Astrocyte-TREM2 alleviates brain injury by regulating reactive astrocyte states following ischemic stroke.

Triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to confer strong neuroprotective effects in acute ischemic stroke (AIS). However, as the vast majority of research findings to date are based on its functions in microglia, the precise role of TREM2 in astrocytes after AIS is unknown. Here, both loss- and gain-of-function experiments were employed to investigate how astrocytic TREM2 influences the pathogenesis of AIS in vivo and in vitro. Our results demonstrated that cerebral ischemia triggered induction of TREM2 expression on reactive astrocytes following AIS. In addition, astrocyte-specific TREM2 knockout mice exhibited much greater brain injury than TREM2 flox/flox controls following AIS, as evidenced by increased cerebral infarct volume, neuronal apoptosis and neurological deficit, which was associated with an increased expression of pro-inflammatory molecule complement component 3 (C3) on reactive astrocytes and activation of microglia/macrophages but decreased expression of S100 calcium binding protein A10 (S100A10) and arginase1 (Arg1) on reactive astrocytes. Mechanistic analyses revealed that astrocytic TREM2 alleviated brain injury by inhibiting detrimental actions of reactive astrocytes but promoting their neuro- and glioprotective actions via the kruppel-like transcription factor-4-nuclear factor-κB axis. Together, this study provides novel evidence for a critical protective role of astrocyte-derived TREM2 in AIS and highlights a potential therapeutic target for the treatment of AIS.

DJ-1 regulates astrocyte activation through miR-155/SHP-1 signaling in cerebral ischemia/reperfusion injury.

Reactive astrocyte activation in the context of cerebral ischemia/reperfusion (I/R) injury gives rise to two distinct subtypes: the neurotoxic A1 type and the neuroprotective A2 type. DJ-1 (Parkinson disease protein 7, PARK7), originally identified as a Parkinson's disease-associated protein, is a multifunctional anti-oxidative stress protein with molecular chaperone and signaling functions. SHP-1 (Src homology 2 domain-containing phosphatase-1) is a protein tyrosine phosphatase closely associated with cellular signal transduction. miR-155 is a microRNA that participates in cellular functions by regulating gene expression. Recent studies have uncovered the relationship between DJ-1 and astrocyte-mediated neuroprotection, which may be related to its antioxidant properties and regulation of signaling molecules such as SHP-1. Furthermore, miR-155 may exert its effects by influencing SHP-1, providing a potential perspective for understanding the molecular mechanisms of stroke. A middle cerebral artery occlusion/reperfusion (MCAO/R) model and an oxygen-glucose deprivation/reperfusion (OGD/R) model were established to simulate focal cerebral I/R injury in vivo and in vitro, respectively. The in vivo interaction between DJ-1 and SHP-1 has been experimentally validated through immunoprecipitation. Overexpression of DJ-1 attenuates I/R injury and suppresses miR-155 expression. In addition, inhibition of miR-155 upregulates SHP-1 expression and modulates astrocyte activation phenotype. These findings suggest that DJ-1 mediates astrocyte activation via the miR-155/SHP-1 pathway, playing a pivotal role in the pathogenesis of cerebral ischemia-reperfusion injury. Our results provide a potential way for exploring the pathogenesis of ischemic stroke and present promising targets for pharmacological intervention.

MiR-155-5p Aggravated Astrocyte Activation and Glial Scarring in a Spinal Cord Injury Model by Inhibiting Ndfip1 Expression and PTEN Nuclear Translocation.

