Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Rb, H, R, M, Po, Ch, B||WB, ICC, IHC, IH(P)||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
Store the reconstituted antibody at 2-8°C for up to 6 months after date of receipt.
DO NOT FREEZE.
|Material Size||40 µg|
References | 60 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Genetic ablation of Nrf2/antioxidant response pathway in Alexander disease mice reduces hippocampal gliosis but does not impact survival. |
Tracy L Hagemann,Emily M Jobe,Albee Messing
PloS one 7 2012
In Alexander disease (AxD) the presence of mutant glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, triggers protein aggregation, with marked induction of a stress response mediated by the transcription factor, Nrf2. To clarify the role of Nrf2 in AxD, we have crossed Gfap mutant and transgenic mouse models into an Nrf2 null background. Deletion of Nrf2 eliminates the phase II stress response normally present in mouse models of AxD, but causes no change in body weight or lifespan, even in a severe lethal model. AxD astrocytes without Nrf2 retain features of reactivity, such as expression of the endothelin-B receptor, but have lower Gfap levels, a decrease in p62 protein and reduced iron accumulation, particularly in hippocampus. Microglial activation, indicated by Iba1 expression, is also diminished. Although the Nrf2 response is generally considered beneficial, these results show that in the context of AxD, loss of the antioxidant pathway has no obvious negative effects, while actually decreasing Gfap accumulation and pathology. Given the attention Nrf2 is receiving as a potential therapeutic target in AxD and other neurodegenerative diseases, it will be interesting to see whether induction of Nrf2, beyond the endogenous response, is beneficial or not in these same models.
|Hypoxia-inducible factor 1 protects hypoxic astrocytes against glutamate toxicity. |
Yomna Badawi,Prabhu Ramamoorthy,Honglian Shi
ASN neuro 4 2012
Stroke is a major neurological disorder characterized by an increase in the Glu (glutamate) concentration resulting in excitotoxicity and eventually cellular damage and death in the brain. HIF-1 (hypoxia-inducible factor-1), a transcription factor, plays an important protective role in promoting cellular adaptation to hypoxic conditions. It is known that HIF-1α, the regulatable subunit of HIF-1, is expressed by astrocytes under severe ischaemia. However, the effect of HIF-1 on astrocytes following Glu toxicity during ischaemia has not been well studied. We investigated the role of HIF-1 in protecting ischaemic astrocytes against Glu toxicity. Immunostaining with GFAP (glial fibrillary acidic protein) confirmed the morphological modification of astrocytes in the presence of 1 mM Glu under normoxia. Interestingly, when the astrocytes were exposed to severe hypoxia (0.1% O2), the altered cell morphology was ameliorated with up-regulation of HIF-1α. To ascertain HIF-1's protective role, effects of two HIF-1α inhibitors, YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzylindazole] and 2Me2 (2-methoxyoestradiol), were tested. Both the inhibitors decreased the recovery in astrocyte morphology and increased cell death. Given that ischaemia increases ROS (reactive oxygen species), we examined the role of GSH (reduced glutathione) in the mechanism for this protection. GSH was increased under hypoxia, and this correlated with an increase in HIF-1α stabilization in the astrocytes. Furthermore, inhibition of GSH with BSO (l-butathione sulfoximine) decreased HIF-1α expression, suggesting its role in the stabilization of HIF-1α. Overall, our results indicate that the expression of HIF-1α under hypoxia has a protective effect on astrocytes in maintaining cell morphology and viability in response to Glu toxicity.
|Overexpression of HGF attenuates the degeneration of Purkinje cells and Bergmann glia in a knockin mouse model of spinocerebellar ataxia type 7. |
Satsuki Noma,Wakana Ohya-Shimada,Masaaki Kanai,Keiji Ueda,Toshikazu Nakamura,Hiroshi Funakoshi
Neuroscience research 73 2012
Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder associated with cerebellar neurodegeneration caused by expansion of a CAG repeat in the ataxin-7 gene. Hepatocyte growth factor (HGF), a pleiotrophic growth factor, displays highly potent neurotrophic activities on cerebellar neurons. A mutant c-met/HGF receptor knockin mouse model has revealed a role for HGF in the postnatal development of the cerebellum. The present study was designed to elucidate the effect of HGF on cerebellar neurodegeneration in a knockin mouse model of SCA7 (SCA7-KI mouse). SCA7-KI mice were crossed with transgenic mice overexpressing HGF (HGF-Tg mice) to produce SCA7-KI/HGF-Tg mice that were used to examine the phenotypic differences following HGF overexpression. The Purkinje cellular degeneration is thought to occur via cell-autonomous and non-cell autonomous mechanisms mediated by a reduction of glutamate transporter levels in Bergmann glia. The Purkinje cellular degeneration and reduced expression of glutamate transporters in the cerebellum of SCA7-KI mice were largely attenuated in the SCA7-KI/HGF-Tg mice. Moreover, phenotypic impairments exhibited by SCA7-KI mice during rotarod tests were alleviated in SCA7-KI/HGF-Tg mice. The bifunctional nature of HGF on both Purkinje cells and Bergmann glia highlight the potential therapeutic utility of this molecule for the treatment of SCA7 and related disorders.
|Effect of curcumin in a mouse model of Pelizaeus-Merzbacher disease. |
Li-Hua Yu,Toshifumi Morimura,Yurika Numata,Ryoko Yamamoto,Naoko Inoue,Barbara Antalfy,Yu-Ichi Goto,Kimiko Deguchi,Hitoshi Osaka,Ken Inoue
Molecular genetics and metabolism 106 2012
PLP1 amino acid substitutions cause accumulation of misfolded protein and induce endoplasmic reticulum (ER) stress, causing Pelizaeus-Merzbacher disease (PMD), a hypomyelinating disorder of the central nerve system. Currently no effective therapy is available for PMD. Promoted by its curative effects in other genetic disease models caused by similar molecular mechanisms, we tested if curcumin, a dietary compound, can rescue the lethal phenotype of a PMD mouse model (myelin synthesis deficient, msd). Curcumin was administered orally to myelin synthesis deficit (msd) mice at 180 mg·kg(-1)·day(-1) from the postnatal day 3. We evaluated general and motor status, changes in myelination and apoptosis of oligodendrocytes by neuropathological and biochemical examination, and transcription levels for ER-related molecules. We also examined the pharmacological effect of curcumin in cell culture system. Oral curcumin treatment resulted in 25% longer survival (p<0.01). In addition, oligodendrocytes undergoing apoptosis were reduced in number (p<0.05). However, no apparent improvement in motor function, neurological phenotype, and myelin formation was observed. Curcumin treatment did not change the expression of ER stress markers and subcellular localization of the mutant protein in vitro and/or in vivo. Curcumin partially mitigated the clinical and pathological phenotype of msd mice, although molecular mechanisms underlying this curative effect are yet undetermined. Nonetheless, curcumin may serve as a potential therapeutic compound for PMD caused by PLP1 point mutations.
|Toll-like receptors 2 and 3 agonists differentially affect oligodendrocyte survival, differentiation, and myelin membrane formation. |
Malika Bsibsi,Anita Nomden,Johannes M van Noort,Wia Baron
Journal of neuroscience research 90 2012
Toll-like receptors (TLRs) play a key role in controlling innate immune responses to a wide variety of pathogen-associated molecules as well as endogenous signals. In addition, TLR expression within nonimmune cells has been recognized as as modulator of cell behavior. In this study we have addressed the question of whether functional TLRs are expressed on oligodendrocytes, the myelinating cells of the central nervous system. Primary cultures of rat oligodendrocytes at different maturation stages were found to express TLR2 and, to lesser extent, TLR3. Immunocytochemical analysis revealed that both TLRs were localized at the cell body and primary processes and were excluded from myelin-like membranes. Interestingly, innate immune receptor ligands were able to modulate oligodendrocyte survival, differentiation, and myelin-like membrane formation, indicating that TLRs on oligodendrocytes are functional. In highly purified oligodendrocytes cultures, the TLR2 agonist zymosan promoted survival, differentiation, and myelin-like membrane formation, whereas poly-I:C, a TLR3 ligand, was a potent inducer of apoptosis. Together, these data indicate that, in addition to other neural cell types, also oligodendrocytes express functional TLRs, which play a role in regulating various aspects of oligodendrocyte behavior.
|Neuroprotective function for ramified microglia in hippocampal excitotoxicity. |
Jonathan Vinet,Hilmar Rj van Weering,Annette Heinrich,Roland E Kälin,Anja Wegner,Nieske Brouwer,Frank L Heppner,Nico van Rooijen,Hendrikus Wgm Boddeke,Knut Biber,Hilmar R J van Weering,Nico van Rooijen,Hendrikus W G M Boddeke
Journal of neuroinflammation 9 2012
Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.
|An optimized protocol for the acute isolation of human microglia from autopsy brain samples. |
Marta Olah,Divya Raj,Nieske Brouwer,Alexander H De Haas,Bart J L Eggen,Wilfred F A Den Dunnen,Knut P H Biber,Hendrikus W G M Boddeke
Glia 60 2012
Microglia are increasingly recognized to be crucially involved in the maintenance of tissue homeostasis of the brain and spinal cord. Not surprisingly is therefore the growing scientific interest in the microglia phenotypes associated with various physiological and pathological processes of the central nervous system. Until recently the investigation of these phenotypes was hindered by the lack of an isolation protocol that (without an extended culturing period) would offer a microglia population of high purity and yield. Thus, our objective was to establish a rapid and efficient method for the isolation of human microglia from postmortem brain samples. We tested multiple elements of already existing protocols (e.g., density separation, immunomagnetic bead separation) and combined them to minimize preparation time and maximize yield and purity. The procedure presented in this article enables acute isolation of human microglia from autopsy (and biopsy) samples with a purity and yield that is suitable for downstream applications, such as protein and gene expression analysis and functional assays. Moreover, the present protocol is appropriate for the isolation of microglia from autopsy samples irrespective of the neurological state of the brain or specific brain regions and (with minor modification) could be even used for the isolation of microglia from human glioma tissue.
