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Objective: Recent evidence supports a link between increased TDP-43 burden and the presence of an APOE4 gene allele in Alzheimer's disease (AD); however, it is difficult to conclude the direct effect of APOE on TDP-43 pathology due to the presence of mixed AD pathologies. The goal of this study is to address how APOE isoforms impact TDP-43 pathology and related neurodegeneration in the absence of typical AD pathologies., Methods: We overexpressed human TDP-43 via viral transduction in humanized APOE2, APOE3, APOE4 mice, and murine Apoe-knockout (Apoe-KO) mice. Behavior tests were performed across ages. Animals were harvested at 11 months of age and TDP-43 overexpression-related neurodegeneration and gliosis were assessed. To further address the human relevance, we analyzed the association of APOE with TDP-43 pathology in 160 postmortem brains from autopsy-confirmed amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND) in the Mayo Clinic Brain Bank., Results: We found that TDP-43 overexpression induced motor function deficits, neuronal loss, and gliosis in the motor cortex, especially in APOE2 mice, with much milder or absent effects in APOE3, APOE4, or Apoe-KO mice. In the motor cortex of the ALS and FTLD-MND postmortem human brains, we found that the APOE2 allele was associated with more severe TDP-43-positive dystrophic neurites., Interpretation: Our data suggest a genotype-specific effect of APOE on TDP-43 proteinopathy and neurodegeneration in the absence of AD pathology, with the strongest association seen with APOE2. ANN NEUROL 2023;93:830-843., (© 2022 American Neurological Association.)

Background and Objective: Mitochondrial dysfunction has been implicated in several neurodegenerative diseases. Creatine administration increases concentration of the energy buffer phosphocreatine, exerting protective effects in the brain. We evaluate whether a creatine-enriched diet would be beneficial for a mouse model of spinocerebellar ataxia type 3, a genetically defined neurodegenerative disease for which no treatment is available., Methods: We performed 2 independent preclinical trials using the CMVMJD135 mouse model (treating 2 groups of animals with different disease severity) and wild-type mice, to which 2% creatine was provided for 19 (preclinical trial 1) or 29 (preclinical trial 2) weeks, starting at a presymptomatic age. Motor behavior was evaluated at several time points from 5 to 34 weeks of age, and neuropathological studies were performed at the end of each trial., Results: Creatine supplementation led to an overall improvement in the motor phenotype of CMVMJD135 mice in both trials, rescuing motor balance and coordination and also restored brain weight, mitigated astrogliosis, and preserved Calbindin-positive cells in the cerebellum. Moreover, a reduction of mutant ataxin-3 aggregates occurred despite maintained steady-state levels of the protein and the absence of autophagy activation. Creatine treatment also restored the expression of the mitochondrial mass marker Porin and reduced the expression of antioxidant enzymes Heme oxygenase 1 (HO1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), suggesting a beneficial effect at the level of mitochondria and oxidative stress., Conclusions: Creatine slows disease progression and improves motor dysfunction as well as ameliorates neuropathology of the CMVMJD135 animals, supporting this as a useful strategy to slow the progression of spinocerebellar ataxia type 3. © 2018 International Parkinson and Movement Disorder Society., (© 2018 International Parkinson and Movement Disorder Society.)

Defects in repair of damaged brain accumulate injury and contribute to slow-developing neurodegeneration. Here, we report that a deficiency of DJ-1, a Parkinson's disease (PD) gene, delays repair of brain injury due to destabilization of Sox9, a positive regulator of astrogliosis. Stereotaxic injection of ATP into the brain striatum produces similar size of acute injury in wild-type and DJ-1-knockout (KO) mice. However, recovery of the injury is delayed in KO mice, which is confirmed by 9.4T magnetic resonance imaging and tyrosine hydroxylase immunostaining. DJ-1 regulates neurite outgrowth from damaged neurons in a non-cell autonomous manner. In DJ-1 KO brains and astrocytes, Sox9 protein levels are decreased due to enhanced ubiquitination, resulting in defects in astrogliosis and glial cell-derived neurotrophic factor/ brain-derived neurotrophic factor expression in injured brain and astrocytes. These results indicate that DJ-1 deficiency causes defects in astrocyte-mediated repair of brain damage, which may contribute to the development of PD., (© 2017 Wiley Periodicals, Inc.)

Stimulation of Na + /H + exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1 flox/flox (Nhe1 f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1 f/f mice with Gfap-Cre ERT2 Cre-recombinase mice. Gfap-Cre ERT2+/- ;Nhe1 f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-Cre ERT2+/- ;Nhe1 f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage., (© 2017 Wiley Periodicals, Inc.)

Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long-term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca 2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone-induced demyelination. This toxin-induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen-activated protein kinase pathways, as well as transcription factor c-Jun and a proapoptotic Bcl-2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca 2+ concentration modulate astrocyte functions, and influence the pro or anti-apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166-2180., (© 2016 Wiley Periodicals, Inc.)

