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Background: The study investigated the effect of co-administration of curcumin and donepezil on several markers of cognitive function (such as spatial memory, astrocyte activation, cholinesterase expressions) in the brain cortex and hippocampus of scopolamine-treated rats., Method and Results: For seven consecutive days, a pre-treatment of curcumin (50 mg/kg) and/or donepezil (2.5 mg/kg) was administered. On the seventh day, scopolamine (1 mg/kg) was administered to elicit cognitive impairment, 30 min before memory test was conducted. This was followed by evaluating changes in spatial memory, cholinesterase, and adenosine deaminase (ADA) activities, as well as nitric oxide (NO) level were determined. Additionally, RT-qPCR for glial fibrillary acidic protein (GFAP) and cholinesterase gene expressions was performed in the brain cortex and hippocampus. Also, GFAP immunohistochemistry  of the brain tissues for neuronal injury were performed in the brain cortex and hippocampus. In comparison to the control group, rats given scopolamine had impaired memory, higher levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ADA activities, as well as elevated markers of oxidative stress. In addition to enhanced GFAP immunoreactivity, there was also overexpression of the GFAP and BChE genes in the brain tissues. The combination of curcumin and donepezil was, however, observed to better ameliorate these impairments in comparison to the donepezil-administered rat group., Conclusion: Hence, this evidence provides more mechanisms to support the hypothesis that the concurrent administration of curcumin and donepezil mitigates markers of cognitive dysfunction in scopolamine-treated rat model., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Astrocytes play a role in healthy cognitive function and Alzheimer's disease (AD). The transcriptional factor nuclear factor-κB (NF-κB) drives astrocyte diversity, but the mechanisms are not fully understood. By combining studies in human brains and animal models and selectively manipulating NF-κB function in astrocytes, we deepened the understanding of the role of astrocytic NF-κB in brain health and AD. In silico analysis of bulk and cell-specific transcriptomic data revealed the association of NF-κB and astrocytes in AD. Confocal studies validated the higher level of p50 NF-κB and phosphorylated-p65 NF-κB in glial fibrillary acidic protein (GFAP) + -astrocytes in AD versus non-AD subjects. In the healthy mouse brain, chronic activation of astrocytic NF-κB disturbed the proteomic milieu, causing a loss of mitochondrial-associated proteins and the rise of inflammatory-related proteins. Sustained NF-κB signaling also led to microglial reactivity, production of pro-inflammatory mediators, and buildup of senescence-related protein p16 INK4A in neurons. However, in an AD mouse model, NF-κB inhibition accelerated β-amyloid and tau accumulation. Molecular biology studies revealed that astrocytic NF-κB activation drives the increase in GFAP and inflammatory proteins and aquaporin-4, a glymphatic system protein that assists in mitigating AD. Our investigation uncovered fundamental mechanisms by which NF-κB enables astrocytes' neuroprotective and neurotoxic responses in the brain., (© 2024. The Author(s).)

Reproduction is safeguarded by multiple, often cooperative, regulatory networks. Kisspeptin signaling, via KISS1R, plays a fundamental role in reproductive control, primarily by regulation of hypothalamic GnRH neurons. We disclose herein a pathway for direct kisspeptin actions in astrocytes that contributes to central reproductive modulation. Protein-protein interaction and ontology analyses of hypothalamic proteomic profiles after kisspeptin stimulation revealed that glial/astrocyte markers are regulated by kisspeptin in mice. This glial-kisspeptin pathway was validated by the demonstrated expression of Kiss1r in mouse astrocytes in vivo and astrocyte cultures from humans, rats, and mice, where kisspeptin activated canonical intracellular signaling-pathways. Cellular coexpression of Kiss1r with the astrocyte markers GFAP and S100-β occurred in different brain regions, with higher percentage in Kiss1- and GnRH-enriched areas. Conditional ablation of Kiss1r in GFAP-positive cells in the G-KiR-KO mouse altered gene expression of key factors in PGE2 synthesis in astrocytes and perturbed astrocyte-GnRH neuronal appositions, as well as LH responses to kisspeptin and LH pulsatility, as surrogate marker of GnRH secretion. G-KiR-KO mice also displayed changes in reproductive responses to metabolic stress induced by high-fat diet, affecting female pubertal onset, estrous cyclicity, and LH-secretory profiles. Our data unveil a nonneuronal pathway for kisspeptin actions in astrocytes, which cooperates in fine-tuning the reproductive axis and its responses to metabolic stress.

Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter-AAV9-gfaABC(1)D-EIF2B5-thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure., Competing Interests: Declaration of interests J.L.B. is on the board of wFluidx, Inc., owns stock in Orchard Therapeutics, and has consulted for Bluebird bio, Calico Life Sciences, Denali Therapeutics, Enzyvant, and Neurogene. A.M.B. is a beneficiary of a licensing agreement with Axovant Gene Therapies (royalties) and Neurogene (royalties) and has received income from Neurogene (consulting and honorarium). A.M.B., K.M.F., J.L.B., N.K.P., and J.A.H. are inventors on a provisional patent: Materials and Methods for the Treatment of EIF2B5 Mutations and Diseases Resulting Therefrom. Provisional Patent 63/316,241., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

We compared the effects of two different high-caloric diets administered to 4-week-old rats for 12 weeks: a diet rich in sugar (30% sucrose) and a cafeteria diet rich in sugar and high-fat foods. We focused on the hippocampus, particularly on the gamma-aminobutyric acid (GABA)ergic system, including the Ca 2+ -binding proteins parvalbumin (PV), calretinin (CR), calbindin (CB), and the neuropeptides somatostatin (SST) and neuropeptide Y (NPY). We also analyzed the density of cholinergic varicosities, brain-derived neurotrophic factor ( BDNF ), reelin ( RELN ), and cyclin-dependent kinase-5 ( CDK-5 ) mRNA levels, and glial fibrillary acidic protein (GFAP) expression. The cafeteria diet reduced PV-positive neurons in the granular layer, hilus, and CA1, as well as NPY-positive neurons in the hilus, without altering other GABAergic populations or overall GABA levels. The high-sugar diet induced a decrease in the number of PV-positive cells in CA3 and an increase in CB-positive cells in the hilus and CA1. No alterations were observed in the cholinergic varicosities. The cafeteria diet also reduced the relative mRNA expression of RELN without significant changes in BDNF and CDK5 levels. The cafeteria diet increased the number but reduced the length of the astrocyte processes. These data highlight the significance of determining the mechanisms mediating the observed effects of these diets and imply that the cognitive impairments previously found might be related to both the neuroinflammation process and the reduction in PV, NPY, and RELN expression in the hippocampal formation.

