Human Glial Fibrillary Acidic Protein Research Articles

Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons

Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1Hz applied 30min later. This induction of DP was inhibited by application of either 100nM adenosine or 0.5nM N6-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice.

Fluorescent protein-expressing neural progenitor cells as a tool for transplantation studies.

The purpose of this study was to generate quadruple fluorescent protein (QFP) transgenic mice as a source for QFP-expressing neural stem and progenitor cells (NSCs/NPCs) that could be utilized as a tool for transplantation research. When undifferentiated, these NSCs only express cyan fluorescent protein (CFP); however, upon neuronal differentiation, the cells express yellow fluorescent protein (YFP). During astrocytic differentiation, the cells express green fluorescent protein (GFP), and during oligodendrocytic differentiation, the cells express red fluorescent protein (DsRed). Using immunocytochemistry, immunoblotting, flow cytometry and electrophysiology, quadruple transgenic NPCs (Q-NPCs) and GFP-sorted NPCs were comprehensively characterized in vitro. Overall, the various transgenes did not significantly affect proliferation and differentiation of transgenic NPCs in comparison to wild-type NPCs. In contrast to a strong CFP and GFP expression in vitro, NPCs did not express YFP and dsRed either during proliferation or after differentiation in vitro. GFP-positive sorted NPCs, expressing GFP under the control of the human GFAP promoter, demonstrated a significant improvement in astroglial differentiation in comparison to GFP-negative sorted NPCs. In contrast to non-sorted and GFP-positive sorted NPCs, GFP-negative sorted NPCs demonstrated a high proportion of neuronal differentiation and proved to be functional in vitro. At 6 weeks after the intracerebroventricular transplantation of Q-NPCs into neonatal wild-type mice, CFP/DCX (doublecortin) double-positive transplanted cells were observed. The Q-NPCs did not express any other fluorescent proteins and did not mature into neuronal or glial cells. Although this model failed to visualize NPC differentiation in vivo, we determined that activation of the NPC glial fibrillary acid protein (GFAP) promoter, as indicated by GFP expression, can be used to separate neuronal and glial progenitors as a valuable tool for transplantation studies.

Astrocytic TDP-43 Pathology in Alexander Disease

Alexander disease (AxD) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic inclusions known as Rosenthal fibers (RFs) within astrocytes, and is caused by dominant mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP). GFAP is the major astrocytic intermediate filament, and in AxD patient brain tissue GFAP is a major component of RFs. TAR DNA binding protein of 43 kDa (TDP-43) is the major pathological protein in almost all cases of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and ∼50% of frontotemporal lobar degeneration (FTLD), designated as FTLD-TDP. In ALS and FTLD-TDP, TDP-43 becomes insoluble, ubiquitinated, and pathologically phosphorylated and accumulates in cytoplasmic inclusions in both neurons and glia of affected brain and spinal cord regions. Previously, TDP-43 was detected in RFs of human pilocytic astrocytomas; however, involvement of TDP-43 in AxD has not been determined. Here we show that TDP-43 is present in RFs in AxD patient brains, and that insoluble phosphorylated full-length and high molecular weight TDP-43 accumulates in white matter of such brains. Phosphorylated TDP-43 also accumulates in the detergent-insoluble fraction from affected brain regions of Gfap(R236H/+) knock-in mice, which harbor a GFAP mutation homologous to one that causes AxD in humans, and TDP-43 colocalizes with astrocytic RF pathology in Gfap(R236H/+) mice and transgenic mice overexpressing human wild-type GFAP. These findings suggest common pathogenic mechanisms in ALS, FTLD, and AxD, and this is the first report of TDP-43 involvement in a neurological disorder primarily affecting astrocytes.

The Serum S100B Level as a Biomarker of Enteroglial Activation in Patients with Ulcerative Colitis

Objective. Recent studies have demonstrated that enteric glial cells (EGC) participate in the homeostasis of the gastrointestinal tract. This study investigated whether enteroglial markers, including S100B protein and glial fibrillary acidic protein (GFAP), can serve as noninvasive indicators of EGC activation and disease activity in UC patients. Methods. This clinical prospective study included 35 patients with UC and 40 age- and sex-matched controls. The diagnosis of UC was based on standard clinical, radiological, endoscopic, and histological criteria. Clinical disease activity was evaluated using the Modified Truelove-Witts Severity Index. Serum samples were analyzed for human GFAP and S100B using commercial enzyme-linked immunosorbent assay kits. Results. GFAP was not detected in the serum of either UC patients or controls (P > 0.05). However, we found a significant (P < 0.001) decrease in the serum S100B levels in the UC patients. No correlation between the serum S100B level and the disease activity or duration was observed (P > 0.05). The serum S100B levels did not differ between UC patients with active disease (24 patients, 68.6%) or in remission (11 patients, 31.4%) (P > 0.05). Conclusions. Ulcerative colitis patients had significantly lower serum S100B levels, while GFAP was of no diagnostic value in UC patients.