Astrocyte activation is a hallmark of central nervous system injuries resulting in glial scar formation (astrogliosis). The activation of astrocytes involves metabolic and morphological changes with complex underlying mechanisms, which should be defined to provide targets for astrogliosis intervention. Astrogliosis is usually accompanied by an upregulation of glial fibrillary acidic protein (GFAP). Using an in vitro scratch injury model, we scratched primary cultures of cerebral cortical astrocytes and observed an influx of calcium in the form of waves spreading away from the wound through gap junctions. Using the calcium blocker BAPTA-AM and the JNK inhibitor SP600125, we demonstrated that the calcium wave triggered the activation of JNK, which then phosphorylated the transcription factor c-Jun to facilitate the binding of AP-1 to the GFAP gene promoter to switch on GFAP upregulation. Blocking calcium mobilization with BAPTA-AM in an in vivo stab wound model reduced GFAP expression and glial scar formation, showing that the calcium signal, and the subsequent regulation of downstream signaling molecules, plays an essential role in brain injury response. Our findings demonstrated that traumatic scratch injury to astrocytes triggered a calcium influx from the extracellular compartment and activated the JNK/c-Jun/AP-1 pathway to switch on GFAP expression, identifying a previously unreported signaling cascade that is important in astrogliosis and the physiological response following brain injury., (Copyright © 2013 Wiley Periodicals, Inc.)

Müller glia are the resident radial glia in the vertebrate retina. The response of mammalian Müller glia to retinal damage often results in a glial scar and no functional replacement of lost neurons. Adult zebrafish Müller glia, in contrast, are considered tissue-specific stem cells that can self-renew and generate neurogenic progenitors to regenerate all retinal neurons after damage. Here, we demonstrate that regulation of TGFβ signaling by the corepressors Tgif1 and Six3b is critical for the proliferative response to photoreceptor destruction in the adult zebrafish retina. When function of these corepressors is disrupted, Müller glia and their progeny proliferate less, leading to a significant reduction in photoreceptor regeneration. Tgif1 expression and regulation of TGFβ signaling are implicated in the function of several types of stem cells, but this is the first demonstration that this regulatory network is necessary for regeneration of neurons., (Copyright © 2013 Wiley Periodicals, Inc.)

A large body of literature suggests that amyotrophic lateral sclerosis (ALS) pathology is intimately linked with neuroinflammation, specifically activation and recruitment of microglia and astrocytes. The actual cause of gliosis is unclear. Extracellular Cu/Zn superoxide dismutase (SOD1) has recently been shown to activate microglia in a CD14 dependant mechanism providing one potential pathway by which glial cells become activated. As protein inclusions are thought to be an important part of ALS pathology and are associated with all forms of ALS, we sought to determine if aggregated SOD1 would activate microglia. Recombinant SOD1 was aggregated and this, or monomeric forms of SOD1 were then added to EOC.13 microglial cells or primary microglial cells in culture. Although monomeric mutant SOD1 has been shown to promote microglial activation in the past, we found that aggregated SOD1 was able to much more efficiently activate microglia in culture when compared with the unaggregated form of mutant SOD1. In addition to CD14 dependant pathways, aggregated SOD1 also bound to the surface of glial cells and was internalized in a lipid raft and scavenger receptor dependent manner. We have for the first time shown that aggregated mutant SOD1 potently activates microglia. These results suggest that there may be a potential link between protein aggregation and microglial activation in ALS., (Copyright © 2012 Wiley Periodicals, Inc.)

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively., (Copyright © 2012 Wiley Periodicals, Inc.)

Calmodulin-regulated spectrin-associated protein 1 (Camsap1) has been recognized as a new marker for astrocytic lineage cells and is expressed on mature astrocytes in the adult brain (Yamamoto et al. [2009] J. Neurosci. Res. 87:503–513). In the present study, we found that newly born Camsap1-expressing cells exhibited regional heterogeneity in an early phase after stab injury of the mouse brain. In the surrounding area of the lesion site, Camsap1 was expressed on quiescent astrocytes. At 3 days after injury, Camsap1 immunoreactivity was upregulated on glial fibrillary acidic protein-immunoreactive (GFAP-ir) astrocytes. Some of these astrocytes incorporated bromodeoxyuridine (BrdU) together with re-expression of the embryonic cytoskeleton protein nestin. In the neighboring region of the lesion cavity, Camsap1 was expressed on GFAP-negative cells. At 3 days after injury, GFAP-ir astrocytes were absent around the lesion cavity. At this stage, NG2-ir cells immunopositive for Camsap1 and immunonegative for GFAP were distributed in border of the lesion cavity. By 10 days, Camsap1 immunoreactivity was exclusively detected on GFAP-ir reactive astrocytes devoid of NG2 immunoreactivity. BrdU pulse-chase labeling assay suggested the differentiation of Camsap1+/NG2+ cells into Camsap1+/GFAP+ astrocytes. In the subependymal zone of the lateral ventricle, Camsap1-ir cells increased after injury. Camsap1 immunoreactivity was distributed on ependymal and subependymal cells bearing various astrocyte markers, and BrdU incorporation was enhanced on such Camsap1-ir cells after injury. These results suggest that newly born reactive astrocytes are derived from heterogeneous Camsap1-expressing cells in the injured brain.

The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/-) and wild-type mice received thoracic crush SCI. Compared with wild-type controls, GLT1+/- mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/- mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared with wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment., (Copyright © 2011 Wiley‐Liss, Inc.)

Astrocytes are fundamental for brain homeostasis and are at the fulcrum of neurological diseases including Alzheimer's disease (AD). Here, we monitored changes in astroglia morphology throughout the age-dependent progression of AD. We used an immunohistochemical approach that allows us to determine the domain of glial cytoskeleton, by measuring the surface, volume, and the relationship between astrocytes and neuritic plaques. We investigated astroglia in the hippocampus of a triple transgenic mouse model of AD (3xTg-AD) that mimics the progression of the human disease. The numerical density of astrocytes is affected neither by AD nor by age. We found reduction of surface and volume of GFAP profiles from early ages (6 months; 43.84 and 52.76%, respectively), persisting at 12 (40.73 and 45.39%) and 18 months (64.80 and 71.95%) in the dentate gyrus (DG) of 3xTg-AD, whereas in CA1 it appears at 18 months (29.42 and 32.74%). This cytoskeleton atrophy is accompanied by a significant reduction of glial somata volume in DG at 12 and 18 months (40.46 and 75.55%, respectively), whereas in CA1 it is significant at 18 months (42.81%). However, while astroglial atrophy appears as a generalized process, astrocytes surrounding plaques are clearly hypertrophic as revealed by increased surface (48.06%; 66.66%), and volume (57.10%; 71.06%) of GFAP profiles in DG and CA1, respectively, at 18 months. We suggest differential effects of AD on astroglial populations depending on their association with plaques accounting for the progressive disruption of neural networks connectivity and neurotransmitters imbalance which underlie mnesic and cognitive impairments observed in AD.