Toxoplasma gondii (T. gondii) is a protozoan parasite that infects approximately one-third of the global human population, often leading to chronic infection. While acute T. gondii infection can cause neural damage in the central nervous system and result in toxoplasmic encephalitis, the consequences of T. gondii chronic infection (TCI) are generally asymptomatic. However, emerging evidence suggests that TCI may be linked to behavioral changes or mental disorders in hosts. Astrocyte polarization, particularly the A1 subtype associated with neuronal apoptosis, has been identified in various neurodegenerative diseases. Nevertheless, the role of astrocyte polarization in TCI still needs to be better understood. This study aimed to establish a mouse model of chronic TCI and examine the transcription and expression levels of glial fibrillary acidic protein (GFAP), C3, C1q, IL-1α, and TNF-α in the brain tissues of the mice. Quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay, and Western blotting were employed to assess these levels. Additionally, the expression level of the A1 astrocyte-specific marker C3 was evaluated using indirect fluorescent assay (IFA). In mice with TCI, the transcriptional and expression levels of the inflammatory factors C1q, IL-1α, and TNF-α followed an up-down-up pattern, although they remained elevated compared to the control group. These findings suggest a potential association between astrocyte polarization towards the A1 subtype and synchronized changes in these three inflammatory mediators. Furthermore, immunofluorescence assay (IFA) revealed a significant increase in the A1 astrocytes (GFAP + C3 + ) proportion in TCI mice. This study provides evidence that TCI can induce astrocyte polarization, a biological process that may be influenced by changes in the levels of three inflammatory factors: C1q, IL-1α, and TNF-α. Additionally, the release of neurotoxic substances by A1 astrocytes may be associated with the development of TCI., (© 2024. The Author(s).)

We determined the genetic association between specific human leucocyte antigen (HLA) loci and autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy. Our results showed that autoimmune GFAP astrocytopathy was associated with HLA-A*3303 (odds ratio [OR] = 2.02, 95% confidence interval [CI] = 1.32-3.06, p = 0.00072, p adj.  = 0.046) and HLA-DBP1*0501 (OR = 0.51, 95% CI = 0.36-0.71, p = 0.000048, p adj.  = 0.0062). Moreover, HLA-A*3303 carriers with the disease had a longer hospital stay (p = 0.0005) than non-carriers. This study for the first time provides evidence for a role of genetic factor in the development of autoimmune GFAP astrocytopathy. ANN NEUROL 2024;95:901-906., (© 2024 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed in specific types of glial cells in the nervous system. The expression of GFAP is highly regulated during brain development and in neurological diseases. The presence of distinct GFAP-isoforms in various cell types, developmental stages, and diseases indicates that GFAP (post-)transcriptional regulation has a role in glial cell physiology and pathology. GFAP-isoforms differ in sub-cellular localisation, IF-network assembly properties, and IF-dynamics which results in distinct molecular interactions and mechanical properties of the IF-network. Therefore, GFAP (post-)transcriptional regulation is likely a mechanism by which radial glia, astrocytes, and glioma cells can modulate cellular function., Competing Interests: Declaration of competing interest Nothing to declare., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Astrocytes constitute a major part of the central nervous system and the delineation of their activity patterns is conducive to a better understanding of brain network dynamics. This study aimed to develop a magnetic resonance imaging (MRI)-based method in order to monitor the brain-wide or region-specific astrocytes in live animals. Adeno-associated virus (AAVs) vectors carrying the human glial fibrillary acidic protein (GFAP) promoter driving the EGFP-AQP1 (Aquaporin-1, an MRI reporter) fusion gene were employed. The following steps were included: constructing recombinant AAV vectors for astrocyte-specific expression, detecting MRI reporters in cell culture, brain regions, or whole brain following cell transduction, stereotactic injection, or tail vein injection. The astrocytes were detected by both fluorescent imaging and Diffusion-weighted MRI. The novel AAV mutation (Site-directed mutagenesis of surface-exposed tyrosine (Y) residues on the AAV5 capsid) significantly increased fluorescence intensity (p < 0.01) compared with the AAV5 wild type. Transduction of the rAAV2/5 carrying AQP1 induced the titer-dependent changes in MRI contrast in cell cultures (p < 0.05) and caudate-putamen (CPu) in the brain (p < 0.05). Furthermore, the MRI revealed a good brain-wide alignment between AQP1 levels and ADC signals, which increased over time in most of the transduced brain regions. In addition, the rAAV2/PHP.eB serotype efficiently introduced AOP1 expression in the whole brain via tail vein injection. This study provides an MRI-based approach to detect dynamic changes in astrocytes in live animals. The novel in vivo tool could help us to understand the complexity of neuronal and glial networks in different pathophysiological conditions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

We describe three cases of overlapping Epstein-Barr virus (EBV) Encephalitis and Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy (GFAP-A). The three cases all presented with initial symptoms of fever, headache, coma, and posture tremor of the upper limbs, then followed by limb weakness and dysuria. All of the three cases were on ventilators. Case 1 and 2 improved dramatically after intravenous methylprednisoloneand immunoglobulin treatment. However, case 3 presented dyspneic, and died from gastrointestinal hemorrhage. The GFAP-A triggered by EBV intracranial infection could initially masquerade as EBV encephalitis only, and the detection of GFAP antibody is essential for differentiation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

Acute brain injuries evoke various response cascades directing the formation of the glial scar. Here, we report that acute lesions associated with hemorrhagic injuries trigger a re-programming of oligodendrocytes. Single-cell RNA sequencing highlighted a subpopulation of oligodendrocytes activating astroglial genes after acute brain injuries. By using PLP-DsRed1/GFAP-EGFP and PLP-EGFP mem /GFAP-mRFP1 transgenic mice, we visualized this population of oligodendrocytes that we termed AO cells based on their concomitant activity of astro- and oligodendroglial genes. By fate mapping using PLP- and GFAP-split Cre complementation and repeated chronic in vivo imaging with two-photon laser-scanning microscopy, we observed the conversion of oligodendrocytes into astrocytes via the AO cell stage. Such conversion was promoted by local injection of IL-6 and was diminished by IL-6 receptor-neutralizing antibody as well as by inhibiting microglial activation with minocycline. In summary, our findings highlight the plastic potential of oligodendrocytes in acute brain trauma due to microglia-derived IL-6., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hypothalamic astrocytes are particularly affected by energy-dense food consumption. How the anatomical location of these glial cells and their spatial molecular distribution in the arcuate nucleus of the hypothalamus (ARC) determine the cellular response to a high caloric diet remains unclear. In this study, we investigated their distinctive molecular responses following exposure to a high-fat high-sugar (HFHS) diet, specifically in the ARC. Using RNA sequencing and proteomics, we showed that astrocytes have a distinct transcriptomic and proteomic profile dependent on their anatomical location, with a major proteomic reprogramming in hypothalamic astrocytes. By ARC single-cell sequencing, we observed that a HFHS diet dictates time- and cell- specific transcriptomic responses, revealing that astrocytes have the most distinct regulatory pattern compared to other cell types. Lastly, we topographically and molecularly characterized astrocytes expressing glial fibrillary acidic protein and/or aldehyde dehydrogenase 1 family member L1 in the ARC, of which the abundance was significantly increased, as well as the alteration in their spatial and molecular profiles, with a HFHS diet. Together, our results provide a detailed multi-omics view on the spatial and temporal changes of astrocytes particularly in the ARC during different time points of adaptation to a high calorie diet., (© 2022 The Authors. GLIA published by Wiley Periodicals LLC.)