Central nervous injury and regeneration repair have always been a hot and difficult scientific questions in neuroscience, such as spinal cord injury (SCI) caused by a traffic accident, fall injury, and war. After SCI, astrocytes further migrate to the injured area and form dense glial scar through proliferation, which not only limits the infiltration of inflammatory cells but also affects axon regeneration. We aim to explore the effect and underlying mechanism of miR-155-5p overexpression promoted astrocyte activation and glial scarring in an SCI model. MiR-155-5p mimic (50 or 100 nm) was used to transfect CTX-TNA2 rat brain primary astrocyte cell line. MiR-155-5p antagonist and miR-155-5p agomir were performed to treat SCI rats. MiR-155-5p mimic dose-dependently promoted astrocyte proliferation, and inhibited cell apoptosis. MiR-155-5p overexpression inhibited nuclear PTEN expression by targeting Nedd4 family interacting protein 1 (Ndfip1). Ndfip1 overexpression reversed astrocyte activation which was induced by miR-155-5p mimic. Meanwhile, Ndfip1 overexpression abolished the inhibition effect of miR-155-5p mimic on PTEN nuclear translocation. In vivo, miR-155-5p silencing improved SCI rat locomotor function and promoted astrocyte activation and glial scar formation. And miR-155-5p overexpression showed the opposite results. MiR-155-5p aggravated astrocyte activation and glial scarring in a SCI model by targeting Ndfip1 expression and inhibiting PTEN nuclear translocation. These findings have ramifications for the development of miRNAs as SCI therapeutics.

Sirt1 attenuates astrocyte activation via modulating Dnajb1 and chaperone-mediated autophagy after closed head injury.

Our previous study indicates that Silent information regulator 1 (Sirt1) is involved in macroautophagy by upregulating light chain 3 (LC3) expression in astrocyte to exert a neuroprotective effect. Chaperon-mediated autophagy (CMA), another form of autophagy, is also upregulated after brain injury. However, little is known about the role of Sirt1 in regulation of the CMA. In the present study, an in vivo model of closed head injury (CHI) and an in vitro model of primary cortical astrocyte stimulated with interleukin-1ß were employed to mimic the astrocyte activation induced by traumatic brain injury. Lentivirus carrying target complementary DNA (cDNA) or short hairpin RNA (shRNA) sequence was used to overexpress Sirt1 or knockdown DnaJ heat shock protein family member B1 (Dnajb1) (a molecular chaperone). We found that Sirt1 overexpression ameliorated neurological deficits, reduced tissue loss, and attenuated astrocyte activation after CHI, which was reversed by Dnajb1-shRNA administration. The upregulation of CMA activity induced by CHI in vivo and in vitro was inhibited after Dnajb1 knockdown. Sirt1 potently promoted CMA activity via upregulating Dnajb1 expression. Mechanically, Sirt1 could interact with Dnajb1 and modulate the deacetylation and ubiquitination of Dnajb1. These findings collectively suggest that Sirt1 plays a protective role against astrocyte activation, which may be associated with the regulation of the CMA activity via modulating the deacetylation and ubiquitination of Dnajb1 after CHI.

Fragmentation of rest periods, astrocyte activation, and cognitive decline in older adults with and without Alzheimer's disease.

INTRODUCTION: Sleep disruption is associated with astrocyte activation and impaired cognition in model organisms. However, the relationship among sleep, astrocyte activation, and cognition in humans is uncertain. METHODS: We used RNA-seq to quantify the prefrontal cortex expression of a panel of human activated astrocyte marker genes in 1076 older adults in the Religious Orders Study and Rush Memory and Aging Project, 411 of whom had multi-day actigraphy prior to death. We related this to rest fragmentation, a proxy for sleep fragmentation, and to longitudinal cognitive function. RESULTS: Fragmentation of rest periods was associated with higher expression of activated astrocyte marker genes, which was associated with a lower level and faster decline of cognitive function. DISCUSSION: Astrocyte activation and fragmented rest are associated with each other and with accelerated cognitive decline. If experimental studies confirm a causal relationship, targeting sleep fragmentation and astrocyte activation may benefit cognition in older adults. HIGHLIGHTS: Greater fragmentation of rest periods, a proxy for sleep fragmentation, is associated with higher composite expression of a panel of genes characteristic of activated astrocytes. Increased expression of genes characteristic of activated astrocytes was associated with a lower level and more rapid decline of cognitive function, beyond that accounted for by the burden of amyloid and neurofibrillary tangle pathology. Longitudinal and experimental studies are needed to delineate the causal relationships among sleep, astrocyte activation, and cognition.