Upregulation of expression of the close homolog of adhesion molecule L1 (CHL1) by reactive astrocytes in the glial scar reduces axonal regeneration and inhibits functional recovery after spinal cord injury (SCI). Here, we investigate the molecular mechanisms underlying upregulation of CHL1 expression by analyzing the signal transduction pathways in vitro. We show that astrogliosis stimulated by bacterial lipopolysaccharide (LPS) upregulates CHL1 expression in primary cultures of mouse cerebral astrocytes, coinciding with elevated protein synthesis and translocation of protein kinase delta (PKCdelta) from cytosol to the membrane fraction. Blocking PKCdelta activity pharmacologically and genetically attenuates LPS-induced elevation of CHL1 protein expression through a phosphatidylinositol 3-kinase (PI3K) dependent pathway. LPS induces extracellular signal-regulated kinases (ERK1/2) phosphorylation through PKCdelta and blockade of ERK1/2 activation abolishes upregulation of CHL1 expression. LPS-triggered upregulation of CHL1 expression mediated through translocation of nuclear factor kappaB (NF-kappaB) to the nucleus is blocked by a specific NF-kappaB inhibitor and by inhibition of PI3K, PKCdelta, and ERK1/2 activities, implicating NF-kappaB as a downstream target for upregulation of CHL1 expression. Furthermore, the LPS-mediated upregulation of CHL1 expression by reactive astrocytes is inhibitory for hippocampal neurite outgrowth in cocultures. Although the LPS-triggered NO-guanylate cyclase-cGMP pathway upregulates glial fibrillary acid protein expression in cultured astrocytes, we did not observe this pathway to mediate LPS-induced upregulation of CHL1 expression. Our results indicate that elevated CHL1 expression by reactive astrocytes requires activation of PI3K/PKCdelta-dependent pathways and suggest that reduction of PI3K/PKCdelta activity represents a therapeutic target to downregulate CHL1 expression and thus benefit axonal regeneration after SCI., ((c) 2009 Wiley-Liss, Inc.)

Alzheimer's disease (AD) is the most common progressive dementia and is pathologically characterized by brain deposition of amyloid-beta (Abeta) peptide as senile plaques. Inflammatory and immune response pathways are chronically activated in AD patient brains at low levels, and likely play a role in disease progression. Like microglia, activated astrocytes produce numerous acute-phase reactants and proinflammatory molecules in the AD brain. One such molecule, S100B, is highly expressed by reactive astrocytes in close vicinity of beta-amyloid deposits. We have previously shown that augmented and prolonged activation of astrocytes has a detrimental impact on neuronal survival. Furthermore, we have implicated astrocyte-derived S100B as a candidate molecule responsible for this deleterious effect. To evaluate a putative relationship between S100B and AD pathogenesis, we crossed transgenic mice overexpressing human S100B (TghuS100B mice) with the Tg2576 mouse model of AD, and examined AD-like pathology. Brain parenchymal and cerebral vascular beta-amyloid deposits and Abeta levels were increased in bigenic Tg2576-huS100B mice. These effects were associated with increased cleavage of the beta-C-terminal fragment of amyloid precursor protein (APP), elevation of the N-terminal APP cleavage product (soluble APPbeta), and activation of beta-site APP cleaving enzyme 1. In addition, double transgenic mice showed augmented reactive astrocytosis and microgliosis, high levels of S100 expression, and increased levels of proinflammatory cytokines as early as 7-9 months of age. These results provide evidence that (over)-expression of S100B acts to accelerate AD-like pathology, and suggest that inhibiting astrocytic activation by blocking S100B biosynthesis may be a promising therapeutic strategy to delay AD progression.., ((c) 2009 Wiley-Liss, Inc.)

Objective: MicroRNAs (miRNAs) regulate gene expression and have many roles in the brain, but a role in oligodendrocyte (OL) function has not been demonstrated., Methods: A Dicer floxed conditional allele was crossed with the proteolipid protein promoter-driven inducible Cre allele to generate inducible, OL-specific Dicer-floxed mice., Results: OL-specific Dicer mutants show demyelination, oxidative damage, inflammatory astrocytosis and microgliosis in the brain, and eventually neuronal degeneration and shorter lifespan. miR-219 and its target ELOVL7 (elongation of very long chain fatty acids protein 7) were identified as the main molecular components that are involved in the development of the phenotype in these mice. Overexpressing ELOVL7 results in lipid accumulation, which is suppressed by miR-219 co-overexpression. In Dicer mutant brain, excess lipids accumulate in myelin-rich brain regions, and the peroxisomal beta-oxidation activity is dramatically reduced., Interpretation: Postnatal Dicer ablation in mature OLs results in inflammatory neuronal degeneration through increased demyelination, lipid accumulation, and peroxisomal and oxidative damage, and therefore indicates that miRNAs play an essential role in the maintenance of lipids and redox homeostasis in mature OLs that are necessary for supporting axonal integrity as well as the formation of compact myelin.

