and continuous neurodegenerative process.,Full Text Article
|The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. |
PC Chen, LN Qin, XM Li, BJ Walters, JA Wilson, L Mei, SM Wilson
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 10909-19 2009
Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer's, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient axJ mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the axJ mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.Full Text Article
|Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells. |
Borland, MK; Trimmer, PA; Rubinstein, JD; Keeney, PM; Mohanakumar, K; Liu, L; Bennett, JP
Molecular neurodegeneration 3 21 2008
Parkinson's disease, the most common adult neurodegenerative movement disorder, demonstrates a brain-wide pathology that begins pre-clinically with alpha-synuclein aggregates ("Lewy neurites") in processes of gut enteric and vagal motor neurons. Rostral progression into substantia nigra with death of dopamine neurons produces the motor impairment phenotype that yields a clinical diagnosis. The vast majority of Parkinson's disease occurs sporadically, and current models of sporadic Parkinson's disease (sPD) can utilize directly infused or systemic neurotoxins.We developed a differentiation protocol for human SH-SY5Y neuroblastoma that yielded non-dividing dopaminergic neural cells with long processes that we then exposed to 50 nM rotenone, a complex I inhibitor used in Parkinson's disease models. After 21 days of rotenone, ~60% of cells died. Their processes retracted and accumulated ASYN-(+) and UB-(+) aggregates that blocked organelle transport. Mitochondrial movement velocities were reduced by 8 days of rotenone and continued to decline over time. No cytoplasmic inclusions resembling Lewy bodies were observed. Gene microarray analyses showed that the majority of genes were under-expressed. qPCR analyses of 11 mtDNA-encoded and 10 nDNA-encoded mitochondrial electron transport chain RNAs' relative expressions revealed small increases in mtDNA-encoded genes and lesser regulation of nDNA-encoded ETC genes.Subacute rotenone treatment of differentiated SH-SY5Y neuroblastoma cells causes process retraction and partial death over several weeks, slowed mitochondrial movement in processes and appears to reproduce the Lewy neuritic changes of early Parkinson's disease pathology but does not cause Lewy body inclusions. The overall pattern of transcriptional regulation is gene under-expression with minimal regulation of ETC genes in spite of rotenone's being a complex I toxin. This rotenone-SH-SY5Y model in a differentiated human neural cell mimics changes of early Parkinson's disease and may be useful for screening therapeutics for neuroprotection in that disease stage.
|Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation. |
Kim, MO; Chawla, P; Overland, RP; Xia, E; Sadri-Vakili, G; Cha, JH
The Journal of neuroscience : the official journal of the Society for Neuroscience 28 3947-57 2008
Although transcriptional dysregulation is a critical pathogenic mechanism in Huntington's disease (HD), it is still not known how mutant huntingtin causes it. Here we show that alteration of histone monoubiquitylation is a key mechanism. Disrupted interaction of huntingtin with Bmi-1, a component of the hPRC1L E3 ubiquitin ligase complex, increases monoubiquityl histone H2A (uH2A) levels in a cell culture model of HD. Genes with expression that is repressed in transgenic R6/2 mouse brain have increased uH2A and decreased uH2B at their promoters, whereas actively transcribed genes show the opposite pattern. Reduction in uH2A reverses transcriptional repression and inhibits methylation of histone H3 at lysine 9 in cell culture. In contrast, reduction in uH2B induces transcriptional repression and inhibits methylation of histone H3 at lysine 4. This is the first report to demonstrate hPRC1L as a huntingtin-interacting histone modifying complex and a crucial role for histone monoubiquitylation in mammalian brain gene expression, which broadens our understanding of histone code. These findings also provide a rationale for targeting histone monoubiquitylation for therapy in HD.
|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.
|Caspase-cleaved TAR DNA-binding protein-43 is a major pathological finding in Alzheimer's disease. |
Brain research 1228 189-98 2008
The TAR DNA-binding protein-43 (TDP-43) has been identified as a major constituent of inclusions found in frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). To determine a possible role for TDP-43 in Alzheimer's disease (AD), a site-directed caspase-cleavage antibody to TDP-43 based upon a known caspase-3 cleavage consensus site within TDP- 43 at position D219 was designed. In vitro, this antibody labeled the predicted 25 kDa caspase-cleavage fragment of TDP-43 without labeling full-length TDP-43 following digestion of recombinant TDP-43 with caspase-3 or treatment of HeLa cells with staurosporine. Application of this antibody in postmortem brain sections indicated the presence of caspase-cleaved TDP-43 in Hirano bodies, tangles, reactive astrocytes and neuritic plaques of the AD brain. Caspase-cleaved TDP-43 also co-localized with ubiquitin labeled neurons as well as dystrophic neurites within plaque regions. These results suggest that caspase-cleaved TDP-43 is a major pathological finding in AD and may contribute to the neurodegeneration associated with this disease.Full Text Article
|Limbic structures are prone to age-related impairments in proteasome activity and neuronal ubiquitinated inclusions in SAMP10 mouse: a model of cerebral degeneration. |
A Shimada, H Keino, N Kawamura, Y Chiba, M Hosokawa, A Shimada, H Keino, N Kawamura, Y Chiba, M Hosokawa
Neuropathology and applied neurobiology 34 33-51 2008
AIMS: Neurodegenerative diseases are characterized by ubiquitinated inclusions in selective brain regions. Here we investigated whether the dysfunction of the ubiquitin proteasome system might be involved in the pathogenesis and regional selectivity of neuronal ubiquitinated inclusions using the SAMP10 strain of mouse, an inbred model of age-related cerebral degeneration. METHODS: By comparing SAMP10 mice at various ages with SAMR1 and C57BL mice as normal brain ageing controls, we studied morphological features and distribution of inclusions. We measured tissue proteasome activity in different brain regions of mice at various ages by fluorogenic substrate assays. We induced inclusions in cultured neurones by inhibiting the proteasome and analysed changes in the dendritic morphology. RESULTS: Inclusions were formed in association with lipofuscin in neuronal perikarya and occurred most frequently in the limbic-related forebrain structures. There were sparse inclusion-bearing neurones in the non-limbic forebrain. In aged SAMR1 and C57BL, there were far fewer inclusions in the limbic-related forebrain than in aged SAMP10. The proteasome activity in the limbic-related forebrain decreased much more rapidly and remarkably upon ageing (26% activity was detected in 17-month-old compared with 3-month-old mice) in SAMP10 than in SAMR1. The proteasome activity in the non-limbic forebrain did not change significantly with advancing age in either SAMP10 or SAMR1. Proteasomal inhibition enhanced the formation of ubiquitinated inclusions in cultured neurones. Neurones bearing inclusions had shortened neurites. CONCLUSIONS: We propose that the regional selectivity of proteasomal impairment is causally related to the selectivity of inclusion formation and associated dendritic degeneration in neurones of ageing SAMP10 mice.