The GFAP Monoclonal Antibody GA-5 Identifies Astrocyte Remodeling and Glio-Vascular Uncoupling During the Evolution of EAE.

To examine how astrocyte activation is regulated at different phases of relapsing-remitting EAE, we performed an immunofluorescent analysis of the spinal cord using the anti-glial fibrillary acidic protein (GFAP) monoclonal antibody GA-5. In keeping with previous studies, gray matter astrocytes showed strongly increased GFAP expression during the peak phase of disease (14 days post-immunization), which remained elevated during the remission phase (21-28 days post-immunization). In sharp contrast, during the peak phase of disease, the GA-5 signal in sub-meningeal white matter transiently disappeared in areas containing high levels of infiltrating leukocytes, but during the remission phase, the GFAP signal was fully restored. Parallel staining of the same sections with a polyclonal GFAP antibody confirmed elevated GFAP expression in the gray matter but no loss of signal in white matter. Interestingly, loss of GA-5 signal in sub-meningeal white matter was strongly associated with vascular disruption as defined by extravascular fibrinogen leak and by glio-vascular uncoupling, as defined by dissociation of AQP4-positive astrocyte endfeet and CD31-positive blood vessels. GA-5-negative areas were also associated with demyelination. These findings demonstrate a novel staining pattern of a GFAP antibody during EAE progression and suggest that the GFAP epitope recognized by the GA-5 monoclonal antibody transiently disappears as white matter astrocytes undergo remodeling during the peak phase of EAE. They also suggest that the GA-5 antibody provides a novel tool to identify astrocyte remodeling in other neurological conditions.

Exposure to short-chain chlorinated paraffins induces astrocyte activation via JAK2/STAT3 signaling pathway.

In the last few decades, short-chain chlorinated paraffins (SCCPs) have become the most heavily produced monomeric organohalogen compounds, and have been reported to induce multiple organ toxicity. However, the effects of SCCPs on the central nervous system are unknown. In the present study, we show that SCCP exposure induced astrocyte proliferation and increased the expression of two critical markers of astrocyte activation, glial fibrillary acidic protein and inducible nitric oxide synthase, in vivo and in vitro. SCCP exposure also increased inflammatory factory gene expression. Moreover, SCCP treatment triggered Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signalling, as shown by increased phosphorylation and STAT3 translocation to the nucleus. Both JAK2 and STAT3 inhibition effectively attenuated SCCP-induced astrocyte activation. Finally, JAK2 inhibition significantly rescued STAT3 phosphorylation and nuclear translocation. Taken together, JAK2/STAT3 pathway activation contributed to SCCP-induced astrocyte activation. These data will help elucidate the molecular mechanism underlying SCCP-induced neurotoxicity.

Role of chronic neuroinflammation in neuroplasticity and cognitive function: A hypothesis.

OBJECTIVE: Evaluating the efficacy of 3,6'-dithioPomalidomide in 5xFAD Alzheimer's disease (AD) mice to test the hypothesis that neuroinflammation is directly involved in the development of synaptic/neuronal loss and cognitive decline. BACKGROUND: Amyloid-ß (Aß) or tau-focused clinical trials have proved unsuccessful in mitigating AD-associated cognitive impairment. Identification of new drug targets is needed. Neuroinflammation is a therapeutic target in neurodegenerative disorders, and TNF-α a pivotal neuroinflammatory driver. NEW HYPOTHESIS: AD-associated chronic neuroinflammation directly drives progressive synaptic/neuronal loss and cognitive decline. Pharmacologically mitigating microglial/astrocyte activation without altering Aß generation will define the role of neuroinflammation in AD progression. MAJOR CHALLENGES: Difficulty of TNF-α-lowering compounds reaching brain, and identification of a therapeutic-time window to preserve the beneficial role of neuroinflammatory processes. LINKAGE TO OTHER MAJOR THEORIES: Microglia/astroglia are heavily implicated in maintenance of synaptic plasticity/function in healthy brain and are disrupted by Aß. Mitigation of chronic gliosis can restore synaptic homeostasis/cognitive function.

