Dysfunction of astrocytic glycophagy exacerbates reperfusion injury in ischemic stroke.

Glycophagy has evolved from an alternative glycogen degradation pathway into a multifaceted pivot to regulate cellular metabolic hemostasis in peripheral tissues. However, the pattern of glycophagy in the brain and its potential therapeutic impact on ischemic stroke remain unknown. Here, we observed that the dysfunction of astrocytic glycophagy was caused by the downregulation of the GABA type A receptor-associated protein like 1 (GABARAPL1) during reperfusion in ischemic stroke patients and mice. PI3K-Akt pathway activation is involved in driving GABARAPL1 downregulation during cerebral reperfusion. Moreover, glycophagy dysfunction-induced glucosamine deficiency suppresses the nuclear translocation of specificity protein 1 and TATA binding protein, the transcription factors for GABARAPL1, by decreasing their O-GlcNAcylation levels, and accordingly feedback inhibits GABARAPL1 in astrocytes during reperfusion. Restoring astrocytic glycophagy by overexpressing GABARAPL1 decreases DNA damage and oxidative injury in astrocytes and improves the survival of surrounding neurons during reperfusion. In addition, a hypocaloric diet in the acute phase after cerebral reperfusion can enhance astrocytic glycophagic flux and accelerate neurological recovery. In summary, glycophagy in the brain links autophagy, metabolism, and epigenetics together, and glycophagy dysfunction exacerbates reperfusion injury after ischemic stroke.

Gastrodin Ameliorates Post-Stroke Depressive-Like Behaviors Through Cannabinoid-1 Receptor-Dependent PKA/RhoA Signaling Pathway.

Post-stroke depression (PSD) is a significant complication in stroke patients, increases long-term mortality, and exaggerates ischemia-induced brain injury. However, the underlying molecular mechanisms and effective therapeutic targets related to PSD have remained elusive. Here, we employed an animal behavioral model of PSD by combining the use of middle cerebral artery occlusion (MCAO) followed by spatial restraint stress to study the molecular underpinnings and potential therapies of PSD. Interestingly, we found that sub-chronic application of gastrodin (Gas), a traditional Chinese medicinal herb Gastrodia elata extraction, relieved depression-related behavioral deficits, increased the impaired expression of synaptic transmission-associated proteins, and restored the altered spine density in hippocampal CA1 of PSD animals. Furthermore, our results indicated that the anti-PSD effect of Gas was dependent on membrane cannabinoid-1 receptor (CB1R) expression. The contents of phosphorated protein kinase A (p-PKA) and phosphorated Ras homolog gene family member A (p(ser188)-RhoA) were decreased in the hippocampus of PSD-mice, which was reversed by Gas treatment, and CB1R depletion caused a diminished efficacy of Gas on p-PKA and p-RhoA expression. In addition, the anti-PSD effect of Gas was partially blocked by PKA inhibition or RhoA activation, indicating that the anti-PSD effect of Gas is associated with the CB1R-mediated PKA/RhoA signaling pathway. Together, our findings revealed that Gas treatment possesses protective effects against the post-stroke depressive-like state; the CB1R-involved PKA/RhoA signaling pathway is critical in mediating Gas's anti-PSD potency, suggesting that Gas application may be beneficial in the prevention and adjunctive treatment of PSD.

Estrogen receptor GPR30 in the anterior cingulate cortex mediates exacerbated neuropathic pain in ovariectomized mice.

Menopausal women experience neuropathic pain 63% more frequently than men do, which may attribute to the estrogen withdrawal. However, the underlying mechanisms remain unclear. Here, the role of estrogen receptors (ERs) in ovariectomized (OVX) female mice following chronic constriction injury (CCI) was investigated. With 17ß-estradiol (E2) supplemented, aggravated mechanical allodynia in OVX mice could be significantly alleviated, particularly after intra-anterior cingulate cortex (ACC) E2 delivery. Pharmacological interventions further demonstrated that the agonist of G-protein-coupled estrogen receptor 30 (GPR30), rather than ERα or ERß in the ACC, exhibited the similar analgesic effect as E2, whereas antagonist of GPR30 exacerbated allodynia. Furthermore, OVX surgery reduced GPR30 expression in the ACC, which could be restored with estrogen supplementation. Selective downregulation of GPR30 in the ACC of naïve female mice induces mechanical allodynia, whereas GPR30 overexpression in the ACC remarkedly alleviated OVX-exacerbated allodynia. Collectively, estrogen withdrawal could downregulate the ACC GPR30 expression, resulting in exacerbated neuropathic pain. Our findings highlight the importance of GPR30 in the ACC in aggravated neuropathic pain during menopause, and offer a potential therapeutic candidate for neuropathic pain management in menopausal women.