|Frontotemporal dementia mimicking dementia with Lewy bodies. |
Daniel O Claassen, Joseph E Parisi, Caterina Giannini, Bradley F Boeve, Dennis W Dickson, Keith A Josephs, Daniel O Claassen, Joseph E Parisi, Caterina Giannini, Bradley F Boeve, Dennis W Dickson, Keith A Josephs
Cognitive and behavioral neurology : official journal of the Society for Behavioral and Cognitive Neurology 21 157-63 2008
BACKGROUND: Some patients with frontotemporal dementia (FTD) have concomitant extrapyramidal symptoms and psychosis and may simultaneously meet consensus criteria for FTD and for dementia with Lewy bodies (DLB). Clinicopathologic studies are helpful in understanding the underlying neurodegenerative process in such cases. OBJECTIVE: To describe clinical and pathologic features of 6 patients with signs and symptoms suggestive of both a diagnosis of FTD and DLB at first clinical presentation, of which 2 patients have now undergone autopsy, and to compare them with autopsy-confirmed FTD and Lewy body disease patients. RESULTS: All 6 patients met published consensus criteria for a diagnosis of both FTD and DLB (5 probable and 1 possible). Clinical symptoms of FTD included personality and behavioral changes, whereas those suggestive of DLB included Parkinsonism, fluctuating cognition, parasomnia, and hallucinations. Five patients underwent single photon emission computed tomography ((99m)Tc) imaging, which showed varying degrees of frontal lobe hypoperfusion. Magnetic resonance imaging, electroencephalogram, and electromyogram were not helpful in differentiating FTD from DLB. Histologic examination of the 2 autopsy cases was consistent with a pathologic diagnosis of TDP-43 proteinopathy; specifically frontotemporal lobar degeneration with ubiquitin-only immunoreactive changes, type 1. There were significant differences between these 6 patients and the 2 groups of autopsy confirmed FTD and Lewy body disease patients. CONCLUSIONS: We have identified a novel group of FTD patients with clinical features that overlap with DLB, yet seem to be different from both typical FTD and typical Lewy body disease.
|Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha. |
Krämer, OH; Müller, S; Buchwald, M; Reichardt, S; Heinzel, T
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 22 1369-79 2008
The chromosomal translocation products AML1-ETO and PML-RARalpha contribute to the pathogenesis of leukemias. Here, we demonstrate that both AML1-ETO and PML-RARalpha are degraded by the ubiquitin-proteasome system and that their turnover critically depends on the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Contrary to its role in HDAC2 degradation, the E3-ligase RLIM does not target AML1-ETO and PML-RARalpha for ubiquitin-dependent elimination. RLIM rather is a substrate of SIAH-1, which indicates that these E3-ligases operate in a hierarchical order. Remarkably, proteasomal degradation of leukemia fusion proteins, in addition to the block of histone deacetylase (HDAC) enzymatic activity is a consequence of HDAC-inhibitor treatment. The former requires the induction of UbcH8 expression and each of these processes might be beneficial for leukemia treatment. Our observations shed light on the mechanism determining the interplay between E2-conjugases, E3-ligases, and their substrates and suggest a strategy for utilizing the ubiquitylation machinery in a therapeutic setting.
|Basophilic inclusion body disease and neuronal intermediate filament inclusion disease: a comparative clinicopathological study. |
Osamu Yokota, Kuniaki Tsuchiya, Seishi Terada, Hideki Ishizu, Hirotake Uchikado, Manabu Ikeda, Kiyomitsu Oyanagi, Imaharu Nakano, Shigeo Murayama, Shigetoshi Kuroda, Haruhiko Akiyama
Acta neuropathologica 115 561-75 2008
While both neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD) show frontotemporal lobar degeneration and/or motor neuron disease, it remains unclear whether, and how, these diseases differ from each other. Here, we compared the clinicopathological characteristics of four BIBD and two NIFID cases. Atypical initial symptoms included weakness, dysarthria, and memory impairment in BIBD, and dysarthria in NIFID. Dementia developed more than 1 year after the onset in some BIBD and NIFID cases. Upper and lower motor neuron signs, parkinsonism, and parietal symptoms were noted in both diseases, and involuntary movements in BIBD. Pathologically, severe caudate atrophy was consistently found in both diseases. Cerebral atrophy was distributed in the convexity of the fronto-parietal region in NIFID cases. In both BIBD and NIFID, the frontotemporal cortex including the precentral gyrus, caudate nucleus, putamen, globus pallidus, thalamus, amygdala, hippocampus including the dentate gyrus, substantia nigra, and pyramidal tract were severely affected, whereas lower motor neuron degeneration was minimal. While alpha-internexin-positive inclusions without cores were found in both NIFID cases, one NIFID case also had alpha-internexin- and neurofilament-negative, but p62-positive, cytoplasmic spherical inclusions with eosinophilic p62-negative cores. These two types of inclusions frequently coexisted in the same neuron. In three BIBD cases, inclusions were tau-, alpha-synuclein-, alpha-internexin-, and neurofilament-negative, but occasionally p62-positive. These findings suggest that: (1) the clinical features and distribution of neuronal loss are similar in BIBD and NIFID, and (2) an unknown protein besides alpha-internexin and neurofilament may play a pivotal pathogenetic role in at least some NIFID cases.
|RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells. |
Doi, H; Okamura, K; Bauer, PO; Furukawa, Y; Shimizu, H; Kurosawa, M; Machida, Y; Miyazaki, H; Mitsui, K; Kuroiwa, Y; Nukina, N
The Journal of biological chemistry 283 6489-500 2008
Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.
|Voxel-based morphometry in autopsy proven PSP and CBD. |
Josephs, KA; Whitwell, JL; Dickson, DW; Boeve, BF; Knopman, DS; Petersen, RC; Parisi, JE; Jack, CR
Neurobiology of aging 29 280-9 2008
The aim of this study was to compare the patterns of grey and white matter atrophy on MRI in autopsy confirmed progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), and to determine whether the patterns vary depending on the clinical syndrome. Voxel-based morphometry was used to compare patterns of atrophy in 13 PSP and 11 CBD subjects and 24 controls. PSP and CBD subjects were also subdivided into those with a dominant dementia or extrapyramidal syndrome. PSP subjects showed brainstem atrophy with involvement of the cortex and underlying white matter. Frontoparietal grey and subcortical grey matter atrophy occurred in CBD. When subdivided, PSP subjects with an extrapyramidal syndrome had more brainstem atrophy and less cortical atrophy than CBD subjects with an extrapyramidal syndrome. PSP subjects with a dementia syndrome had more subcortical white matter atrophy than CBD subjects with a dementia syndrome. These results show regional differences between PSP and CBD that are useful in predicting the underlying pathology, and help to shed light on the in vivo distribution of regional atrophy in PSP and CBD.Full Text Article
|Neuropathology of primary adult-onset dystonia. |
J L Holton, S A Schneider, T Ganesharajah, S Gandhi, C Strand, P Shashidharan, J Barreto, N W Wood, A J Lees, K P Bhatia, T Revesz
Neurology 70 695-9 2008
BACKGROUND: Idiopathic adult-onset primary dystonia usually affects the upper body and remains focal. Underlying mechanisms are unknown, and there are only limited neuropathologic studies in the literature. Recently, ubiquitinated perinuclear inclusion bodies were found in the brainstem of patients with DYT1-related dystonia. In X-linked recessive dystonia-parkinsonism, neuronal loss in the striosome compartment of the striatum has been described. However, it was unclear whether these changes are characteristic of these particular disorders or an epiphenomenon of dystonic conditions in general. METHODS: Six cases of adult-onset dystonia and four controls were studied using immunohistochemistry to determine the presence of inclusion bodies immunoreactive for torsinA, ubiquitin, and laminA/C in the brainstem. The distribution of calcineurin expressing neurons in the striatum was also determined to ascertain whether there is loss of neurons in the striosome compartment. RESULTS: In contrast to early-onset dystonia, neuronal inclusions immunoreactive for torsinA, ubiquitin, and laminA/C were not present in the brainstem nuclei. There was no apparent loss of the striatal striosome compartment. CONCLUSION: Our findings suggest that the underlying mechanism in the adult-onset primary torsion dystonia is different from that of early-onset DYT1-related dystonia and also DYT3 X-linked recessive dystonia-parkinsonism. Alternative mechanisms may underpin the pathophysiology of adult-onset primary dystonia.