Acute Colon Inflammation Triggers Primary Motor Cortex Glial Activation, Neuroinflammation, Neuronal Hyperexcitability, and Motor Coordination Deficits.

Neuroinflammation underlies neurodegenerative diseases. Herein, we test whether acute colon inflammation activates microglia and astrocytes, induces neuroinflammation, disturbs neuron intrinsic electrical properties in the primary motor cortex, and alters motor behaviors. We used a rat model of acute colon inflammation induced by dextran sulfate sodium. Inflammatory mediators and microglial activation were assessed in the primary motor cortex by PCR and immunofluorescence assays. Electrophysiological properties of the motor cortex neurons were determined by whole-cell patch-clamp recordings. Motor behaviors were examined using open-field and rotarod tests. We show that the primary motor cortex of rats with acute colon inflammation exhibited microglial and astrocyte activation and increased mRNA abundance of interleukin-6, tumor necrosis factor-alpha, and both inducible and neuronal nitric oxide synthases. These changes were accompanied by a reduction in resting membrane potential and rheobase and increased input resistance and action potential frequency, indicating motor neuron hyperexcitability. In addition, locomotion and motor coordination were impaired. In conclusion, acute colon inflammation induces motor cortex microglial and astrocyte activation and inflammation, which led to neurons' hyperexcitability and reduced motor coordination performance. The described disturbances resembled some of the early features found in amyotrophic lateral sclerosis patients and animal models, suggesting that colon inflammation might be a risk factor for developing this disease.

Astrocyte-Derived Estrogen Regulates Reactive Astrogliosis and is Neuroprotective following Ischemic Brain Injury.

Expression of the 17ß-estradiol (E2) synthesis enzyme aromatase is highly upregulated in astrocytes following brain injury. However, the precise role of astrocyte-derived E2 in the injured brain remains unclear. In the current study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse model to deplete astrocyte-derived E2 in the brain and determine its roles after global cerebral ischemia (GCI) in male and female mice. GFAP-ARO-KO mice were viable and fertile, with normal gross brain structure, normal morphology, intensity and distribution of astrocytes, normal aromatase expression in neurons, and normal cognitive function basally. In contrast, after GCI, GFAP-ARO-KO mice: (1) lacked the normal elevation of astrocyte aromatase and hippocampal E2 levels; (2) had significantly attenuated reactive astrogliosis; and (3) displayed enhanced neuronal damage, microglia activation, and cognitive deficits. RNA-sequencing (RNA-seq) analysis revealed that the ischemic GFAP-ARO-KO mouse hippocampus failed to upregulate the "A2" panel of reactive astrocyte genes. In addition, the JAK-STAT3 pathway, which is critical for the induction of reactive astrogliosis, was significantly downregulated in the GFAP-ARO-KO hippocampus following GCI. Finally, exogenous E2 administration fully rescued the compromised JAK-STAT3 pathway and reactive astrogliosis, and reversed the enhanced neuronal damage and microglial activation in the GFAP-ARO-KO mice after GCI, suggesting that the defects in the KO mice are because of a loss of E2 rather than an increase in precursor androgens. In conclusion, the current study provides novel genetic evidence for a beneficial role of astrocyte-derived E2 in reactive astrogliosis, microglial activation, and neuroprotection following an ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, reactive astrocytes express the enzyme aromatase and produce 17ß-estradiol (E2), although the precise role of astrocyte-derived E2 is poorly understood. In this study, we generated a glial fibrillary acidic protein (GFAP) promoter-driven aromatase knock-out (GFAP-ARO-KO) mouse to deplete astrocyte-derived E2 and elucidate its roles after global cerebral ischemia (GCI). The GFAP-ARO-KO mice exhibited significantly attenuated reactive astrogliosis, as well as enhanced microglial activation, neuronal damage, and cognitive dysfunction after GCI. Transcriptome analysis further revealed that astrocyte-derived E2 was critical for the induction of the JAK-STAT3 signaling pathway, as well as the A2 reactive astrocyte phenotype after ischemia. Collectively, these findings indicate that astrocyte-derived E2 has a key role in the regulation of reactive astrogliosis, microglial activation, and neuroprotection after cerebral ischemia.