Potentiation of the lateral habenula-ventral tegmental area pathway underlines the susceptibility to depression in mice with chronic pain.

Chronic pain often develops severe mood changes such as depression. However, how chronic pain leads to depression remains elusive and the mechanisms determining individuals' responses to depression are largely unexplored. Here we found that depression-like behaviors could only be observed in 67.9% of mice with chronic neuropathic pain, leaving 32.1% of mice with depression resilience. We determined that the spike discharges of the ventral tegmental area (VTA)-projecting lateral habenula (LHb) glutamatergic (Glu) neurons were sequentially increased in sham, resilient and susceptible mice, which consequently inhibited VTA dopaminergic (DA) neurons through a LHbGlu-VTAGABA-VTADA circuit. Furthermore, the LHbGlu-VTADA excitatory inputs were dampened via GABAB receptors in a pre-synaptic manner. Regulation of LHb-VTA pathway largely affected the development of depressive symptoms caused by chronic pain. Our study thus identifies a pivotal role of the LHb-VTA pathway in coupling chronic pain with depression and highlights the activity-dependent contribution of LHbGlu-to-VTADA inhibition in depressive behavioral regulation.

Sexual dimorphic distribution of G protein-coupled receptor 30 in pain-related regions of the mouse brain.

Sex differences in pain sensitivity have contributed to the fact that medications for curing chronic pain are unsatisfactory. However, the underlying mechanism remains to be elucidated. Brain-derived estrogen participates in modulation of sex differences in pain and related emotion. G protein-coupled receptor 30 (GPR30), identified as a novel estrogen receptor with a different distribution than traditional receptors, has been proved to play a vital role in regulating pain affected by estrogen. However, the contribution of its distribution to sexually dimorphic pain-related behaviors has not been fully explored. In the current study, immunofluorescence assays were applied to mark the neurons expressing GPR30 in male and female mice (in metestrus and proestrus phase) in pain-related brain regions. The neurons that express CaMKIIα or VGAT were also labeled to observe overlap with GPR30. We found that females had more GPR30-positive (GPR30+ ) neurons in the primary somatosensory (S1) and insular cortex (IC) than males. In the lateral habenula (LHb) and the nucleus tractus solitarius (NTS), males had more GPR30+ neurons than females. Moreover, within the LHb, the expression of GPR30 varied with estrous cycle phase; females in metestrus had fewer GPR30+ neurons than those in proestrus. In addition, females had more GPR30+ neurons, which co-expressed CaMKIIα in the medial preoptic nucleus (mPOA) than males, while males had more than females in the basolateral complex of the amygdala (BLA). These findings may partly explain the different modulatory effects of GPR30 in pain and related emotional phenotypes between sexes and provide a basis for comprehension of sexual dimorphism in pain related to estrogen and GPR30, and finally provide new targets for exploiting new treatments of sex-specific pain.

RIPK3 promotes hantaviral replication by restricting JAK-STAT signaling without triggering necroptosis.