|The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172. |
Peter J Wookey,Catriona A McLean,Peter Hwang,Sebastian G B Furness,Sandy Nguyen,Angela Kourakis,David L Hare,Jeffrey V Rosenfeld
Histopathology 60 2012
Previous studies have indicated that expression of calcitonin receptor (CTR) could be induced in a proinflammatory environment. In the present study, CTR-immunoreactivity (CTR-ir) was investigated in brain tissue from patients with glioblastoma multiforme (GBM).
|Indirect immunofluorescence staining of cultured neural cells. |
Massimo Barbierato,Carla Argentini,Stephen D Skaper
Methods in molecular biology (Clifton, N.J.) 846 2012
Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluorescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immunofluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a primary antibody. This chapter describes procedures for the application of indirect immunofluorescence staining to neural cells in culture.
|Young coconut juice, a potential therapeutic agent that could significantly reduce some pathologies associated with Alzheimer\'s disease: novel findings. |
Radenahmad N, Saleh F, Sawangjaroen K, Vongvatcharanon U, Subhadhirasakul P, Rundorn W, Withyachumnarnkul B, Connor JR
The British journal of nutrition 105 738-46. Epub 2010 Nov 30. 2011
Brains from ovariectomised (ovx) rats can display features similar to those observed in menopausal women with Alzheimer\'s disease (AD), and oestrogen seems to play a key role. Preliminary studies on young coconut juice (YCJ) have reported the presence of oestrogen-like components in it. The aim of the study was to investigate the effects of YCJ on the AD pathological changes in the brains of ovx rats. Rat groups included sham-operated, ovx, ovx+oestradiol benzoate (EB) and ovx+YCJ. Brain sections (4 μm) were taken and were immunostained with β-amyloid (Aβ) 1-42, glial fibrillary acidic protein (GFAP) (an intermediate neurofilament of astrocytes) and Tau-1 antibodies. Aβ 1-42, GFAP and Tau-1 are considered as reliable biomarkers of amyloidosis, astrogliosis and tauopathy (neurofibrillary tangles), respectively, which in turn are characteristic features associated with AD. The serum oestradiol (E2) level was measured using a chemiluminescent immunoassay technique. YCJ restored the serum E2 to levels significantly (P < 0·001) higher than that of the ovx group, and even that of the sham group. Aβ deposition was significantly (P < 0·0001) reduced in the cerebral cortex of the YCJ group, as compared with the ovx group and with the sham and ovx+EB groups (P < 0·01). A similar trend was observed in relation to GFAP expression in the cerebral cortex and to Tau-1 expression in the hippocampus. This is a novel study demonstrating that YCJ could have positive future implications in the prevention and treatment of AD in menopausal women.
|Interleukin-1β biosynthesis inhibition reduces acute seizures and drug resistant chronic epileptic activity in mice. |
Maroso, Mattia, et al.
Neurotherapeutics, 8: 304-15 (2011) 2011
Experimental evidence and clinical observations indicate that brain inflammation is an important factor in epilepsy. In particular, induction of interleukin-converting enzyme (ICE)/caspase-1 and activation of interleukin (IL)-1β/IL-1 receptor type 1 axis both occur in human epilepsy, and contribute to experimentally induced acute seizures. In this study, the anticonvulsant activity of VX-765 (a selective ICE/caspase-1 inhibitor) was examined in a mouse model of chronic epilepsy with spontaneous recurrent epileptic activity refractory to some common anticonvulsant drugs. Moreover, the effects of this drug were studied in one acute model of seizures in mice, previously shown to involve activation of ICE/caspase-1. Quantitative analysis of electroencephalogram activity was done in mice exposed to acute seizures or those developing chronic epileptic activity after status epilepticus to assess the anticonvulsant effects of systemic administration of VX-765. Histological and immunohistochemical analysis of brain tissue was carried out at the end of pharmacological experiments in epileptic mice to evaluate neuropathology, glia activation and IL-1β expression, and the effect of treatment. Repeated systemic administration of VX-765 significantly reduced chronic epileptic activity in mice in a dose-dependent fashion (12.5-200 mg/kg). This effect was observed at doses ≥ 50 mg/kg, and was reversible with discontinuation of the drug. Maximal drug effect was associated with inhibition of IL-1β synthesis in activated astrocytes. The same dose regimen of VX-765 also reduced acute seizures in mice and delayed their onset time. These results support a new target system for anticonvulsant pharmacological intervention to control epileptic activity that does not respond to some common anticonvulsant drugs.
|Differentiation of induced pluripotent stem cells into functional oligodendrocytes. |
Czepiel M, Balasubramaniyan V, Schaafsma W, Stancic M, Mikkers H, Huisman C, Boddeke E, Copray S
Glia 59 882-92 2011
The technology to generate autologous pluripotent stem cells (iPS cells) from almost any somatic cell type has brought various cell replacement therapies within clinical research. Besides the challenge to optimize iPS protocols to appropriate safety and GMP levels, procedures need to be developed to differentiate iPS cells into specific fully differentiated and functional cell types for implantation purposes. In this article, we describe a protocol to differentiate mouse iPS cells into oligodendrocytes with the aim to investigate the feasibility of IPS stem cell-based therapy for demyelinating disorders, such as multiple sclerosis. Our protocol results in the generation of oligodendrocyte precursor cells (OPCs) that can develop into mature, myelinating oligodendrocytes in-vitro (co-culture with DRG neurons) as well as in-vivo (after implantation in the demyelinated corpus callosum of cuprizone-treated mice). We report the importance of complete purification of the iPS-derived OPC suspension to prevent the contamination with teratoma-forming iPS cells. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.
|Ezh2 expression in astrocytes induces their dedifferentiation toward neural stem cells. |
Sher, Falak, et al.
Cell Reprogram, 13: 1-6 (2011) 2011
Recently, we have demonstrated the expression of the polycomb group protein Ezh2 in embryonic and adult neural stem cells. Although Ezh2 remained highly expressed when neural stem cells differentiate into oligodendrocyte precursor cells, it is downregulated during the differentiation into neurons or astrocytes. This is in accordance with the differentiation repressive role Ezh2 is thought to play in the maintenance and self-renewal of stem cells. To establish the importance of downregulation of Ezh2 for becoming astrocytes, we have studied the effect of forced Ezh2 expression in postnatal mouse astrocytes. Upon forced expression of this polycomb group protein, cultured astrocytes retracted their cell extensions and became proliferating round/bipolar cells that occasionally formed small neurosphere-like clusters. Analysis of the expression profile of these Ezh2-expressing astrocytes reveal downregulation of typical astrocytic genes, like GFAP and S100, and upregulation of genes that are generally expressed in neural stem cells, like nestin, Sox2, musashi, and CD133. However, these neural stem cell-like cells lack a differentiation potential, indicating that overexpression of Ezh2 alone is insufficient for a complete dedifferentiation.
|Spatiotemporal nuclear factor interleukin-6 expression in the rat brain during lipopolysaccharide-induced fever is linked to sustained hypothalamic inflammatory target gene induction. |
Damm J, Luheshi GN, Gerstberger R, Roth J, Rummel C
J Comp Neurol 519 480-505. 2011
Rats injected with lipopolysaccharide (LPS) show brain-controlled sickness symptoms, including fever. In these animals, early genomic activation of brain cells was previously monitored by immunohistochemical detection of transcription factors such as nuclear factor (NF)-κB or signal transducer and activator of transcription (STAT)3 and was linked to the initiation or maintenance of the febrile response. To investigate whether NF-IL6 might be another important transcription factor implicated in this kind of immune-to-brain signaling, rats were injected with LPS (100 μg/kg, intraperitoneally) or phosphate-buffered saline, and brains were analyzed by immunohistochemistry, real-time PCR, or Western blot 4, 6, 8, and 10 hours later. Moderate to strong LPS-induced nuclear NF-IL6 immunoreactivity (IR) occurred in a time-dependent manner within circumventricular organs, namely, the vascular organ of the lamina terminalis, the subfornical organ, the area postrema, and the median eminence, brain structures with a leaky blood-brain barrier. Furthermore, nuclear NF-IL6-IR was observed in the pituitary gland, the choroid plexus, and the meninges as well as blood vessels throughout the entire brain. Endothelial, microglial, and ependymal cells, astrocytes, perivascular macrophages, and neurons exhibited LPS-induced nuclear NF-IL6-IR; mRNA levels of NF-IL6, responsive inflammatory genes, and NF-IL6 protein levels were significantly elevated. As opposed to observations on STAT3 or NFκB, the percentage of NF-IL6-reactive cells increased in parallel to late phases of the febrile response. In conclusion, these results suggest a potential role for NF-IL6 in the maintenance or possibly the termination of LPS-induced fever. Moreover, we propose NF-IL6 to be a delayed brain cell activation marker.© 2010 Wiley-Liss, Inc.
|Astroglial loss and edema formation in the rat piriform cortex and hippocampus following pilocarpine-induced status epilepticus. |
Ji-Eun Kim,Seong-Il Yeo,Hea Jin Ryu,Min-Ju Kim,Duk-Soo Kim,Seung-Mook Jo,Tae-Cheon Kang
The Journal of comparative neurology 518 2010
In the present study we analyzed aquaporin-4 (AQP4) immunoreactivity in the piriform cortex (PC) and the hippocampus of pilocarpine-induced rat epilepsy model to elucidate the roles of AQP4 in brain edema following status epilepticus (SE). In non-SE-induced animals, AQP4 immunoreactivity was diffusely detected in the PC and the hippocampus. AQP4 immunoreactivity was mainly observed in the endfeet of astrocytes. Following SE the AQP4-deleted area was clearly detected in the PC, not in the hippocampus. Decreases in dystrophin and α-syntrophin immunoreactivities were followed by reduction in AQP4 immunoreactivity. These alterations were accompanied by the development of vasogenic edema and the astroglial loss in the PC. In addition, acetazolamide (an AQP4 inhibitor) treatment exacerbated vasogenic edema and astroglial loss both in the PC and in the hippocampus. These findings suggest that SE may induce impairments of astroglial AQP4 functions via disruption of the dystrophin/α-syntrophin complex that worsen vasogenic edema. Subsequently, vasogenic edema results in extensive astroglial loss that may aggravate vasogenic edema.