Germ-Line Recombination Activity of the Widely Used hGFAP-Cre and Nestin-Cre Transgenes

Herein we demonstrate with PCR, immunodetection and reporter gene approaches that the widely used human Glial Fibrillary Acidic Protein (hGFAP)-Cre transgene exhibits spontaneous germ-line recombination activity in leading to deletion in brain, heart and tail tissue with high frequency. The ectopic activity of hGFAP-Cre requires a rigorous control. We likewise observed that a second widely used nestin-Cre transgene shows germ-line deletion. Here we describe procedures to identify mice with germ-line recombination mediated by the hGFAP-Cre and nestin-Cre transgenes. Such control is essential to avoid pleiotropic effects due to germ-line deletion of loxP-flanked target genes and to maintain the CNS-restricted deletion status in transgenic mouse colonies.

S100B as a glial cell marker in diabetic peripheral neuropathy

Evidence suggests that acute and chronic hyperglycemia can cause oxidative stress in the peripheral nervous system which, in turn, can promote the development of diabetic neuropathy. Recent studies have found increased expression of glial fibrillary acidic protein (GFAP) and S100B, both of which are indicators of glial reactivity, in the neural and retinal tissues of diabetic rats. For the first time in the literature, the serum levels of GFAP and S100B were assessed in patients with diabetes to evaluate the potential of these factors to serve as peripheral glial biomarkers of diabetes and to investigate their relationship to diabetic peripheral neuropathy. This prospective clinical study included 72 patients with type 2 diabetes mellitus and 50 age- and sex-matched control subjects. All diabetic patients were assessed with respect to diabetes-related microvascular complications, such as peripheral neuropathy, retinopathy, and nephropathy. Serum samples were analyzed for human GFAP and S100B using a commercially available Enzyme-linked Immuno Sorbent Assay kit. GFAP was not detected in the serum samples of either diabetic or control patients (p>0.05). However, we found a statistically significant decrease in S100B serum levels in patients with diabetes compared with control participants (p<0.001). No associations between serum S100B levels and the presence of diabetic peripheral neuropathy or other microvascular complications were observed (p>0.05). The findings of markedly decreased serum levels of S100B may possibly indicate a neuroprotective effect of S100B, whereas GFAP may be of no diagnostic value in human patients with diabetes.

128. Selective contributions of neuronal and astroglial interleukin-1 receptor 1 to behavioral immune responses

Immune challenges alter central nervous system (CNS)-mediated processes; however, the mechanisms by which the CNS regulates behavior and physiology during immune responses are not fully understood. We hypothesize that neuronal-glial interactions, mediated by cytokines, contribute to altered behavior during immune challenges. To this end, we engineered two transgenic mouse lines that express interleukin-1 receptor 1 (IL1R1) only in the CNS and selectively on neurons or astrocytes. These mice express murine IL1R1 cDNA under transcriptional control of the neuron-specific enolase (NSE) or the human glial fibrillary acidic protein (gfa2) promoters. The transgenes were bred onto a genetic background lacking endogenous IL1R1 expression centrally and peripherally. Animals with selective neuronal IL1R1 expression are referred to as NSE-IL1R1, and strains expressing IL1R1 on astrocytes are called gfa2-IL1R1. Transgenic mice instrumented with cranial electroencephalographic electrodes and a thermistor were injected intracerebroventricularly with 50 ng IL-1 beta or intraperitoneally with lipopolysaccharide (LPS; 0.40 mg/kg). Preliminary data from ongoing studies suggest that IL-1 beta induces rapid eye movement sleep (REMS) suppression in NSE-IL1R1 mice that is of greater magnitude than REMS responses of gfa2-IL1R1 mice. Similarly, early results indicate systemic challenge with LPS induces fever and suppresses REMS to a greater extent in NSE-IL1R1 mice than in gfa2-IL1R1 mice. These encouraging results suggest a potential role for neurons as primary effectors of cytokine activity in mediating behavioral responses to immune challenge.

Identification and characterization of citrulline-modified brain proteins by combining HCD and CID fragmentation.