Hantaan virus (HTNV) is a rodent-borne virus that causes hemorrhagic fever with renal syndrome (HFRS), resulting in a high mortality rate of 15%. Interferons (IFNs) play a critical role in the anti-hantaviral immune response, and IFN pretreatment efficiently restricts HTNV infection by triggering the expression of a series of IFN-stimulated genes (ISGs) through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT) pathway. However, the tremendous amount of IFNs produced during late infection could not restrain HTNV replication, and the mechanism remains unclear. Here, we demonstrated that receptor-interacting protein kinase 3 (RIPK3), a crucial molecule that mediates necroptosis, was activated by HTNV and contributed to hantavirus evasion of IFN responses by inhibiting STAT1 phosphorylation. RNA-seq analysis revealed the upregulation of multiple cell death-related genes after HTNV infection, with RIPK3 identified as a key modulator of viral replication. RIPK3 ablation significantly enhanced ISGs expression and restrained HTNV replication, without affecting the expression of pattern recognition receptors (PRRs) or the production of type I IFNs. Conversely, exogenously expressed RIPK3 compromised the host's antiviral response and facilitated HTNV replication. RIPK3-/- mice also maintained a robust ability to clear HTNV with enhanced innate immune responses. Mechanistically, we found that RIPK3 could bind STAT1 and inhibit STAT1 phosphorylation dependent on the protein kinase domain (PKD) of RIPK3 but not its kinase activity. Overall, these observations demonstrated a noncanonical function of RIPK3 during viral infection and have elucidated a novel host innate immunity evasion strategy utilized by HTNV.

Chaperone-mediated autophagy (CMA) alleviates cognitive impairment by reducing neuronal death in sepsis-associated encephalopathy (SAE).

Sepsis-associated encephalopathy (SAE) is a common and severe complication of sepsis, which causes long-term neurological deficits, such as cognitive impairment. Despite extensive research, there is still lack of specific treatments for SAE. Chaperone-mediated autophagy (CMA), a selective type of autophagy, has been reported to be related to cognitive dysfunctions in many neurodegenerative diseases. The aim of this study was to investigate the alteration of CMA activity in the hippocampus of SAE mice and explore the neuroprotective effect of enhanced CMA. Cecal ligation and puncture (CLP) was conducted to induce SAE. In the contextual fear conditioning test, the ratio of freezing time of CLP mice significantly decreased compared with that of the mice in the Sham group, indicating cognitive impairment in SAE mice. The expression of lysosome-associated membrane protein type 2A (Lamp2a) and chaperone heat shock cognate 71 kDa protein (Hsc70), positive markers for CMA activity, decreased in hippocampal neurons of SAE mice. Although overexpression of Lamp2a in neurons via adeno-associated virus injection in the hippocampus had little effect on the mortality of septic mice, this intervention significantly alleviated the memory impairments in contextual fear conditioning test, Y-maze test and novel objective recognition test, and attenuated the neural death observed in SAE mice. We further demonstrated that the overexpression of Lamp2a in the hippocampus increased the expression of phosphorylated cyclic-AMP response element binding protein (p-CREB), brain-derived neurotrophic factor (BDNF) and B-cell lymphoma-2 (Bcl-2), and suppressed the expression of cleaved caspase-3. Taken together, our study results suggested that the upregulation of CMA activity ameliorated cognitive impairments and neuron loss in SAE mice partially through the p-CREB-BDNF/Bcl-2 signaling pathways, providing a potential therapeutic target for SAE.

Adiponectin Promotes Neurogenesis After Transient Cerebral Ischemia Through STAT3 Mediated BDNF Upregulation in Astrocytes.

Newborn neurons from the subventricular zone (SVZ) are essential to functional recovery following ischemic stroke. However, the number of newly generated neurons after stroke is far from enough to support a potent recovery. Adiponectin could increase neurogenesis in the dentate gyrus of hippocampus in neurodegenerative diseases. However, the effect of adiponectin on the neurogenesis from SVZ and the functional recovery after ischemic stroke was unknown, and the underlying mechanism was not specified either. The middle cerebral artery occlusion model of mice was adopted and adiponectin was administrated once a day from day 3 to 7 of reperfusion. The levels of BDNF and p-STAT3 were detected by western blotting on day 7 of reperfusion. The virus-encoded BDNF shRNA with GFAP promoter and a STAT3 inhibitor Stattic were used, respectively. Neurogenesis was evidenced by the expression of doublecortin and 5-bromo-2'-deoxyuridine (BrdU) labelling and brain atrophy was revealed by Nissl staining on day 28 of reperfusion. Neurological functional recovery was assessed by the adhesive removal test and the forepaw grip strength. We found that adiponectin increased both the doublecortin-positive cells and NeuN/BrdU double-positive cells around the injured area on day 28 of reperfusion, along with the improved long-term neurological recovery. Mechanistically, adiponectin increased the protein levels of p-STAT3 and BDNF in astrocytes on day 7 of reperfusion, while silencing BDNF diminished the adiponectin-induced neurogenesis and functional recovery. Moreover, inhibition of STAT3 not only prevented the increase of BDNF but also the improved neurogenesis and functional recovery after stroke. In conclusion, adiponectin enhances neurogenesis and functional recovery after ischemic stroke via STAT3/BDNF pathway in astrocytes.

