Filament Glial Fibrillary Acidic Protein Research Articles

Lithium Decreases Glial Fibrillary Acidic Protein in a Mouse Model of Alexander Disease.

Alexander disease is a fatal neurodegenerative disease caused by mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP). The disease is characterized by elevated levels of GFAP and the formation of protein aggregates, known as Rosenthal fibers, within astrocytes. Lithium has previously been shown to decrease protein aggregates by increasing the autophagy pathway for protein degradation. In addition, lithium has also been reported to decrease activation of the transcription factor STAT3, which is a regulator of GFAP transcription and astrogliogenesis. Here we tested whether lithium treatment would decrease levels of GFAP in a mouse model of Alexander disease. Mice with the Gfap-R236H point mutation were fed lithium food pellets for 4 to 8 weeks. Four weeks of treatment with LiCl at 0.5% in food pellets decreased GFAP protein and transcripts in several brain regions, although with mild side effects and some mortality. Extending the duration of treatment to 8 weeks resulted in higher mortality, and again with a decrease in GFAP in the surviving animals. Indicators of autophagy, such as LC3, were not increased, suggesting that lithium may decrease levels of GFAP through other pathways. Lithium reduced the levels of phosphorylated STAT3, suggesting this as one pathway mediating the effects on GFAP. In conclusion, lithium has the potential to decrease GFAP levels in Alexander disease, but with a narrow therapeutic window separating efficacy and toxicity.

Elusive roles for reactive astrocytes in neurodegenerative diseases.

Astrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes (hypertrophy, remodeling of processes) and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND.

Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber.

Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity.

Astrocytosis in infantile neuronal ceroid lipofuscinosis: friend or foe?

Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is an inherited paediatric neurodegenerative disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1) and is thus classified as a lysosomal storage disease. Pathological examination of both human and murine INCL brains reveals progressive, widespread neuroinflammation. In fact, astrocyte activation appears to be the first histological sign of disease. However, the role of astrocytosis in INCL was poorly understood. The hallmark of astrocyte activation is the up-regulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) and vimentin. The role of astrocytosis in INCL was studied in a murine model lacking PPT1 and the intermediate filaments GFAP and vimentin (triple-knockout). This murine model of INCL with attenuated astrocytosis had an exacerbated pathological and clinical phenotype. The triple-knockout mouse had a significantly shortened lifespan, and accelerated cellular and humoural neuroinflammatory response compared with the parental PPT1(-/-) mouse. The data obtained from the triple-knockout mouse strongly suggest that astrocyte activation plays a beneficial role in early INCL disease progression. A more thorough understanding of the glial responses to lysosomal enzyme deficiencies and the accumulation of undergraded substrates will be crucial to developing effective therapeutics.

Phosphatase and actin regulator 4 is associated with intermediate filaments in adult neural stem cells and their progenitor astrocytes.

Phosphatase and actin regulator 4 (Phactr4) is a newly discovered protein that inhibits protein phosphatase 1 and shows actin-binding activity. We previously found that Phactr4 is expressed in the neurogenic niche in adult mice, although its precise subcellular localization and possible function in neural stem cells (NSCs) is not yet understood. Here, we show that Phactr4 formed punctiform clusters in the cytosol of subventricular zone-derived adult NSCs and their progeny in vitro. These Phactr4 signals were not associated with F-actin fibers but were closely associated with intermediate filaments such as nestin and glial fibrillary acidic protein (GFAP) fibers. Direct binding of Phactr4 with nestin and GFAP filaments was demonstrated using Duolink protein interaction analyses and immunoprecipitation assays. Interestingly, when nestin fibers were de-polymerized during the mitosis or by the phosphatase inhibitor, Phactr4 appeared to be dissociated from nestin, suggesting that their protein interaction is regulated by the protein phosphorylation. These results suggest that Phactr4 forms functional associations with intermediate filament networks in adult NSCs.