|Cell surface targeting of VPAC1 receptors: evidence for implication of a quality control system and the proteasome. |
Ingrid Langer, Karelle Leroy, Nathalie Gaspard, Jean-Pierre Brion, Patrick Robberecht, Ingrid Langer, Karelle Leroy, Nathalie Gaspard, Jean-Pierre Brion, Patrick Robberecht
Biochimica et biophysica acta 1783 1663-72 2008
Like for most transmembrane proteins, translation of G protein-coupled receptors (GPCRs) mRNA takes place at the endoplasmic reticulum (ER) where they are synthesized, folded and assembled. The molecular mechanisms involved in the transport process of GPCRs from ER to the plasma membrane are poorly investigated. Here we studied the mechanisms involved in glycosylation-dependent cell surface expression and quality control of the receptor for Vasoactive Intestinal Polypeptide (VIP) VPAC1, a member of the B family of GPCRs. Using biochemical and pharmacological techniques and fluorescence microscopy, we have shown that only a fraction of newly synthesized VPAC1 attains properly conformation that allows their cell surface targeting. Misfolded or immature VPAC1 are taken in charge by co- and post-translational quality control that involves: 1) calnexin-dependent folding strictly through a glycan-dependent mechanism, 2) BiP-dependant folding, 3) translocation to the cytoplasm and proteasome-dependent degradation of improper proteins, and 4) post-ER quality control check points. Our data suggest that VPAC1 expression/trafficking pathways are under the control of complex and precise molecular mechanisms to ensure that only proper VPAC1 reaches the cell surface.
|White matter lesions in the brain with frontotemporal lobar degeneration with motor neuron disease: TDP-43-immunopositive inclusions co-localize with p62, but not ubiquitin. |
Masanori Hiji, Tetsuya Takahashi, Hiromasa Fukuba, Hiroshi Yamashita, Tatsuo Kohriyama, Masayasu Matsumoto
Acta neuropathologica 116 183-91 2008
Recently, TDP-43 was established as a major component of the ubiquitinated inclusions found in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND). However, differences in the underlying pathogenesis between ALS and FTLD-MND remain yet to be elucidated. Originally, TDP-43-immunopositive inclusions were found in neuronal cells and reported to be ubiquitinated. This study shows that TDP-43-positive inclusions were distributed throughout the subcortical white matter except for the occipital lobe in the FTLD-MND brain, but not in the ALS brain. TDP-43-positive inclusions were also prominent features of pathologically proven FTLD-MND cases (p-FTLD-MND) without history of apparent clinical cognitive decline. A substantial fraction of these inclusions was also p62-immunoreactive, and another noteworthy feature was that those inclusions did not stain positively for ubiquitin. Significant correlations between immunoreactivity for TDP-43 and p62 were observed, particularly in p-FTLD-MND (Pearson correlation coefficient, 0.976). Furthermore, TDP-43 extracted from white matter appeared to be uncleaved. These results indicate that pathological changes might take place within the white matter also in the brain with FTLD-MND, but in a different manner than within the gray matter.
|Differential effects of Usp14 and Uch-L1 on the ubiquitin proteasome system and synaptic activity. |
B J Walters,S L Campbell,P C Chen,A P Taylor,D G Schroeder,L E Dobrunz,K Artavanis-Tsakonas,H L Ploegh,J A Wilson,G A Cox,S M Wilson
Molecular and cellular neurosciences 39 2008
The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and Usp14, result in neurological phenotypes in mice. We identified a new mutation in Uch-L1 and compared the roles of Uch-L1 and Usp14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or Usp14 result in decreased levels of ubiquitin, suggesting that they both regulate ubiquitin stability in the nervous system. However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the Usp14-deficient mice, and changes in hippocampal synaptic transmission were only observed in Usp14-deficient mice. In addition, while Usp14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and Usp14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.Full Text Article
|Increased level of active GSK-3beta in Alzheimer's disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration |
Leroy, K, et al
Neuropathol Appl Neurobiol, 33:43-55 (2007) 2007
|Hippocampal sclerosis in tau-negative frontotemporal lobar degeneration. |
Keith A Josephs, Dennis W Dickson
Neurobiology of aging 28 1718-22 2007
Tau-negative frontotemporal lobar degeneration (FTLD) can be divided into those with motor neuron disease (FTLD-MND), and those without MND, but with ubiquitin-immunoreactive neuronal inclusions (FTLD-U). Some authors group FTLD-U and FTLD-MND together as tau-negative FTLD, but others separate them on the basis of clinical, pathologic and imaging differences. In 103 cases of pathologically confirmed, tau-negative FTLD (FTLD-MND and FTLD-U), we assessed the frequency of hippocampal sclerosis defined as neuronal loss in the subicular or CA1 regions of the hippocampus. The subjects in the FTLD-U group were older at death and had longer disease duration. After adjusting for age at death and disease duration, we found a significant difference in the frequency of hippocampal sclerosis in the FTLD-U group (79%) compared to FTLD-MND group (26%) (p=0.02). The difference in frequency of HpScl in FTLD-U compared to FTLD-MND is further evidence that they are separate clinicopathologic entities.
|PIASy-mediated sumoylation of Yin Yang 1 depends on their interaction but not the RING finger. |
Deng, Z; Wan, M; Sui, G
Molecular and cellular biology 27 3780-92 2007
As a multifunctional protein, Yin Yang 1 (YY1) has been demonstrated to regulate both gene expression and protein posttranslational modifications. However, gaps still exist in our knowledge of how YY1 can be modified and what the consequences of its modifications are. Here we report that YY1 protein can be sumoylated both in vivo and in vitro. We have identified lysine 288 as the major sumoylation site of YY1. We also discovered that PIASy, a SUMO E3 ligase, is a novel YY1-interacting protein and can stimulate the sumoylation of YY1 both in vitro and in vivo. Importantly, the effects of PIASy mutants on in vivo YY1 sumoylation correlate with the YY1-PIASy interaction but do not depend on the RING finger domain of PIASy. This regulation is unique to YY1 sumoylation because PIASy-mediated p53 sumoylation still relies on the integrity of PIASy, which is also true of all of the previously identified substrates of PIASy. In addition, PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 in vivo, and differentially regulates YY1 transcriptional activity on different target promoters. This study demonstrates that YY1 is a target of SUMOs and reveals a novel feature of a SUMO E3 ligase in the PIAS family that selectively stimulates protein sumoylation independent of the RING finger domain.Full Text Article
|Appearance pattern of TDP-43 in Japanese frontotemporal lobar degeneration with ubiquitin-positive inclusions. |
Shinji Higashi, Eizo Iseki, Ryoko Yamamoto, Michiko Minegishi, Hiroaki Hino, Koshiro Fujisawa, Takashi Togo, Omi Katsuse, Hirotake Uchikado, Yoshiko Furukawa, Kenji Kosaka, Heii Arai
Neuroscience letters 419 213-8 2007
TAR-DNA-binding protein 43 (TDP-43) was identified as a major component of ubiquitin-positive intracellular inclusions from brains of patients with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Here, we immunohistochemically investigated the appearance pattern of TDP-43 to compare the distribution of TDP-43-positive structures with that of ubiquitin-positive structures in brains of seven patients with Japanese FTLD-U, five of atypical Pick's disease (aPiD) and two of dementia with motor neuron disease (D-MND), as well as two patients with PiD as control. TDP-43-immunoreactivity generally colocalized to ubiquitin-immunoreactivity in both neuronal cytoplasmic inclusions and neurites in FTLD-U brains, but TDP-43-immunoreactivity alone or ubiquitin-immunoreactivity alone was also observed. In five aPiD cases, double-immunostaining with TDP-43 and ubiquitin demonstrated that diffuse neuronal cytoplasmic immunostaining for ubiquitin did not always display TDP-43-immunoreactivity. In contrast, ubiquitin-positive neuronal cytoplasmic inclusions usually displayed TDP-43-immunoreactivity in two D-MND cases, although most glial inclusions in one of two cases were immunostained only for TDP-43. TDP-43-positive structures were not detected in two PiD cases. Thus, the ratio in the appearance pattern of TDP-43 and ubiquitin was different between aPiD and D-MND, leading to the hypothesis that this difference may be associated with the two pathogenic variants related to clinical and pathological heterogeneity in FTLD-U.
|Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases. |
Hanae Nakashima-Yasuda, Kunihiro Uryu, John Robinson, Sharon X Xie, Howard Hurtig, John E Duda, Steven E Arnold, Andrew Siderowf, Murray Grossman, James B Leverenz, Randy Woltjer, Oscar L Lopez, Ronald Hamilton, Debby W Tsuang, Douglas Galasko, Eliezer Masliah, Jeffrey Kaye, Christopher M Clark, Thomas J Montine, Virginia M-Y Lee, John Q Trojanowski
Acta neuropathologica 114 221-9 2007
Here, we investigated if TAR-DNA-binding protein-43 (TDP-43), the disease protein in frontotemporal lobar degeneration and ubiquitin inclusions with or without motor neuron disease as well as amyotrophic lateral sclerosis, also formed inclusions in Lewy body (LB) disorders including Parkinson's disease (PD) without or with dementia (PDD), and dementia with LBs (DLB) alone or in association with Alzheimer's disease (AD). Immunohistochemical analyses of TDP-43 in clinically well characterized and pathologically confirmed cases of DLB + AD, PD and PDD demonstrated TDP-43 pathology in the following percentage of cases: DLB + AD = 25/80 (31.3%); PD = 5/69 (7.2%); PDD = 4/21 (19%), while DLB and normal controls exhibited no (0/10, 0%) and one cases (1/33, 3%) presenting TDP-43 pathology, respectively. Significant differences in the prevalence of TDP-43 lesions were noted between disease versus normal brains (P 0.001) as well as demented versus non-demented brains (P 0.001). Statistical analyses revealed a positive relationship between TDP-43 lesions and several clinical and pathological parameters in these disorders suggesting the TDP-43 pathology may have co-morbid effects in LB diseases. This study expands the concept of TDP-43 proteinopathies by implicating TDP-43 lesions in mechanisms of neurodegeneration in LB disorders.
|Early axonopathy preceding neurofibrillary tangles in mutant tau transgenic mice. |
Karelle Leroy, Alexis Bretteville, Katharina Schindowski, Emmanuel Gilissen, Michèle Authelet, Robert De Decker, Zehra Yilmaz, Luc Buée, Jean-Pierre Brion
The American journal of pathology 171 976-92 2007
Neurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction.Full Text Article
|Ubiquitin and ubiquitin-conjugated protein expression in the rat cerebral cortex and hippocampus following traumatic brain injury (TBI). |
Xianglan Yao, Jiong Liu, Joseph T McCabe
Brain research 1182 116-22 2007
Regulation of protein turnover is essential to the survival of eukaryotic cells. This important cellular process is partly regulated by the ubiquitin-proteasome system through posttranslational modification by the conjugation of ubiquitin chains to proteins targeted for degradation by proteasomes. The present study examined ubiquitin mRNA and protein expression in the CNS of rats that sustained traumatic brain injury (TBI). Quantitative real-time polymerase chain reaction results indicated that mRNA levels of ubA52, ubB and ubC in the ipsilateral cerebral cortex were significantly decreased on Day 1 post-TBI, that ubC mRNA levels also were significantly lower than control on Day 3 post-TBI, but that by Day 7 post-TBI, ubA52, ubB and ubC mRNA levels had all returned to control levels. In the ipsilateral hippocampus, ubA52 mRNA levels were significantly lower on Days 1-7 post-TBI, while ubB and ubC mRNA levels were less only on Day 1 post-TBI. Western blotting found that free ubiquitin protein levels were significantly reduced in both ipsilateral cerebral cortex and hippocampus on Days 1-7 post-TBI, while there was markedly increased ubiquitin-conjugated protein in ipsilateral cerebral cortex on Day 7 and in hippocampus on Days 3-7 post-TBI. Our study suggests that altered ubiquitin system function in the CNS contributes to the pathological outcomes of TBI.
|Rates of cerebral atrophy in autopsy-confirmed progressive supranuclear palsy. |
Keith A Josephs, Jennifer L Whitwell, Bradley F Boeve, Maria M Shiung, Jeffrey L Gunter, Joseph E Parisi, Dennis W Dickson, Clifford R Jack
Annals of neurology 59 200-3 2006
OBJECTIVE: To determine the rates of cerebral atrophy and ventricular expansion in six patients with autopsy confirmed progressive supranuclear palsy (PSP) and multiple antemortem volumetric head MRI scans. METHODS: This study uses the boundary shift integral to determine rates of cerebral atrophy and ventricular expansion in six patients with pathologically confirmed PSP compared with six age- and sex-matched control subjects. RESULTS: Patients with PSP had a rate of cerebral atrophy and ventricular expansion of 1.3 and 7.0% per year compared with 0.4 and 1.8% in control subjects. INTERPRETATION: These rates provide a benchmark that can be used to monitor future treatment response in PSP.
|Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. |
Neumann, Manuela, et al.
Science, 314: 130-3 (2006) 2006
Ubiquitin-positive, tau- and alpha-synuclein-negative inclusions are hallmarks of frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Although the identity of the ubiquitinated protein specific to either disorder was unknown, we showed that TDP-43 is the major disease protein in both disorders. Pathologic TDP-43 was hyper-phosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments and was recovered only from affected central nervous system regions, including hippocampus, neocortex, and spinal cord. TDP-43 represents the common pathologic substrate linking these neurodegenerative disorders.
|Frontotemporal lobar degeneration without lobar atrophy. |
Keith A Josephs, Jennifer L Whitwell, Clifford R Jack, Joseph E Parisi, Dennis W Dickson
Archives of neurology 63 1632-8 2006
BACKGROUND: Frontotemporal lobar degeneration with ubiquitin-only-immunoreactive neuronal inclusions (FTLD-U) is the most common form of frontotemporal dementia. Neuronal loss and gliosis in cornu ammonis 1 and the subiculum of the hippocampus are features of hippocampal sclerosis (HpScl), which occurs in many cases of FTLD-U. OBJECTIVE: To determine if there were any clinical or magnetic resonance imaging correlates of HpScl in FTLD-U. DESIGN: We reviewed demographics and clinical features of 24 cases of FTLD-U and subjectively assessed the severity of neuronal loss and frequency of ubiquitin-positive neuronal lesions in the frontal and temporal cortices and the dentate gyrus of the hippocampus. SETTING: Mayo Clinic, Rochester, Minn. Patients Twenty-six cases were identified from the medical records linkage system query that met clinical criteria and had autopsy material available for additional studies. Two cases were excluded from further analysis after pathologic studies revealed coexisting Alzheimer disease, leaving 24 cases included in the study. Cases were subdivided based on the presence or absence of HpScl. MAIN OUTCOME MEASURES: Patterns of gray matter atrophy were assessed in cases of FTLD-U with and without HpScl using voxel-based morphometry. RESULTS: Six of the 24 cases of FTLD-U did not have HpScl. No differences were found in demographic or clinical features, including disease duration, between cases with and without HpScl; however, voxel-based morphometry analysis revealed minimal cortical atrophy in cases without HpScl, which was significantly different from the pattern of moderate to severe frontal and temporal lobe atrophy in FTLD-U with HpScl. This finding was in keeping with histopathologic observations. CONCLUSIONS: Despite similar clinical features, cases of FTLD-U with HpScl differ from those without HpScl with respect to pathologic findings and structural imaging. Specifically, FTLD-U without HpScl showed on average minimal or no cortical atrophy, even at end-stage disease. Consequently, FTLD-U without HpScl does not conform to the proposed FTLD staging scheme, is underrecognized, and may have different genetic and environmental underpinnings.