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N -acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.

Physical exercise is considered an adjuvant treatment to Parkinson's disease (PD) patients, possibly reducing inflammatory responses in the brain. Studies have stated that physical exercise protects dopaminergic neurons in PD models produced by neurotoxins. However, few studies focused on immunohistochemically reacted astrocytes and morphometric analyses of these cells in a PD mouse model submitted to physical exercise. We investigated the effects of treadmill training on striatal astrocytes of a PD mouse model combining immunohistochemistry and western-blotting for glial fibrillary acidic protein (GFAP) with morphometric analyses. Male Swiss mice were divided into 4 groups: sedentary control (SEDCONT), exercise control (EXERCONT), sedentary Parkinson (SEDPD), and exercise Parkinson (EXERPD). Stereotaxic bilateral injections of 6-hydroxydopamine into the striatum were adopted for PD groups. Striatal astrocytes showed increased GFAP in EXERPD, and we observed a higher level of GFAP in EXERPD than SEDPD. The number of primary and secondary processes was similar in striatal astrocytes of control groups and EXERPD. The astrocyte primary processes of SEDPD were larger than those of EXERPD, EXERCONT and SEDCONT. Cell body diameters and areas showed no difference between groups. We concluded that physical exercise influences striatal astrocytes in exercised parkinsonian mice., (Copyright © 2022 Elsevier B.V. All rights reserved.)

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mitochondrial superoxide (O 2 - ) production is implicated in aging, neurodegenerative disease, and most recently epilepsy. Yet the specific contribution of neuronal O 2 - to these phenomena is unclear. Here, we selectively deleted superoxide dismutase-2 (SOD2) in neuronal basic helix-loop-helix transcription factor (NEX)-expressing cells restricting deletion to a subset of excitatory principle neurons primarily in the forebrain (cortex and hippocampus). This resulted in nSOD2 KO mice that lived into adulthood (2-3 months) with epilepsy, selective loss of neurons, metabolic rewiring and a marked mitohormetic gene response. Surprisingly, expression of an astrocytic gene, glial fibrillary acidic protein (GFAP) was significantly increased relative to WT. Further studies in rat primary neuron-glial cultures showed that increased mitochondrial O 2 - , specifically in neurons, was sufficient to upregulate GFAP. These results suggest that neuron-specific mitochondrial O 2 - is sufficient to drive a complex and catastrophic epileptic phenotype and highlights the ability of SOD2 to act in a cell-nonautonomous manner to influence an astrocytic response., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Astrocytes act as neural stem cells (NSCs) that have the potential to self-renew and differentiate into other neuronal cells. The protein expression of these astrocytes depends on the stage of differentiation, showing sequential expression of multiple proteins such as octamer-binding transcription factor 4 (Oct4), nestin, glial fibrillary acidic protein (GFAP), and aldehyde dehydrogenase 1 family member L1 (aldh1L1). Photobiomodulation (PBM) affects cell apoptosis, proliferation, migration, and adhesion. We hypothesized that astrocyte proliferation and differentiation would be modulated by PBM. We used an optimized astrocyte culture method and a 660-nanometer light-emitting diode (LED) to enhance the biological actions of many kinds of cells. We determined that the 660-nanometer LED promoted the biological actions of cultured astrocytes by increasing the reactive oxygen species levels. The overall viability of the cultured cells, which included various cells other than astrocytes, did not change after LED exposure; however, astrocyte-specific proliferation was observed by the increased co-expression of GFAP and bromodeoxyuridine (BrdU)/Ki67. Furthermore, the 660-nanometer LED provides evidence of differentiation, as shown by the decreased Oct4 and GFAP co-expression and increased nestin and aldh1L1 expression. These results demonstrate that a 660-nanometer LED can modify astrocyte proliferation, which suggests the efficacy of the therapeutic application of LED in various pathological states of the central nervous system.

Astrocytes are the most abundant cell type in the central nervous system (CNS) and their major function is to maintain homeostasis of the CNS by exerting various functions. Simultaneously, reactive astrocytes are well known to be involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD). Reactive astrocytes, induced by amyloid beta peptide (Aβ), the main component of the neuritic plaques found in AD, induce neuroinflammation, producing cytokines that lead to neuronal cell death in AD. Phloroglucinol,a polyphenol monomer and a component of phlorotannin, is found at sufficient levels in Ecklonia cava of the Laminariaceae family. Recently, several studies have reported that phloroglucinol has the ability to trap free radicals in lung fibroblasts or cancer cells. However, the effects of phloroglucinol in astrocytes have not yet been studied. Here, we found that phloroglucinol inhibits the generation of ROS induced by oligomeric Aβ 1 - ) treatment in primary astrocytes. Futhermore, phloroglucinol was shown to ameliorate the protein expression of glial fibrillary acidic protein, a marker of reactive astrocytes, after treatment with oAβ 42 . These results indicate that phloroglucinol exerts antioxidant effects in primary cultured astrocytes and attenuates the astrocytic activation induced by oAβ 1 - 42 ) treatment in primary astrocytes. Futhermore, phloroglucinol was shown to ameliorate the protein expression of glial fibrillary acidic protein, a marker of reactive astrocytes, after treatment with oAβ 1 - 42 . These results indicate that phloroglucinol exerts antioxidant effects in primary cultured astrocytes and attenuates the astrocytic activation induced by oAβ 1 - 42 ., Competing Interests: Declaration of competing interest Not applicable., (Copyright © 2021 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases., Competing Interests: Competing interest statement: E.K. is listed as inventor on pending patent covering the targeting of cysteine-291 in glial fibrillary acidic protein as intervention strategy for treating neurodegenerative diseases., (Copyright © 2021 the Author(s). Published by PNAS.)