Induction of a reactive state in perineuronal satellite glial cells akin to that produced by nerve injury is linked to the level of p75NTR expression in adult sensory neurons

Satellite glial cells (SGCs) surrounding primary sensory neurons are similar to astrocytes of the central nervous system in that they buffer the extracellular environment via potassium and calcium channels and express the intermediate filament glial fibrillary acidic protein (GFAP). Peripheral nerve injury induces a reactive state in SGCs that includes SGC proliferation, increased SGC/SGC coupling via gap junctions, decreased inward rectifying potassium channel 4.1 (Kir 4.1) expression and increased expression of GFAP and the common neurotrophin receptor, p75NTR. In contrast, neuronal p75NTR expression, normally detected in ∼80% of adult rat sensory neurons, decreases in response to peripheral axotomy. Given the differential regulation of p75NTR expression in neurons versus SGCs with injury, we hypothesized that reduced signaling via neuronal p75NTR contributes to the induction of a reactive state in SGCs. We found that reducing neuronal p75NTR protein expression in uninjured sensory neurons by intrathecal subarachnoid infusion of p75NTR-selective anti-sense oligodeoxynucleotides for one week was sufficient to induce a "reactive-like" state in the perineuronal SGCs akin to that normally observed following peripheral nerve injury. This reactive state included significantly increased SGC p75NTR, GFAP and gap junction protein connexin-43 protein expression, increased numbers of SGCs surrounding individual sensory neurons and decreased SGC Kir 4.1 channel expression. Collectively, this supports the tenet that reductions in target-derived trophic support leading to, or as a consequence of, reduced neuronal p75NTR expression plays a critical role in switching the SGC to a reactive state.

Identification and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

Protein Changes in Immunodepleted Cerebrospinal Fluid from a Transgenic Mouse Model of Alexander Disease Detected Using Mass Spectrometry

Cerebrospinal fluid (CSF) is a low protein content biological fluid with a dynamic range spanning at least 9 orders of magnitude in protein content and is in direct contact with the brain. A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the low volumes of CSF that are obtainable from mice. As a model system in which to test this approach, we utilized transgenic mice that overexpress the intermediate filament glial fibrillary acidic protein (GFAP). These mice are models for Alexander disease (AxD), a severe leukodystrophy in humans. From the CSF of control and transgenic mice we report the identification of 289 proteins, with relative quantification of 103 proteins. Biological and technical triplicates were performed to address animal variability as well as reproducibility in mass spectrometric analysis. Relative quantitation was performed using distributive normalized spectral abundance factor (dNSAF) spectral counting analysis. A panel of biomarker proteins with significant changes in the CSF of GFAP transgenic mice has been identified with validation from enzyme-linked immunosorbent assay (ELISA) and microarray data, demonstrating the utility of our methodology and providing interesting targets for future investigations on the molecular and pathological aspects of AxD.

Immunological Demyelination Triggers Macrophage/Microglial Cells Activation without Inducing Astrogliosis

The glial scar formed by reactive astrocytes and axon growth inhibitors associated with myelin play important roles in the failure of axonal regeneration following central nervous system (CNS) injury. Our laboratory has previously demonstrated that immunological demyelination of the CNS facilitates regeneration of severed axons following spinal cord injury. In the present study, we evaluate whether immunological demyelination is accompanied with astrogliosis. We compared the astrogliosis and macrophage/microglial cell responses 7 days after either immunological demyelination or a stab injury to the dorsal funiculus. Both lesions induced a strong activated macrophage/microglial cells response which was significantly higher within regions of immunological demyelination. However, immunological demyelination regions were not accompanied by astrogliosis compared to stab injury that induced astrogliosis which extended several millimeters above and below the lesions, evidenced by astroglial hypertrophy, formation of a glial scar, and upregulation of intermediate filaments glial fibrillary acidic protein (GFAP). Moreover, a stab or a hemisection lesion directly within immunological demyelination regions did not induced astrogliosis within the immunological demyelination region. These results suggest that immunological demyelination creates a unique environment in which astrocytes do not form a glial scar and provides a unique model to understand the putative interaction between astrocytes and activated macrophage/microglial cells.

Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network formation with the potential to both activate caspase 3 and decrease cell viability

Alexander disease is a primary genetic disorder of astrocyte caused by dominant mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). While most of the disease-causing mutations described to date have been found in the conserved α-helical rod domain, some mutations are found in the C-terminal non-α-helical tail domain. Here, we compare five different mutations (N386I, S393I, S398F, S398Y and D417M14X) located in the C-terminal domain of GFAP on filament assembly properties in vitro and in transiently transfected cultured cells. All the mutations disrupted in vitro filament assembly. The mutations also affected the solubility and promoted filament aggregation of GFAP in transiently transfected MCF7, SW13 and U343MG cells. This correlated with the activation of the p38 stress-activated protein kinase and an increased association with the small heat shock protein (sHSP) chaperone, αB-crystallin. Of the mutants studied, D417M14X GFAP caused the most significant effects both upon filament assembly in vitro and in transiently transfected cells. This mutant also caused extensive filament aggregation coinciding with the sequestration of αB-crystallin and HSP27 as well as inhibition of the proteosome and activation of p38 kinase. Associated with these changes were an activation of caspase 3 and a significant decrease in astrocyte viability. We conclude that some mutations in the C-terminus of GFAP correlate with caspase 3 cleavage and the loss of cell viability, suggesting that these could be contributory factors in the development of Alexander disease.

Oligomers of Mutant Glial Fibrillary Acidic Protein (GFAP) Inhibit the Proteasome System in Alexander Disease Astrocytes, and the Small Heat Shock Protein αB-Crystallin Reverses the Inhibition

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G(0)/G(1) arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.

Apolipoprotein E4 Domain Interaction Induces Endoplasmic Reticulum Stress and Impairs Astrocyte Function

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase

S100B is a Ca(2+)-binding protein of the EF-hand type that is abundantly expressed in astrocytes and has been implicated in the regulation of several intracellular activities, including proliferation and differentiation. We show here that reducing S100B levels in the astrocytoma cell line GL15 and the Müller cell line MIO-M1 by small interference RNA technique results in a rapid disassembly of stress fibers, collapse of F-actin onto the plasma membrane and reduced migration, and acquisition of a stellate shape. Also, S100B-silenced GL15 and MIO-M1 Müller cells show a higher abundance of glial fibrillary acidic protein filaments, which mark differentiated astrocytes, compared with control cells. These effects are dependent on reduced activation of the phosphatidylinositol 3-kinase (PI3K) downstream effectors, Akt and RhoA, and consequently elevated activity of GSK3beta and Rac1 and decreased activity of the RhoA-associated kinase. Also, rat primary astrocytes transiently down-regulate S100B expression when exposed to the differentiating agent dibutyryl cyclic AMP and re-express S100B at later stages of dibutyryl cyclic AMP-induced differentiation. Moreover, reducing S100B levels results in a remarkably slow resumption of S100B expression, suggesting the S100B might regulate its own expression. Finally, we show that S100B interacts with Src kinase, thereby stimulating the PI3K/Akt and PI3K/RhoA pathways. These results suggest that S100B might contribute to reduce the differentiation potential of cells of the astrocytic lineage and participate in the astrocyte activation process in the case of brain insult and in invasive properties of glioma cells.

The effect of long-term lactobacilli (lactic acid bacteria) enteral treatment on the central nervous system of growing rats