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of beta1-integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up-regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin-C and the DSD-1 proteoglycan. In addition, we also observed to a certain degree a non-cell autonomous activation of microglial cells after conditional beta1-integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of beta1-integrin-mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron-specific deletion of beta1-integrin, we conclude that beta1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in beta1-integrin-mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury., ((c) 2009 Wiley-Liss, Inc.)

Objective: Mutations in the X-linked adrenoleukodystrophy (X-ALD) protein cause accumulation of unbranched saturated very-long-chain fatty acids, particularly in brain and adrenal cortex. In humans, the genetic defect causes progressive inflammatory demyelination in the brain, where very-long-chain fatty acids accumulate within phospholipid fractions such as lysophosphatidylcholine., Methods: To address mechanisms of inflammation, we studied microglial activation in human ALD (10 autopsies) and lysophosphatidylcholine (C24:0) injection into the parietal cortex of mice., Results: Unexpectedly, we found a zone lacking microglia within perilesional white matter, immediately beyond the actively demyelinating lesion edge. Surrounding this zone we observed clusters of activated and apoptotic microglia within subcortical white matter. Lysophosphatidylcholine (C24:0) injection in mice led to widespread microglial activation and apoptosis., Interpretation: Our data suggest that the distinct mononuclear phagocytic cell response seen in cerebral X-ALD results, at least in part, from aberrant signaling to cognate receptors on microglia. Our findings support a hypothesis that microglial apoptosis in perilesional white matter represents an early stage in lesion evolution and may be an appropriate target for intervention in X-ALD patients with evidence of cerebral demyelination.

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination-remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS-specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte-specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPbeta in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPbeta expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPbeta in untreated mixed glial cultures. C/EBPbeta is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS-induced upregulation of glial C/EBPbeta is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPbeta nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS-induced increase in microglial C/EBPbeta is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPbeta as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPbeta expression in activated glial cells in vivo. These findings support a role for C/EBPbeta in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPbeta-regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation. (c) 2006 Wiley-Liss, Inc.

Inducible gene modification using the Cre/loxP system provides a valuable tool for the analysis of gene function in the active animal. GFAP-Cre transgenic mice have been developed to achieve gene recombination in astrocytes, the most abundant cells of the central nervous system, with pivotal roles during brain function and pathology. Unfortunately, these mice displayed neuronal recombination as well, since the GFAP promoter is also active in embryonic radial glia, which possess a substantial neurogenic potential. To enable the temporal control of gene deletions in astrocytes only, we generated a transgenic mouse with expression of CreERT2, a fusion protein of the DNA recombinase Cre and a mutated ligand-binding domain of the estrogen receptor, under the control of the human GFAP promoter. In offspring originating from crossbreedings of GFAP-CreERT2-transgenic mice with various Cre-sensitive reporter mice, consecutive intraperitoneal injections of tamoxifen induced genomic recombination selectively in astrocytes of almost all brain regions. In Bergmann glia, which represent the main astroglial cell population of the cerebellum, virtually all cells showed successful gene recombination. When adult mice received cortical stab wound lesions, simultaneously given tamoxifen induced substantial recombination in reactive glia adjacent to the site of injury. These transgenic GFAP-CreERT2 mice will allow the functional analysis of loxP-modified genes in astroglia of the postnatal and adult brain., (Copyright 2005 Wiley-Liss, Inc.)

To identify proteins implicated in peripheral nerve regeneration, we performed two-dimensional polyacrylamide gel electrophoresis and subsequent mass spectrometry analysis using nerve-injured hypoglossal nuclei of rat. We have identified annexin III (ANX III/ANX A3) as an induced protein after rat hypoglossal nerve injury. ANX III is known as a Ca2+-dependent phospholipid-binding protein, but its physiological function is mostly unknown. By in situ hybridization and immunohistochemistry, we demonstrated that ANX III expression was induced specifically in activated (axotomy-stimulated) microglia after nerve injury. ANX III was the most prominent ANX expressed in microglia of the major ANX family members (ANX I-VI). Hybridization signals for other ANX mRNAs (II, IV, V, and VI) were mainly observed in neuronal cells, and no significant hybridization signal for ANX I mRNA was detected in hypoglossal nuclei. In cultured microglia, ATP treatment induced ANX III translocation to the ruffling membrane where F-actin was accumulated. Further in vitro studies revealed that ANX III was not secreted and had F-actin binding activity in a Ca2+-dependent manner. These results suggest that ANX III may be a Ca2+-dependent mediator between phospholipids and F-actin in microglia stimulated by peripheral nerve injury., (Copyright 2006 Wiley-Liss, Inc.)

Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low-molecular-weight NF subunit [NFL (-/-)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild-type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein-70 (HSP-70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (-/-) mice demonstrated significant astrogliosis and HSP-70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non-neuronal cells may differ significantly depending on the etiology of the NF aggregate itself., ((c) 2005 Wiley-Liss, Inc.)

Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro-inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll-like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron-glia cultures with antibodies against F4/80 revealed that while TLR4(+/+) microglia were activated the low concentration of 1 ng/ml of LPS, TLR4(-/-) microglia exhibit activated morphology in response to LPS only at higher concentrations (100-1,000 ng/ml). Additionally, tumor necrosis factor-alpha (TNF-alpha) was only produced from higher concentrations (100-1,000 ng/ml) of LPS in TLR4(-/-) enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF-alpha production from TLR4(-/-) microglia. The influence of TLR4 on LPS-induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4(-/-) and TLR4(+/+) microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1-1,000 ng/ml). These data indicate that LPS-induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-alpha response of both the TLR4-dependent and independent pathway., ((c) 2005 Wiley-Liss, Inc.)