|Partial depletion of CREB-binding protein reduces life expectancy in a mouse model of Huntington disease. |
Klevytska AM, Tebbenkamp AT, Savonenko AV, Borchelt DR
J Neuropathol Exp Neurol 69 396-404. 2010
Previous studies have reported that mutant huntingtin (htt) interferes with cyclic AMP response element binding protein binding protein (CBP)-mediated transcription, possibly by inhibiting the acetylation of histones. In Drosophila models that express fragments of mutant htt, histone deacetylase inhibitors reverse deficits in histone acetylation, rescue photoreceptor degeneration, and prolong their survival. These compounds also improve motor deficits in a transgenic mouse model of Huntington disease (HD). To determine whether endogenous CBP depletion contributes to HD pathogenesis, we crossed HD-N171-82Q transgenic mice with mice harboring a disrupted CBP gene and produced mice with partial (50%) depletion of CBP. This reduction of CBP levels decreased the life expectancy of the HD-N171-82Q Line 6 mouse model. The loss of CBP had no obvious impact on the severity of motor impairment, degeneration of the striatum, mutant htt inclusion formation, or global levels of acetylated histones H3 or H4 in brain. In cell models, we confirmed that mutant htt inclusions recruit human CBP but found no evidence for interactions between soluble forms of mutant htt and CBP. Although we identified no neurological explanation for the decreased life expectancy of HD-N171-82Q mice with partial depletion of CBP, the data are consistent with the notion that CBP function mitigates mutant htt toxicity by a currently unidentified mechanism.
|Proliferation inhibition, DNA damage, and cell-cycle arrest of human astrocytoma cells after acrylamide exposure. |
Chen JH, Tsou TC, Chiu IM, Chou CC
Chem Res Toxicol 23 1449-58. 2010
Acrylamide (ACR) has been recognized as a neurological and reproductive toxin in humans and laboratory animals. This study aimed to determine the effects of ACR-induced DNA damage on cell cycle regulation in human astrocytoma cell lines. Treatment of U-1240 MG cells with 2 mM ACR for 48 h resulted in a significant inhibition of cell proliferation as evaluated by Ki-67 protein expression and MTT assay. The analysis of DNA damage with the comet assay showed that treatment of the cells with 0.5, 1, and 2 mM ACR for 48 h caused significant increases in DNA damage by 3.5-, 4-, and 14-fold, respectively. Meanwhile, analysis of cell-cycle arrest with flow cytometry revealed that the ACR treatments resulted in significant increases in the G(0)/G(1)-arrested cells in a time- and dose-dependent manner. Expression of DNA damage-associated/checkpoint-related signaling molecules, including phosphorylated-p53 (pp53), p53, p21, p27, Cdk2, and cyclin D(1), in three human astrocytoma cell lines (U-1240 MG, U-251 MG, and U-87 MG) was also analyzed by immunoblotting. Treatment of the three cell lines with 2 mM ACR for 48 h caused marked increases in pp53 and Cdk2, as well as decreases in cyclin D(1) and p27. Moreover, increases in p53 and p21 were detected in both U-1240 and U-87 MG cells, whereas no marked change in p53 and a decrease in p21 were observed in U-251 MG cells. To address the involvement of ataxia telangiectasia mutated/ATM-Rad3-related (ATM/ATR) kinase in the signaling of ACR-induced G(0)/G(1) arrest, caffeine was used to block the ATM/ATR pathway in U-1240 MG cells. Caffeine significantly attenuated the ACR-induced G(0)/G(1) arrest as well as the expression of DNA damage-associated/checkpoint-related signaling molecules in a dose-dependent manner. This in vitro study clearly demonstrates the critical role of ATM/ATR in the signaling of ACR-induced cell-cycle arrest in astrocytoma cells.
|Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures. |
Maroso, Mattia, et al.
Nat. Med., 16: 413-9 (2010) 2010
Brain inflammation is a major factor in epilepsy, but the impact of specific inflammatory mediators on neuronal excitability is incompletely understood. Using models of acute and chronic seizures in C57BL/6 mice, we discovered a proconvulsant pathway involving high-mobility group box-1 (HMGB1) release from neurons and glia and its interaction with Toll-like receptor 4 (TLR4), a key receptor of innate immunity. Antagonists of HMGB1 and TLR4 retard seizure precipitation and decrease acute and chronic seizure recurrence. TLR4-defective C3H/HeJ mice are resistant to kainate-induced seizures. The proconvulsant effects of HMGB1, like those of interleukin-1beta (IL-1beta), are partly mediated by ifenprodil-sensitive N-methyl-d-aspartate (NMDA) receptors. Increased expression of HMGB1 and TLR4 in human epileptogenic tissue, like that observed in the mouse model of chronic seizures, suggests a role for the HMGB1-TLR4 axis in human epilepsy. Thus, HMGB1-TLR4 signaling may contribute to generating and perpetuating seizures in humans and might be targeted to attain anticonvulsant effects in epilepsies that are currently resistant to drugs.
|CCR7 is expressed in astrocytes and upregulated after an inflammatory injury. |
Gomez-Nicola, Diego, et al.
J. Neuroimmunol., 227: 87-92 (2010) 2010
Neurodegenerative or autoimmune diseases are frequently regulated by chemokines and their receptors, controlling both glial activation and immune cell infiltration. CCL19 and CCL21 have been described to mediate crucial functions during CNS pathological states, regulating both immune cell traffic to the CNS and communication between glia and neurons. Here, we describe the expression pattern and cellular sources of CCR7, receptor of CCL19 and CCL21, in the normal mouse brain. Moreover, we found that CCR7 is upregulated in reactive astrocytes upon intracerebral LPS, regulating early glial reactivity through its ligands CCL19 and CCL21. Our results indicate that CCR7 is playing an important role for the intercellular communication during the inflammatory activation in the CNS.
|Functional characterization of mouse organic anion transporting peptide 1a4 in the uptake and efflux of drugs across the blood-brain barrier. |
Ose A, Kusuhara H, Endo C, Tohyama K, Miyajima M, Kitamura S, Sugiyama Y
Drug metabolism and disposition: the biological fate of 38 168-176 2010
This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.
|CX3CR1 promotes recruitment of human glioma-infiltrating microgliamacrophages (GIMs). |
Held-Feindt J, Hattermann K, MÃ¼erkÃ¶ster SS, Wedderkopp H, Knerlich-Lukoschus F, Ungefroren H, Mehdorn HM, Mentlein R
Experimental cell research 2010
The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets. Copyright Â© 2010. Published by Elsevier Inc.
|Alteration of glial-neuronal metabolic interactions in a mouse model of Alexander disease. |
Tore Wergeland Meisingset,Øystein Risa,Michael Brenner,Albee Messing,Ursula Sonnewald
Glia 58 2010
Alexander disease is a rare and usually fatal neurological disorder characterized by the abundant presence of protein aggregates in astrocytes. Most cases result from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP), but how these mutations lead to aggregate formation and compromise function is not known. A transgenic mouse line (Tg73.7) over-expressing human GFAP produces astrocytic aggregates indistinguishable from those seen in the human disease, making them a model of this disorder. To investigate possible metabolic changes associated with Alexander disease Tg73.7 mice and controls were injected simultaneously with [1-(13)C]glucose to analyze neuronal metabolism and [1,2-(13)C]acetate to monitor astrocytic metabolism. Brain extracts were analyzed by (1)H magnetic resonance spectroscopy (MRS) to quantify amounts of several key metabolites, and by (13)C MRS to analyze amino acid neurotransmitter metabolism. In the cerebral cortex, reduced utilization of [1,2-(13)C]acetate was observed for synthesis of glutamine, glutamate, and GABA, and the concentration of the marker for neuronal mitochondrial metabolism, N-acetylaspartate (NAA) was decreased. This indicates impaired astrocytic and neuronal metabolism and decreased transfer of glutamine from astrocytes to neurons compared with control mice. In the cerebellum, glutamine and GABA content and labeling from [1-(13)C]glucose were increased. Evidence for brain edema was found in the increased amount of water and of the osmoregulators myo-inositol and taurine. It can be concluded that astrocyte-neuronal interactions were altered differently in distinct regions.Full Text Article
|Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels. |
Mareschi, Katia, et al.
Cytotherapy, 11: 534-47 (2009) 2009
BACKGROUND AIMS: Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro. METHODS: AF was harvested during routine pre-natal amniocentesis at 14-16 weeks of pregnancy. AF sample pellets were plated in alpha-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction. RESULTS: Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na(+) channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na(+) channels. CONCLUSIONS: These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.
|Acrylamide-induced astrogliotic and apoptotic responses in human astrocytoma cells. |
Jong-Hang Chen, Kuen-Yuh Wu, Ing-Ming Chiu, Tsui-Chun Tsou, Chin-Cheng Chou, Jong-Hang Chen, Kuen-Yuh Wu, Ing-Ming Chiu, Tsui-Chun Tsou, Chin-Cheng Chou
Toxicology in vitro : an international journal published in association with BIBRA 23 855-61 2009
This study was to clarify whether acrylamide (ACR) will induce apoptosis and astrogliosis in an astrocytic cell line in vitro. Different time- and dose-dependent cytotoxic studies were conducted upon neuronal (SH-SY5Y) and glial cell lines (U-1240 MG) under exposure to ACR up to 72h. We showed that SH-SY5Y cells were more sensitive in cytotoxic assays than U-1240 MG cells, and significantly decreased cell viability was observed at concentrations higher than 1mM with increased lactate dehydrogenase leakage observed only at 5 and 10mM in U-1240 MG cells. The ACR-induced apoptotic responses and phosphorylation of p53 protein at Ser15 for U-1240 MG cells were identified at 48h. The increase of glial fibrillary acidic protein (GFAP) as a chemical-induced astrogliotic response was found to be associated with different ACR concentrations and exposure times, particularly at or=48h of or=2mM. In addition, immunocytochemical staining at 36h of 5 and 10mM treatments had significantly higher density of GFAP than the control. Thus, ACR-induced effects can be seen in neuronal and astrocytic cells. These results suggest that ACR exposure may lead to apoptotic and astrogliotic effects in human astrocytoma cells in vitro in a time- and dose-dependent manner.
|Age-dependent vascular changes induced by status epilepticus in rat forebrain: implications for epileptogenesis. |
Marcon, Jessica, et al.
