Intranasal administration of recombinant prosaposin attenuates neuronal apoptosis through GPR37/PI3K/Akt/ASK1 pathway in MCAO rats.

Studies have reported that Prosaposin (PSAP) is neuroprotective in cerebrovascular diseases. We hypothesized that PSAP would reduce infarct volume by attenuating neuronal apoptosis and promoting cell survival through G protein-coupled receptor 37(GPR37)/PI3K/Akt/ASK1 pathway in middle cerebral artery occlusion (MCAO) rats. Two hundred and thirty-five male and eighteen female Sprague-Dawley rats were used. Recombinant human PSAP (rPSAP) was administered intranasally 1 h (h) after reperfusion. PSAP small interfering ribonucleic acid (siRNA), GPR37 siRNA, and PI3K specific inhibitor LY294002 were administered intracerebroventricularly 48 h before MCAO. Infarct volume, neurological score, immunofluorescence staining, Western blot, Fluoro-Jade C (FJC) and TUNEL staining were examined. The expression of endogenous PSAP and GPR37 were increased after MCAO. Intranasal administration of rPSAP reduced brain infarction, neuronal apoptosis, and improved both short- and long-term neurological function. Knockdown of endogenous PSAP aggravated neurological deficits. Treatment with exogenous rPSAP increased PI3K expression, Akt and ASK1 phosphorylation, and Bcl-2 expression; phosphorylated-JNK and Bax levels were reduced along with the number of FJC and TUNEL positive neurons. GPR37 siRNA and LY294002 abolished the anti-apoptotic effect of rPSAP at 24 h after MCAO. In conclusion, rPSAP attenuated neuronal apoptosis and improved neurological function through GPR37/PI3K/Akt/ASK1 pathway after MCAO in rats. Therefore, further exploration of PSAP as a potential treatment option in ischemic stroke is warranted.

Macrophage Infiltration Reduces Neurodegeneration and Improves Stroke Recovery after Delayed Recanalization in Rats.

Background: Recent cerebrovascular recanalization therapy clinical trials have validated delayed recanalization in patients outside of the conventional window. However, a paucity of information on the pathophysiology of delayed recanalization and favorable outcomes remains. Since macrophages are extensively studied in tissue repair, we anticipate that they may play a critical role in delayed recanalization after ischemic stroke. Methods: In adult male Sprague-Dawley rats, two ischemic stroke groups were used: permanent middle cerebral artery occlusion (pMCAO) and delayed recanalization at 3 days following middle cerebral artery occlusion (rMCAO). To evaluate outcome, brain morphology, neurological function, macrophage infiltration, angiogenesis, and neurodegeneration were reported. Confirming the role of macrophages, after their depletion, we assessed angiogenesis and neurodegeneration after delayed recanalization. Results: No significant difference was observed in the rate of hemorrhage or animal mortality among pMCAO and rMCAO groups. Delayed recanalization increased angiogenesis, reduced infarct volumes and neurodegeneration, and improved neurological outcomes compared to nonrecanalized groups. In rMCAO groups, macrophage infiltration contributed to increased angiogenesis, which was characterized by increased vascular endothelial growth factor A and platelet-derived growth factor B. Confirming these links, macrophage depletion reduced angiogenesis, inflammation, neuronal survival in the peri-infarct region, and favorable outcome following delayed recanalization. Conclusion: If properly selected, delayed recanalization at day 3 postinfarct can significantly improve the neurological outcome after ischemic stroke. The sanguineous exposure of the infarct/peri-infarct to macrophages was essential for favorable outcomes after delayed recanalization at 3 days following ischemic stroke.

S100A8 regulates autophagy-dependent ferroptosis in microglia after experimental subarachnoid hemorrhage.