Down-regulation of circular RNA CDC14A peripherally ameliorates brain injury in acute phase of ischemic stroke.

BACKGROUND: Inflammation is integral to the pathophysiology of ischemic stroke and a prime target for the development of new stroke therapies. The aim of the present study is to seek out the regulatory mechanism of circCDC14A in neuroinflammatory injury in tMCAO mice. METHODS: The expression level of circCDC14A in peri-infarct cortex and plasma of mice were detected by qPCR. The localization of circCDC14A in peripheral blood cells and peri-infarct cortex of tMCAO mice were explored by in situ hybridization and immunofluorescence colocalization staining. Lentivirus were microinjected into lateral ventricular of brain or injected into tail vein to interfere with the expression of circCDC14A, thus their effects on behavior, morphology, and molecular biology of tMCAO mice were analyzed. RESULTS: The expression of circCDC14A in plasma and peri-infarct cortex of tMCAO mice significantly increased, and circCDC14A was mainly localized in neutrophils peripherally while in astrocytes in peri-infarct cortex centrally. Tail vein injection of lentivirus to interfere with the expression of circCDC14A significantly reduced the infarct volume (P < 0.01) at 72 h after reperfusion and density of activated astrocytes in peri-infarct cortex at 3 days, 5 days and 7 days after tMCAO modeling (all P < 0.0001). Moreover, mNSS (P < 0.0001) and survival rate (P < 0.001) were significantly improved within 7 days in si-circCDC14A group compared to circCon group. Additionally, morphology analysis showed the volume and surface area of each activated astrocytes significantly decreased (P < 0.0001). Quantification analysis measured the percentage of N2 phenotype among infiltrated neutrophils in brain sections and found N2 ratio was significantly higher in si-circCDC14A group compared to circCon group (P < 0.001). CONCLUSION: Knocking down the expression of circCDC14A in peripheral blood cells relieved astrocytes activation in peri-infarct cortex, thereby relieved brain damage in the acute phase of ischemic stroke.

The Dual Role of the GABAA Receptor in Peripheral Inflammation and Neuroinflammation: A Study in Hyperammonemic Rats.

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1ß and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.

Astrocytes in Multiple Sclerosis-Essential Constituents with Diverse Multifaceted Functions.

In multiple sclerosis (MS), astrocytes respond to the inflammatory stimulation with an early robust process of morphological, transcriptional, biochemical, and functional remodeling. Recent studies utilizing novel technologies in samples from MS patients, and in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), exposed the detrimental and the beneficial, in part contradictory, functions of this heterogeneous cell population. In this review, we summarize the various roles of astrocytes in recruiting immune cells to lesion sites, engendering the inflammatory loop, and inflicting tissue damage. The roles of astrocytes in suppressing excessive inflammation and promoting neuroprotection and repair processes is also discussed. The pivotal roles played by astrocytes make them an attractive therapeutic target. Improved understanding of astrocyte function and diversity, and the mechanisms by which they are regulated may lead to the development of novel approaches to selectively block astrocytic detrimental responses and/or enhance their protective properties.