The aim of this study was to explore the relationship between consumption of large doses of lactic acid bacteria (LAB) and the behaviour and brain morphobiochemistry of normal growing rats. Four groups of rats were treated with LAB cultures twice daily for 6 months. The control group received 1 ml of saline per treatment, while two experimental groups received 1 ml of living bacteria ( Lactobacillus plantarum and Lactobacillus fermentum, respectively) and the remaining group received a heat-treated (inactivated) L. fermentum culture. After 2 and 6 months of treatment, respectively, eight animals from each group were sacrificed, and specimens were taken for further analyses. The behaviour of the rats was evaluated five times in an open-field test at monthly intervals throughout the study. Lactobacilli treatment for 2 months induced changes in the motoric behaviour of the rats. The concentration of the astrocytesoluble and filament glial fibrillary acidic protein (GFAP) decreased in the posterior part of the hemispheres, including the thalamus, hippocampus and cortex of the rats treated with L. fermentum. A greater decrease in filament GFAP (up to 50%) was shown in the group receiving the live form of L. fermentum. In contrast, the GFAP in the live L. plantarum-treated group increased, showing elevated levels of the soluble and filament forms of GFAP in the posterior part of the hemispheres. A 60–66% decrease in the amount of the astrocyte-specific Ca-binding protein S-100b was shown in the posterior parts of the hemispheres and in the hindbrain of rats given LAB for 2 months. Prolonged feeding with LAB for 4 months up to full adulthood led to a further decrease in astrocyte reaction, reflected as an additional decrease in the amount of soluble GFAP and locomotor activity in all experimental groups. The changes in filament GFAP and S-100b appeared to disappear after prolonged feeding (total of 6 months) with LAB. In summary, LAB dietary treatment affected the ontogenetic development of the astrocytes, with the highest intensity observed in the early stages of rat development. It can be postulated that LAB treatment may play a preventive role in neurological diseases by decreasing astrocyte reaction and, consequently, lowering locomotor activity.

Glial Fibrillary Acidic Protein Filaments Can Tolerate the Incorporation of Assembly-compromised GFAP-δ, but with Consequences for Filament Organization and αB-Crystallin Association

The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-α, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-δ. GFAP-δ has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-α, assembly was restored providing GFAP-δ levels were kept low (∼10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-δ is naturally part of the endogenous intermediate filaments, although levels were low (∼10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease–causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-δ and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of αB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-δ splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein– and filament–filament associations as well as Jnk phosphorylation.

Alteration of Glial Fibrillary Acidic Proteins Immunoreactivity in Astrocytes of the Spinal Cord Diabetic Rats

Diabetes affects retinal and nervous glial cells, especially the astrocytes. A key indicator of this response is the alteration in the level of intermediate filament glial fibrillary acidic protein (GFAP) and number of GFAP-immunoreactive astrocytes. To date, no study has investigated the effect of diabetes on the distribution of GFAP-immunoreactive astrocytes in the spinal cord. Therefore, the present study investigated the effect of diabetes on the number of GFAP-immunoreactive astrocytes in the gray matter of the spinal cord of streptozotocin-induced diabetic Wistar rats. Animals were divided into six groups (n = 7); 6 weeks and 12 weeks diabetic duration groups and their respective age-matched normal control and sham control groups. Our results demonstrated a significant (P < 0.001) decrease in the number of GFAP-immunoreactive astrocytes in different areas of the spinal cord sections of the 6 weeks and 12 weeks long diabetic rats when compared with the spinal cord of normal and sham control groups of comparable age. The mean percentage in total number of GFAP-immunoreactive astrocytes in the whole gray matter areas of the spinal cord of the 6 and 12 weeks diabetic groups were approximately 28% and 41% less than control groups. Furthermore, the 12 weeks diabetic group showed a significant (P < 0.001) reduction in the number of GFAP-immunoreactive astrocytes when compared with the 6 weeks diabetic animals. These results suggest that the induction of diabetes is associated with a reduction in GFAP-positive astrocytes in the spinal cord, which may affect the functional support and role of astrocytic cells in the nervous tissue. This in turn may contribute to the pathological changes associated with diabetic state in the central nervous system.