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

Genetic Background Affects Human Glial Fibrillary Acidic Protein Promoter Activity

The human glial fibrillary acidic protein (hGFAP) promoter has been used to generate numerous transgenic mouse lines, which has facilitated the analysis of astrocyte function in health and disease. Here, we evaluated the expression levels of various hGFAP transgenes at different ages in the two most commonly used inbred mouse strains, FVB/N (FVB) and C57BL/6N (B6N). In general, transgenic mice maintained on the B6N background displayed weaker transgene expression compared with transgenic FVB mice. Higher level of transgene expression in B6N mice could be regained by crossbreeding to FVB wild type mice. However, the endogenous murine GFAP expression was equivalent in both strains. In addition, we found that endogenous GFAP expression was increased in transgenic mice in comparison to wild type mice. The activities of the hGFAP transgenes were not age-dependently regulated. Our data highlight the importance of proper expression analysis when non-homologous recombination transgenesis is used.

Delta-like 1 regulates Bergmann glial monolayer formation during cerebellar development

BackgroundBergmann glia (BG) are unipolar cerebellar astrocytes. The somata of mature BG reside in the Purkinje cell layer and extend radially arranged processes to the pial surface. BG have multiple branched processes, which enwrap the synapses of Purkinje cell dendrites. They migrate from the ventricular zone and align next to the Purkinje cell layer during development. Previously, we reported that Notch1, Notch2, and RBPj genes in the BG play crucial roles in the monolayer formation and morphogenesis of BG. However, it remains to be determined which ligand activates Nocth1 and Notch 2 on BG. Delta-like 1 (Dll1) is a major ligand of Notch receptors that is expressed in the developing cerebellum.ResultsIn this study, we used human glial fibrillary acidic protein (hGFAP) promoter-driven Cre-mediated recombination to delete Dll1 in BG. Dll1-conditional mutant mice showed disorganization of Bergmann fibers, ectopic localization of BG in the molecular layer and a reduction in the number of BG.ConclusionThese results suggest that Dll1 is required for the formation of the BG layer and its morphological maturation, apparently through a Notch1/2-RBPj dependent signaling pathway.

RNA-binding protein QKI regulates Glial fibrillary acidic protein expression in human astrocytes

Linkage, association and expression studies previously pointed to the human QKI, KH domain containing, RNA-binding (QKI) as a candidate gene for schizophrenia. Functional studies of the mouse orthologue Qk focused mainly on its role in oligodendrocyte development and myelination, while its function in astroglia remained unexplored. Here, we show that QKI is highly expressed in human primary astrocytes and that its splice forms encode proteins targeting different subcellular localizations. Uncovering the role of QKI in astrocytes is of interest in light of growing evidence implicating astrocyte dysfunction in the pathogenesis of several disorders of the central nervous system. We selectively silenced QKI splice variants in human primary astrocytes and used RNA sequencing to identify differential expression and splice variant composition at the genome-wide level. We found that an mRNA expression of Glial fibrillary acidic protein (GFAP), encoding a major component of astrocyte intermediate filaments, was down-regulated after QKI7 splice variant silencing. Moreover, we identified a potential QKI-binding site within the 3' untranslated region of human GFAP. This sequence was not conserved between mice and humans, raising the possibility that GFAP is a target for QKI in humans but not rodents. Haloperidol treatment of primary astrocytes resulted in coordinated increases in QKI7 and GFAP expression. Taken together, our results provide the first link between QKI and GFAP, two genes with alterations previously observed independently in schizophrenic patients. Our findings for QKI, together with its well-known role in myelination, suggest that QKI is a hub regulator of glia function in humans.

Inactivation of Cdc42 in embryonic brain results in hydrocephalus with ependymal cell defects in mice

The establishment of a polarized cellular morphology is essential for a variety of processes including neural tube morphogenesis and the development of the brain. Cdc42 is a Ras-related GTPase that plays an essential role in controlling cell polarity through the regulation of the actin and microtubule cytoskeleton architecture. Previous studies have shown that Cdc42 plays an indispensable role in telencephalon development in earlier embryo developmental stage (before E12.5). However, the functions of Cdc42 in other parts of brain in later embryo developmental stage or in adult brain remain unclear. Thus, in order to address the role of Cdc42 in the whole brain in later embryo developmental stage or in adulthood, we used Cre/loxP technology to generate two lines of tissue-specific Cdc42-knock-out mice. Inactivation of Cdc42 was achieved in neuroepithelial cells by crossing Cdc42/ flox mice with Nestin-Cre mice and resulted in hydrocephalus, causing death to occur within the postnatal stage. Histological analyses of the brains from these mice showed that ependymal cell differentiation was disrupted, resulting in aqueductal stenosis. Deletion of Cdc42 in the cerebral cortex also induced obvious defects in interkinetic nuclear migration and hypoplasia. To further explore the role of Cdc42 in adult mice brain, we examined the effects of knocking-out Cdc42 in radial glial cells by crossing Cdc42/flox mice with human glial fibrillary acidic protein (GFAP)-Cre mice. Inactivation of Cdc42 in radial glial cells resulted in hydrocephalus and ependymal cell denudation. Taken together, these results highlight the importance of Cdc42 for ependymal cell differentiation and maintaining, and suggest that these functions likely contribute to the essential roles played by Cdc42 in the development of the brain.

