Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke

Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury.Low-density lipoprotein receptor,a classic cholesterol regulatory receptor,has been found to inhibit NLR family pyrin domain containing protein 3(NLRP3)inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer’s disease.However,little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke.To address this issue in the present study,we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models.First,we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis.We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation.Second,we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus.Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype.Finally,we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin,an NLRP3 agonist,restored the neurotoxic astrocyte phenotype.These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

Impacts of PI3K/protein kinase B pathway activation in reactive astrocytes: from detrimental effects to protective functions

Astrocytes are the most abundant type of glial cell in the central nervous system.Upon injury and inflammation,astrocytes become reactive and undergo morphological and functional changes.Depending on their phenotypic classification as A1 or A2,reactive astrocytes contribute to both neurotoxic and neuroprotective responses,respectively.However,this binary classification does not fully capture the diversity of astrocyte responses observed across different diseases and injuries.Transcriptomic analysis has revealed that reactive astrocytes have a complex landscape of gene expression profiles,which emphasizes the heterogeneous nature of their reactivity.Astrocytes actively participate in regulating central nervous system inflammation by interacting with microglia and other cell types,releasing cytokines,and influencing the immune response.The phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway is a central player in astrocyte reactivity and impacts various aspects of astrocyte behavior,as evidenced by in silico,in vitro,and in vivo results.In astrocytes,inflammatory cues trigger a cascade of molecular events,where nuclear factor-κB serves as a central mediator of the pro-inflammatory responses.Here,we review the heterogeneity of reactive astrocytes and the molecular mechanisms underlying their activation.We highlight the involvement of various signaling pathways that regulate astrocyte reactivity,including the PI3K/AKT/mammalian target of rapamycin(mTOR),αvβ3 integrin/PI3K/AKT/connexin 43,and Notch/PI3K/AKT pathways.While targeting the inactivation of the PI3K/AKT cellular signaling pathway to control reactive astrocytes and prevent central nervous system damage,evidence suggests that activating this pathway could also yield beneficial outcomes.This dual function of the PI3K/AKT pathway underscores its complexity in astrocyte reactivity and brain function modulation.The review emphasizes the importance of employing astrocyte-exclusive models to understand their functions accurately and these models are essential for clarifying astrocyte behavior.The findings should then be validated using in vivo models to ensure real-life relevance.The review also highlights the significance of PI3K/AKT pathway modulation in preventing central nervous system damage,although further studies are required to fully comprehend its role due to varying factors such as different cell types,astrocyte responses to inflammation,and disease contexts.Specific strategies are clearly necessary to address these variables effectively.

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress–induced anxiety-like behavior

The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions.Specifically, astrocytes in the basolateral amygdala have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which basolateral amygdala astrocytes regulate chronic stress–induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress, the expression of excitatory amino acid transporter 2 was upregulated in the basolateral amygdala. Interestingly, our findings indicate that the targeted knockdown of excitatory amino acid transporter 2 specifically within the basolateral amygdala astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of excitatory amino acid transporter 2 in the basolateral amygdala, whether achieved through intracranial administration of excitatory amino acid transporter 2agonists or through injection of excitatory amino acid transporter 2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of excitatory amino acid transporter 2 specifically in astrocytes in the basolateral amygdala. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the basolateral amygdala of mice subjected to chronic stress was higher compared with normal mice.After knocking down the expression of excitatory amino acid transporter 2 in the basolateral amygdala, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an excitatory amino acid transporter 2 inhibitor in the basolateral amygdala yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that basolateral amygdala astrocytic excitatory amino acid transporter 2 plays a role in in the regulation of unpredictable chronic mild stress-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting excitatory amino acid transporter 2 in the basolateral amygdala holds therapeutic promise for addressing anxiety disorders.

Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

Invasive inflammation and excessive scar formation are the main reasons for the difficulty in repairing nervous tissue after spinal cord injury.Microglia and astrocytes play key roles in the spinal cord injury micro-environment and share a close interaction.However,the mechanisms involved remain unclear.In this study,we found that after spinal cord injury,resting microglia(M0)were polarized into pro-inflammatory phenotypes(MG1 and MG3),while resting astrocytes were polarized into reactive and scar-forming phenotypes.The expression of growth arrest-specific 6(Gas6)and its receptor Axl were significantly down-regulated in microglia and astrocytes after spinal cord injury.In vitro experiments showed that Gas6 had negative effects on the polarization of reactive astrocytes and pro-inflammatory microglia,and even inhibited the cross-regulation between them.We further demonstrated that Gas6 can inhibit the polarization of reactive astrocytes by suppressing the activation of the Yes-associated protein signaling pathway.This,in turn,inhibited the polarization of pro-inflammatory microglia by suppressing the activation of the nuclear factor-κB/p65 and Janus kinase/signal transducer and activator of transcription signaling pathways.In vivo experiments showed that Gas6 inhibited the polarization of pro-inflammatory microglia and reactive astrocytes in the injured spinal cord,thereby promoting tissue repair and motor function recovery.Overall,Gas6 may play a role in the treatment of spinal cord injury.It can inhibit the inflammatory pathway of microglia and polarization of astrocytes,attenuate the interaction between microglia and astrocytes in the inflammatory microenvironment,and thereby alleviate local inflammation and reduce scar formation in the spinal cord.

Regulation of sleep by astrocytes in the hypothalamic ventrolateral preoptic nucleus

Astrocytes are functionally dynamic cells that support neurons in multiple ways throughout an organism’s lifespan.The astrocytic regulation of neuronal activity has been increasingly recognized in recent years.Astrocytes are now recognized as playing a more complex role than mere bystanders in the central nervous system.However,their role in regulating the sleep neurocircuitry is not well understood.From this perspective,we highlight the role of astrocytes in sleep modulation,with a particular focus on regulatory mechanisms related to the ventrolateral preoptic nucleus(VLPO)of the hypothalamus.We briefly discuss recent literature reporting the role of VLPO astrocytes in regulating sleep-associated behaviors.

Astrocytes, reactive astrogliosis, and glial scar formation in traumatic brain injury

Traumatic brain injury is a global health crisis,causing significant death and disability worldwide.Neuroinflammation that follows traumatic brain injury has serious consequences for neuronal survival and cognitive impairments,with astrocytes involved in this response.Following traumatic brain injury,astrocytes rapidly become reactive,and astrogliosis propagates from the injury core to distant brain regions.Homeostatic astroglial proteins are downregulated near the traumatic brain injury core,while pro-inflammatory astroglial genes are overexpressed.This altered gene expression is considered a pathological remodeling of astrocytes that produces serious consequences for neuronal survival and cognitive recovery.In addition,glial scar formed by reactive astrocytes is initially necessary to limit immune cell infiltration,but in the long term impedes axonal reconnection and functional recovery.Current therapeutic strategies for traumatic brain injury are focused on preventing acute complications.Statins,cannabinoids,progesterone,beta-blockers,and cerebrolysin demonstrate neuroprotective benefits but most of them have not been studied in the context of astrocytes.In this review,we discuss the cell signaling pathways activated in reactive astrocytes following traumatic brain injury and we discuss some of the potential new strategies aimed to modulate astroglial responses in traumatic brain injury,especially using cell-targeted strategies with miRNAs or lncRNA,viral vectors,and repurposed drugs.

Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)have shown potential for brain injury repair in central nervous system diseases.In this study,we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism.Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits,enhanced blood-brain barrier integrity,and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage.Additionally,hiPSC-NSC-Exos decreased immune cell infiltration,activated astrocytes,and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1,macrophage inflammatory protein-1α,and tumor necrosis factor-αpost-intracerebral hemorrhage,thereby improving the inflammatory microenvironment.RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion,thereby improving blood-brain barrier integrity.Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects.In summary,our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity,in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

Resident immune responses to spinal cord injury:role of astrocytes and microglia

Spinal cord injury can be traumatic or non-traumatic in origin,with the latter rising in incidence and prevalence with the aging demographics of our society.Moreove r,as the global population ages,individuals with co-existent degenerative spinal pathology comprise a growing number of traumatic spinal cord injury cases,especially involving the cervical spinal cord.This makes recovery and treatment approaches particula rly challenging as age and comorbidities may limit regenerative capacity.For these reasons,it is critical to better understand the complex milieu of spinal cord injury lesion pathobiology and the ensuing inflammatory response.This review discusses microglia-specific purinergic and cytokine signaling pathways,as well as microglial modulation of synaptic stability and plasticity after injury.Further,we evaluate the role of astrocytes in neurotransmission and calcium signaling,as well as their border-forming response to neural lesions.Both the inflammatory and reparative roles of these cells have eluded our complete understanding and remain key therapeutic targets due to their extensive structural and functional roles in the nervous system.Recent advances have shed light on the roles of glia in neurotransmission and reparative injury responses that will change how interventions are directed.Understanding key processes and existing knowledge gaps will allow future research to effectively target these cells and harness their regenerative potential.