Mild functional effects of a novel GFAP mutant allele identified in a familial case of adult-onset Alexander disease

Alexander disease is a neurological genetic disorder characterized by progressive white-matter degeneration, with astrocytes containing cytoplasmic aggregates, called Rosenthal fibers, including the intermediate filament glial fibrillary acidic protein (GFAP). The age of onset of the disease defines three different forms, infantile, juvenile and adult, all due to heterozygous GFAP mutations and characterized by a progressive less severe phenotype from infantile to adult forms. In an Italian family with a recurrent mild adult onset of Alexander disease, we have identified two GFAP mutations, coupled on a same allele, leading to p.[R330G; E332K]. Functional studies on this complex allele revealed less severe aggregation patterns compared to those observed with p.R239C GFAP mutant, associated with a severe Alexander disease phenotype. Moreover, in addition to confirming the involvement of the ubiquitin-proteasome system in cleaning cells from aggregates and a dominant effect of the novel mutant protein, in cells expressing the mild p.[R330G; E332K] mutant we have observed that indirect alphaB-crystallin overexpression, induced by high extracellular potassium concentration, could completely rescue the correct filament organization while, under the same experimental conditions, in cells expressing the severe p.R239C mutant only a partial rescue effect could be achieved.

Dietary Taurine Supplementation Ameliorates Diabetic Retinopathy via Anti-excitotoxicity of Glutamate in Streptozotocin-induced Sprague-Dawley Rats

The purpose of this study was to investigate whether taurine ameliorate the diabetic retinopathy, and to further explore the underlying mechanisms. The Sprague-Dawley rats were injected with streptozotocin to establish experimental diabetic model, then fed without or with 1.2% taurine for additional 4-12 weeks. After that, the protective effects of dietary taurine supplementation on diabetic retinopathy were estimated. Our results showed that chronic taurine supplement effectively improved diabetic retinopathy as changes of histopathology and ultrastructure. The supplementation could not lower plasma glucose concentration (P > 0.05), but caused an elevation in taurine content and a decline in levels of glutamate and gamma-aminobutyric acid (GABA) in diabetic retina (P < 0.05). Moreover, chronic taurine supplementation increased glutamate transporter (GLAST) expression (P < 0.05), decreased intermediate filament glial fibrillary acidic protein (GFAP) and N-methyl-D: -aspartate receptor subunit 1 (NR1) expression in diabetic retina (P < 0.05). These results demonstrated that chronic taurine supplementation ameliorates diabetic retinopathy via anti-excitotoxicity of glutamate in rats.

ARG3.1/ARC expression in hippocampal dentate gyrus astrocytes: ultrastructural evidence and co‐localization with glial fibrillary acidic protein

Synaptic efficacy following long-term potentiation (LTP) and memory consolidation is associated with changes in the expression of immediate early genes (IEGs). These changes are often accompanied by increased expression of glial fibrillary acidic protein (GFAP). While the protein products of the majority of IEGs are mainly restricted to the cell body, Arg3.1/Arc product is rapidly delivered to dendrites, where it accumulates close to synaptic sites. Arg3.1/Arc protein was originally considered neurone specific; however, we have recently found Arg3.1/Arc immunoreactivity (Arg3.1/Arc-IR) within glial cells and demonstrated its increased expression after LTP in the hippocampal dentate gyrus (DG). Here, we have further investigated this novel finding, using electron microscopic immunocytochemistry to determine the localization and sub-cellular distribution of Arg3.1/Arc protein in GFAP positive glia (GFAP-IR) in the DG. Arg3.1/Arc labelling was seen prominently in GFAP-IR glial cell bodies and in large- and medium-sized glial filamentous processes. GFAP-labelled medium–small peri-synaptic glial profiles also displayed Arg3.1/Arc-IR; however, the very thin and distal glial filaments only displayed Arc-IR. Arc-IR was distributed throughout the cytoplasm, often associated with GFAP filaments, and along the plasma membrane of glial processes. Peri-synaptic glial Arg3.1/Arc-IR processes were apposed to pre- and/or post-synaptic profiles at asymmetric axospinous synapses. These data, taken with our earlier study which provided evidence for an increase in astrocytic Arg3.1/Arc-IR after the induction of LTP, suggest a role for glial Arg3.1/Arc in structural and synaptic plasticity which may be critical for the maintenance of cognitive functions.