S-100B is an astrocyte-derived protein that is increased in focal areas of the brain most severely affected by neuropathological changes in Alzheimer's disease (AD). Cell-based and clinical studies have implicated S-100B in progression of a pathologic, glial-mediated pro-inflammatory state in the CNS. However, the relationship between S-100B levels and susceptibility to AD-relevant neuroinflammation and neuronal dysfunction in vivo has not been determined. To test the hypothesis that overexpression of S-100B increases vulnerability to beta-amyloid (Abeta)-induced damage, we used S-100B-overexpressing transgenic (Tg) and S-100B knockout (KO) mice in a mouse model that involves intracerebroventricular infusion of human oligomeric Abeta1-42. This model mimics many features of AD, including robust neuroinflammation, Abeta plaques, synaptic damage and neuronal loss in the hippocampus. S-100B Tg, KO, and wild-type (WT) mice were infused with Abeta for 28 days, sacrificed at 60 days, and hippocampal endpoints analyzed. We found that Tg mice showed increased vulnerability to Abeta-induced neuropathology relative to either WT or KO mice. Specifically, Tg mice exhibited enhanced glial activation and neuroinflammation, increased nitrotyrosine staining (a marker of glial-induced neuronal damage), and more pronounced loss of synaptic markers. Interestingly, Tg mice showed no significant differences in Abeta plaque burden compared with WT or KO mice, suggesting that, as in the human situation, the severity of neuronal dysfunction did not correlate with amyloid deposition. Our data are consistent with a model in which S-100B overexpression in AD enhances glial activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.

Axoglial interactions underlie the clustering of ion channels and of cell adhesion molecules, regulate gene expression, and control cell survival. We report that Cnp1-null mice, lacking expression of the myelin protein cyclic nucleotide phosphodiesterase (CNP), have disrupted axoglial interactions in the central nervous system (CNS). Nodal sodium channels (Nav) and paranodal adhesion proteins (Caspr) are initially clustered normally, but become progressively disorganized with age. These changes are characterized by mislocalized Caspr immunostaining, combined with a decrease of clustered Na+ channels, and occur before axonal degeneration and microglial invasion, both prominent in older Cnp1-null mice. We suggest that CNP is a glial protein required for maintaining the integrity of paranodes and that disrupted axoglial signaling at this site underlies progressive axonal degeneration, observed later in the CNS of Cnp1-null mice., (2005 Wiley-Liss, Inc.)

We have studied possible differences in glial activation between cells from wild-type and p21Cip1-/- mice. We compared the effect of serum mitogenic stimulation on proliferation rate and on the total number of glial cells after 7 days of culture. No differences between wild-type and p21Cip1-/- glial cells were observed. We also compared the effect of lipopolysaccharide (LPS) from Escherichia coli, an agent widely used to induce glial activation. Nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) release, and nuclear factor kappa-B (NF-kappaB) activation were evaluated as indicators of glial activation. We observed an attenuation of NO release and NF-kappaB activation in p21Cip1-/- glial cells when compared with glial cells from wild-type mice. In contrast, TNF-alpha release was enhanced in p21Cip1-/- glia. These results suggest that the cell cycle inhibitor p21Cip1 plays a role in the inflammatory response induced by LPS., (copyright (c) 2004 Wiley-Liss, Inc.)

Tumor necrosis factoralpha (TNFalpha) and lymphotoxin-alpha (Ltalpha) are upregulated in and around multiple sclerosis plaques and are proposed to play a role during chronic inflammation in demyelinating disease. Despite the perceived detrimental role of these cytokines, human clinical trials inhibiting TNFalpha signaling has led to worsening of symptoms in multiple sclerosis (MS) patients. Our laboratory has verified a role for TNFalpha in the exacerbation of demyelination but, more importantly, has demonstrated a novel role for TNFalpha in reparative remyelination in a cuprizone-induced demyelination model. This may explain the worsening of symptoms experienced by MS patients. In view of the cross-talk in TNF family signaling, the aim of this study is to understand the role of Ltalpha in demyelination and remyelination in hopes of improving therapeutic strategies for MS. Using the same model, we show that mice lacking Ltalpha exhibit a delay in demyelination that is greater than that exhibited by TNFalpha null mice. In this model, Ltalpha is expressed primarily by astroglia. The delay in demyelination is accompanied by a delay in the loss of mature GSTpi-positive oligodendrocytes in Ltalpha-/- mice compared with wild-type mice. Ltalpha-/- mice have decreased numbers of microglia at the site of insult during demyelination, although the number of astrocytes present is similar between strains. In contrast to TNFalpha the lack of Ltalpha did not alter the time course of remyelination, or the number of mature oligodendrocytes during the remyelination phase. Since Ltalpha is detrimental in inflammation and demyelination, but not necessary for remyelination and repair, inhibiting Ltalpha signaling may represent a promising strategy to treat MS., (copyright (c) 2004 Wiley-Liss, Inc.)