The functions of exosomes targeting astrocytes and astrocyte-derived exosomes targeting other cell types

Astrocytes are the most abundant glial cells in the central nervous system;they participate in crucial biological processes,maintain brain structure,and regulate nervous system function.Exosomes are cell-derived extracellular vesicles containing various bioactive molecules including proteins,peptides,nucleotides,and lipids secreted from their cellular sources.Increasing evidence shows that exosomes participate in a communication network in the nervous system,in which astrocyte-derived exosomes play important roles.In this review,we have summarized the effects of exosomes targeting astrocytes and the astrocyte-derived exosomes targeting other cell types in the central nervous system.We also discuss the potential research directions of the exosome-based communication network in the nervous system.The exosome-based intercellular communication focused on astrocytes is of great significance to the biological and/or pathological processes in different conditions in the brain.New strategies may be developed for the diagnosis and treatment of neurological disorders by focusing on astrocytes as the central cells and utilizing exosomes as communication mediators.

General anesthetic agents induce neurotoxicity through astrocytes

Neuroscientists have recognized the importance of astrocytes in regulating neurological function and their influence on the release of glial transmitters.Few studies,however,have focused on the effects of general anesthetic agents on neuroglia or astrocytes.Astrocytes can also be an important target of general anesthetic agents as they exert not only sedative,analgesic,and amnesic effects but also mediate general anesthetic-induced neurotoxicity and postoperative cognitive dysfunction.Here,we analyzed recent advances in understanding the mechanism of general anesthetic agents on astrocytes,and found that exposure to general anesthetic agents will destroy the morphology and proliferation of astrocytes,in addition to acting on the receptors on their surface,which not only affect Ca^(2+)signaling,inhibit the release of brain-derived neurotrophic factor and lactate from astrocytes,but are even involved in the regulation of the pro-and anti-inflammatory processes of astrocytes.These would obviously affect the communication between astrocytes as well as between astrocytes and neighboring neurons,other neuroglia,and vascular cells.In this review,we summarize how general anesthetic agents act on neurons via astrocytes,and explore potential mechanisms of action of general anesthetic agents on the nervous system.We hope that this review will provide a new direction for mitigating the neurotoxicity of general anesthetic agents.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

In situ direct reprogramming of astrocytes to neurons via polypyrimidine tract-binding protein 1 knockdown in a mouse model of ischemic stroke

In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1(PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.e B-GFAP-sh PTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-sh PTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells

Objective To investigate whether hypertonic saline （HS） can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. Methods Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat （1 day）. The astrocytes were randomly divided into five groups： isotonic （IS） and HS groups, astrocytes were incubated by IS and HS （320 mosM NaCl） medium, respectively, for 1, 3, 5, 10 or 15 rain; carbenoxolone （CBX） ＋IS and CBX＋HS groups, astrocytes were pre-treated with CBX （100 mmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2＋＋HS group, astrocytes were pre-incubated with Ca2＋ （1 000 μmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein （GFAP） and anti-glutamate. The C6 cells were divided into four groups： IS, HS, CBX＋IS and CBX＋HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry （FCM）. Results （1） Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. （2） The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 rain （P 〈 0.01 vs the 5 min of HS group）. In CBX＋HS group, the glutamate intensity was higher than that in CBX＋IS and HS groups. （3） The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min （P 〈 0.01 vs 1 min and 3 min）. On the contrary, the medium glutamate concentrations in the CBX＋HS or Ca2＋＋HS group were significant lower than that in the HS group （P 〈 0.01）. （4） FCM showed HS and CBX＋HS induced glutamate increase in C6 cells. Conclusion HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

Immortalized hippocampal astrocytes from 3xTg-AD mice,a new model to study disease-related astrocytic dysfunction:a comparative review