Pioglitazone attenuates ischaemic stroke aggravation by blocking PPARγ reduction and inhibiting chronic inflammation in diabetic mice.

Diabetes can cause vascular remodelling and is associated with worse outcome after ischaemic stroke. Pioglitazone is a commonly used anti-diabetic agent. However, it is not known whether pioglitazone use before ischaemia could reduce brain ischaemic injury. Pioglitazone was administered to 5-week-old db+ or db/db mice. Cerebral vascular remodelling was examined at the age of 9 weeks. Expression of peroxisome proliferator-activated receptor-γ (PPARγ), p-PPARγ (S112 and S273), nucleotide-binding domain (NOD)-like receptor protein 3 (Nlrp3), interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) was evaluated in the somatosensory cortex of mice. Neurological outcome was evaluated 24 h after brain ischaemia. Results showed that early pioglitazone treatment provided a long-lasting effect of euglycaemia but enhanced hyperlipidaemia in the db/db mice. Diabetic mice exhibited increased vascular tortuosity, narrower middle cerebral artery (MCA) width and IgG leakage in the brain. These changes were blocked by early pioglitazone treatment. In diabetic animals, PPARγ expression was reduced, and p-PPARγ at S273 but not S112, Nlrp3, IL-1ß and TNF-α were increased in the somatosensory cortex. PPARγ decrease and Nlrp3 increase were mainly in the neurons of the diabetic brain, which was reversed by early pioglitazone treatment. Pioglitazone attenuated the aggravated neurological outcome after stroke in diabetic mice. But this protective effect was abolished through restoring cerebral inflammation by intracerebroventricular administration of IL-1ß and TNF-α in pioglitazone-treated diabetic mice before MCAO. In summary, early pioglitazone treatment attenuates cerebral vascular remodelling and ischaemic brain injury possibly via blocking chronic neuroinflammation in the db/db mice.

Neuronal GPER Participates in Genistein-Mediated Neuroprotection in Ischemic Stroke by Inhibiting NLRP3 Inflammasome Activation in Ovariectomized Female Mice.

Estrogen replacement therapy (ERT) is potentially beneficial for the prevention and treatment of postmenopausal cerebral ischemia but inevitably increases the risk of cerebral hemorrhage and breast cancer when used for a long period of time. Genistein, a natural phytoestrogen, has been reported to contribute to the recovery of postmenopausal ischemic stroke with reduced risks. However, the underlying mechanism of genistein-mediated neuroprotection remains unclear. We reported that genistein exerted significant neuroprotective effects by enhancing the expression of neuronal G protein-coupled estrogen receptor (GPER) in the ischemic penumbra after cerebral reperfusion in ovariectomized (OVX) mice, and this effect was achieved through GPER-mediated inhibition of nod-like receptor protein 3 (NLRP3) inflammasome activation. In addition, we found that peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) was the pivotal molecule that participated in GPER-mediated inhibition of NLRP3 inflammasome activation in OVX mice after ischemia/reperfusion (I/R) injury. Our data suggest that the neuronal GPER/PGC-1α pathway plays an important role in genistein-mediated neuroprotection against I/R injury in OVX mice.

Sex-specific cannabinoid 1 receptors on GABAergic neurons in the ventrolateral periaqueductal gray mediate analgesia in mice.