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75 NTR ). The activation of p75 NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75 NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75 NTR , in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75 NTR . Infected mice showed higher abundance of p75 NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75 NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75 NTR -mediated neurodegeneration. In contrast, p75 NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75 NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Objective: Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation., Methods: Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR)., Results: Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation in vivo and in vitro . Antimony exposure consistently upregulated the expression of inflammatory factors. Moreover, it induced the NF-κB signaling, indicated by increased p65 phosphorylation and translocation to the nucleus. NF-κB inhibition effectively attenuated antimony-induced astrocyte activation. Furthermore, antimony phosphorylated TGF-β-activated kinase 1 (TAK1), while TAK1 inhibition alleviated antimony-induced p65 phosphorylation and subsequent astrocyte activation., Conclusion: Antimony activated astrocytes by activating the NF-κB signaling pathway., (Copyright © 2020 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Expression of the GFAP gene has attracted considerable attention because its onset is a marker for astrocyte development, its upregulation is a marker for reactive gliosis, and its predominance in astrocytes provides a tool for their genetic manipulation. The literature on GFAP regulation is voluminous, as almost any perturbation of development or homeostasis in the CNS will lead to changes in its expression. In this review, we limit our discussion to mechanisms proposed to regulate GFAP synthesis through a direct interaction with its gene or mRNA. Strengths and weaknesses of the supportive experimental findings are described, and suggestions made for additional studies. This review covers 15 transcription factors, DNA and histone methylation, and microRNAs. The complexity involved in regulating the expression of this intermediate filament protein suggests that GFAP function may vary among both astrocyte subtypes and other GFAP-expressing cells, as well as during development and in response to perturbations.

Objectives: Postherpetic neuralgia (PHN) is the most common complication of herpes zoster, but the mechanism of PHN is still unclear. Activation of spinal astrocytes is involved in PHN. Our study aims to explore whether lncRNA KCNA2 antisense RNA (KCNA2-AS) regulates spinal astrocytes in PHN through signal transducers and activators of transcription 3 (STAT3)., Methods: Varicella zoster virus (VZV)-infected CV-1 cells were injected into rats to construct a PHN model. Primary spinal cord astrocytes were activated using S-Nitrosoglutathione (GSNO). Glial fibrillary acidic protein (GFAP; marker of astrocyte activation), phosphorylated STAT3 (pSTAT3), and KCNA2-AS were analyzed by immunofluorescence and RNA fluorescence in situ hybridization. RNA pull-down and RNA immunoprecipitation were used to detect binding of KCNA2-AS to pSTAT3., Results: KCNA2-AS was highly expressed in the spinal cord tissue of PHN model rats, and was positively correlated with GFAP expression. GFAP was significantly increased in GSNO-induced cells, but the knockdown of KCNA2-AS reversed this result. Meanwhile, pSTAT3 was significantly increased in GSNO-induced cells, but knockdown of KCNA2-AS reduced pSTAT3 within the nucleus while the total pSTAT3 did not change significantly. pSTAT3 bound to KCNA2-AS and this binding increased with GSNO treatment. Furthermore, knockdown of KCNA2-AS in PHN model rats relieved mechanical allodynia., Conclusion: Down-regulation of KCNA2-AS alleviates PHN partly by reducing the translocation of pSTAT3 cytoplasm to the nucleus and then inhibiting the activation of spinal astrocytes.

Astrocyte activation is one of the crucial hallmarks of Alzheimer's disease (AD) along with amyloid-β (Aβ) plaques, neurofibrillary tangles and neuron death. Glial scar and factors secreted from activated astrocytes have important contribution on neuronal health in AD. In this study, we investigated the mechanisms of astrocyte activation both in in vitro and in vivo models of AD. In this regard, mitogen activated protein kinase (MAPK) signalling cascades that control several fundamental and stress related cellular events, has been implicated in astrocyte activation in various neurological diseases. We checked activation of different MAPKs by western blot and immunocytochemistry and found that both JNK and p38K, but not ERK pathways are activated in Aβ-treated astrocytes in culture and in Aβ-infused rat brain cortex. Next, to investigate the downstream consequences of these two MAPKs (JNK and p38K) in Aβ-induced astrocyte activation, we individually blocked these pathways by specific inhibitors in presence and absence of Aβ and checked Aβ-induced cellular proliferation, morphological changes and glial fibrillary acidic protein (GFAP) upregulation. We found that activation of both JNK and p38K signalling cascades are involved in astrocyte proliferation evoked by Aβ, whereas only p38K pathway is implicated in morphological changes and GFAP upregulation in astrocytes exposed to Aβ. To further validate the implication of p38K pathway in Aβ-induced astrocyte activation, we also observed that transcription factor ATF2, a downstream phosphorylation substrate of p38, is phosphorylated upon Aβ treatment. Taken together, our study indicates that p38K and JNK pathways mediate astrocyte activation and both the pathways are involved in cellular proliferation but only p38K pathway contributes in morphological changes triggered by Aβ., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The important role of astrocytes in the central control of energy balance and glucose homeostasis has recently been recognized. Changes in thermoregulation can lead to metabolic dysregulation, but the role of astrocytes in this process is not yet clear. Therefore, we generated mice congenitally lacking insulin receptors (Ir) in astrocytes (IrKOGFAP mice) to investigate the involvement of astrocyte insulin signaling. IrKOGFAP mice displayed significantly lower energy expenditure and a strikingly lower basal and fasting body temperature. When exposed to cold, however, they were able to mount a thermogenic response. IrKOGFAP mice displayed sex differences in metabolic function and thermogenesis that may contribute to the development of obesity and type II diabetes as early as 2 months of age. While brown adipose tissue exhibited higher adipocyte size in both sexes, more apoptosis was seen in IrKOGFAP males. Less innervation and lower BAR3 expression levels were also observed in IrKOGFAP brown adipose tissue. These effects have not been reported in models of astrocyte Ir deletion in adulthood. In contrast, body weight and glucose regulatory defects phenocopied such models. These findings identify a novel role for astrocyte insulin signaling in the development of normal body temperature control and sympathetic activation of BAT. Targeting insulin signaling in astrocytes has the potential to serve as a novel target for increasing energy expenditure.