Neurobiol. Dis., 34: 121-32 (2009) 2009
Brain inflammation, angiogenesis and increased blood-brain barrier (BBB) permeability occur in adult rodent and human epileptogenic brain tissue. We addressed the role of these events in epileptogenesis using a developmental approach since the propensity to develop spontaneous seizures, therefore the induction of epileptogenesis, is age-dependent and increases with brain maturation. Inflammation, angiogenesis and BBB permeability were studied in postnatal day (PN)9 and PN21 rats, 1 week and 4 months after pilocarpine-induced status epilepticus. Brain inflammation was evaluated by interleukin(IL)-1beta immunohistochemistry; angiogenesis was quantified by measuring the density of microvessels identified by an anti-laminin antibody or by the intraluminal signal of FITC-albumin; BBB integrity was assessed by extravascular IgG immunostaining or detection of parenchymal extravasation of FITC-albumin. Neither inflammation nor angiogenesis or changes in BBB permeability were detected in PN9 rats after status epilepticus, and these rats did not develop spontaneous seizures in adulthood as assessed by video-EEG monitoring. Differently, status epilepticus in PN21 rats induced chronic inflammation, angiogenesis and BBB leakage in the hippocampus in 62% of rats, while in the remaining rats only transient inflammation in forebrain was observed. Epilepsy developed in about 62% of PN21 rats exposed to SE and these epileptic rats showed the three phenomena concomitantly in the hippocampus. PN21 rats that did not develop epilepsy 4 months after status epilepticus, as assessed by video-EEG monitoring, they did not show inflammation, angiogenesis or BBB damage in forebrain at this time. Our data show that age-dependent vascular changes and brain inflammation induced by status epilepticus are associated with epileptogenesis, suggesting that these phenomena are implicated in the mechanisms underlying the occurrence of spontaneous seizures.
|Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain. |
D Ballhausen,L Mittaz,O Boulat,L Bonafé,O Braissant
Neuroscience 164 2009
Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.
|Overexpression of CXCL16 and its receptor CXCR6/Bonzo promotes growth of human schwannomas. |
Janka Held-Feindt, Brigitte Rehmke, Rolf Mentlein, Kirsten Hattermann, Friederike Knerlich, Heinz-Hermann Hugo, Andreas Ludwig, H Maximilian Mehdorn
Glia 56 764-74 2008
Chemokines and their receptors play a decisive role in tumor progression and metastasis. Here, we describe the expression of the CXCL16-CXCR6-system in human schwannomas of different localization and in malignant peripheral nerve sheath tumors. The transmembrane chemokine CXCL16 and its receptor CXCR6/Bonzo were overexpressed on the mRNA and protein levels in all tumor samples investigated as compared with normal peripheral or 8th cranial nerve tissues. Chromogenic immunostaining and confocal laser microscopy revealed that CXCL16 and CXCR6 were localized mainly on S-100 positive schwannoma cells. Cultured schwannoma cells responded to CXCL16-stimulation by phosphorylation of kinases p42/44 (Erk 2/1) that could be inhibited by the MEK1/2-inhibitor U0126 indicating an involvement of the mitogen-activated protein kinase signal transduction pathway. As a biological response, CXCL16 increased proliferation and induced migration of schwannomas. Hence, CXCL16 appears to be a novel growth factor for schwannomas of different localization.
|Constitutive activation of Raf-1 induces glioma formation in mice. |
Yelena Lyustikman, Hiroyuki Momota, William Pao, Eric C Holland
Neoplasia (New York, N.Y.) 10 501-10 2008
In human glioblastoma multiforme (GBM), RAS activity is upregulated in the majority of the tumors. Furthermore, the levels of phospho-mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), a downstream effector of RAS, are also increased. In mice, activated KRas cooperates with the loss of INK4a-ARF locus or with activated Akt to induce gliomas, confirming an important role for this pathway in glioma biology. However, to correctly target therapies against the RAS signaling pathway, it is necessary to identify the effectors that contribute to RAS-mediated gliomagenesis. In this study, we investigated the contribution of RAF signaling in glioma oncogenesis. We find that the levels of RAF-1 and BRAF proteins and RAF kinase activity are increased in human GBM samples. We confirm the importance of this finding by demonstrating a causal role for a constitutively active Raf-1 mutant in glioma formation in mice. Specifically, we find that activated Raf-1 cooperates with Arf loss or Akt activation to generate gliomas similar to activated KRas under the same conditions. Our study suggests that the oncogenic effect of KRas in glioma formation may be transduced at least in part through Raf signaling and that therapeutic targeting of this pathway may be beneficial in glioma treatment.Full Text Article
|Interleukin Converting Enzyme inhibition impairs kindling epileptogenesis in rats by blocking astrocytic IL-1beta production. |
Ravizza, Teresa, et al.
Neurobiol. Dis., 31: 327-33 (2008) 2008
An enhanced production of IL-1beta in glia is a typical feature of epileptogenic tissue in experimental models and in human drug-refractory epilepsy. We show here that the selective inhibition of Interleukin Converting Enzyme (ICE), which cleaves the biologically active form of IL-1beta using VX-765, blocks kindling development in rats by preventing IL-1beta increase in forebrain astrocytes, without interfering with glia activation. The average afterdischarge duration was not altered significantly by VX-765. Up to 24 h after kindling completion and drug washout, kindled seizures could not be evoked in treated rats. VX-765 did not affect seizures or afterdischarge duration in fully kindled rats. These data indicate an antiepileptogenic effect mediated by ICE inhibition and suggest that specific anti-IL-1beta pharmacological strategies can be envisaged to interfere with epileptogenic mechanisms.
|Caspase-cleaved TAR DNA-binding protein-43 is a major pathological finding in Alzheimer's disease. |
Troy T Rohn
Brain research 1228 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
|The effect of activated microglia on astrogliosis parameters in astrocyte cultures. |
Claudia Röhl, Ralph Lucius, Jobst Sievers
Brain research 1129 43-52 2007
In the diseased central nervous system, astrogliosis is accompanied by microglial activation. Depending on the context of their activation, reactive astrocytes are involved in neuronal survival and regeneration in an either protective or impedimental way. Major reactive changes of astrocytes in vivo are the upregulation of the intermediate filaments GFAP (glial fibrillary acidic protein) and vimentin with accompanying cellular hypertrophy and/or hyperplasia. To examine the involvement of activated microglia in the onset and maintenance of astrogliosis, we used an in vitro model of purified cultures of astrocytes and assessed as parameters for astrogliosis GFAP, vimentin, astroglial hypertrophy and cell growth after treatment with medium conditioned by LPS (lipopolysaccarides)-stimulated microglia. Furthermore, IL-6 as a typically upregulated cytokine in proinflammatory processes in the brain was determined in treated astrocytes. GFAP, the classical marker for astrogliosis, was downregulated on its protein and in parallel with vimentin on its mRNA level. The expression of actin, another cytoskeleton protein used as control, remained unchanged. Ultrastructural studies of astroglial intermediate filaments supported these findings. No hypertrophy was found. Nevertheless, LPS-activated microglia stimulated astrocytes as demonstrated by an increased cell number and an enhanced mRNA expression of IL-6. Resting microglia did not change any of the determined parameters. Our results suggest that the role of activated microglia in astrogliotic processes following injury of the brain has to be reevaluated, as microglia in their activated state might support the onset of astrogliosis on the one hand, but might delay or reduce subsequent glial scar formation on the other hand.
|Early decrease of mitochondrial DNA repair enzymes in spinal motor neurons of presymptomatic transgenic mice carrying a mutant SOD1 gene. |
Tetsuro Murakami, Makiko Nagai, Kazunori Miyazaki, Nobutoshi Morimoto, Yasuyuki Ohta, Tomoko Kurata, Yasushi Takehisa, Tatsushi Kamiya, Koji Abe
Brain research 1150 182-9 2007
Growing evidence has recently shown that mutant SOD1 accumulate in the mitochondria and cause vacuolation in transgenic mice carrying mutant SOD1, an animal model of amyotrophic lateral sclerosis (ALS). In this study, the expressions of DNA repair enzymes, oxoguanine glycosylase 1 (ogg1), DNA polymerase beta (polbeta), and DNA polymerase gamma (polgamma) were examined in transgenic mice with an ALS-linked mutant SOD1 gene, a valuable model for human ALS. In presymptomatic Tg mice, the nuclear form of ogg1 was upregulated, whereas mitochondrial ogg1 remained at the same level. DNA polymerase was selectively downregulated in the mitochondria. This study suggests an impaired protective mechanism against oxidative stress in mitochondria. The expressions of these enzymes are predominant in spinal motor neurons, suggesting a mechanism of selective motor neuron death in this animal model of ALS.
|Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons. |
Xianrong Mao,Shao-Hua Yang,James W Simpkins,Steven W Barger
Journal of neurochemistry 100 2007
Sp-family transcription factors (Sp1, Sp3 and Sp4) contain a zinc-finger domain that binds to DNA sequences rich in G-C/T. As assayed by RT-PCR analysis of mRNA, western-blot analysis, immunofluorescence, and antibody-dependent supershift of DNA-binding assays, the prominent Sp-family factors in cerebral neurons were identified as Sp3 and Sp4. By contrast, glial cells were found to express Sp1 and Sp3. We previously showed that the pattern of G-C/T binding activity of Sp-family factors is rapidly and specifically altered by the calcium influx accompanying activation of glutamate receptors. Here, we demonstrate that Sp-factor activity is also lost after a cerebral ischemia/reperfusion injury in vivo. Consistent with its calcium-dependent nature, we found that glutamate's effect on Sp-family factors could be blocked by inhibitors of calpains, neutral cysteine proteases activated by calcium. Purified calpain I cleaved Sp3 and Sp4 into products that retained G-C/T-binding activity, consistent with species observed in glutamate-treated neurons. These data provide details of an impact of glutamate-receptor activation on molecular events connected to gene expression.Full Text Article
|Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor. |
Liu, Hua-Qing, et al.