Sex differences in analgesic effects have gradually attracted public attention in preclinical and clinical studies. Both human and animal females are more sensitive to cannabinoid antinociception than males. Expression of the cannabinoid 1 receptor (CB1 R) and the function of the endocannabinoid system have been explored in both male and female mice and CB1 Rs in the ventrolateral periaqueductal gray (vlPAG) participate in antinociception. However, whether there are cell-type- and sex-specific patterns of vlPAG CB1 R expression that affect analgesia is unknown. In the current study, we either activated or inhibited CB1 Rs in the vlPAG and found that female mice produced stronger analgesia or developed more robust mechanical allodynia than males did. Specific deletion of GABAergic CB1 Rs in the vlPAG promoted stronger mechanical allodynia in female mice than that in male mice. However, no sex differences in cannabinoid antinociception were found following chemogenetic inhibition of GABAergic neurons. Using fluorescence in situ hybridization, we found that the sex difference in cannabinoid antinociception was due to females having higher expression of GABAergic CB1 Rs in the vlPAG than males. Furthermore, activation of CB1 Rs in the vlPAG significantly reduced the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents recorded in vGlut2-tdTomato positive neurons in both sexes. This effect was greater in females than males and this reduction was closely related to CB1 R expression difference between sexes. Our work indicates that vlPAG GABAergic CB1 Rs modulate cannabinoid-mediated analgesia in a sex-specific manner, which may provide a potential explanation of sex difference found in the analgesic effect of cannabinoids.

N-myc Downstream-Regulated Gene 2 (Ndrg2): A Critical Mediator of Estrogen-Induced Neuroprotection Against Cerebral Ischemic Injury.

Growing evidence indicates that estrogen plays a pivotal role in neuroprotection against cerebral ischemia, but the molecular mechanism of this protection is still elusive. N-myc downstream-regulated gene 2 (Ndrg2), an estrogen-targeted gene, has been shown to exert neuroprotective effects against cerebral ischemia in male mice. However, the role of Ndrg2 in the neuroprotective effect of estrogen remains unknown. In this study, we first detected NDRG2 expression levels in the cortex and striatum in both female and male mice with western blot analyses. We then detected cerebral ischemic injury by constructing middle cerebral artery occlusion and reperfusion (MCAO-R) models in Ndrg2 knockout or conditional knockdown female mice. We further implemented estrogen, ERα, or ERß agonist replacement in the ovariectomized (OVX) Ndrg2 knockout or conditional knockdown female mice, then tested for NDRG2 expression, glial fibrillary acidic protein (GFAP) expression, and extent of cerebral ischemic injury. We found that NDRG2 expression was significantly higher in female than in male mice in both the cortex and striatum. Ndrg2 knockouts and conditional knockdowns showed significantly aggravated cerebral ischemic injury in female mice. Estrogen and ERß replacement treatment (DPN) led to NDRG2 upregulation in both the cortex and striatum of OVX mice. Estrogen and DPN also led to GFAP upregulation in OVX mice. However, the effect of estrogen and DPN in activating astrocytes was lost in Ndrg2 knockout OVX mice and primary cultured astrocytes, but partially retained in conditional knockdown OVX mice. Most importantly, we found that the neuroprotective effects of E2 and DPN against cerebral ischemic injury were lost in Ndrg2 knockout OVX mice but partially retained in conditional knockdown OVX mice. These findings demonstrate that estrogen alleviated cerebral ischemic injury via ERß upregulation of Ndrg2, which could activate astrocytes, indicating that Ndrg2 is a critical mediator of E2-induced neuroprotection against cerebral ischemic injury.

Astrocytic glycogen mobilization participates in salvianolic acid B-mediated neuroprotection against reperfusion injury after ischemic stroke.

Astrocytic glycogen serves as an important glucose reserve, and its degradation provides extra support for neighboring neurons during energy deficiency. Salvianolic acid B (SAB) exerts a neuroprotective effect on reperfusion insult after cerebrovascular occlusion, but the effect of SAB on astrocytic glycogen and its relationship with neuroprotection are not completely understood. Here, we knocked down astrocyte-specific glycogen phosphorylase (GP, the rate-limiting enzyme in glycogenolysis) in vitro and in vivo and investigated the changes in key enzymes in glycogen metabolism by performing immunoblotting in vitro and immunofluorescence in vivo. Neurobehavioral and morphological assessments were conducted to uncover the outcomes during brain reperfusion. SAB accelerated astrocytic glycogenolysis by upregulating GP activity but not GP expression after reperfusion. Suppression of astrocytic glycogenolysis weakened SAB-mediated neuroprotection against the reperfusion insult. In addition, activation of glycogenolysis by SAB contributed to the survival of astrocytes and surrounding neurons by increasing antioxidant levels in astrocytes. Our data reveal that astrocytic GP represents an important metabolic target in SAB-induced protection against brain damage after cerebrovascular recanalization.

