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•Sb activation CREB, ERK, as well as p38 MAPK signaling in astrocytes.•CREB activation mediated Sb-induced astrocyte activation.•Both ERK and p38 MAPK activation contributed to ...Sb-stimulated CREB phosphorylation.
Recent studies suggest that the chemical element antimony (Sb) is neurotoxic; however, the molecular mechanisms behind Sb-related neuronal damage are currently unknown. In this study, we found that Sb exposure promoted astrocyte proliferation and increased the expression of inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), two key protein markers of reactive astrogliosis, at both the gene and protein level, suggesting that Sb induced astrocyte activation. Moreover, the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-related kinase (ERK) pathways were activated following Sb exposure. Inhibition of p38 MAPK reduced Sb-induced iNOS and GFAP upregulation, while inhibiting ERK reduced GFAP expression only, in Sb-exposed C6 cells. Sb treatment also induced the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), and the inhibition of CREB caused a reduction in Sb-induced GFAP and iNOS expression. Furthermore, inhibiting both p38 MAPK and ERK effectively alleviated CREB phosphorylation in Sb-exposed C6 cells. Taken together, our results suggest that p38 MAPK and ERK activation mediate Sb-induced astrocyte activation through CREB phosphorylation. These results help to clarify the molecular mechanisms underlying Sb-associated neurotoxicity.
Circular RNAs (circRNAs) are highly expressed in the central nervous system and are involved in the regulation of physiological and pathophysiological processes. However, the potential role of ...circRNAs in stroke remains largely unknown. Here, using a circRNA microarray, we showed that circular RNA Hectd1 (circHectd1) levels were significantly increased in ischemic brain tissues in transient middle cerebral artery occlusion (tMCAO) mouse stroke models and further validated this finding in plasma samples from acute ischemic stroke (AIS) patients. Knockdown of circHectd1 expression significantly decreased infarct areas, attenuated neuronal deficits, and ameliorated astrocyte activation in tMCAO mice. Mechanistically, circHECTD1 functions as an endogenous MIR142 (microRNA 142) sponge to inhibit MIR142 activity, resulting in the inhibition of TIPARP (TCDD inducible polyADP-ribose polymerase) expression with subsequent inhibition of astrocyte activation via macroautophagy/autophagy. Taken together, the results of our study indicate that circHECTD1 and its coupling mechanism are involved in cerebral ischemia, thus providing translational evidence that circHECTD1 can serve as a novel biomarker of and therapeutic target for stroke.
Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AIS: acute ischemic stroke; AS: primary mouse astrocytes; BECN1: beclin 1, autophagy related; BMI: body mass index; circHECTD1: circRNA HECTD1; circRNAs: circular RNAs; CBF: cerebral blood flow; Con: control; DAPI: 4ʹ,6-diamidino-2-phenylindole; ECA: external carotid artery; FISH: fluorescence in situ hybridization; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; Gdna: genomic DNA; GFAP: glial fibrillary acidic protein; GO: gene ontology; HDL: high-density lipoprotein; IOD: integrated optical density; LDL: low-density lipoprotein; LPA: lipoprotein(a); MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MIR142: microRNA 142; mNSS: modified neurological severity scores; MRI: magnetic resonance imaging; NIHSS: National Institute of Health Stoke Scale; OGD-R: oxygen glucose deprivation-reperfusion; PCR: polymerase chain reaction; PFA: paraformaldehyde; SQSTM1: sequestosome 1; TIPARP: TCDD inducible poly(ADP-ribose) polymerase; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; UTR: untranslated region; WT: wild type
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‐β (Aβ) 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 Aβ 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 Aβ. Mitigation of chronic gliosis can restore synaptic homeostasis/cognitive function.