|Amyotrophic lateral sclerosis with dementia: an autopsy case showing many Bunina bodies, tau-positive neuronal and astrocytic plaque-like pathologies, and pallido-nigral degeneration. |
Osamu Yokota, Kuniaki Tsuchiya, Tatsurou Oda, Takeshi Ishihara, Rohan de Silva, Andrew J Lees, Tetsuaki Arai, Toshiki Uchihara, Hideki Ishizu, Shigetoshi Kuroda, Haruhiko Akiyama
Acta neuropathologica 112 633-45 2006
We report the case of a 54-year-old woman with mental retardation who developed frontotemporal dementia and amyotrophic lateral sclerosis (ALS) in the presenium. She presented with dementia at age 48, and motor neuron signs developed at age 53. She had no family history of dementia or ALS. Postmortem examination disclosed histopathological features of ALS, including pyramidal tract degeneration, mild loss of motor neurons, and many Bunina bodies immunoreactive for cystatin C, but not ubiquitin-positive inclusions. Unusual features of this case included severe neuronal loss in the substantia nigra and medial globus pallidus. The subthalamic nucleus, limbic system, and cerebral cortex were well preserved. In addition, neurofibrillary tangles (NFTs) were found in the frontal, temporal, insular, and cingulate cortices, nucleus basalis of Meynert, and locus coeruleus, and to a lesser degree, in the dentate nucleus, cerebellum, hippocampus, and amygdala. No ballooned neurons, tufted astrocytes, or astrocytic plaques were found. Tau immunostaining demonstrated many pretangles rather than NFTs and glial lesions resembling astrocytic plaques in the frontal and temporal cortices. This glial tau pathology predominantly developed in the middle to deep layers in the primary motor cortex, and was frequently associated with the walls of blood vessels. NFTs were immunolabeled with 3-repeat and 4-repeat specific antibodies against tau, respectively. Although the pathophysiological relationship between tau pathology and the selective involvement of motor neurons, substantia nigra, and globus pallidus was unclear, we considered that it might be more than coincidental.
|Heterogeneous inclusions in neurofilament inclusion disease. |
Hirotake Uchikado, Ang Li, Wen-Lang Lin, Dennis W Dickson
Neuropathology : official journal of the Japanese Society of Neuropathology 26 417-21 2006
Neurofilament inclusion disease (NFID) is a rare disease, whose pathogenesis remains to be elucidated. Immunoreactivity of ubiquitin-binding protein p62 has been reported in various neurodegenerative diseases, but it has not been studied in NFID. In this report we show p62 immunoreactivity in neuronal perikaryon of three cases of NFID. We found inclusions in NFID to be heterogenous based on immunoreactivity for alpha-internexin, phosphorylated neurofilament-H, p62 and ubiquitin. Moreover, we showed both p62- and alpha-internexin-immunoreactive inclusions within the perikarya of the same neuron. Electron microscopy findings support the notion that inclusions in NFID are heterogenous. The present study extends the list of proteins that have been identified as components of neuronal inclusions in NFID, and may help account for the pathogenesis of NFID.
|Positive immunoreactivity for vesicular monoamine transporter 2 in Lewy bodies and Lewy neurites in substantia nigra. |
Shoji Yamamoto, Jiro Fukae, Hideo Mori, Yoshikuni Mizuno, Nobutaka Hattori
Neuroscience letters 396 187-91 2006
Vesicular monoamine transporter 2 (VMAT2) is responsible for packing dopamine into vesicles, and reduces the effects of neurotoxins by sequestering them into vesicles. In this report, we tested the hypothesis that VMAT2 is associated with Lewy body (LB) formation by immunohistochemical staining of midbrain and cortical sections of autopsied brains of patients with Parkinson's disease (PD) and diffuse Lewy body disease (DLBD) for VMAT2 using a polyclonal antibody against VMAT2. LBs in the substantia nigra (SN) of PD and DLBD were immunoreactive for VMAT2, especially in the peripheral zone. Previous electron microscopic studies also revealed the presence of numerous dense core vesicles around the LBs, suggesting that these vesicles are related to LB formation. Indeed, the presence of a few vesicle-linked proteins such as synaptophysin and chromogranin A in LBs has been reported. Together with the low expression of VMAT2 in the SN of PD, the involvement of VMAT2 in LBs of the SN suggests the association of this protein in the neurodegeneration of nigral neurons in PD.
|Clinicopathologic analysis of frontotemporal and corticobasal degenerations and PSP. |
K A Josephs, R C Petersen, D S Knopman, B F Boeve, J L Whitwell, J R Duffy, J E Parisi, D W Dickson
Neurology 66 41-8 2006
OBJECTIVE: To examine the relationship between early clinical features, pathologies, and biochemistry of the frontotemporal lobar degenerations (FTLDs), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). METHODS: The authors conducted pathologic reexamination with the most recent immunohistochemistry of all cases diagnosed with FTLD, PSP, and CBD between 1970 and 2004. The authors also reviewed the early clinical features for clinical diagnosis and application of published research criteria. RESULTS: Of 127 cases analyzed, 57 had a pathologic diagnosis of FTLD, 49 PSP, and 21 CBD. Of these, 38 were clinically reclassified as frontal variant frontotemporal dementia (FTD), 13 as progressive non-fluent aphasia (PNFA), 21 as CBD-like, 33 as PSP-like, and 13 with frontotemporal dementia with coexisting motor neuron disease (FTD-MND). The authors were unable to classify nine cases. All cases of FTD-MND were tau-negative and had pathologic evidence of motor neuron degeneration. All cases classified as PSP-like or CBD-like had tau-positive pathology. Of the 13 cases with PNFA, PSP and CBD accounted for almost 70% of the cases, while FTD was almost equally divided between tau-positive and tau-negative diseases. CONCLUSION: Frontotemporal lobar degeneration, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP) have overlapping clinical features. The prediction of tau-positive pathology from a CBD or PSP-like presentation is good, while the frontotemporal dementia (FTD)-motor neuron disease syndrome almost certainly predicts motor neuron degeneration. Surprisingly, PSP and CBD accounted for most cases classified as progressive non-fluent aphasia. Frontal variant FTD is an unpredictable disease in terms of its biochemistry.