Alzheimer's disease(AD)is characterized by complex etiology,long-lasting pathogenesis,and celltype-specific alterations.Currently,there is no cure for AD,emphasizing the urgent need for a comprehensive understanding of cell-specific pathology.Astrocytes,principal homeostatic cells of the central nervous system,are key players in the pathogenesis of neurodegenerative diseases,including AD.Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways.Tumor-derived and immortalized astrocytic cell lines,alongside the emerging technology of adult induced pluripotent stem cells,are widely used to study cellular dysfunction in AD.Surprisingly,no stable cell lines were available from genetic mouse AD models.Recently,we established immortalized hippocampal astroglial cell lines from amyloid-βprecursor protein/presenilin-1/Tau triple-transgenic(3xTg)-AD mice(denominated as wild type(WT)-and 3Tg-iAstro cells)using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection,thereby maintaining natural heterogeneity of primary cultures.Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling,mitochondrial dysfunctions,disproteostasis,altered homeostatic and signaling support to neurons,and blood-brain barrier models.Here we provide a comparative overview of the most used models to study astrocytes in vitro,such as primary culture,tumor-derived cell lines,immortalized astroglial cell lines,and induced pluripotent stem cell-derived astrocytes.We conclude that immortalized WT-and 3Tg-iAstro cells provide a noncompetitive but complementary,low-cost,easy-to-handle,and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery.

M1-type microglia can induce astrocytes to deposit chondroitin sulfate proteoglycan after spinal cord injury

After spinal cord injury(SCI),astrocytes gradually migrate to and surround the lesion,depositing chondroitin sulfate proteoglycan-rich extracellular matrix and forming astrocytic scar,which limits the spread of inflammation but hinders axon regeneration.Meanwhile,microglia gradually accumulate at the lesion border to form microglial scar and can polarize to generate a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype.However,the effect of microglia polarization on astrocytes is unclear.Here,we found that both microglia(CX3 CR1^(+))and astrocytes(GFAP^(+))gathered at the lesion border at 14 days post-injury(dpi).The microglia accumulated along the inner border of and in direct contact with the astrocytes.M1-type microglia(i NOS^(+)CX3 CR1^(+))were primarily observed at 3 and 7 dpi,while M2-type microglia(Arg1^(+)CX3 CR1^(+))were present at larger numbers at 7 and 14 dpi.Transforming growth factor-β1(TGFβ1)was highly expressed in M1 microglia in vitro,consistent with strong expression of TGFβ1 by microglia in vivo at 3 and 7 dpi,when they primarily exhibited an M1 phenotype.Furthermore,conditioned media from M1-type microglia induced astrocytes to secrete chondroitin sulfate proteoglycan in vitro.This effect was eliminated by knocking down sex-determining region Y-box 9(SOX9)in astrocytes and could not be reversed by treatment with TGFβ1.Taken together,our results suggest that microglia undergo M1 polarization and express high levels of TGFβ1 at 3 and 7 dpi,and that M1-type microglia induce astrocytes to deposit chondroitin sulfate proteoglycan via the TGFβ1/SOX9 pathway.The study was approved by the Institutional Animal Care and Use Committee of Anhui Medical University,China(approval No.LLSC20160052)on March 1,2016.

Astrocytes: a double-edged sword in neurodegenerative diseases

Astrocytes play multifaceted and vital roles in maintaining neurophysiological function of the central nervous system by regulating homeostasis, increasing synaptic plasticity, and sustaining neuroprotective effects. Astrocytes become activated as a result of inflammatory responses during the progression of pathological changes associated with neurodegenerative disorders. Reactive astrocytes(neurotoxic A1 and neuroprotective A2) are triggered during disease progression and pathogenesis due to neuroinflammation and ischemia. However, only a limited body of literature describes morphological and functional changes of astrocytes during the progression of neurodegenerative diseases. The present review investigated the detrimental and beneficial roles of astrocytes in neurodegenerative diseases reported in recent studies, as these cells have promising therapeutic potential and offer new approaches for treatment of neurodegenerative diseases.

Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury

Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.