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OBJECTIVES: To explore the effect of intrathecal administration of exogenous noggin (NOG) on the pain behavior in the neuropathic pain (NP) rats through L5 spinal nerve ligation (SNL), and to examine the regulative role of NOG in astrocyte activation, inflammatory cytokines and downstream signals. METHODS: A total of 40 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: a control group (n=10), a SNL group (SNL+intrathecal injection of artificial cerebrospinal fluid, n=15), and a SNL+NOG group (SNL+intrathecal injection of recombinant NOG protein, n=15). Von-Frey filaments were used to test the changes of paw withdrawal threshold (PWT) at Day 1 before operation, and Day 1, Day 4, Day 7 and Day 14 after operation in each group. Immunofluorescence was used to observe the activation of astrocyte located in the dorsal horn of spinal cord in the 3 groups. Western blotting was conducted to detect the expression levels of glial fibrillary acidic protein (GFAP), interleukin-6 (IL-6), signal transducer and activator of transcription (STAT3) and phosphorylation STAT3 (p-STAT3). RESULTS: Compared with the control group, the PWT in the SNL group was markedly decreased at each time point, together with the increase in GFAP, IL-6 and the ratio of p-STAT3/STAT3 (all P<0.05). Meanwhile, compared with the SNL group, the PWT in the lumbar swelling of spinal cord in the SNL+NOG group was elevated at Day 4 and lasted to Day 14 (P<0.05), accompanied by the decrease in GFAP, IL-6 and the ratio of p-STAT3/STAT3 (all P<0.05). CONCLUSIONS: The intrathecal administration of NOG may alleviate NP in the SNL rats through inhibiting astrocyte activation and down-regulating the STAT3 signal pathway.

Neuron-generated thrombin induces a protective astrocyte response via protease activated receptors.

Astrocytes protect neurons during cerebral injury through several postulated mechanisms. Recent therapeutic attention has focused on enhancing or augmenting the neuroprotective actions of astrocytes but in some instances astrocytes can assume a neurotoxic phenotype. The signaling mechanisms that drive astrocytes toward a protective versus toxic phenotype are not fully known but cell-cell signaling via proteases acting on cell-specific receptors underlies critical mechanistic steps in neurodevelopment and disease. The protease activated receptor (PAR), resides in multiple brain cell types, and most PARs are found on astrocytes. We asked whether neuron-generated thrombin constituted an important astrocyte activation signal because our previous studies have shown that neurons contain prothrombin gene and transcribed protein. We used neuron and astrocyte mono-cell cultures exposed to oxygen-glucose deprivation and a model of middle cerebral artery occlusion. We found that ischemic neurons secrete thrombin into culture media, which leads to astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin. Media from prothrombin-deficient neurons failed to activate astrocytes and adding thrombin to such media restored activation. Astrocytes lacking PAR1 did not respond to neuron-generated thrombin. Induced astrocyte activation was antagonized dose-dependently with thrombin inhibitors or PAR1 antagonists. Ischemia-induced astrocyte activation in vivo was inhibited after neuronal prothrombin knockout, resulting in larger strokes. Restoring prothrombin to neurons with a lentiviral gene vector restored astrocyte activation and reduced stroke damage. We conclude that neuron-generated thrombin, released during ischemia, acts via PAR1 and may cause astrocyte activation and paracrine neuroprotection.

Resveratrol Downregulates STAT3 Expression and Astrocyte Activation in Primary Astrocyte Cultures of Rat.

Astrocytes respond to all forms of central nervous system (CNS) insults by a process referred to as reactive astrogliosis. Inhibition of astrocyte growth and activation is an important strategy for promoting injured CNS repair. STAT3 (signal transducer and activator of transcription 3) is reported to be a critical regulator of astrogliosis, and resveratrol (RES, a dietary polyphenol) is considered to be a natural inhibitor of STAT3 expression and phosphorylation. In this study, we investigated the effects of RES on STAT3 expression and phosphorylation, and then on the proliferation and activation of astrocytes, a critical process in reactive astrogliosis, in rat primary cultured astrocytes and an in vitro scratch-wound model. RES downregulated the expression levels of STAT3, P-STAT3 and GFAP (glial fibrillary acidic protein) in cultured astrocytes. The positive index of Ki67 was apparently reduced in cultured astrocytes after RES treatment. Meanwhile, cultured astrocyte proliferation and activation were attenuated by RES. Moreover, in the established in vitro scratch-wound model the increased expression levels of STAT3, P-STAT3 and GFAP induced by scratching injury were also clearly inhibited by RES. In addition, the inhibitory effect of RES on cell proliferation was similar to that of AG490 (a selective inhibitor of STAT3 phosphorylation) and abrogated by Colivelin (a STAT3 activator) stimuli. Taken together, our data suggest that RES is able to inhibit reactive astrocyte proliferation and activation mainly via deactivating STAT3 pathway. So RES may have a therapeutic benefit for the treatment of the injured CNS.