This work investigates the effects and mechanisms of inhibiting TRPC6 (a non-selective cation channel) downregulation on rat astrocyte activation and proliferation following spinal cord injury (SCI) ...by suppressing AQP4 expression. We used HYP9 (TRPC6-specific agonist) and TGN-020 (AQP4-specific inhibitor) to explore the relationship between TRPC6 and AQP4 and their probable protective effects on SCI. Methods: In a rat SCI model, we randomly assigned female Sprague-Dawley rats into the following four groups: Sham, SCI, SCI+HYP9, and SCI+TGN-020. Western blotting and immunofluorescence staining were used to determine protein expression among groups following SCI. TUNEL and immunofluorescence staining were used to identify changes in the rate of apoptosis and the fraction of surviving neurons after SCI. The Basso-Beattie-Bresnahan open-field locomotor scale was used to identify changes in motor function after SCI. In vitro astrocyte scratch model, we first used the CCK8 assay to test the effects of varying doses of HYP9 or TGN-020 on astrocytes and then split the astrocytes into four groups: Con, Scratch, Scratch+HYP9, and Scratch+TGN-020. Western blotting and immunofluorescence were used to identify changes in the expression of target proteins. Results: In vivo and in vitro models, SCI dramatically decreased TRPC6 while considerably upregulating AQP4, glial fibrillary acidic protein (GFAP), and proliferating cell nuclear antigen (PCNA) expression. However, HYP9 or TGN-020 significantly suppressed activation of astrocytes, promoted neurons survival in the anterior horn of the spinal cords, and benefited the recovery of motor function in the hind limbs of rats following SCI. Interestingly, TRPC6 agonists dramatically suppressed AQP4 overexpression, indicating that the probable mechanism of HYP9 benefiting alleviation of SCI may be connected to AQP4 inhibition and astrocyte activation and proliferation reduction. Conclusion: we discovered for the first time that HYP9 inhibits astrocyte activation and proliferation by inhibiting AQP4 in SCI rats in vivo and in vitro models and that it preserves neuronal survival and functional recovery after SCI.
•HYP9 (TRPC6-specific agonist) inhibits astrocyte activation and proliferation in SCI.•TGN-020 (AQP4-specific inhibitor) inhibits astrocyte activation and proliferation in SCI.•HYP9 inhibits AQP4 expression in SCI.•HYP9 or TGN-020 promote neuronal survival in the spinal cord and facilitate recovery of hindlimb motor function in rats after SCI.
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.
•SCCP exposure triggered astrocyte activation in vivo and in vitro.•SCCP exposure activated JAK2/STAT3 signaling pathway and increased STAT3 phosphorylation and translocation to the nucleus.•Both JAK2 and STAT3 inhibition effectively attenuated SCCP-induced astrocyte activation.
Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs), which are potent mediators of intercellular communication between ...microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs) propagate a robust inflammatory reaction among astrocytes and microglia
and in mice with subclinical neuroinflammation (Verderio et al., 2012). However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.
Parkinson’s disease (PD) pathology is characterized by the abnormal accumulation and aggregation of the pre-synaptic protein α-synuclein in the dopaminergic neurons as Lewy bodies (LBs). Curcumin, ...which plays a neuroprotective role in various animal models of PD, was found to directly modulate the aggregation of α-synuclein in in vitro as well as in in vivo studies. While curcumin has been shown to exhibit strong anti-oxidant and anti-inflammatory properties, there are a number of other possible mechanisms by which curcumin may alter α-synuclein aggregation which still remains obscure. Therefore, the present study was designed to understand such concealed mechanisms behind neuroprotective effects of curcumin. An animal model of PD was established by injecting lipopolysaccharide (LPS, 5 µg/5 µl PBS) into the substantia nigra (SN) of rats which was followed by curcumin administration (40 mg/kg b.wt (i.p.)) daily for a period of 21 days. Modulatory functions of curcumin were evident from the inhibition of astrocytic activation (GFAP) by immunofluorescence and NADPH oxidase complex activation by RT-PCR. Curcumin supplementation prevented the LPS-induced upregulation in the protein activity of transcription factor NFκB, proinflammatory cytokines (TNF-α, IL-1β, and IL-1α), inducible nitric oxide synthase (iNOS) as well as the regulating molecules of the intrinsic apoptotic pathway (Bax, Bcl-2, Caspase 3 and Caspase 9) by ELISA. Curcumin also resulted in significant improvement in the glutathione system (GSH, GSSG and redox ratio) and prevented iron deposition in the dopaminergic neurons as depicted from atomic absorption spectroscopy (AAS) and Prussian blue staining, respectively. Curcumin also prevented α-synuclein aggregates in the dopaminergic neurons as observed from gene as well as protein activity of α-synuclein using RT-PCR and IHC. Collectively, our results suggest that curcumin can be further pursued as a candidate drug in the molecules targeted therapy for PD and other related synucleopathies.