Br. J. Pharmacol., 148: 314-25 (2006) 2006
1. This study examined whether Paeoniflorin (PF), the major active components of Chinese herb Paeoniae alba Radix, has neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). 2. Subcutaneous administration of PF (2.5 and 5 mg kg(-1)) for 11 days could protect tyrosine hydroxylase (TH)-positive substantia nigra neurons and striatal nerve fibers from death and bradykinesia induced by four-dose injection of MPTP (20 mg kg(-1)) on day 8. 3. When given at 1 h after the last dose of MPTP, and then administered once a day for the following 3 days, PF (2.5 and 5 mg kg(-1)) also significantly attenuated the dopaminergic neurodegeneration in a dose-dependent manner. Post-treatment with PF (5 mg kg(-1)) significantly attenuated MPTP-induced proinflammatory gene upregulation and microglial and astrocytic activation. 4. Pretreatment with 0.3 mg kg(-1) 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor (A1AR) antagonist, 15 min before each dose of PF, reversed the neuroprotective and antineuroinflammatory effects of PF. 5. In conclusion, this study demonstrated that PF could reduce the MPTP-induced toxicity by inhibition of neuroinflammation by activation of the A1AR, and suggested that PF might be a valuable neuroprotective agent for the treatment of PD.
|Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system. |
Ravizza, T and Vezzani, A
Neuroscience, 137: 301-8 (2006) 2006
Interleukin-1beta is rapidly synthesized by glia after the induction of seizures. Recent evidence shows that endogenous IL-1beta has proconvulsant actions mediated by interleukin-1 receptor type I. This receptor also mediates interleukin-1beta effects on neuronal susceptibility to neurotoxic insults. In this study, we investigated the basal and seizure-induced expression of interleukin-1 receptor type I in rat forebrain to identify the cells targeted by interleukin-1beta during epileptic activity. Self-sustained limbic status epilepticus was induced in rats by electrical stimulation of the ventral hippocampus. Interleukin-1 receptor type I immunoreactivity was barely detectable in neurons in control brain tissue. During status epilepticus, interleukin-1 receptor type I was induced in the hippocampal neurons firstly, and several hours later in astrocytes localized in limbic and extralimbic areas. Neuronal interleukin-1 receptor type I expression in the hippocampus outlasted the duration of spontaneous electroencephalographic seizure and was not observed in degenerating neurons. Astrocytic expression occurred transiently, between six and 18 h after the induction of status epilepticus and was invariably found in regions of neuronal damage. These time-dependent, cell- and region-specific changes in interleukin-1 receptor type I expression during status epilepticus suggest that interleukin-1 receptor type I in neurons mediates interleukin-1beta-induced fast changes in hippocampal excitability while interleukin-1 receptor type I receptors in astrocytes may mediate interleukin-1beta effects on neuronal survival in hostile conditions.
|Dynein light chain Tctex-1 identifies neural progenitors in adult brain. |
Carlos Dedesma, Jen-Zen Chuang, Peter D Alfinito, Ching-Hwa Sung
The Journal of comparative neurology 496 773-86 2006
The identity and biology of stem cells and progenitors in the adult brain are of considerable interest, because these cells hold great promise for the development of novel therapies for damaged brain tissue in human diseases. This research field critically needs biological markers that specifically identify the resident precursors in the germinal zones of the adult central nervous system so that the discovery of regulatory influences for adult neurogenesis may be facilitated. In this study, by using a combination of in situ hybridization, bromodeoxyuridine incorporation, immunocolocalization, and ultrastructural studies, we show that in rodents Tctex-1, a cytoplasmic dynein light chain, is selectively enriched in almost all cycling progenitors and young neuronal progeny, but not in mature granular cells and astrocytes, in the subgranular zone of the adult dentate gyrus. Tctex-1 is also selectively abundant in cells closely resembling previously described immature progenitors and migrating neuroblasts at the subventricular zone of the lateral ventricle. Our results suggest that Tctex-1 serves as a novel marker for the identification of neural progenitors of the adult brain.
|Paeoniflorin attenuates chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rats. |
Jing Liu, Dao-Zhong Jin, Liang Xiao, Xing-Zu Zhu
Brain research 1089 162-70 2006
Chronic cerebral hypoperfusion, a mild ischemic condition, is associated with the cognitive deficits of AD. Paeoniflorin (PF), a major constituent of peony root, was proved to be neuroprotective in middle cerebral artery occlusion model. In this study, we investigated whether PF could attenuate chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rat. Seven weeks after permanent bilateral occlusion of the common carotid arteries, the rats were tested in the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglias were labeled with immunocytochemistry in hippocampus. PF at the dose of 2.5 mg/kg ameliorated cerebral hypoperfusion-related learning dysfunction and prevented CA1 neuron damage. Chronic cerebral hypoperfusion increased the immunoreactivity of astrocytes and microglias in hippocampus. The increase was prevented by PF at the dose of 2.5 mg/kg. Cerebral hypoperfusion also increased expression of nuclear factor-kappaB (NF-kappaB), mostly in astrocytes, but not in neurons. With the treatment of PF (2.5 mg/kg), NF-kappaB immunostaining was diminished in hippocampus. Our results demonstrated that PF could attenuate cognitive deficit and brain damage induced by chronic cerebral hypoperfusion and that suppression of neuroinflammatory reaction in brain might be involved in PF-induced neuroprotection.
|Alexander disease-associated glial fibrillary acidic protein mutations in mice induce Rosenthal fiber formation and a white matter stress response. |
Tracy L Hagemann, Jolien X Connor, Albee Messing
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 11162-73 2006
Mutations in the gene for the astrocyte specific intermediate filament, glial fibrillary acidic protein (GFAP), cause the rare leukodystrophy Alexander disease (AxD). To study the pathology of this primary astrocyte defect, we have generated knock-in mice with missense mutations homologous to those found in humans. In this report, we show that mice with GFAP-R76H and -R236H mutations develop Rosenthal fibers, the hallmark protein aggregates observed in astrocytes in AxD, in the hippocampus, corpus callosum, olfactory bulbs, subpial, and periventricular regions. Astrocytes in these areas appear reactive and total GFAP expression is elevated. Although general white matter architecture and myelination appear normal, when crossed with an antioxidant response element reporter line, the mutant mice show a distinct pattern of reporter-gene induction that is especially prominent in the corpus callosum, and histochemical staining reveals accumulation of iron in the same region. The mutant mice have a normal lifespan and show no overt behavioral defects, but are more susceptible to kainate-induced seizures. Although these mice demonstrate increased GFAP expression by themselves, further elevation of GFAP via crosses to GFAP transgenic animals leads to a shift in GFAP solubility, an increased stress response, and ultimately death. The mice do not display the full spectrum of pathology observed in human infantile AxD, but may more closely resemble the adult form of the disease. These studies provide formal proof linking GFAP mutations with Rosenthal fibers and oxidative stress, and correlate gliosis and GFAP protein levels to the severity of the disease.
|Possible role of scavenger receptor SRCL in the clearance of amyloid-beta in Alzheimer's disease. |
Kenji Nakamura, Wakana Ohya, Hiroshi Funakoshi, Gaku Sakaguchi, Akira Kato, Masatoshi Takeda, Takashi Kudo, Toshikazu Nakamura
Journal of neuroscience research 84 874-90 2006
Accumulation of beta-amyloid protein (Abeta) in the brain is a hallmark of Alzheimer's disease (AD), and Abeta-mediated pathogenesis could result from increased production of Abeta or insufficient Abeta clearance by microglia, astrocytes, or the vascular system. Cell-surface receptors, such as scavenger receptors, might play a critical role in the binding and clearing of Abeta; however, the responsible receptors have yet to be identified. We show that scavenger receptor with C-type lectin (SRCL), a member of the scavenger receptor family containing coiled-coil, collagen-like, and C-type lectin/carbohydrate recognition domains, is expressed in cultured astrocytes and microglia. In contrast to the low expression of SRCL in the wild-type mouse brain, in a double transgenic mouse model of AD (Tg-APP/PS1), immunohistochemistry showed that SRCL was markedly induced in Abeta-positive astrocytes and Abeta-positive vascular/perivascular cells, which are associated closely with cerebral amyloid angiopathy. In patients with AD, the distribution of SRCL was similar to that seen in the Tg-APP/PS1 temporal cortex. The presence of a large number of SRCL/Abeta double-positive particles in the intracellular compartments of reactive astrocytes and vascular/perivascular cells in Tg-APP/PS1 mice and AD patients suggests a role for SRCL in Abeta clearance. Moreover, CHO-K1 cells transfected with SRCL isoforms were found to bind fibrillar Abeta(1-42). These findings suggest that SRCL could be the receptor involved in the binding or clearing of Abeta by glial and vascular/perivascular cells in AD.
|ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier. |
Bronger, Holger, et al.