Cryogen spray cooling mitigates inflammation and injury-induced CISD2 decline in rat spinal cord hemisection model.

Therapeutic strategies for traumatic spinal cord injury generally involve rectifying concomitant destruction to the spinal cord from inflammation, mitochondrial dysfunction, and eventual neuronal apoptosis. Elevating the expression of spinal cord injury-attenuated CDGSH iron-sulfur domain-2 has been shown to mitigate the pathologies above. In the current work, hypothermia was induced via continuous cryogen spray cooling in a rat spinal cord hemisection model. Spinal cord injury was shown to elevate the mRNA expression of proinflammatory mediators, including NFκB, iNOS, TNF-α, and regulated upon activation, normal T-cell expressed and secreted as well as lower CDGSH iron-sulfur domain-2 expression. Cryogen spray cooling treatment was shown to attenuate inflammatory reactions and elevate CDGSH iron-sulfur domain-2 expression. Immunohistochemical analysis of the glial fibrillary acidic protein, caspase-3 and NeuN in spinal cord injured rats that underwent cryogen spray cooling treatment revealed notable reductions in injury-induced astrocytic activation, apoptosis, neuronal loss, and decline in CDGSH iron-sulfur domain-2 expression. These results demonstrate the CDGSH iron-sulfur domain-2 preserving effects of cryogen spray cooling, which could contribute to the prevention of astrocytic activation, astrocyte-mediated neuroinflammation, apoptosis, and neuron loss.

Optogenetic Modulation of Neural Progenitor Cells Improves Neuroregenerative Potential.

Neural progenitor cell (NPC) transplantation possesses enormous potential for the treatment of disorders and injuries of the central nervous system, including the replacement of lost cells or the repair of host neural circuity after spinal cord injury (SCI). Importantly, cell-based therapies in this context still require improvements such as increased cell survival and host circuit integration, and we propose the implementation of optogenetics as a solution. Blue-light stimulation of NPCs engineered to ectopically express the excitatory light-sensitive protein channelrhodopsin-2 (ChR2-NPCs) prompted an influx of cations and a subsequent increase in proliferation and differentiation into oligodendrocytes and neurons and the polarization of astrocytes from a pro-inflammatory phenotype to a pro-regenerative/anti-inflammatory phenotype. Moreover, neurons derived from blue-light-stimulated ChR2-NPCs exhibited both increased branching and axon length and improved axon growth in the presence of axonal inhibitory drugs such as lysophosphatidic acid or chondroitin sulfate proteoglycan. Our results highlight the enormous potential of optogenetically stimulated NPCs as a means to increase neuroregeneration and improve cell therapy outcomes for enhancing better engraftments and cell identity upon transplantation in conditions such as SCI.

Rosiglitazone up-regulates glial fibrillary acidic protein via HB-EGF secreted from astrocytes and neurons through PPARγ pathway and reduces apoptosis in high-fat diet-fed mice.