Amyotrophic lateral sclerosis is a fatal, adult-onset motor neuron disease. A subset of cases is caused by mutations of superoxide dismutase 1 (SOD1) gene. The mechanisms how the mutations in this ubiquitous enzyme mediate the highly selective motor neuron degeneration, however, remain poorly understood. Recent results from transgenic animal models suggest a "non-cell autonomous" mechanism; i.e., cells other than neurons play an active role in motor neuron death. To investigate a possible effect of mtSOD1 on microglial cells, we compared primary cultured microglia from mtSOD1-transgenic mice and nontransgenic litter controls at neonatal (3 days) and adult (60 days) age. We found that mtSOD1 expression increases the production of TNF-alpha and attenuates IL-6-release by LPS-activated adult microglia. Neonatal microglia, however, showed no differences. Our findings suggest an increased cytotoxic potential of adult mtSOD1 microglia, which only becomes apparent after microglial activation.

IL-1alpha and IL-1beta are induced immediately after insults to the brain, and signaling through the type 1 IL-1 receptor is essential for a normal microglial and astroglial response to injury. To better understand which genes are induced in astrocytes by IL-1beta, we used the unbiased technique of differential display to analyze mouse astroglial gene expression after IL-1beta treatment. Two novel genes were induced, as well as the gene for ceruloplasmin, a ferroxidase with antioxidant properties. Ceruloplasmin was analyzed further by Northern and Western blot. RNA and protein levels of ceruloplasmin were increased when astrocytes were treated with IL-1beta. To determine whether the IL-1 type 1 receptor (IL-1R1) is essential for the injury-induced expression of ceruloplasmin, a Western blot analysis was performed after a traumatic brain injury on mice that were IL-1R1-deficient. Ceruloplasmin increased significantly above controls after injury; however, injury-induced levels of ceruloplasmin were lower in IL-1R1-deficient (2.7-fold increase) than in the wild-type animals (3.5-fold increase). These data indicate that while IL-1R1 deletion has a slight effect on ceruloplasmin expression, it is not essential for either the basal or the induced expression of ceruloplasmin in vivo. Since ceruloplasmin buffers free copper, oxidizes ferrous iron, and catalyzes the dismutation of free radicals, increased levels of ceruloplasmin likely protect neurons and glia from sustaining damage after injury. Furthermore, as the IL-1R1 has been proposed to be a target for achieving neuroprotection after injury, these data suggest that the protection afforded by ceruloplasmin will be retained even when the IL-1R1 is antagonized., (Copyright 2003 Wiley-Liss, Inc.)

Several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, are associated with immunocompetent microglia, leading to the suggestion that chronic glial-mediated inflammation contributes to the neurodegeneration seen in these diseases. Little direct evidence supports this hypothesis, and no suitable rodent models exist that do not involve the use of blunt trauma or ischaemia, events that are infrequently encountered in the human disease state. In the present study, we report that administration of double-stranded RNA, a classical inducer of interferon-gamma (IFN-gamma), causes rapid and persistent activation of microglia and astrocytes, as well as induction of interleukin-1beta (IL-beta) and nitric oxide synthase. In close temporal succession to glial activation, there is neurodegeneration, with neuron loss involving apoptosis in selected brain regions including the septal nucleus, hippocampus, cortex and thalamus, along with hippocampal atrophy. This neuronal loss is accompanied by punctate deposits of material that are immunoreactive for amyloid precursor protein, beta-amyloid peptide (Abeta), and apolipoprotein E. The findings may have clinical relevance, since the administration of the nonsteroidal antiinflammatory agent (NSAID) ibuprofen markedly reduces the neurodegeneration observed in the absence of significant glial inhibition. These findings may be relevant to the pathogenesis of Alzheimer's disease in particular, and to other neurodegenerative diseases involving inflammation., (Copyright 2003 Wiley-Liss, Inc.)

The adult mammalian central nervous system (CNS) contains a large number of different cell types, which arise from the ventricular (VZ) and subventricular zones during embryonic development. In this study, we used a transgenic mouse expressing Lmo1-LacZ from a randomly inserted promoter/reporter gene construct to identify a glial subpopulation. LMO1 is an LIM domain-containing protein, thought to act in protein-protein interactions. We found first that in the adult transgenic CNS, beta-galactosidase (beta-gal) was expressed in a specific subpopulation of protoplasmic-like cells, which did not express detectable levels of glial fibrilary acidic protein unless a lesion was produced. Secondly, during development, beta-gal(+) cells were found arising from discrete regions of the VZ. Taken together, these results identify a subpopulation of protoplasmic glial cells in the adult CNS and suggest that they arise from a restricted VZ region during CNS development., (Copyright 2003 Wiley-Liss, Inc.)

Reactive astrocytes are one of the main impediments for axonal regeneration in the central nervous system of mammals. Using mice KO for GFAP and vimentin, we show that reinnervation occurs after an hemisection of the spinal cord, mainly through sprouting of controlateral intact serotoninergic and cortico-spinal axons, thanks to the absence of glial reactivity. This reinnervation is paralleled by the restoration of impaired locomotion of the ipselateral hindleg. Future applications to spinal cord injured patients are discussed., (Copyright 2003 Wiley-Liss, Inc.)

GFAP is the major intermediate filament of mature astrocytes, and its relatively specific expression in these cells suggests an important function. To study the role of the GFAP gene, mice have been created carrying null alleles (no protein), modified alleles (altered protein), or added wild type alleles (elevated protein). Surprisingly, absence of GFAP has relatively subtle effects on development. On the other hand, over-expression can be lethal, and led to the discovery that GFAP coding mutations are responsible for most cases of Alexander disease, a devastating neurodegenerative disorder. Here we review what the various GFAP mouse models reveal about GFAP and astrocyte function., (Copyright 2003 Wiley-Liss, Inc.)