Dynamic reactive astrocytes after focal ischemia

Astrocytes are specialized and most numerous glial cell type in the central nervous system and play important roles in physiology. Astrocytes are also critically involved in many neural disorders including focal ischemic stroke, a leading cause of brain injury and human death. One of the prominent pathological features of focal ischemic stroke is reactive astrogliosis and glial scar formation associated with morphological changes and proliferation. This review paper discusses the recent advances in spatial and temporal dynamics of morphology and proliferation of reactive astrocytes after ischemic stroke based on results from experimental animal studies. As reactive astrocytes exhibit stem cell-like properties, knowledge of dynamics of reactive astrocytes and glial scar formation will provide important insiehts for astrocvte-based cell therapy in stroke.

Effects of primary microglia and astrocytes on neural stem cells in in vitro and in vivo models of ischemic stroke

Transplantation of neural stem cells(NSCs) can protect neurons in animal stroke models;however, their low rates of survival and neuronal differentiation limit their clinical application. Glial niches, an important location of neural stem cells, regulate survival, proliferation and differentiation of neural stem cells. However, the effects of activated glial cells on neural stem cells remain unclear. In the present study, we explored the effects of activated astrocytes and microglia on neural stem cells in vitro stroke models. We also investigated the effects of combined transplantation of neural stem cells and glial cells after stroke in rats. In a Transwell co-culture system, primary cultured astrocytes, microglia or mixed glial cells were exposed to glutamate or H_2O_2 and then seeded in the upper inserts, while primary neural stem cells were seeded in the lower uncoated wells and cultured for 7 days. Our results showed that microglia were conducive to neurosphere formation and had no effects on apoptosis within neurospheres, while astrocytes and mixed glial cells were conducive to neurosphere differentiation and reduced apoptosis within neurospheres, regardless of their pretreatment. In contrast, microglia and astrocytes induced neuronal differentiation of neural stem cells in differentiation medium, regardless of their pretreatment, with an exception of astrocytes pretreated with H_2O_2. Rat models of ischemic stroke were established by occlusion of the middle cerebral artery. Three days later, 5 × 10~5 neural stem cells with microglia or astrocytes were injected into the right lateral ventricle. Neural stem cell/astrocyte-treated rats displayed better improvement of neurological deficits than neural stem cell only-treated rats at 4 days after cell transplantation. Moreover, neural stem cell/microglia-, and neural stem cell/astrocyte-treated rats showed a significant decrease in ischemic volume compared with neural stem celltreated rats. These findings indicate that microglia and astrocytes exert different effects on neural stem cells, and that co-transplantation of neural stem cells and astrocytes is more conducive to the recovery of neurological impairment in rats with ischemic stroke. The study was approved by the Animal Ethics Committee of Tongji University School of Medicine, China(approval No. 2010-TJAA08220401) in 2010.

High mobility group box 1 mediates inflammatory response of astrocytes via cyclooxygenase 2/prostaglandin E2 signaling following spinal cord injury

High mobility group box 1(HMGB1) interacts with pattern-recognition receptors of immune cells to activate the inflammatory response. Astrocytes play a positive role in the inflammatory response of the central nervous system by expressing a broad range of patternrecognition receptors. However, the underlying relationship between HMGB1 and the inflammatory reaction of astrocytes remains unclear. In this study, we established rat models of spinal cord injury via laminectomy at the T8–10 level, and the injured spinal cord was subjected to transcriptome sequencing. Our results showed that the HMGB1/Toll-like receptor 4(TLR4) axis was involved in the activation of astrocyte inflammatory response through regulation of cyclooxygenase 2(COX2)/prostaglandin E2(PGE2) signaling. Both TLR4 and COX2 were distributed in astrocytes and showed elevated protein levels following spinal cord injury. Stimulation of primary astrocytes with recombinant HMGB1 showed that COX2 and microsomal PGE synthase(mPGES)-1, rather than COX1, mPGES-2, or cytosolic PGE synthase, were significantly upregulated. Accordingly, PGE2 production in astrocytes was remarkably increased in response to recombinant HMGB1 challenges. Pharmacologic blockade of TLR2/4 attenuated HMGB1-mediated activation of the COX2/PGE2 pathway. Interestingly, HMGB1 did not impact the production of tumor necrosis factor-α or interleukin-1β in astrocytes. Our results suggest that HMGB1 mediates the astrocyte inflammatory response through regulating the COX2/PGE2 signaling pathway. The study was approved by the Laboratory Animal Ethics Committee of Nantong University, China(approval No. 20181204-001) on December 4, 2018.