Cancer Res., 65: 11419-28 (2005) 2005
Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents.
|Tenascin-C regulates proliferation and migration of cultured astrocytes in a scratch wound assay. |
T Nishio, S Kawaguchi, M Yamamoto, T Iseda, T Kawasaki, T Hase
Neuroscience 132 87-102 2005
Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Astrocytes are known to play major roles in wound healing in the CNS. To elucidate the roles of TNC in wound closure by astrocytes, we have examined the morphological changes of cultured astrocytes in a scratch wound assay and measured the content of soluble TNC released into the medium. We have also localized the expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP), vimentin and integrin beta1. After wounding, glial cells rapidly released the largest TNC isoform and proliferated in the border zones. Subsequently, they became polarized with unidirectional processes and finally migrated toward the denuded area. The proliferating border zone cells and pre-migratory cells intensely expressed TNC mRNA, TNC-, vimentin-, GFAP- and integrin beta1-like immunoreactivity, while the migratory cells showed generally reduced expression except the front. Exogenous TNC enhanced cell proliferation and migration, while functional blocking with anti-TNC or anti-integrin beta1 antibody reduced both of them. These results suggest that mechanical injury induces boundary astrocytes to produce and release TNC that promotes cell proliferation and migration via integrin beta1 in an autocrine/paracrine fashion.
|Microglia is activated by astrocytes in trimethyltin intoxication. |
Claudia Röhl, Jobst Sievers
Toxicology and applied pharmacology 204 36-45 2005
Microglia participates in most acute and chronic neuropathologies and its activation appears to involve interactions with neurons and other glial cells. Trimethyltin (TMT)-induced brain damage is a well-characterized model of neurodegeneration, in which microglial activation occurs before neuronal degeneration. The aim of this in vitro study was to investigate the role of astroglia in TMT-induced microgliosis by using nitric oxide (NO), inducible NO synthase (iNOS), and morphological changes as parameters for microglial activation. Our investigation discusses (a) whether microglial cells can be activated directly by TMT; (b) if astroglial cells are capable of triggering or modulating microglial activation; (c) how the morphology and survival of microglia and astrocytes are affected by TMT treatment; and (d) whether microglial-astroglial interactions depend on direct cell contact or on soluble factors. Our results show that microglia are more vulnerable to TMT than astrocytes are and cannot be activated directly by TMT with regard to the examined parameters. In bilayer coculture with viable astroglial cells, microglia produce NO in significant amounts at subcytotoxic concentrations of TMT (20 micromol/l). At these TMT concentrations, microglial cells in coculture convert into small round cells without cell processes, whereas flat, fibroblast-like astrocytes convert into thin process bearing stellate cells with a dense and compact cell body. We conclude that astrocytes trigger microglial activation after treatment with TMT, although the mechanisms of this interaction remain unknown.
|Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice. |
Barbara Ahlemeyer, Eveline Baumgart-Vogt
Journal of neuroscience methods 149 110-20 2005
Knockout mouse models allow preparation of primary neuronal cultures from distinct brain regions in order to investigate the underlying neuronal pathomechanisms of human metabolic diseases associated with severe, regionally distinct brain pathologies (e.g. Zellweger syndrome, the most severe form of a peroxisomal biogenesis disorder). However, homozygous mouse pups with Zellweger syndrome usually die shortly after birth. Therefore, in this study, we established optimized protocols for the simultaneous preparation and cultivation of serum-free primary neuronal cultures from distinct brain regions (medial neocortex, hippocampus and cerebellum) from individual newborn (P0.5) C57Bl/6J mice. For each of the three types of neuronal cultures, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, mode of trituration, seeding density and composition of the culture medium. As indicated by indirect immunofluorescence using antibodies against NeuN, GFAP and CNPase, the purity of the distinct neuronal cultures was high. The percentage of oligodendrocytes was less than 1% in all neuronal cultures. Only 5% astrocytes were present in cortical, 7% in hippocampal and 10% in cerebellar cultures. Cytosine arabinofuranoside (AraC) treatment reduced the percentage of astrocytes only significantly in hippocampal cultures, however, increased the percentage of apoptotic neurons in hippocampal and cortical cultures.
|A role for hypoxia-inducible factor-1alpha in desferoxamine neuroprotection. |
Shannon Elise Goldsmith Hamrick, Patrick Sean McQuillen, Xiangning Jiang, Dezhi Mu, Ashima Madan, Donna Marie Ferriero
Neuroscience letters 379 96-100 2005
The newborn brain has increased vulnerability to hypoxia-ischemia from maturational differences in the oxidative stress response. We hypothesized that desferoxamine (DFO), an iron chelator, would provide protection in an in vitro model of ischemia in part through activation of the hypoxia-inducible gene hypoxia-inducible factor-1alpha (HIF-1alpha). Hippocampal neurons from E16 CD1 mice were exposed to 3 h of oxygen and glucose deprivation with and without pretreatment with 10 mmol/L DFO in the presence and absence of 2 micromol/L antisense oligonucleotides specific for HIF-1alpha (antiHIF-1alpha). DFO pretreatment resulted in 45% reduction in cell death (p = 0.006). This protection was diminished with transfection of antiHIF-1alpha (p = 0.049). Blocking HIF-1alpha reduces DFO protection suggesting that DFO protects through iron chelation and HIF-1alpha induction.
|The protective effect of dantrolene on ischemic neuronal cell death is associated with reduced expression of endoplasmic reticulum stress markers. |
Feng Li, Takeshi Hayashi, Guang Jin, Kentaro Deguchi, Shoko Nagotani, Isao Nagano, Mikio Shoji, Pak H Chan, Koji Abe
Brain research 1048 59-68 2005
The endoplasmic reticulum (ER) plays an important role in ischemic neuronal cell death. In order to determine the effect of dantrolene, a ryanodine receptor antagonist, on ER stress response and ischemic brain injury, we investigated changes in ER stress-related molecules, that is phosphorylated form of double-stranded RNA-activated protein kinase (PKR)-like ER kinase (p-PERK), phosphorylated form of eukaryotic initiation factor 2alpha (p-eIF2alpha), activating transcription factor-4 (ATF-4), and C/EBP-homologous protein (CHOP), as well as terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in the peri-ischemic area and ischemic core region of rat brain after transient middle cerebral artery occlusion (MCAO). In contrast to the cases treated with vehicle, the infarct volume and TUNEL-positive cells were significantly reduced at 24 h of reperfusion by treatment with dantrolene. The immunoreactivities for p-PERK, p-eIF2alpha, ATF-4, and CHOP were increased at the ischemic peripheral region after MCAO, which were partially inhibited by dantrolene treatment. The present results suggest that dantrolene significantly decreased infarct volume and provided neuroprotective effect on rats after transient MCAO by reducing ER stress-mediated apoptotic signal pathway activation in the ischemic area.
|Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina. |
Ward, Michelle M, et al.
Cell Tissue Res., 315: 305-10 (2004) 2004
Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.
|Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer's amyloidogenesis. |
Alpaslan Dedeoglu, Kerry Cormier, Sandra Payton, Katya A Tseitlin, Jonathan N Kremsky, Li Lai, Xiaohua Li, Robert D Moir, Rudolph E Tanzi, Ashley I Bush, Neil W Kowall, Jack T Rogers, Xudong Huang
Experimental gerontology 39 1641-9 2004
A growing body of evidence indicates that dysregulation of cerebral biometals (Fe, Cu, Zn) and their interactions with APP and Abeta amyloid may contribute to the Alzheimer's amyloid pathology, and thus metal chelation could be a rational therapeutic approach for interdicting AD pathogenesis. However, poor target specificity and consequential clinical safety of current metal-complexing agents have limited their widespread clinical use. To develop the next generation of metal chelators, we have designed and synthesized a new bifunctional molecule-XH1, based on a novel 'pharmacophore conjugation' concept. This lipophilic molecule has both amyloid-binding and metal-chelating moieties covalently connected by amide bonds. It achieved a putative binding geometry with Abeta1-40 peptide by the computational chemistry modeling and reduced Zn(II)-induced Abeta1-40 aggregation in vitro as determined by turbidometry. Moreover, our pilot data indicated that XH1 has no significant neurotoxicity at low micromolar concentrations and acute animal toxicity. XH1 specifically reduced APP protein expression in human SH-SY5Y neuroblastoma cells and attenuated cerebral Abeta amyloid pathology in PS1/APP transgenic mice without inducing apparent toxicity and behavior disturbances. Collectively, these preliminary findings carry implication for XH1 being a BBB-permeable lead compound for AD therapeutics targeting Alzheimer's amyloidogenesis, although further studies are needed.
|Gene expression changes in the course of neural progenitor cell differentiation. |
Ulf Gurok, Christine Steinhoff, Bettina Lipkowitz, H-Hilger Ropers, Constance Scharff, Ulrike A Nuber
The Journal of neuroscience : the official journal of the Society for Neuroscience 24 5982-6002 2004
The molecular changes underlying neural progenitor differentiation are essentially unknown. We applied cDNA microarrays with 13,627 clones to measure dynamic gene expression changes during the in vitro differentiation of neural progenitor cells that were isolated from the subventricular zone of postnatal day 7 mice and grown in vitro as neurospheres. In two experimental series in which we withdrew epidermal growth factor and added the neurotrophins Neurotrophin-4 or BDNF, four time points were investigated: undifferentiated cells grown as neurospheres, and cells 24, 48, and 96 hr after differentiation. Expression changes of selected genes were confirmed by semiquantitative RT-PCR. Ten different groups of gene expression dynamics obtained by cluster analysis are described. To correlate selected gene expression changes to the localization of respective proteins, we performed immunostainings of cultured neurospheres and of brain sections from adult mice. Our results provide new insights into the genetic program of neural progenitor differentiation and give strong hints to as yet unknown cellular communications within the adult subventricular zone stem cell niche.
|Infection of human astrocytoma cells with simian-human immunodeficiency virus results in up-regulation of gene expression and altered growth properties. |
Singh, Dinesh K, et al.