Transgenic expression of IL-6 in the CNS under the control of the GFAP gene promoter, glial fibrillary acidic protein-interleukin-6 (GFAP-IL-6) mice, raises an inflammatory response and causes significant brain damage. However, the results obtained in the GFAP-IL-6 mice after a traumatic brain injury, such as a cryolesion, demonstrate a neuroprotective role of IL-6. Thus, the GFAP-IL-6 mice showed faster tissue repair and decreased oxidative stress and apoptosis compared with control litter-mate mice. The neuroprotective factors metallothionein-I+II (MT-I+II) were upregulated by the cryolesion to a higher extent in the GFAP-IL-6 mice, suggesting that they could be related to the neuroprotection afforded by the transgenic expression of IL-6. To examine this possibility, we have crossed GFAP-IL-6 mice with transgenic mice overexpressing MT-I (TgMT), producing double transgenic GFAP-IL-6 TgMT mice. The results obtained after cryolesion in GFAP-IL-6 TgMT mice, as well as in TgMT mice, consistently supported the idea that the increased MT-I+II levels observed in GFAP-IL-6 mice are a fundamental and important mechanism for coping with brain damage. Accordingly, MT-I overexpression regulated the inflammatory response, decreased oxidative stress and apoptosis significantly, and increased brain tissue repair in comparison with either GFAP-IL-6 or control litter-mate mice. Overall, the results demonstrate that brain MT-I+II proteins are fundamental neuroprotective factors., (Copyright 2003 Wiley-Liss, Inc.)

The slit family serves as a repellent for growing axons toward correct targets during neural development. A recent report describes slit mRNAs expressed in various brain regions in adult rats. However, their functions in the adult nervous system remain unknown. In the present study, we investigated whether slit mRNAs were expressed in the cryo-injured brain, using in situ hybridization. All slit family members were expressed at the lesion. Slit2 mRNA was the most intensely expressed in the cells surrounding the necrotic tissue. A double-labeling study showed that slit2 mRNA was expressed in the glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. In addition, glypican-1, a heparan sulfate proteoglycan that serves as a high-affinity receptor for Slit protein, was coexpressed with slit2 mRNA in the reactive astrocytes. These findings suggested that slit2 might prevent regenerating axons from entering into the lesion in concert with glypican-1., (Copyright 2003 Wiley-Liss, Inc.)

After spinal cord injury (SCI), white matter tracts are characterized by demyelination and increased sensitivity to the K(+) channel blocker 4-aminopyridine (4-AP). These effects appear to contribute to neurological impairment after SCI, although the molecular changes in K(+) channel subunit expression remain poorly understood. We examined changes in gene expression of the 4-AP-sensitive voltage-gated K(+) channel Kv 1.4 after chronic SCI in the rat. Quantitative immunoblotting showed that Kv 1.4 protein was significantly increased at 6 weeks, but not at 1 week, after SCI in spinal cord white matter. Kv 1.4 was localized to astrocytes, oligodendrocytes, and oligodendrocyte progenitor cells but not to axons in both the normal and the injured spinal cord white matter. Because glial cells proliferate after SCI, we used immunogold electron microscopy to quantify Kv 1.4 protein in individual glial cells and found a sixfold increase of Kv 1.4 in cells of the oligodendrocyte lineage after chronic injury. Finally, quantitative in situ hybridization showed that Kv 1.4 mRNA was significantly upregulated in spinal cord white matter, but not gray matter, after SCI. In summary, we show that Kv 1.4 is expressed in glial cells and not in axons in the rat spinal cord white matter and that its expression is markedly increased in cells of the oligodendrocyte lineage after chronic SCI. Given that K(+) channels play a role in glial cell proliferation, cells exhibiting changes in Kv 1.4 expression may represent proliferating oligodendroglia in the chronically injured spinal cord., (Copyright 2002 Wiley-Liss, Inc.)

The expression of major histocompatibility complex (MHC) class I and class II in the CNS has received considerable interest because of its importance in neurodegenerative or inflammatory diseases, such as multiple sclerosis (MS). However, at the moment nothing is known about the expression patterns of transcription factors controlling MHC expression in MS lesions. Here, we performed an extensive immunohistochemical analysis on MS affected postmortem brain tissue to determine the cellular localization and distribution of different MHC-controlling transcription factors. We show that phagocytic macrophages in active demyelinating MS lesions displayed a moderate to strong immunostaining of the MHC-specific transcription factors RFX and CIITA, as well as the general transcription factors NF-kappaB, IRF1, STAT1, USF, and CREB, which was congruent with a strongly enhanced expression of HLA-DR, HLA-DQ, HLA-DP, and HLA class I. In the normal-appearing white matter (NAWM), clusters of activated microglial cells forming preactive lesions displayed an overall stronger expression level of these transcription factors, combined with a strong to intense level of MHC class I and class II immunostaining. In general, astrocytes and oligodendrocytes either did not express, or weakly expressed, these transcription factors, correlating with a lack of MHC class II and weak MHC class I expression. Together, the elevated expression level of transcription factors governing expression of MHC class I and class II molecules in activated microglial cells and phagocytic macrophages strongly suggests a general state of microglial cell activation in MS lesions., (Copyright 2001 Wiley-Liss, Inc.)