3. Novel mouse models for the investigation of neuronal–glial immune interactions

Immune challenge alters central nervous system (CNS)-mediated processes and complex behaviors. Pro-inflammatory cytokines such as interleukin-1 beta (IL-1) and tumor necrosis factor alpha (TNF) are well characterized immunomodulators that mediate multiple aspects of the innate immune response. IL-1 and TNF respectively bind to IL-1 receptor 1 (IL-1R1) and TNF p55 receptor (TNFR1) present on neurons and glia. Systemic or central administration of IL-1 or TNF elicits sickness behaviors including sleep–wake behavior alterations, decreased appetite, and social withdrawal. Furthermore, neurons and glia produce cytokines in response to an immune challenge. Collectively, these observations suggest neuronal–glial interactions involving cytokines are crucial to the manifestation of sickness behavior. Nevertheless, the relative contribution of neurons and glia to the modulation of complex behavior and physiology during immune challenge is not known. To address this knowledge gap, we engineered four transgenic mouse lines that express IL-1R1 or TNFR1 only in the CNS and selectively on neurons or astrocytes. These transgenic mice express either IL-1R1 or TNFR1 cDNA under transcriptional control of neuron-specific enolase or human glial fibrillary acidic protein promoters. Our animals provide a unique tool to study CNS responses to immune challenge independent from peripheral actions of IL-1 or TNF. The systematic and selective examination of neuronal–glial cytokine interactions will further our understanding of the central response to immune activation relevant to a spectrum of pathologies characterized by inflammation.

Dicer1 and MiR‐9 are required for proper Notch1 signaling and the Bergmann glial phenotype in the developing mouse cerebellum

MicroRNAs (miRNAs) have important roles in the development of the central nervous system (CNS). Several reports indicate that tissue development and cellular differentiation in the developing forebrain are disrupted in the absence of miRNAs. However, the functions of miRNAs during cerebellar development have not been systematically characterized. Here, we conditionally knocked out the Dicer1 gene under the control of the human glial fibrillary acidic protein (hGFAP) promoter to examine the effect of miRNAs in the developing cerebellum. We particularly focused on the phenotype of Bergmann glia (BG). The hGFAP-Cre activity was detected as early as embryonic day 13.5 (E13.5) at the rhombic lip (RL) in the cerebellar plate, and later in several postnatal cerebellar cell types, including BG. Dicer1 ablation induces a smaller and less developed cerebellum, accompanied by aberrant BG morphology. Notch1 signaling appears to be blocked in Dicer1-ablated BG, with reduced expression of the Notch1 target gene, brain lipid binding protein (BLBP). Using neuronal co-culture assays, we showed an intrinsic effect of Dicer1 on BG morphology and Notch1 target gene expression. We further identified miR-9 as being differentially expressed in BG and showed that miR-9 is a critical, but not the only, miRNA component of the Notch1 signaling pathway in cultured BG cells.

Astrocyte-specific IKK2 activation in mice is sufficient to induce neuroinflammation but does not increase susceptibility to MPTP

A key regulator of inflammatory gene expression is the transcription factor NF-κB that is controlled by the IκB proteins. We used a transgenic mouse model expressing a constitutively active IκB-kinase-2 (IKK2-CA) in astrocytes under control of the human glial fibrillary acidic protein promotor (IKK2-mice) to investigate neuroinflammation, proinflammatory cytokine expression, microglial activation and a potential enhanced susceptibility to the neurotoxin MPTP (4×10mg/kg). Readouts included the determination of cytokines, striatal dopamine (DA), nigral tyrosine hydroxylase (TH) positive neurons, microglial activation and motor activity.IKK2-CA expression in astrocytes conditionally induced by the tet-off system resulted in a widespread neuroinflammation indicated by the increased expression of inflammatory cytokines and the presence of activated microglia and astrogliosis. Additionally, striatal DA concentrations but not nigral TH-positive neurons were reduced in IKK2-mice by 20%. Motor activity of IKK2-mice was not affected. Surprisingly, there was a similar reduction in striatal DA concentrations and the number of nigral TH-positive neurons in IKK2 and control mice after MPTP treatment.In conclusion, although naïve IKK2-mice showed reduced striatal DA concentrations and an increase in inflammatory markers in the brain, a higher susceptibility to MPTP was not observed. This finding argues against a prominent role of astrocyte specific, IKK2-mediated neuroinflammation in MPTP-induced neurodegeneration.