Astrocytes are major glial cells critically in maintaining stability of the central nervous system and functional activation of astrocytes occurs rapidly in various diseased or traumatic events. We are interested in functional changes of astrocytes during the spinal cord injury, and studied expression of nerve growth factor (NGF) in activated astrocytes by mouse model of contused spinal cord injury and cell culture experiment. It revealed that the spinal cord injury resulted in apparent activation of astrocytes and microglial cells and decreased BMS scores. A larger number of astrocytes showed immunoreactivity to proNGF in the injured spinal cord areas, and proNGF expression increased and remained high level at 7 to 14dpi, which was coincided with upregulation of glial fibrillary acidic protein. The proNGF was clearly localized in both exosome-like vesicles and cytoplasm of astrocytes in culture. Electron microscopy confirmed exosome-like vesicles with proNGF-immunoreactivity in diameter sizes of 50-100 nm. Finally, cell culture with lipopolysaccharide (LPS) experiment indicated increasing expression and release of proNGF in the astrocytes with LPS exposure. This study demonstrated that reactive astrocytes increased proNGF expression after spinal cord injury, also suggesting involvement of exosome-like proNGF transport or release in triggering neuronal apoptosis and aggravating progression of spinal cord injury., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

We studied spatial organization and structural characteristics of striatal glial cells in spontaneously hypertensive rats (SHR) in 48 h after 30-min focal ischemia. Immunocytochemical analysis of nestin and glial fibrillar acidic protein (GFAP) revealed 3 types of activated astrocytes: expressing only nestin, only GFAP, or both markers. There were no nestin-immunopositive astrocytes in the striatum of sham-operated rats. In cells expressing nestin and GFAP, localization of these markers did not completely coincide, which can be explained by different functions of these proteins or formation of heterodimers of nestin with other intermediate filament proteins.

The role of astrocytes in the progression of Alzheimer's disease (AD) remains poorly understood. We assessed the consequences of ablating astrocytic proliferation in 9 months old double transgenic APP23/GFAP-TK mice. Treatment of these mice with the antiviral agent ganciclovir conditionally ablates proliferating reactive astrocytes. The loss of proliferating astrocytes resulted in significantly increased levels of monomeric amyloid-β (Aβ) in brain homogenates, associated with reduced enzymatic degradation and clearance mechanisms. In addition, our data revealed exacerbated memory deficits in mice lacking proliferating astrocytes concomitant with decreased levels of synaptic markers and higher expression of pro-inflammatory cytokines. Our data suggest that loss of reactive astrocytes in AD aggravates amyloid pathology and memory loss, possibly via disruption of amyloid clearance and enhanced neuroinflammation., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

The microenvironment and architecture of peritumoral tissue have been suggested to affect permissiveness for infiltration of malignant cells. Astrocytes constitute a heterogeneous population of cells and have been linked to proliferation, migration, and drug sensitivity of glioblastoma (GBM) cells. Through double-immunohistochemical staining for platelet-derived growth factor receptor α (PDGFRα) and glial fibrillary acidic protein (GFAP), this study explored the intercase variability among 45 human GBM samples regarding density of GFAP+ peritumoral astrocytes and a subset of GFAP+ peritumoral astrocyte-like cells also expressing PDGFRα. Large intercase variability regarding the total peritumoral astrocyte density and the density of PDGFRα+/GFAP+ peritumoral astrocyte-like cells was detected. DNA fluorescence in situ hybridization analyses for commonly altered genetic tumor markers supported the interpretation that these cells represented a genetically unaffected host cell subset referred to as PDGFRα+/GFAP+ peritumoral astrocytes. The presence of PDGFRα+/GFAP+ peritumoral astrocytes was significantly positively correlated to older patient age and peritumoral astrocyte density, but not to other established prognostic factors. Notably, presence of PDGFRα+/GFAP+ peritumoral astrocytes, but not peritumoral astrocyte density, was associated with significantly shorter patient overall survival. The prognostic association of PDGFRα+/GFAP+ peritumoral astrocytes was confirmed in multivariable analyses. This exploratory study thus demonstrates previously unrecognized intercase variability and prognostic significance of peritumoral abundance of a novel PDGFRα+ subset of GFAP+ astrocytes. Findings suggest clinically relevant roles of the microenvironment of peritumoral GBM tissue and encourage further characterization of the novel astrocyte subset with regard to origin, function, and potential as biomarker and drug target., (© 2019 Wiley Periodicals, Inc.)

Glial fibrillary acidic protein (GFAP), a type III intermediate filament, is a marker of mature astrocytes. The expression of GFAP gene is regulated by many transcription factors (TFs), mainly Janus kinase-2/signal transducer and activator of transcription 3 cascade and nuclear factor κ-light-chain-enhancer of activated B cell signaling. GFAP expression is also modulated by protein kinase and other signaling molecules that are elicited by neuronal activity and hormones. Abnormal expression of GFAP proteins occurs in neuroinflammation, neurodegeneration, brain edema-eliciting diseases, traumatic brain injury, psychiatric disorders and others. GFAP, mainly in α-isoform, is the major component of cytoskeleton and the scaffold of astrocytes, which is essential for the maintenance of astrocytic structure and shape. GFAP also has highly morphological plasticity because of its quick changes in assembling and polymerizing states in response to environmental challenges. This plasticity and its corresponding cellular morphological changes endow astrocytes the functions of physical barrier between adjacent neurons and stabilizer of extracellular environment. Moreover, GFAP colocalizes and even molecularly associates with many functional molecules. This feature allows GFAP to function as a platform for direct interactions between different molecules. Last, GFAP involves transportation and localization of other functional proteins and thus serves as a protein transport guide in astrocytes. This guiding role of GFAP involves an elastic retraction and extension cytoskeletal network that couples with GFAP reassembling, transporting, and membrane protein recycling machinery. This paper reviews our current understanding of the expression and functions of GFAP as well as their regulation., (© 2019 Wiley Periodicals, Inc.)

Astrocytes respond to central nervous system (CNS) insults with varieties of changes, such as cellular hypertrophy, migration, proliferation, scar formation, and upregulation of glial fibrillary acidic protein (GFAP) expression. While scar formation plays a very important role in wound healing and prevents further bleeding by forming a physical barrier, it is also one of key features of CNS injury, resulting in glial scar formation (astrogliosis), which is closely related to treatment resistant epilepsy, chronic pain, and other devastating diseases. Therefore, slowing the astrocytic activation process may give a time window of axonal growth after the CNS injury. However, the underlying mechanism of astrocytic activation remains unclear, and there is no effective therapeutic strategy to attenuate the activation process. Here, we found that methimazole could effectively inhibit the GFAP expression in physiological and pathological conditions. Moreover, we scratched primary cultures of cerebral cortical astrocytes with and without methimazole pretreatment and investigated whether methimazole could slow the healing process in these cultures. We found that methimazole could inhibit the GFAP protein expression in scratched astrocytes and prolong the latency of wound healing in cultures. We also measured the phosphorylation of extracellular signal-regulated kinase (ERK) in these cultures and found that methimazole could significantly inhibit the scratch-induced GFAP upregulation. For the first time, our study demonstrated that methimazole might be a possible compound that could inhibit the astrocytic activation following CNS injury by reducing the ERK phosphorylation in astrocytes., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Lina Zhao et al.)