Optic nerve injury is a leading cause of irreversible visual impairment worldwide and can even cause blindness. Excessive activation of astrocytes has negative effects on the repair and recovery of ...retinal ganglion cells following optic nerve injury. However, the molecular and cellular mechanisms underlying astrocyte activation after optic nerve injury remain largely unknown. In the present study, we explored the effects of microRNA-21 (miR-21) on axon regeneration and flash visual evoked potential (F-VEP) and the underlying mechanisms of these effects based on astrocyte activation in the rat model of optic nerve crush (ONC). To the best of our knowledge, this article is the first to report that inhibition of miR-21 enhances axonal regeneration and promotes functional recovery in F-VEP in the rat model of ONC. Furthermore, inhibition of miR-21 attenuates excessive astrocyte activation and glial scar formation, thereby promoting axonal regeneration by regulating the epidermal growth factor receptor (EGFR) pathway. In addition, we observed that the expression of tissue inhibitor of metalloproteinase-3, a target gene of miR-21, was inhibited during this process. Taken together, these findings demonstrate that inhibition of miR-21 regulates the EGFR pathway, ameliorating excessive astrocyte activation and glial scar progression and promoting axonal regeneration and alleviating impairment in F-VEP function in a model of ONC. This study's results suggest that miR-21 may represent a therapeutic target for optic nerve injury.
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•Inhibition of miR-21 enhances axon regeneration and promotes functional recovery of F-VEP in a rat model of ONC.•Inhibition of miR-21 attenuates excessive astrocyte activation and glial scar formation, thus promoting axon regeneration.•miR-21-mediated activation of the EGFR/PI3K/AKT pathway is involved in astrocyte activation after ONC.•Tissue inhibitor of metalloproteinase-3, a target gene of miR-21, was inhibited during the procedure.
Depression is a common and potentially life-threatening mental illness, and currently, there is a lack of effective treatment. It has been reported that dehydrocorydaline (DHC) can inhibit monoamine ...transporter uptake in depressed CUMS mice, but more possible mechanisms of action remain to be further studied.
C57BL/6 mice were exposed to chronic unpredictable mild stress (CUMS) for five consecutive weeks. The mice were administrated with dehydrocorydaline or fluoxetine (FLU) for four consecutive weeks. Behavioral tests including sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST) were applied. In parallel, hematoxylin and eosin (H&E) staining and Nissl staining were used to explore the effect of DHC on pathological changes in the hippocampus. The concentrations of depression-related factors (5-HT and DA) and inflammatory factors (TNF-α, IL-6, and IL-1β) in the hippocampus and serum were assessed by ELISA assay. NLRP3 inflammasome pathway-related proteins (NLRP3, IL-18, IL-1 IL-1α, and caspase-1) were detected by western blot. The activation of microglia and astrocytes was subjected to immunofluorescent staining. Additionally, microglia were treated with DHC (100 mg/L) for 24 h following incubation with 100 ng/ml LPS for 12 h. ov-NC or ov-NLRP3 plasmid was transfected into microglia 6 h before LPS induction for exploring the effect of NLRP3 overexpression on DHC-inhibited microglia activation. Then, conditioned media of microglia were collected from each group, followed by intervention of astrocytes for 24 h to explore the effect of NLRP3 overexpression of microglia on astrocyte activation.
administration of DHC was found to ameliorate depressive-like behaviors and attenuate neuron damage of CUMS mice. DHC increased neurotransmitter concentration, reduced the proinflammatory factor levels, attenuated NLRP3 inflammasome activation, and decreased A1 astrocyte and microglia activation in the hippocampus of CUMS mice. Furthermore,
results showed that activated microglia induced activation of A1 astrocytes but not A2 astrocytes.
Taken together, we provided evidence that DHC exhibited antidepressive effects on CUMS mice possibly
NLRP3 inflammasome-mediated astrocyte activation.
Expression of the 17β-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.
Following cerebral ischemia, reactive astrocytes express the enzyme aromatase and produce 17β-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.
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