Cell‐penetrating anti‐GFAP VHH and corresponding fluorescent fusion protein VHH‐GFP spontaneously cross the blood‐brain barrier and specifically recognize astrocytes: application to brain imaging

Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

Hypoxia Regulated Gene Transcription in Human Optic Nerve Lamina Cribrosa Cells in Culture

Vascular hypoperfusion, extracellular matrix remodeling and axon loss are pathological characteristics of the glaucomatous optic nerve head. We report a novel study demonstrating transcriptional responses in optic nerve lamina cribrosa (LC) cells exposed to in vitro hypoxic stress. Primary cultures of human glial fibrillary acid protein (GFAP) negative LC cells were generated from four donors. Cells were exposed to 24 hours of hypoxic stress (1% O2) or normoxia (21% O2). Hypoxia responsive genes were identified using Affymetrix HG-U133A microarrays (n = 3) and validated with real time PCR (n = 3). Secreted protein was measured by ELISA (n = 4) and cellular protein by Western blot (n = 4). Expression data were annotated with NIH DAVID software and putative transcription factor sites in hypoxia-responsive gene promoters were identified using Core_TF software. Hypoxia-sensitive genes included those involved in apoptosis (e.g., BNIP3), neurogenesis (e.g., STC1), extracellular matrix (e.g., MIF, DDR1/TrkE, and IGFR2), mitochondrion (e.g., CYP1B1) and angiogenesis (e.g., VEGF). Real time PCR for selected genes supported the expression changes identified by microarray. ELISA and Western blot validated corresponding changes in protein production. Promoter sequence interrogation revealed putative conserved transcription factor binding sites (e.g., HIF and CREB) in the promoters of the hypoxia responsive genes. Our data show that LC cell gene expression is sensitive to reduced oxygen levels in vitro and provides bioinformatic evidence of the potential transcriptional regulators of this response.

Sex hormone-dependent attenuation of EAE in a transgenic mouse with astrocytic expression of the RNA regulator HuR

In experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative diseases, astrocytes play an important role in promoting or attenuating the inflammatory response through induction of different cytokines and growth factors. HuR plays a major role in regulating many of these factors by modulating RNA stability and translational efficiency. Here, we engineered transgenic mice to express HuR in astrocytes using the human glial fibrillary acidic protein promoter and found that female transgenic mice had significantly less clinical disability and histopathological changes in the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our findings support a role for the astrocyte and posttranscriptional regulation in hormonally-mediated attenuation of EAE.

Regional difference of reactive astrogliosis following traumatic brain injury revealed by hGFAP-GFP transgenic mice

Reactive astrocytes greatly influence the wound healing and neuronal regeneration processes following brain injury. However, the origin and fate of reactive astrocytes appear to be different depending on the type, severity and duration of brain injury. Using the cryogenic traumatic brain injury model, here we comprehensively addressed the regional differences of reactive astrocytes in the injured cortex. In the proximal region of injury site, NG2-expressing and cytoplasmic Olig2-labeled cells were densely localized 3 days after the injury. Next to this proximal layer, most of reactive astrocytes did not express NG2 but exhibited radial glia-like shape with elongated processes. Accordingly, they expressed the progenitor or radial glial markers, such as vimentin, brain lipid binding protein (BLBP) and the green fluorescent protein (GFP) under the control of the human GFAP (hGFAP) promoter. However, only few glial fibrillary acidic protein (GFAP) expressing astrocytes were found in this layer. Distal to the injury site, most of astrocytes strongly expressed GFAP with hypertonic morphology. At day 15 after injury, all layers expressing GFAP and other marker expressions disappeared, indicating the termination of reactive astrogliosis. Taken together, our data suggest that reactive astrogliosis occurs in a regionally segregated manner in the early phase of brain injury.

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K+ and Cl− channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K+ effluxes. Additionally, the inhibition of Na+-K+-Cl− co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K+ channels (Kir4.1), K2P channels (TREK-1 and TWIK-1) and Cl− channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K2P channels.