Ventral root avulsion in the rat leads to a retrograde response, with activation of glia and up-regulation of immunologic cell surface molecules such as major histocompatibility complex (MHC) antigens, and the subsequent degeneration of a large proportion of the lesioned motoneurons. Herein, we examined several inbred congenic rat strains previously known to react differently to experimentally induced autoimmune diseases and demonstrate a substantial genetic diversity in the regulation of glial activation and neuron death in this injury model. The panel of examined inbred rat strains included DA(RT1AV1), PVG.1AV1, LEW.1AV1, LEW.1N, BN(RT1N) and E3(RT1U), and the following parameters were determined: (1) MHC class II expression on glia; (2) expression of glial fibrillary acidic protein, C3 complement, and microglial response factor-1 mRNAs in glia; (3) levels of the tumor necrosis factor-alpha and interleukin-1beta cytokine mRNAs; (4) degree of motoneuron loss. The findings of considerable strain-dependent differences in all parameters studied demonstrate important polymorphisms in the genetic regulation of these events. Furthermore, some of the studied features segregated from each other, suggesting independent regulatory mechanisms. Genes outside of the MHC complex are mainly implicated as being of importance for the phenotypic differences, as significant differences were recorded between the MHC congenic strains differing in the non-MHC genes but not vice versa. These results contribute new important insights into the genetic regulation of glial reactivity and neuron death after mechanical nerve injuries. In addition, the finding of conspicuous strain-dependent differences makes it necessary to consider the genetic background when designing and interpreting animal experiments involving noxious insults to the central nervous system resulting in glial activation and nerve cell loss., (Copyright 2001 Wiley-Liss, Inc.)

We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology.

We report a family with early-onset deafness and progressive dystonia exclusively involving males over two successive generations. There is also evidence of cognitive impairment and corticospinal tract involvement. The pedigree suggests an X-linked inheritance. A similar family was originally described by Scribanu and Kennedy. Tranebjaerg et al. have recently reported two other families with linkage to Xq22 and also proposed a novel X-linked candidate gene. These findings support the existence of a distinct neurodegenerative syndrome principally characterized by early-onset deafness and progressive dystonia. Neuropathology of one case showed a mosaic pattern of neuronal loss and gliosis in the caudate and putamen suggesting that this pattern is not restricted to XDP or Lubag.

Familial frontotemporal dementia (FTD) is a complex disorder with lack of distinctive histopathological markers found in other types of dementia. Most of the linkage reports from FTD families map the disease loci to chromosome 17q21-22. However, FTD is genetically heterogeneous, as linkage also has been reported to chromosome 3. In the present study, we investigated the genetics of a Swedish family with an early-onset type of rapidly progressive FTD, associated with muscular rigidity and akinetic movements. Neuropathological features such as severe frontal lobe degeneration, spongy changes, and gliosis were present in affected family members. We here report probable linkage to chromosome 17q12-21 with a maximum two-point lod score of 2.76 at theta = 0 for marker D17S806, and a peak multipoint lod score of 2.86 for the same marker. Linkage to chromosome 3 was excluded, as two-point lod scores of -2.79, and -2.27 at theta = 0.01 for markers D3S1603 and D3S1552, respectively, were obtained. Sequencing of the translated exons of a strong candidate gene in the linked region of chromosome 17, the tau gene, failed to identify any mutations segregating with the disease.

The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

Dentatorubropallidoluysian atrophy (DRPLA) has been described chiefly in Japan and appears to be rare in Europe. It is of autosomal dominant inheritance. We report the first British family with DRPLA, which contains four affected individuals in two generations. The diagnosis was made at autopsy in one case. The age of onset of symptoms ranged from 15 to 38 years, and clinical features included ataxia, dementia, chorea, and dystonia; three patients had generalized seizures. The three living patients resemble those with early Huntington's disease clinically. Three main phenotypes of DRPLA have been proposed: an ataxo-choreoathetoid type, a pseudo-Huntington type, and a myoclonic epilepsy type. The variation in clinical presentation in our family demonstrates the difficulty in applying such classifications to this and other dominantly inherited disorders with phenotypic variation. DRPLA is likely to be confused with Huntington's disease in European families.

In this article we describe the neuropathological changes in three patients with neuroacanthocytosis and review the neuropathology of the other eight cases reported in the literature. Macroscopically the brains showed enlargement of the lateral ventricles, especially the frontal horns. The most severely and consistently affected brain areas were the caudate nucleus and putamen, which were atrophic and showed by light microscopy marked neuronal loss and gliosis. Small and medium-sized striatal neurons were particularly depleted. The globus pallidus was almost as severely involved as the striatum. In some cases the thalamus, substantia nigra, and anterior horns of the spinal cord showed pathology, mainly neuronal loss and mild gliosis. Brain areas with no pathology included the subthalamic nucleus, cerebral cortex, cerebellum, pons, and medulla. The preservation of these areas may help in the neuropathological distinction of neuroacanthocytosis from Huntington's disease.

We describe a family with nearly 300 members over 8 generations with 32 affected individuals who have an autosomal dominant neurodegenerative disease characterized by progressive parkinsonism with dystonia unrelated to medications, dementia, ocular motility abnormalities, pyramidal tract dysfunction, frontal lobe release signs, perseverative vocalizations, and urinary incontinence. The course is exceptionally aggressive; symptom onset and death consistently occur in the fifth decade. Positron emission tomographic studies with [18F]6-fluoro-L-dopa (6FD) were performed in 4 patients and 7 individuals at risk for development of the disease. All affected subjects had markedly reduced striatal uptake of 6FD (p less than 0.001). All individuals at risk had normal striatal uptake, but high 6FD uptake rate constants were noted in 3 of the 7 studied. Autopsy findings revealed severe neuronal loss with gliosis in substantia nigra, pontine tegmentum, and globus pallidus, with less involvement of the caudate and the putamen. There were no plaques, tangles, Lewy bodies, or amyloid bodies. This kindred appears to represent a neurodegenerative disease not heretofore described. We propose the following name for this new genetic disease: autosomal dominant parkinsonism and dementia with pallido-ponto-nigral degeneration.