Astrocytes are morphologically complex and use local translation to regulate distal functions. To study the distribution of mRNA in astrocytes, we combined mRNA detection via in situ hybridization with immunostaining of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). mRNAs at the level of GFAP-immunolabelled astrocyte somata, and large and fine processes were analysed using AstroDot, an ImageJ plug-in and the R package AstroStat. Taking the characterization of mRNAs encoding GFAP-α and GFAP-δ isoforms as a proof of concept, we showed that they mainly localized on GFAP processes. In the APPswe/PS1dE9 mouse model of Alzheimer's disease, the density and distribution of both α and δ forms of Gfap mRNA changed as a function of the region of the hippocampus and the astrocyte's proximity to amyloid plaques. To validate our method, we confirmed that the ubiquitous Rpl4 (large subunit ribosomal protein 4) mRNA was present in astrocyte processes as well as in microglia processes immunolabelled for ionized calcium binding adaptor molecule 1 (Iba1; also known as IAF1). In summary, this novel set of tools allows the characterization of mRNA distribution in astrocytes and microglia in physiological or pathological settings., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Chronic glial activation is characterized by increased numbers of activated glial cells, secreting free radicals and cytotoxic cytokines, subsequently causing neuronal damage. In order to investigate the anti-inflammatory activity of Longvida ® Optimised Curcumin (LC), we fed 500 ppm of LC to 2-month-old wild type and GFAP-IL6 mice for 6 months. LC feeding led to a significant reduction in the number of Iba-1 + microglia by 26% in the hippocampus and by 48% in the cerebellum, GFAP + astrocytes by 30%, and TSPO + cells by 24% in the hippocampus and by 31% in the cerebellum of the GFAP-IL6 mice. The morphology of the cells was assessed and LC significantly decreased the dendritic length of microglia and the convex area, convex perimeter, dendritic length, nodes and number of processes of astrocytes in the hippocampus while decreasing the soma area and perimeter in the cerebellum, in LC-fed GFAP-IL6 mice. In addition, LC feeding increased pre- and postsynaptic protein levels and improved balance measured by Rotarod. Together, these data suggest that LC is able to attenuate the inflammatory pathology and ameliorate neurodegeneration and motor deficits in GFAP-IL6 mice. For patients with neuro-inflammatory disorders, LC might potentially reverse the detrimental effects of chronic glial activation.

Fifty years have passed since the discovery of glial fibrillary acidic protein (GFAP) by Lawrence Eng and colleagues. Now recognized as a member of the intermediate filament family of proteins, it has become a subject for study in fields as diverse as structural biology, cell biology, gene expression, basic neuroscience, clinical genetics and gene therapy. This review covers each of these areas, presenting an overview of current understanding and controversies regarding GFAP with the goal of stimulating continued study of this fascinating protein.

Lead is a main threat to human health due to its neurotoxicity and the astrocyte is known to be a common deposit site of lead in vivo. However, the detailed mechanisms related to lead exposure in the astrocytes were unclear. In order to deeply investigate this issue, we used Sprague-Dawley (SD) rats and astrocytes isolated from the hippocampus of SD rats to establish the lead-exposed animal and cell models through treating with lead acetate. The expression levels of GFAP, LC3, and p62 in the rat hippocampus were detected by immunofluorescence and Western blot after lead exposure. The effects of autophagy on lead-exposed astrocytes were studied by further autophagy inhibitor 3-methyladenine (3-MA) induction. Transmission electron microscopy was used to observe autophagosomes in astrocytes after lead acetate treatment, followed by assessing related autophagy protein markers. In addition, some inflammatory cytokines and oxidative stress markers were also evaluated after lead exposure and 3-MA administration. We found that lead exposure induced activation of astrocytes, as evidenced by increased GFAP levels and GFAP-positive staining cells in the rat hippocampus. Moreover, lead exposure induced autophagy in astrocytes, as evidenced by increased LC3II and Beclin 1 protein levels and decreased p62 expression in both the rat hippocampus and astrocytes, and it was confirmed that this autophagy was activated through blocking the downstream Akt/target of the rapamycin (mTOR) pathway in astrocytes. Furthermore, it was shown that treatment of lead acetate increased the release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and the accumulation of malondialdehyde (MDA) and myeloperoxidase (MPO) in astrocytes, which could be alleviated by further 3-MA induction. Therefore, we conclude that lead exposure can induce the autophagy of astrocytes via blocking the Akt/mTOR pathway, leading to accelerated release of inflammatory factors and oxidative stress indicators in astrocytes.

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network., Competing Interests: The authors have declared that no competing interests exist.

The accumulation of senescent cells in tissues is causally linked to the development of several age-related diseases; the removal of senescent glial cells in animal models prevents Tau accumulation and cognitive decline. Senescent cells can arise through several distinct mechanisms; one such mechanism is dysregulation of alternative splicing. In this study, we characterised the senescent cell phenotype in primary human astrocytes in terms of SA-β-Gal staining and SASP secretion, and then assessed splicing factor expression and candidate gene splicing patterns. Finally, we assessed associations between expression of dysregulated isoforms and premature cognitive decline in 197 samples from the InCHIANTI study of ageing, where expression was present in both blood and brain. We demonstrate here that senescent astrocytes secrete a modified SASP characterised by increased IL8, MMP3, MMP10, and TIMP2 but decreased IL10 levels. We identified significant changes in splicing factor expression for 10/20 splicing factors tested in senescent astrocytes compared with early passage cells, as well as dysregulation of isoform levels for 8/13 brain or senescence genes tested. Finally, associations were identified between peripheral blood GFAPα, TAU3, and CDKN2A (P14 ARF ) isoform levels and mild or severe cognitive decline over a 3-7-year period. Our data are suggestive that some of the features of cognitive decline may arise from dysregulated splicing of important genes in senescent brain support cells, and that defects in alternative splicing or splicing regulator expression deserve exploration as points of therapeutic intervention in the future.