Neurosci. Lett., 340: 201-4 (2003) 2003
Recent reports of human immunodeficiency virus-1 (HIV-1) infection of astrocytes suggest a role for astrocytes in HIV encephalitis. In this study, we infected a human astrocytoma cell line with a pathogenic simian HIV (SHIV(50OLNV)) and examined growth patterns and immunomodulatory genes. Approximately 1% of uninfected cells in culture expressed glial fibrillary acid protein (GFAP) whereas 40% of the cells expressed GFAP at 7 days post-inoculation along altered growth patterns. Using targeted cytokine cDNA arrays, we found that SHIV(50OLNV) infection resulted in the up-regulation of several genes including metalloproteinase bone morphogenic protein 1 and chemokines monocyte chemoattractant protein 1 and stromal cell derived factor 1alpha. These data suggest that astrocytic activation, altered morphology and up-regulation of immunomodulatory genes in response to SHIV infection may participate in initiation of inflammation and trafficking of infected monocytes/macrophages into the central nervous system, potentiating the development of HIV encephalitis.
|Developmental changes of parameters for astrogliosis during cultivation of purified cerebral astrocytes from newborn rats. |
Claudia Röhl, Janka Held-Feindt, Jobst Sievers
Brain research. Developmental brain research 144 191-9 2003
Astrogliosis is a common phenomenon seen in most neuropathological changes of the central nervous system. Several in vitro models have been used to study the mechanisms and conditions for the induction of astrogliosis, however many do not take into account that the metabolic and structural characteristics of astrocytes change with time in culture. Thus, it appears difficult to attribute changes of, e.g., GFAP to the normal change in vitro as opposed to additional changes due to an astrogliotic reaction. The present study was therefore undertaken to characterize these developmental changes in purified astroglial secondary cultures during cultivation to provide a basis for further investigations of astrogliosis in vitro. During 6 weeks of cultivation (3-43 days) GFAP (ELISA) increased much more (22-fold) than the cell number (2.5-fold) and the total protein (3.5-fold). The GFAP/protein ratio increased during the first 4 weeks of cultivation and reached a plateau thereafter, which was accompanied by a significant increase of GFAP mRNA (Northern blot). At the ultrastructural level (transmission electron microscopy) gliofilaments in the perinuclear region as well as in the cell processes of 4-day-old astrocytes showed a dispersed pattern, whereas an accumulation of gliofilaments was found in 39-day-old cells, which formed large aggregated bundles localized mostly in the cell processes. Our results show that in vitro astrocytes undergo developmental changes in their accumulation of GFAP and intermediate filaments which reach a stable steady state after 4 weeks in culture. These 'normal' developmental changes will have to be taken into account, when experiments with variations of the level of GFAP are performed. Stable culture conditions for experimentation appear to be present after 4 weeks in culture.
|Different glial reactions to hippocampal stab wounds in young adult and aged rats. |
Waner Zhu, Hiroyuki Umegaki, Tadashi Shinkai, Shinobu Kurotani, Yusuke Suzuki, Hidetoshi Endo, Akihisa Iguchi
The journals of gerontology. Series A, Biological sciences and medical sciences 58 117-22 2003
Brain injury induces reactive gliosis. To examine the activation of glial cells after brain injury in young versus aged rats, we used a brain stab-wound model and examined the expression of cells positive for ED1 (ED1(+)) and glial fibrillary acidic protein (GFAP(+)) in the hippocampus in young-mature (3 months) and aged (25 months) Wistar rats at various times following hippocampal stab injury. ED1(+) cells appeared more frequently in the aged rats than in the young-mature rats under control conditions, whereas the number of GFAP(+) cells was not different between two groups. Following the stab wound, there was an increase in ED1 expression that was delayed but stronger in the aged rats and that persisted longer; the increase of the number of GFAP(+) cells also persisted longer. We conclude that different glial reactivity in the aged brain suggests that aging is associated with increased glial responsiveness that may enhance susceptibility to injury and disease in the brain.
|Secretion of tenascin-C by cultured astrocytes: regulation of cell proliferation and process elongation. |
Takeshi Nishio, Saburo Kawaguchi, Tsutomu Iseda, Takayuki Kawasaki, Takao Hase
Brain research 990 129-40 2003
Tenascin-C (TNC), an extracellular matrix glycoprotein, is involved in tissue morphogenesis like embryogenesis, wound healing or tumorigenesis. Quiescent astroglia in long-term primary cultures are known to show rapid morphological changes after subculture and serum deprivation/re-addition (SSDR). To elucidate roles of TNC in the morphogenetic processes of cultured astrocytes, we have revealed morphological changes in association with soluble TNC contents in the medium and expression of TNC mRNA, TNC, glial fibrillary acidic protein (GFAP) and integrin beta1, one of its cell surface receptors, in glial cells after SSDR. Soluble TNC in the medium rapidly increased in amount at 4 h when GFAP-positive cells expressed TNC mRNA, TNC and integrin beta1. Cellular proliferation and growth occurred in colonies expressing TNC mRNA, TNC and integrin beta1 during the first 24 h. During the next 24 h, process elongation and cell migration occurred in association with increased GFAP expression and re-elevation of soluble TNC in the medium. Cell bodies became flat and larger with increased GFAP and reduced TNC expression at 72 h, while cultures became confluent with reduced GFAP and TNC expression at 96 h after SSDR. Functional blocking with anti-TNC antibody reduced cell proliferation and induced morphological change from a process-bearing slender shape to a flat and wide shape presumably due to increased cell adhesion.These findings strongly support the idea that endogenous TNC produced and released by astrocytes in response to serum stimulation induces their proliferation and process elongation through a paracrine/autocrine mechanism.
|Spinal adenosine agonist reduces c-fos and astrocyte activation in dorsal horn of rats with adjuvant-induced arthritis. |
Linda S Sorkin, Karly Maruyama, David L Boyle, Li Yang, Martin Marsala, Gary S Firestein, Linda S Sorkin, Karly Maruyama, David L Boyle, Li Yang, Martin Marsala, Gary S Firestein
Neuroscience letters 340 119-22 2003
Adjuvant-induced arthritis (AIA) is a useful animal model of rheumatoid arthritis (RA). We have previously shown that stimulation of spinal adenosine A1 receptors decreases peripheral signs of AIA. In this study we show that when the final dose of adenosine A1 agonist is given 1 h prior to sacrifice the number of c-Fos positive cells evoked by the AIA in both the superficial and deep dorsal horns decreases by about 50%. In saline-treated AIA animals the increase in c-Fos expression seen in laminae I-IV was accompanied by an increase in glial fibrillary acidic protein immunoreactivity, indicative of astrocyte activation. Astrocyte activation was only partially attenuated by adenosine A1 receptor agonist treatment.
|The primary phase of infection by pathogenic simian-human immunodeficiency virus results in disruption of the blood-brain barrier. |
Edward B Stephens, Dinesh K Singh, M Eric Kohler, Mollie Jackson, Erik Pacyniak, Nancy E J Berman
AIDS research and human retroviruses 19 837-46 2003
Using the simian-human immunodeficiency virus (SHIV), we have investigated whether the blood-brain barrier (BBB) is compromised during the early stages of infection. Five macaques were inoculated with pathogenic SHIV(50OLNV) for 2 weeks at which time macaques were anesthetized, perfused with saline, and sacrificed. The brains were removed and examined for the disruption of the blood-brain barrier by immunohistochemical staining for the plasma protein fibrinogen in the neural parenchyma. Our results indicate a disruption of the BBB in the five of five macaques inoculated with SHIV(50OLNV) for 2 weeks. Zonula occludens 1 (ZO-1), which is a marker for the tight junctions formed by brain vascular endothelial cells, was largely absent in areas that showed fibrinogen deposition in all five macaques. To determine if the BBB integrity correlated with the initial stages of infection, the brains from two macaques were analyzed that had progressed to end-stage disease following inoculation with pathogenic SHIV(50OLNV) but developed no neuropathology and from two macaques that were inoculated with a gene-deleted, nonpathogenic virus (novpuSHIV(KU-1bMC33)) for over 1 year. Our results indicate that unlike the macaques sacrificed during the acute phase of infection, immunohistochemical staining for fibrinogen in the neural parenchyma was negative and ZO-1 staining was readily detected in the endothelial cells of the blood vessels. The results of this study indicate that the transient loss of BBB integrity is a function of the high level of virus replication that occurs during the acute phase of infection and provides important information on the early stages of lentivirus neuroinvasion.
|Pathogenic and nef-interrupted simian-human immunodeficiency viruses traffic to the macaque CNS and cause astrocytosis early after inoculation. |
Singh, Dinesh K, et al.