De novo Lipogenesis in Astrocytes Promotes the Repair of Blood-Brain Barrier after Transient Cerebral Ischemia Through Interleukin-33.

Astrocytes experience significant metabolic shifts in the "sensitive period" of neurological function recovery following cerebral ischemia. However, the changes in astrocyte lipid metabolism and their implications for neurological recovery remain unknown. In the present study, we employed a mouse middle cerebral artery occlusion model to investigate the changes in de novo lipogenesis and interleukin-33 (IL-33) production in astrocytes and elucidate their role in blood-brain barrier (BBB) repair in the subacute phase of cerebral ischemia. Neurological behavior evaluation was used to assess functional changes in mice. Pharmacological inhibition and astrocyte-specific downregulation of fatty acid synthase (FASN) were used to evaluate the role of de novo lipogenesis in brain injury. Intracerebroventricular administration of recombinant IL-33 was performed to study the contribution of IL-33 to BBB disruption. Extravasation of Evans blue dye, dextran and IgG were used to assess BBB integrity. Western blotting of tight junction proteins ZO-1, Occludin, and Claudin-5 were performed at defined time points to evaluate changes in BBB. It was found that de novo lipogenesis was activated, and IL-33 production increased in astrocytes at the subacute stage of cerebral ischemia injury. Inhibition of lipogenesis in astrocytes decreased IL-33 production in the peri-infarct area, deteriorated BBB damage and interfered with neurological recovery. In addition, supplementation of IL-33 alleviated BBB destruction and improved neurological recovery worsened by lipogenesis inhibition. These findings indicate that astrocyte lipogenesis increases the production of IL-33 in the peri-infarct area, which promotes BBB repair in the subacute phase of cerebral ischemia injury and improves long-term functional recovery.

Astrocytic A1/A2 paradigm participates in glycogen mobilization mediated neuroprotection on reperfusion injury after ischemic stroke.

BACKGROUND: Astrocytic glycogen works as an essential energy reserve for surrounding neurons and is reported to accumulate excessively during cerebral ischemia/reperfusion (I/R) injury. Our previous study found that accumulated glycogen mobilization exhibits a neuroprotective effect against I/R damage. In addition, ischemia could transform astrocytes into A1-like (toxic) and A2-like (protective) subtypes. However, the underlying mechanism behind accumulated glycogen mobilization-mediated neuroprotection in cerebral reperfusion injury and its relationship with the astrocytic A1/A2 paradigm is unknown. METHODS: Astrocytic glycogen phosphorylase, the rate-limiting enzyme in glycogen mobilization, was specifically overexpressed and knocked down in mice and in cultured astrocytes. The I/R injury was imitated using a middle cerebral artery occlusion/reperfusion model in mice and an oxygen-glucose deprivation/reoxygenation model in cultured cells. Alterations in A1-like and A2-like astrocytes and the expression of phosphorylated nuclear transcription factor-κB (NF-κB) and phosphorylated signal transducer and activator of transcription 3 (STAT3) were determined by RNA sequencing, immunofluorescence and immunoblotting. Metabolites, including glycogen, NADPH, glutathione and reactive oxygen species (ROS), were analyzed by biochemical analysis. RESULTS: Here, we observed that astrocytic glycogen mobilization inhibited A1-like astrocytes and enhanced A2-like astrocytes after reperfusion in an experimental ischemic stroke model in vivo and in vitro. In addition, glycogen mobilization could enhance the production of NADPH and glutathione by the pentose phosphate pathway (PPP) and reduce ROS levels during reperfusion. NF-κB inhibition and STAT3 activation caused by a decrease in ROS levels were responsible for glycogen mobilization-induced A1-like and A2-like astrocyte transformation after I/R. The astrocytic A1/A2 paradigm is closely correlated with glycogen mobilization-mediated neuroprotection in cerebral reperfusion injury. CONCLUSIONS: Our data suggest that ROS-mediated NF-κB inhibition and STAT3 activation are the key pathways for glycogen mobilization-induced neuroprotection and provide a promising metabolic target for brain reperfusion injury in ischemic stroke.