|Biochemical and pathological characterization of Lrrk2. |
Benoit I Giasson, Jason P Covy, Nancy M Bonini, Howard I Hurtig, Matthew J Farrer, John Q Trojanowski, Vivianna M Van Deerlin
Annals of neurology 59 315-22 2006
OBJECTIVE: Mutations in leucine-rich repeat kinase 2 (LRRK2) recently have been identified as the most common genetic cause of late-onset sporadic and familial Parkinson's disease (PD). The studies herein explore the biological and pathological properties of Lrrk2. METHODS: Genetic analysis was performed to identify autopsied patients with the most common Lrrk2 mutation (G2019S). Using an antibody specific to Lrrk2, the biochemical and immunocytochemical distribution of Lrrk2 was assessed. RESULTS: Three patients with the G2019S Lrrk2 mutation were identified. Two patients demonstrated classic PD with Lewy bodies, although concurrent pathological changes consistent with Alzheimer's disease were also present in one of these individuals. The third patient was characterized by parkinsonism without Lewy bodies but demonstrated dystrophic neurites in the substantia nigra intensely stained for Lrrk2. Lrrk2 accumulations were unique to this patient and Lrrk2 was not detected in other types of pathological inclusions. Biochemical analysis showed that Lrrk2 is predominantly a soluble approximately 250 kDa cytoplasmic protein expressed throughout the brain but also in many other organs. INTERPRETATION: The reasons for the selective predisposition of patients with mutations in LRRK2 to develop parkinsonism remains unclear, but Lrrk2 mutations may prime select neuronal populations to cellular insults that can lead to aberrant protein aggregation.
|Clinicopathological and imaging correlates of progressive aphasia and apraxia of speech. |
Josephs, KA; Duffy, JR; Strand, EA; Whitwell, JL; Layton, KF; Parisi, JE; Hauser, MF; Witte, RJ; Boeve, BF; Knopman, DS; Dickson, DW; Jack, CR; Petersen, RC
Brain : a journal of neurology 129 1385-98 2006
Apraxia of speech (AOS) is a motor speech disorder characterized by slow speaking rate, abnormal prosody and distorted sound substitutions, additions, repetitions and prolongations, sometimes accompanied by groping, and trial and error articulatory movements. Although AOS is frequently subsumed under the heading of aphasia, and indeed most often co-occurs with aphasia, it can be the predominant or even the sole manifestation of a degenerative neurological disease. In this study we determine whether the clinical classifications of aphasia and AOS correlated with pathological diagnoses and specific biochemical and anatomical structural abnormalities. Seventeen cases with initial diagnoses of a degenerative aphasia or AOS were re-classified independently by two speech-language pathologists--blinded to pathological and biochemical findings--into one of five operationally defined categories of aphasia and AOS. Pathological diagnoses in the 17 cases were progressive supranuclear palsy in 6, corticobasal degeneration in 5, frontotemporal lobar degeneration with ubiquitin-only-immunoreactive changes in 5 and Pick's disease in 1. Magnetic resonance imaging analysis using voxel-based morphometry (VBM), and single photon emission tomography were completed, blinded to the clinical diagnoses, and clinicoimaging and clinicopathological associations were then sought. Interjudge clinical classification reliability was 87% (kappa = 0.8) for all evaluations. Eleven cases had evidence of AOS, of which all (100%) had a pathological diagnosis characterized by underlying tau biochemistry, while five of the other six cases without AOS did not have tau biochemistry (P = 0.001). A majority of the 17 cases had more than one yearly evaluation, demonstrating the evolution of the speech and language syndromes, as well as motor signs. VBM revealed the premotor and supplemental motor cortices to be the main cortical regions associated with AOS, while the anterior peri-sylvian region was associated with non-fluent aphasia. Refining the classification of the degenerative aphasias and AOS may be necessary to improve our understanding of the relationships among behavioural, pathological and imaging correlations.Full Text Article
|Ubiquitin-specific protease 2 as a tool for quantification of total ubiquitin levels in biological specimens. |
Kwon-Yul Ryu, Rohan T Baker, Ron R Kopito
Analytical biochemistry 353 153-5 2006
|Survival in two variants of tau-negative frontotemporal lobar degeneration: FTLD-U vs FTLD-MND. |
K A Josephs, D S Knopman, J L Whitwell, B F Boeve, J E Parisi, R C Petersen, D W Dickson
Neurology 65 645-7 2005
Pathologic diagnoses in frontotemporal lobar degeneration (FTLD) include tau-positive FTLD and tau-negative FTLD. Two variants of tau-negative FTLD are FTLD with motor neuron disease (FTLD-MND) and FTLD with motor neuron disease type inclusions but without motor neuron disease (FTLD-U). An analysis of patient outcomes in these cases reveals that FTLD-MND has significantly shorter survival than FTLD-U, suggesting that FTLD-MND is a more aggressive disease process.
|Loss of Usp14 results in reduced levels of ubiquitin in ataxia mice. |
Christopher Anderson, Stephen Crimmins, Julie A Wilson, Greg A Korbel, Hidde L Ploegh, Scott M Wilson
Journal of neurochemistry 95 724-31 2005
The ataxia (ax(J)) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitin-specific protease 14, Usp14. Mice homozygous for the ax(J) mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax(J) mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wild-type mice. Proteasomes isolated from the ax(J) brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax(J) tissues examined. These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease.
|Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets. |
Mayor, T; Lipford, JR; Graumann, J; Smith, GT; Deshaies, RJ
Molecular & cellular proteomics : MCP 4 741-51 2005
The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10Delta) and identified 54 proteins that were uniquely recovered from rpn10Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely in abundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.
|Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies. |
Trimmer, Patricia A, et al.
J. Neurochem., 88: 800-12 (2004) 2004
Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.
|Trigeminal neuralgia due to pontine infarction. |
Selcuk Peker, Gur Akansel, Ibrahim Sun, Necmettin M Pamir
Headache 44 1043-5 2004
Trigeminal neuralgia (TN) is most commonly caused by vascular compression of the trigeminal nerve root entry zone. Secondary trigeminal neuralgia due to ischemic lesion of the pons is very rare. Here we report a patient with a pontine infarct transecting the central trigeminal pathways resulting with trigeminal neuralgia.
|Protein ubiquitination in postsynaptic densities after transient cerebral ischemia. |
Liu, CL; Martone, ME; Hu, BR
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 24 1219-25 2004
The mechanisms underlying neurologic deficits and delayed neuronal death after ischemia are not fully understood. In the present study, we report that transient cerebral ischemia induces accumulation of ubiquitinated proteins (ubi-proteins) in postsynaptic densities (PSDs). By immunoelectron microscopy, we demonstrated that ubi-proteins were highly accumulated in PSD structures after ischemia. On Western blots, ubi-proteins were markedly increased in purified PSDs at 30 minutes of reperfusion, and the increase persisted until cell death in the CA1 region after ischemia. In the resistant DG area, however, the changes were transient and significantly less pronounced. Deposition of ubi-proteins in PSDs after ischemia correlates well with PSD structural damage in the CA1 region as viewed by electron microscopy. These results suggest that the ubiquitin-proteasome system fails to repair and remove damaged proteins in PSDs. The changes may demolish synaptic neurotransmission, contribute to neurologic deficits, and eventually lead to delayed neuronal death after transient cerebral ischemia.