Virology, 296: 39-51 (2002) 2002
Several studies have shown that deletion of the nef gene of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) results in attenuated viruses. However, studies have not critically examined trafficking of attenuated viruses to the central nervous system (CNS) at early stages after inoculation. In this study, we investigated the colocalization of pathogenic and vpu-negative, nef-interrupted SHIVs at early stages following inoculation. The first virus, designated SHIV(50OLNV), was isolated from the lymph node of a pig-tailed macaque which developed severe CD4+ T cell loss and neurological disease. The second virus was a molecularly cloned virus in which the vpu gene was deleted and the gene for the enhanced green fluorescent protein from the jellyfish Aequoria victora had been inserted in-frame within the nef gene of the pathogenic SHIV(KU-1bMC33) (designated SHIV(KU-1bEGFP)). Three pig-tailed macaques were inoculated intravenously with equivalent amounts of two viruses, two macaques were inoculated with SHIV(KU-1bEGFP), and two macaques were inoculated with SHIV(50OLNV). The peripheral blood mononuclear cells (PBMCs) were isolated from bleeds obtained 3, 7, 10, and 14 days postinoculation and monitored for syncytia-inducing virus and for fluorescent cells. Virus was detected in the PBMCs as early as 3 days postinoculation and was present throughout the course of this short-term study. At 14 days postinoculation, the macaques were sacrificed and examined for virus in lymphoid tissues and different regions of the CNS following necropsy. Our results revealed the presence of both viruses in lymphoid and CNS tissues, although SHIV(50OLNV) was present to a much greater extent. Histological examination revealed that one macaque displayed signs of meningitis and all three macaques developed massive cortical astrocyte activation as demonstrated by immunostaining for glial fibrillary acidic protein, but only limited microglial activation. In the two macaques inoculated with SHIV(50OLNV), astrocyte activation similar to that in the macaques inoculated with both viruses was observed while no astrocyte activation was observed in macaques inoculated with SHIV(KU-1bEGFP). Thus, this study demonstrates that SHIVs with an intact nef(SHIV(50OLNV)) as well as those lacking a vpu gene and with a nonfunctional nef gene (SHIV(KU-1bEGFP)) are capable of invading the CNS and that pathogenic SHIVs are capable of causing reactive astrocytosis early after inoculation.
|Ink4a-Arf loss cooperates with KRas activation in astrocytes and neural progenitors to generate glioblastomas of various morphologies depending on activated Akt. |
Lene Uhrbom, Chengkai Dai, Joseph C Celestino, Marc K Rosenblum, Gregory N Fuller, Eric C Holland
Cancer research 62 5551-8 2002
Deletion of the INK4a-ARF locus is found in the majority of human malignant gliomas. However, the role of INK4a-ARF loss in gliomagenesis is unclear. Animal modeling has shown that mice with targeted deletions in the Ink4a-Arf gene do not develop spontaneous gliomas. We have previously reported that combined KRas and Akt signaling could induce glioblastoma (GBM) formation from neural progenitor cells but had no effect in differentiated astrocytes. In this investigation, we have studied the effects of Ink4a-Arf loss on the formation of GBM induced by KRas and Akt gene transfer into neural progenitor cells and astrocytes. We show here that Ink4a-Arf deficiency allows for GBM formation from astrocytes and that it enhances tumor incidence in neural progenitor cells. Furthermore, KRas alone can cooperate with deletion of the Ink4a-Arf locus in tumor formation from both neural progenitor cells and astrocytes. The resulting tumors were nestin positive and resembled a spectrum of glioma morphologies ranging in astrocytic character depending on cell-of-origin and presence of activated Akt. Our data strongly supports the view that one role of loss of Ink4a-Arf in gliomagenesis could be to sensitize astrocytes to transformation through dedifferentiation in response to the appropriate oncogenic stimuli.
|Leptin-induced nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei involved in body weight regulation. |
T Hübschle, E Thom, A Watson, J Roth, S Klaus, W Meyerhof, T Hübschle, E Thom, A Watson, J Roth, S Klaus, W Meyerhof
The Journal of neuroscience : the official journal of the Society for Neuroscience 21 2413-24 2001
Leptin is involved in the hypothalamic control of food intake and body weight. Fos immunohistochemistry has been used to functionally map leptin target neurons involved in these regulatory processes. However, only a subset of hypothalamic neurons expressing the long form of the leptin receptor (Ob-Rb) also coexpress the neuronal activation marker Fos after leptin stimulation. To functionally map all leptin target neurons, regardless of whether leptin-mediated neuronal activation or inhibition occurs, we immunohistochemically investigated the leptin-induced nuclear translocation of the signal transducer and activator of transcription molecule STAT3, which represents a crucial step in the regulation of leptin-dependent gene expression. As proven by colocalization studies with the nuclear 4',6-diamidino-2-phenylindole dilactate stain, intracerebroventricular leptin treatment, but not intracerebroventricular application of pyrogen-free saline, induced a time-dependent nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei, with strong nuclear STAT3 signals detectable in the arcuate nucleus, the lateral hypothalamus, and the ventromedial and dorsomedial hypothalamic nuclei. This leptin-induced STAT3 translocation pattern proved to be distinct from that induced by interleukin-6, another cytokine using STAT3 in its signaling pathway. Combined immunohistochemical STAT3 and Fos detection after leptin treatment revealed a higher number of STAT3-positive than Fos-positive cell nuclei in the aforementioned hypothalamic structures and showed that Fos immunoreactivity colocalized only in a subset of all leptin-responsive STAT3 nuclei. These results suggest that the detection of nuclear STAT3 immunoreactivity represents a new neuroanatomical tool to functionally map central leptin actions. They further support the importance of ventrally located caudal hypothalamic structures representing the main leptin targets involved in body weight regulation.
|Long-term intrathecal catheterization in the rat. |
L Jasmin, P T Ohara
Journal of neuroscience methods 110 81-9 2001
We report an intrathecal (i.t.) catheter system that permits repeated administration of volumes of 10 microl or more in the awake rat over many months. A small skin incision is made and a 32 ga polyurethane catheter is inserted in the sacral subarachnoid space using a modified 22 ga needle. The other end of the catheter is tunneled subcutaneously to the flank and exteriorized through a titanium port. The device is well tolerated, does not cause sensory or motor deficits, and does not interfere with behavioral testing. Rats equipped with this device can be housed with other rats. Over the 9 month observation period the function of the catheter was verified by repeated injection of 15 microl of 2% lidocaine that caused temporary paraplegia. Out of 12 implanted rats, the number of fully functional catheters was 10 at 3 months, seven at 6 months, and six at 9 months. At 3 months, i.t. injection of anti-dopamine-beta-hydroxylase antibodies conjugated to saporin (DBH-SAP, 5 microg/10 microl) resulted in noradrenergic denervation of the spinal cord in all rats (n=10). We propose that intrathecal catheterization is well suited for long term behavioral and pharmacological studies.
|Expression of glial filament protein (GFP) in nerve sheaths and non-neural cells re-examined using monoclonal antibodies, with special emphasis on the co-expression of GFP and cytokeratins in epithelial cells of human salivary gland and pleomorphic adenomas. |
Achstätter, T, et al.
Differentiation, 31: 206-27 (1986) 1986
We describe two novel monoclonal antibodies specific for glial filament protein (GFP), i.e., GF12.23 and GF12.24 (both IgG2a]. These cross-react over a broad range of species with epitopes located in the alpha-helical rod domain typical of all intermediate filament (IF) proteins. These monoclonal antibodies were used, in conjunction with other monoclonal GFP antibodies, rabbit antiserum to GFP, and various antibodies to other cytoskeletal proteins, to examine the occurrence of GFP in cells outside of the central nervous system of rodents, cows, and humans. We detected some scattered GFP-containing cells in the neural sheaths in some species but not in others, and we obtained different results when comparing the rabbit antisera with the monoclonal GFP antibodies. In the enteric glia of rats, we observed GFP-positive cells with all of the antibodies used, whereas in human intestine, the various monoclonal antibodies showed no reaction with any intestinal cells. Similarly, no GFP was detected in surface cells of the lens of cows and rats using any of the GFP antibodies, whereas some reaction was seen in murine lens tissue. We were also unable to detect GFP-positive cells in human, bovine, or rat liver with any of the monoclonal antibodies, which is in contrast to the reactivity of the rabbit GFP antisera with some stellate perisinusoidal cells of rat but not bovine or human liver. The possible reasons for the discrepancies between the different species and the different antibody preparations used are discussed. In addition, using double-label immunofluorescence microscopy, we showed that normal human parotid glands contain a certain type of epithelial cell that co-expresses cytokeratins and desmosomal proteins with GFP. The histological distribution of these GFP-positive cells suggests that they represent a subset of the myoepithelial cells present in this tissue. Cells co-expressing cytokeratins and GFP - in some cases, apparently together with vimentin as the third IF protein present - were also identified in tumors derived from this salivary-gland epithelium, i.e., pleomorphic adenomas, in which GFP-positive cells were relatively frequent in the myxoid and chondroid components, thus confirming the work of other investigators. Possible implications for the concept of histogenesis of these tumor cells are discussed, as are possible mechanisms resulting in the co-expression of IF proteins.
|Molecular interactions in intermediate-sized filaments revealed by chemical cross-linking. Heteropolymers of vimentin and glial filament protein in cultured human glioma cells. |
Quinlan, R A and Franke, W W
Eur. J. Biochem., 132: 477-84 (1983) 1983
Certain glia cells, notably astrocytes and tumor cells derived therefrom, express simultaneously two types of proteins of intermediate-sized filaments, vimentin and glia filament protein (GFP). We have used an established human glioma (astrocytoma) cell culture line (U 333 CG/343 MG) in which both proteins are seen in partly overlapping fibrillar structures by immunofluorescence microscopy, to examine the possible existence of heteropolymer filaments of these two proteins by using reversible oxidative cross-linking facilitated by the 1,10-phenanthroline-cupric ion complex. Dimeric cross-link products are characterized by one-dimensional and two-dimensional gel electrophoresis under non-reducing and reducing conditions as well as by peptide mapping. The relatively large proportions of heterodimers of vimentin and GFP obtained in cytoskeletal filaments cross-linked in this way, demonstrate the frequency of heteropolymer filaments in this cell as well as the frequency of face-to-face 'pairs' of GFP and vimentin in such filaments. Together with our related observations on heteropolymer filaments between vimentin and desmin in some smooth muscle cells [Quinlan, R. A. and Franke, W. W. (1982) Proc. Natl Acad. Sci. USA, 79, 3452-3456], we discuss this as evidence for common principles of molecular arrangements of vimentin, GFP and desmin, at least in the cysteine-containing surface domains. The results are also discussed in relation to cytoskeletal changes during glial differentiation.
|Anti-Glial Fibrillary Acidic Protein, clone GA5 - Data Sheet|