Aim: Thalidomide is one of the first line therapies in cancer pain management. Previous study has shown that thalidomide decreases the expression of tumor necrosis factor alpha in the mouse spinal cord. However, the exact cellular and molecular mechanism underlying the effect of thalidomide remains unclear. Here, we investigated the effect of thalidomide on the expression level of NF-κB as well as glial fibrillary acidic protein (GFAP) in the spinal cord astrocyte in a mice model. Materials and methods: MC57G fibrosarcoma cells were intramedullary injected into the right femurs of C57/BL mice to induce behaviors related to bone cancer pain. Postoperative thalidomide was administered intraperitoneally to the mice at dose of 100 mg/kg/day for 7 days. The effect of thalidomide on pain hypersensitivity was checked by behavioral testing. The expression levels of NF-κB and GFAP in spinal cord were evaluated by using Western blotting and Immunohistochemistry. Results: Compared with the controls, the tumor-bearing mice showed substantial pain-related behaviors. Furthermore, the expression levels of both NF-κB and GFAP increased significantly in the spinal cord astrocytes of the tumor-bearing mice. Treating the tumor-bearing mice with thalidomide results in a dramatic reduction in pain behaviors and a significant decrease of NF-κB and GFAP expressions. Conclusions: Thalidomide alleviates the pain behaviors probably by down-regulating the expression of NF-κB and GFAP.

Vimentin (VIM) is an intermediate filament (nanofilament) protein expressed in multiple cell types, including astrocytes. Mice with VIM mutations of serine sites phosphorylated during mitosis (VIM SA/SA ) show cytokinetic failure in fibroblasts and lens epithelial cells, chromosomal instability, facilitated cell senescence, and increased neuronal differentiation of neural progenitor cells. Here we report that in vitro immature VIM SA/SA astrocytes exhibit cytokinetic failure and contain vimentin accumulations that co-localize with mitochondria. This phenotype is transient and disappears with VIM SA/SA astrocyte maturation and expression of glial fibrillary acidic protein (GFAP); it is also alleviated by the inhibition of cell proliferation. To test the hypothesis that GFAP compensates for the effect of VIM SA/SA in astrocytes, we crossed the VIM SA/SA and GFAP -/- mice. Surprisingly, the fraction of VIM SA/SA immature astrocytes with abundant vimentin accumulations was reduced when on GFAP -/- background. This indicates that the disappearance of vimentin accumulations and cytokinetic failure in mature astrocyte cultures are independent of GFAP expression. Both VIM SA/SA and VIM SA/SA GFAP -/- astrocytes showed normal mitochondrial membrane potential and vulnerability to H 2 O 2 , oxygen/glucose deprivation, and chemical ischemia. Thus, mutation of mitotic phosphorylation sites in vimentin triggers formation of vimentin accumulations and cytokinetic failure in immature astrocytes without altering their vulnerability to oxidative stress., Competing Interests: The authors declare no conflict of interest.

Knowledge on trauma survival time prior to death following a lethal traumatic brain injury (TBI) may be essential for legal purposes. Immunohistochemistry studies might allow to narrow down this survival interval. The biomarkers interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) are well known in the clinical setting for their usability in TBI prediction. Here, both proteins were chosen in forensics to determine whether neuronal or glial expression in various brain regions may be associated with the cause of death and the survival time prior to death following TBI. IL-6 positive neurons, glial cells and GFAP positive astrocytes all concordantly increase with longer trauma survival time, with statistically significant changes being evident from three days post-TBI (p < 0.05) in the pericontusional zone, irrespective of its definite cortical localization. IL-6 staining in neurons increases significantly in the cerebellum after trauma, whereas increasing GFAP positivity is also detected in the cortex contralateral to the focal lesion. These systematic chronological changes in biomarkers of pericontusional neurons and glial cells allow for an estimation of trauma survival time. Higher numbers of IL-6 and GFAP-stained cells above threshold values in the pericontusional zone substantiate the existence of fatal traumatic changes in the brain with reasonable certainty.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP) + astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5 + /GFAP + , and serglycin + /GFAP + cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Astrocyte is considered to be a type of passive supportive cells that preserves neuronal activity and survival. The dysfunction of astrocytes is involved in the pathological processes of major depression. Recent studies implicate sigma-1 receptors as putative therapeutic targets for current available antidepressant drugs. However, it is absent of direct evidences whether sigma-1 receptor could promote activation of astrocyte. In the present study, we took advantage of primary astrocyte culture and a highly selective agonist of sigma-1 receptor, (+)SKF-10047 to determine the effect of sigma-1 receptor on Brdu (bromodeoxyuridine) labeling positive cells, migration as well as GFAP (glial fibrillary acidic protein) expression of astrocyte. The results showed that (+)SKF-10047 notably increased the number of Brdu labeling positive cells, migration, and the expression of GFAP in primary astrocytes, which were blocked by antagonist of sigma-1 receptor. Moreover, we also found that (+)SKF-10047 increased the phosphorylation of ERK1/2 (extracellular signal-regulated kinases 1/2) and GSK3β (glycogen synthase kinase 3β) (ser 9) in the primary astrocytes. In addition, pharmacological inhibition of ERK1/2 and GSK3β (ser 9) abolished sigma-1 receptor-promoted activation of astrocyte. Therefore, sigma-1 receptor could be considerate as a new pattern for modulating astrocytic function might emerge as therapeutic strategies.

Enhanced glial fibrillary acidic protein (GFAP) expression occurs in most diseases of the central nervous system. Thus far, little is known about the effect that GFAP exerts on astrocyte cell signaling. In the present study, we observed that silencing GFAP expression in isolated astrocytes leads to enhanced CCL2 and CXCL10 release, whereas overexpression of GFAP in astrocytes results in a significantly reduced CXCL10 release in vitro. Additionally, we analyzed transgenic mice carrying a full-length copy of the wild-type human GFAP gene. We demonstrate that a persistent GFAP increase alters the astrocytic cell signaling profile, thereby protecting oligodendrocytes, myelin and, subsequently, axons from cuprizone-induced demyelination. Our study revealed that reduced CXCL10 mRNA was accompanied by reduced NF-κB expression in astrocytes. Furthermore, analysis of human tissue from a patient with Alexander disease showed NF-κB activation in astrocytes to be almost completely absent. Our findings indicate that regulation of GFAP expression in astrocytes is crucial for astrocyte signaling and function. Understanding the role of the cytoskeletal protein, GFAP is thus of importance as it is highly regulated in diseases of the central nervous system., (© 2019 Wiley Periodicals, Inc.)