|Identification of ubiquitin-interacting proteins in purified polyglutamine aggregates. |
Hiroshi Doi, Kenichi Mitsui, Masaru Kurosawa, Yoko Machida, Yoshiyuki Kuroiwa, Nobuyuki Nukina
FEBS letters 571 171-6 2004
Nuclear aggregates of enhanced green fluorescent protein and nuclear localization signal-fused truncated N-terminal huntingtin containing 150 repeats of glutamine residue were purified from ecdysine-inducible mutant neuro2A cell line by sequential extraction of nuclear soluble proteins. To analyze the aggregate-interacting proteins, we subjected the nuclear aggregates to high performance liquid chromatography-mass spectrometry analysis. The resulting data revealed the presence of three new putative aggregate-interacting proteins: ubiquilin 1, ubiquilin 2 and Tollip. These proteins also associated with neuronal intranuclear inclusions in a mouse model of Huntington disease (HD). These aggregate-interacting proteins contain ubiquitin-interacting motifs, suggesting that they are recruited to the aggregates where they may lose their normal function.
|Chaperones, protein aggregation, and brain protection from hypoxic/ischemic injury. |
Giffard, RG; Xu, L; Zhao, H; Carrico, W; Ouyang, Y; Qiao, Y; Sapolsky, R; Steinberg, G; Hu, B; Yenari, MA
The Journal of experimental biology 207 3213-20 2004
Chaperones, especially the stress inducible Hsp70, have been studied for their potential to protect the brain from ischemic injury. While they protect from both global and focal ischemia in vivo and cell culture models of ischemia/reperfusion injury in vitro, the mechanism of protection is not well understood. Protein aggregation is part of the etiology of chronic neurodegenerative diseases such as Huntington's and Alzheimer's, and recent data demonstrate protein aggregates in animal models of stroke. We now demonstrate that overexpression of Hsp70 in hippocampal CA1 neurons reduces evidence of protein aggregation under conditions where neuronal survival is increased. We have also demonstrated protection by the cochaperone Hdj-2 in vitro and demonstrated that this is associated with reduced protein aggregation identified by ubiquitin immunostaining. Hdj-2 can prevent protein aggregate formation by itself, but can only facilitate protein folding in conjunction with Hsp70. Pharmacological induction of Hsp70 was found to reduce both apoptotic and necrotic astrocyte death induced by glucose deprivation or oxygen glucose deprivation. Protection from ischemia and ischemia-like injury by chaperones thus involves at least anti-apoptotic, anti-necrotic and anti-protein aggregation mechanisms.
|Ubiquitin and ubiquitin-related proteins in neurons and dendrites of brains of atypical Pick's disease without Pick bodies. |
Yoshiko Furukawa, Eizo Iseki, Hiroaki Hino, Akiko Kanai, Toshinari Odawara, Kenji Kosaka
Neuropathology : official journal of the Japanese Society of Neuropathology 24 38-45 2004
Nine cases of atypical Pick's disease without Pick bodies were investigated immunohistochemically. Ubiquitin (ub)-positive and tau-negative structures were mainly found in the cerebral cortex and hippocampal dentate gyrus. In the cerebral cortex, most of the ub-positive structures had ub-positive dendrites in the neuropil, although some also showed diffuse ub-positive staining in the neuronal cytoplasm. These ub-positive structures were distributed throughout layers II-IIIab and layers V-VI. Granular cells of the dentate gyrus had ub-positive intraneuronal inclusions. When the numbers of ub-positive neurons and dendrites were evaluated in relation to the degree of neuronal loss in the cerebral cortex, the number of ub-positive neurons was significantly lower in regions showing very mild neuronal loss and higher in regions showing moderate neuronal loss. In contrast, ub-positive dendrites were detected even in cortical regions showing very mild neuronal loss. Immunoelectron-microscopically, ub-positive structures contained ub-positive ribosome-like granular components in the neuronal cytoplasm and dendrites, which were occasionally related to the rough endoplasmic reticulum and accompanied by a few filamentous components. Almost all ub-positive structures were positive for ub-binding protein p62 in double-immunostaining method. Some ub-positive or negative neurons in the cerebral cortex were positively immunolabeled with anti-ub ligase (Parkin) and anti-ub C-terminal hydrolase antibodies, whereas dendrites were not labeled by these antibodies. From the present study, it is suggested that in the cerebral cortex, these ubiquitinated proteins may firstly accumulate in the dendrites at the onset of neuronal degeneration, then appear in the neuronal cytoplasm before finally disappearing with neuronal loss.
|A pathogenic peripherin gene mutation in a patient with amyotrophic lateral sclerosis. |
Conrad L Leung, Cui Zhen He, Petra Kaufmann, Steven S Chin, Ali Naini, Ronald K H Liem, Hiroshi Mitsumoto, Arthur P Hays
Brain pathology (Zurich, Switzerland) 14 290-6 2004
Peripherin is a neuronal intermediate filament protein that is expressed chiefly in motor neurons and other nerve cells that project into the peripheral nervous system. Transgenic mice that over-express peripherin develop motor neuron degeneration, suggesting that mutations in peripherin could contribute to the development of motor neuron disease. In this paper, we report the identification of a homozygous mutation in the peripherin gene (PRPH) in a patient with amyotrophic lateral sclerosis (ALS). The mutation resulted in a substitution of aspartate with tyrosine at amino acid position 141, which is located within the first linker region of the rod domain. Immunocytochemical analysis of the spinal cord of the patient upon autopsy revealed distinctive large aggregates within the cell bodies of residual spinal motor neurons that contained peripherin and was also immunoreactive with antibodies to the neurofilament proteins. In order to study the effect of the mutation on peripherin assembly, we performed transient transfections. Unlike wild-type peripherin, which self-assembles to form a filamentous network, the mutant peripherin was prone to form aggregates in transfected cells, indicating that the mutation adversely affects peripherin assembly. Moreover, the neurofilament light (NF-L) protein was not able to rescue the mutant protein from forming aggregates. These data imply that mutation of PRPH is a contributing factor for ALS.
|Brainstem pathology in DYT1 primary torsion dystonia. |
Kevin St P McNaught, Alexander Kapustin, Tehone Jackson, Toni-Ann Jengelley, Ruth Jnobaptiste, Pullanipally Shashidharan, Daniel P Perl, Pedro Pasik, C Warren Olanow
Annals of neurology 56 540-7 2004
DYT1 dystonia is a severe form of young-onset dystonia caused by a mutation in the gene that encodes for the protein torsinA, which is thought to play a role in protein transport and degradation. We describe, for the first time to our knowledge, perinuclear inclusion bodies in the midbrain reticular formation and periaqueductal gray in four clinically documented and genetically confirmed DYT1 patients but not in controls. The inclusions were located within cholinergic and other neurons in the pedunculopontine nucleus, cuneiform nucleus, and griseum centrale mesencephali and stained positively for ubiquitin, torsinA, and the nuclear envelope protein lamin A/C. No evidence of inclusion body formation was detected in the substantia nigra pars compacta, striatum, hippocampus, or selected regions of the cerebral cortex. We also noted tau/ubiquitin-immunoreactive aggregates in pigmented neurons of the substantia nigra pars compacta and locus coeruleus in all four DYT1 dystonia cases, but not in controls. This study supports the notion that DYT1 dystonia is associated with impaired protein handling and the nuclear envelope. The role of the pedunculopontine and cuneiform nuclei, and related brainstem brainstem structures, in mediating motor activity and controlling muscle tone suggests that alterations in these structures could underlie the pathophysiology of DYT1 dystonia [corrected]
|Yin Yang 1 is a negative regulator of p53. |
Guangchao Sui, El Bachir Affar, Yujiang Shi, Chrystelle Brignone, Nathan R Wall, Peng Yin, Mary Donohoe, Margaret P Luke, Dominica Calvo, Steven R Grossman, Yang Shi
Cell 117 859-72 2004
Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degradation. Significantly, recombinant YY1 is sufficient to induce Hdm2-mediated p53 polyubiquitination in vitro, suggesting that this function of YY1 is independent of its transcriptional activity. We identify direct physical interactions of YY1 with Hdm2 and p53 and show that the basis for YY1-regulating p53 ubiquitination is its ability to facilitate Hdm2-p53 interaction. Importantly, the tumor suppressor p14ARF compromises the Hdm2-YY1 interaction, which is important for YY1 regulation of p53. Taken together, these findings identify YY1 as a potential cofactor for Hdm2 in the regulation of p53 homeostasis and suggest a possible role for YY1 in tumorigenesis.