The anti-diabetic drug and peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, rosiglitazone, alters astrocyte activation; however, its mechanism remains less-known. We hypothesized participation of epidermal growth factor receptor (EGFR), known to control astrocyte reactivity. We first detected that rosiglitazone promoted glial fibrillary acidic protein (GFAP) expression in primary astrocytes as well as the mouse cerebral cortex, associated with increased EGFR activation. Screening for EGFR ligands revealed a rosiglitazone-mediated increase of heparin-binding epidermal growth factor (HB-EGF) in astrocytes, resulting in HB-EGF release into culture medium and mouse cerebrospinal fluid too. Treatment with HB-EGF-siRNA and EGFR inhibitors showed that the rosiglitazone-induced HB-EGF and p-EFGR were interdependent, which participated in GFAP increase. Interestingly, we observed that rosiglitazone could induce cellular and secreted-HB-EGF in neurons also, contributing toward the activated EGFR-induced GFAP in astrocytes. Probing whether these effects of rosiglitazone were PPARγ-linked, revealed potential PPARγ-responsive elements within HB-EGF gene. Moreover, gel-shift, site-directed mutagenesis, chromatin-immunoprecipitation and luciferase-reporter assays demonstrated a PPARγ-dependent HB-EGF transactivation. Subsequently, we examined effects of rosiglitazone in a high-fat diet-fed diabetes mouse model, and supporting observations in the normal cortical cells, identified a rosiglitazone-induced GFAP, astrocyte and neuronal HB-EGF and secreted-HB-EGF in the cerebral cortex of diabetic mice. Moreover, assessing relevance of increased HB-EGF and GFAP revealed an anti-apoptotic role of rosiglitazone in the cerebral cortex, supported by a GFAP-siRNA as well as HB-EGF-siRNA-mediated increase in cleaved-caspase 3 and 9 levels in the rosiglitazone-treated astrocyte-neuron coculture. Overall, our study indicates that rosiglitazone may protect the brain, via a PPARγ-dependent HB-EGF/EGFR signaling and increased GFAP.

Soluble epoxide hydrolase modulates immune responses in activated astrocytes involving regulation of STAT3 activity.

BACKGROUND: Astrocyte activation is a common pathological feature in many brain diseases with neuroinflammation, and revealing the underlying mechanisms might shed light on the regulatory processes of the diseases. Recently, soluble epoxide hydrolase (sEH) has been proposed to affect neuroinflammation in brain injuries. However, the roles of astrocytic sEH in brains with neurodegeneration remain unclear. METHODS: The expression of astrocytic sEH in the brains of APPswe/PSEN1dE9 (APP/PS1) mice developing Alzheimer's disease (AD)-like pathology was evaluated by confocal imaging. LPS-activated primary astrocytes with mRNA silencing or overexpression of sEH were used to investigate its regulatory roles in astrocyte activation and the induction of pro-inflammatory markers. Primary astrocytes isolated from a sEH knockout (sEH-/-) background were also applied. RESULTS: The immunoreactivity of sEH was increased in activated astrocytes in parallel with the progression of AD in APP/PS1 mice. Our data from primary astrocyte cultures further demonstrate that the overexpression of sEH ameliorated, while the silencing of sEH mRNA enhanced, the lipopolysaccharides (LPS)-induced expression of pro-inflammatory markers, such as inducible nitric oxide, cyclooxygenase 2 (COX-2), and pro-inflammatory cytokines. These findings suggest that sEH negatively regulates astrocyte immune responses. Enhanced immune responses found in LPS-activated sEH-/- astrocytes also support the notion that the expression of sEH could suppress the immune responses during astrocyte activation. Similarly, sEH-/- mice that received intraperitoneal injection of LPS showed exacerbated astrocyte activation in the brain, as observed by the elevated expression of glial fibrillary acidic protein (GFAP) and pro-inflammatory markers. Moreover, our data show that the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was upregulated in activated astrocytes from sEH mouse brains, and the pharmacological blockade of STAT3 activity alleviated the pro-inflammatory effects of sEH deletion in LPS-activated primary astrocytes. CONCLUSIONS: Our results provide evidence, for the first time, showing that sEH negatively regulates astrocytic immune responses and GFAP expression, while the underlying mechanism at least partly involves the downregulation of STAT3 phosphorylation. The discovery of a novel function for sEH in the negative control of astrocytic immune responses involving STAT3 activation confers further insights into the regulatory machinery of astrocyte activation during the development of neurodegeneration.