Adeno-associated viral (AAV) vectors are a promising system for transgene delivery into the central nervous system (CNS) based on their safety profile and long-term gene expression. Gene delivery to the CNS has largely been neuron centric but advances in AAV technology are facilitating the development of approaches to enable transduction of glial cells. Considering the role of astrocytes in the on-going secondary damage in spinal cord injury (SCI), an AAV vector that targets astrocytes could show benefit as a potential treatment. Transduction efficiency, transgene expression and cellular tropism were compared for the AAV serotypes AAV5, AAV9 and AAVRec2 whereby destabilised yellow fluorescent protein (dYFP) was controlled by the GFAP or the truncated GfaABC 1 D promoter. The vectors were tested in primary spinal cord astrocyte cell culture, spinal cord slice culture and an in vivo model of SCI contusion. AAV5 resulted in greater transduction efficiency, transgene expression and astrocyte tropism compared with AAV9 and AAVRec2. In a rodent model of SCI, robust transgene expression by AAV5-GFAP/GfaABC 1 D-dYFP was observed through 12 mm of spinal cord tissue and expression was largely restricted to astrocytes. Thus, AAV5-GFAP/GfaABC 1 D carries the potential as a potential gene therapy vector, particularly for transducing astrocytes in the damaged spinal cord.

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca 2+ signaling. Using the genetically encoded Ca 2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca 2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies., (© 2018 Wiley Periodicals, Inc.)

Reovirus encephalitis in mice was used as a model system to investigate astrocyte activation (astrogliosis) following viral infection of the brain. Reovirus infection resulted in astrogliosis, as evidenced by increased expression of glial fibrillary acidic protein (GFAP), and the upregulation of genes that have been previously associated with astrocyte activation. Astrocyte activation occurred in regions of the brain that are targeted by reovirus but extended beyond areas of active infection. Astrogliosis also occurred following reovirus infection of ex vivo brain slice cultures (BSCs), demonstrating that factors intrinsic to the brain are sufficient to activate astrocytes and that this process can occur in the absence of any contribution from the peripheral immune response. In agreement with previous reports, reovirus antigen did not colocalize with GFAP in infected brains, suggesting that reovirus does not infect astrocytes. Reovirus-infected neurons produce interferon beta (IFN-β). IFN-β treatment of primary astrocytes resulted in both the upregulation of GFAP and cytokines that are associated with astrocyte activation. In addition, the ability of media from reovirus-infected BSCs to activate primary astrocytes was blocked by anti-IFN-β antibodies. These results suggest that IFN-β, likely released from reovirus-infected neurons, results in the activation of astrocytes during reovirus encephalitis. In areas where infection and injury were pronounced, an absence of GFAP staining was consistent with activation-induced cell death as a mechanism of inflammation control. In support of this, activated Bak and cleaved caspase 3 were detected in astrocytes within reovirus-infected brains, indicating that activated astrocytes undergo apoptosis. IMPORTANCE Viral encephalitis is a significant cause of worldwide morbidity and mortality, and specific treatments are extremely limited. Virus infection of the brain triggers neuroinflammation; however, the role of neuroinflammation in the pathogenesis of viral encephalitis is unclear. Initial neuroinflammatory responses likely contribute to viral clearance, but prolonged exposure to proinflammatory cytokines released during neuroinflammation may be deleterious and contribute to neuronal death and tissue injury. Activation of astrocytes is a hallmark of neuroinflammation. Here, we show that reovirus infection of the brain results in the activation of astrocytes via an IFN-β-mediated process and that these astrocytes later die by Bak-mediated apoptosis. A better understanding of neuroinflammatory responses during viral encephalitis may facilitate the development of new treatment strategies for these diseases., (Copyright © 2019 American Society for Microbiology.)

Objective: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before., Results: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.

Background: HIV-associated neurocognitive disorders (HANDs) afflict more than half of HIV-1-positive individuals. The transactivator of transcription (Tat) produced by HIV virus elicits inflammatory process and is a major neurotoxic mediator that induce neuron damage during HAND pathogenesis. Activated astrocytes are important cells involved in neuroinflammation and neuronal damage. Purinergic receptors expressed in astrocytes participate in a positive feedback loop in virus-induced neurotoxicity. Here, we investigated that whether P2Y4R, a P2Y receptor subtype, that expressed in astrocyte participates in Tat-induced neuronal death in vitro and in vivo., Methods: Soluble Tat protein was performed to determine the expression of P2Y4R and proinflammatory cytokines in astrocytes using siRNA technique via real-time PCR, Western blot, and immunofluorescence assays. Cytometric bead array was used to measure proinflammatory cytokine release. The TUNEL staining and MTT cell viability assay were analyzed for HT22 cell apoptosis and viability, and the ApopTag® peroxidase in situ apoptosis detection kit and cresyl violet staining for apoptosis and death of hippocampal neuron in vivo., Results: We found that Tat challenge increased the expression of P2Y4R in astrocytes. P2Y4R signaling in astrocytes was involved in Tat-induced inflammatory cytokine production via PI3K/Akt- and ERK1/2-dependent pathways. Knockdown of P2Y4R expression significantly reduced inflammatory cytokine production and relieved Tat-mediated neuronal apoptosis in vitro. Furthermore, in vivo challenged with Tat, P2Y4R knockdown mice showed decreased inflammation and neuronal damage, especially in hippocampal CA1 region., Conclusions: Our data provide novel insights into astrocyte-mediated neuron damage during HIV-1 infection and suggest a potential therapeutic target for HANDs.

When central nervous system (CNS) homeostasis is altered, microglial cells become rapidly activated, proliferate and release a broad range of molecules. Among the plethora of molecules involved in the regulation of microglial activation, cytokines are considered crucial. Although production of interleukin-10 (IL-10) has been demonstrated after different types of CNS injuries and associated with protective functions, the specific role played by IL-10 modulating microglial cells remains unclear. Hence, the objective of this study was to evaluate the effects of transgenic astrocyte IL-10 production on microglial activation associated with axonal anterograde degeneration. To address it, the hippocampal area subjected to perforant pathway transection (PPT) was analyzed by immunohistochemistry (IHC), flow cytometry and protein microarray in transgenic (GFAP-IL10Tg) mice and their corresponding wild types (WT) littermates. Our results demonstrated increased microglial/macrophages density in nonlesioned and PPT-lesioned GFAP-IL10Tg animals when compared with nonlesioned and lesioned WT, respectively. This increase was not due to proliferation, as GFAP-IL10Tg mice showed a reduced proliferation of microglial cells, but was related to an increased population of CD11b+/CD45 high monocyte/macrophages. Despite this higher number, the microglia/macrophage population in transgenic animals displayed a downregulated phenotype characterized by lower MHCII, ICOSL, and CD11c. Moreover, a sustained T-cell infiltration was found in transgenic animals. We strongly suggest these modifications must be associated with indirect effects derived from the influence of IL-10 on astrocytes and/or neurons, which express IL-10R. We finally suggested that TGF-β produced by astrocytes, along with IL-2 and CXCL10 might be crucial molecules mediating the effects of transgenic IL-10., (© 2018 Wiley Periodicals, Inc.)