|Characterisation of cytoskeletal abnormalities in mice transgenic for wild-type human tau and familial Alzheimer's disease mutants of APP and presenilin-1. |
Allal Boutajangout, Michèle Authelet, Véronique Blanchard, N Touchet, Gunter Tremp, Laurent Pradier, Jean-Pierre Brion
Neurobiology of disease 15 47-60 2004
To study the role of Abeta amyloid deposits in the generation of cytoskeletal lesions, we have generated a transgenic mouse line coexpressing in the same neurons a wild-type human tau isoform (0N3R), a mutant form of APP (751SL) and a mutant form of PS1 (M146L). These mice developed early cerebral extracellular deposits of Abeta, starting at 2.5 months. A somatodendritic neuronal accumulation of transgenic tau protein was observed in tau only and in tau/PS1/APP transgenic mice, including in neurons adjacent to Abeta deposits. The phosphorylation status of this somatodendritic tau was similar in the two transgenic lines. The Abeta deposits were surrounded by a neuritic reaction composed of axonal dystrophic processes, immunoreactive for many phosphotau epitopes and for the human tau transgenic protein. Ultrastructural observation showed in these dystrophic neurites a disorganisation of the microtubule and the neurofilament network but animals that were observed up to 18 months of age did not develop neurofibrillary tangles. These results indicate that overexpression of mutant PS1, mutant APP and of wild-type human tau were not sufficient per se to drive the formation of neurofibrillary tangles in a transgenic model. The Abeta deposits, however, were associated to marked changes in cytoskeletal organisation and in tau phosphorylation in adjacent dystrophic neurites.
|Increased expression of neuronal cyclooxygenase-2 in the hippocampus in amyotrophic lateral sclerosis both with and without dementia. |
Osamu Yokota, Seishi Terada, Hideki Ishizu, Takeshi Ishihara, Hanae Nakashima, Aki Kugo, Kuniaki Tsuchiya, Kenji Ikeda, Toshiyuki Hayabara, Yuko Saito, Shigeo Murayama, Kenji Uéda, Frédéric Checler, Shigetoshi Kuroda
Acta neuropathologica 107 399-405 2004
The pathophysiological basis of cognitive dysfunction, including frontotemporal dementia (FTD), in patients with amyotrophic lateral sclerosis (ALS) and ALS with dementia (ALSD) remains unclear. On the other hand, increased expression of cyclooxygenase-2 (COX-2) in the spinal cord is thought to play a pivotal role in motor neuron degeneration in ALS. In this study, to assess the relationship between the neuronal COX-2 expression in the cerebrum, the formation of tau- and alpha-synuclein-negative but ubiquitin-positive neuronal inclusions (UPIs), and dementia in motor neuron disease (MND), we examined neuronal COX-2 immunoreactivity in the frontal cortex and hippocampus of patients with non-demented ALS without UPIs ( n=11), ALSD with UPIs ( n=6), and normal controls ( n=24) using a quantitative immunohistochemical technique. Neuronal COX-2 expression in all CA1-4 in the hippocampus was significantly up-regulated in the ALSD group, and, to lesser degree but significantly, in the ALS group. Neuronal COX-2 expression in the frontal cortex was also significantly up-regulated in the ALSD group but not in the ALS group. These findings suggest that (1) the frontal cortex and hippocampus of MND are involved in the same pathogenic process associated with COX-2 induction that has been observed in spinal anterior horn cells, (2) COX-2 induction in the cerebrum is a pathogenic process that can occur even in the absence of UPI formation in MND, and (3) COX-2 expression in the cerebrum may be associated with cognitive dysfunction in MND.
|Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. |
Drisaldi, B; Stewart, RS; Adles, C; Stewart, LR; Quaglio, E; Biasini, E; Fioriti, L; Chiesa, R; Harris, DA
The Journal of biological chemistry 278 21732-43 2003
The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.
|Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast. |
Kominami, K; Seth-Smith, H; Toda, T
The EMBO journal 17 5388-99 1998
Many eukaryotic cells arrest the cell cycle at G1 phase upon nutrient deprivation. In fission yeast, during nitrogen starvation, cells divide twice and arrest at G1. We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G2. Three loci (apc10, ste9/srw1 and rum1) were identified. The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation. apc10(+) is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins. Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex. A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC). A second gene, ste9(+)/srw1(+), encodes a member of the fizzy-related family, also regulators of the APC. Finally, Rum1 is a cyclin-dependent kinase (CDK) inhibitor which exists only in G1. The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G1.
|Amyloid plaques in cerebellar cortex and the integrity of Purkinje cell dendrites. |
Y T Li, D S Woodruff-Pak, J Q Trojanowski, Y T Li, D S Woodruff-Pak, J Q Trojanowski
Neurobiology of aging 15 1-9 1994
We probed serial and near serial sections of cerebellum from 13 Alzheimer's disease (AD), 10 older Down's syndrome (DS) patients, and 9 age-matched, non-AD controls, using single and double labeling immunohistochemistry to investigate the pathologic consequences of beta-amyloid or A4 (A beta) deposits in cerebellum and their relationship to Purkinje cells (PCs). Our data showed that A beta deposits in cerebellum of AD and older DS adults only form diffuse or preamyloid plaques and the density of A beta lesions per unit area of molecular layer correlated with the number of PCs per unit length of the subjacent PC layer in double immunostained sections (r = 0.85; p 0.001). About 65% of these cerebellar A beta deposits were in physical contact with PC dendrites. No A beta plaques were found in the cerebellum of controls. Despite the abundance of A beta deposits in the cerebellar cortex of AD and older DS patients, neither PC bodies nor PC dendrites in physical contact with A beta lesions showed evidence of structural abnormalities.
|Molecular features of hypothalamic plaques in Alzheimer's disease. |
Standaert, DG; Lee, VM; Greenberg, BD; Lowery, DE; Trojanowski, JQ
The American journal of pathology 139 681-91 1991
The pathology of Alzheimer's disease (AD) involves subcortical as well as cortical structures. The authors have used immunohistochemical methods to study the molecular composition of AD plaques in the hypothalamus. In contrast to previous studies using histochemical methods, the authors observed large numbers of diffuse plaques in the AD hypothalamus labeled with an antiserum to the beta-amyloid, or A4 peptide, of the beta-amyloid precursor proteins (beta APPs), whereas A4-immunoreactive plaques were uncommon in the hypothalamus of patients without AD. Unlike plaques in the cortex and hippocampus of AD patients, hypothalamic plaques did not contain epitopes corresponding to other regions of the beta APPs, nor did they contain tau-, neurofilament-, or microtubule-associated protein-reactive epitopes, and did not disrupt the neuropil or produce astrogliosis. These findings demonstrate that there are substantial molecular and cellular differences in the pathologic features of AD in the hypothalamus compared with those observed in hippocampal and cortical structures, which may provide insight into the pathogenetic mechanisms of AD.
|Anti-Ubiquitin, clone Ubi-1 (aka 042691GS) - Data Sheet|