Targeting microglial activation has been shown to ameliorate early brain injury (EBI) after subarachnoid hemorrhage (SAH). Ferroptosis is a new form of programmed cell death after SAH, but these molecular features were not recognized as evidence of microglial function so far. In this study, we constructed microglial S100A8-specific knockdown and established the SAH model in vivo and in vitro. Multi-technology strategies, including high throughput sequencing, adeno-associated virus gene gene-editing and several molecular biotechnologies to validate the effects of S100A8 on microglial autophagy and ferroptosis after SAH. Our results revealed that the expression of S100A8 was significantly increased in brain tissue after SAH. Targeted microglial S100A8 inhibition improved neural function and neuronal apoptosis in mice after SAH. Further mechanism exploration found that favourable effects of S100A8 depletion in EBI may be through the inhibition of microglia autophagy-dependent ferroptosis. In conclusion, S100A8 may be a potential intervention target for microglial ferroptosis in EBI after SAH.

Circular RNA circPHKA2 Relieves OGD-Induced Human Brain Microvascular Endothelial Cell Injuries through Competitively Binding miR-574-5p to Modulate SOD2.

BACKGROUND: Circular RNA phosphorylase kinase regulatory subunit alpha 2 (circPHKA2; hsa_circ_0090002) has a significantly, specifically different expression in acute ischemic stroke (AIS) patients' blood. Here, we intended to investigate the role and mechanism of circPHKA2 in oxygen-glucose deprivation- (OGD-) induced stoke model in human brain microvascular endothelial cells (HBMEC). METHODS: Expression of circPHKA2, microRNA- (miR-) 574-5p, and superoxide dismutase-2 (SOD2) was detected by quantitative PCR and western blotting. Cell injury was measured by detecting cell proliferation (EdU assay and CCK-8 assay), migration (transwell assay), neovascularization (tube formation assay), apoptosis (flow cytometry and western blotting), endoplasmic reticulum stress (western blotting), and oxidative stress (assay kits). Direct intermolecular interaction was determined by bioinformatics algorithms, dual-luciferase reporter assay, biotin-labelled miRNA capture, and argonaute 2 RNA immunoprecipitation. RESULTS: circPHKA2 was downregulated in AIS patients' blood in SOD2-correlated manner. Reexpressing circPHKA2 rescued EdU incorporation, cell viability and migration, tube formation, B cell lymphoma-2 (Bcl-2) expression, and SOD activity of OGD-induced HBMEC and alleviate apoptotic rate and levels of Bcl-2-associated protein (Bax), glucose-regulated protein 78 kD (GRP78), C/EBP-homologous protein (CHOP), caspase-12, reactive oxygen species (ROS), and malondialdehyde (MDA). Additionally, blocking SOD2 partially attenuated these roles of circPHKA2 overexpression. Molecularly, circPHKA2 upregulated SOD2 expression via interacting with miR-574-5p, and miR-574-5p could target SOD2. Similarly, allied to neurovascular protection of circPHKA2 was the downregulation of miR-574-5p. CONCLUSION: circPHKA2 could protect HBMEC against OGD-induced cerebral stroke model via the miR-574-5p/SOD2 axis, suggesting circPHKA2 as a novel and promising candidate in ischemic brain injury.

RNA-Seq Reveals Underlying Transcriptomic Mechanisms of Bone Marrow-Derived Mesenchymal Stem Cells in the Regulation of Microglia-Mediated Neuroinflammation After Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a life-threatening cerebrovascular disease with high rates of morbidity and mortality. Microglia, the resident immune cells of the central nervous system, are involved in initiating inflammatory response post-SAH through releasing a variety of inflammatory mediators. Regulation of neuroinflammation triggered by activated microglia has become a promising therapeutic strategy for SAH. Recent studies reported that bone marrow-derived mesenchymal stem cells (BM-MSCs) have therapeutic effects, resulting from the regulation of microglia activation and production of inflammatory cytokines post-SAH. However, the underlying molecular mechanisms of BM-MSCs in targeting microglia-mediated neuroinflammation after SAH are still unclear. In this study, we used murine microglia cell line BV2 treated with oxyhemoglobin (OxyHb) to mimic the SAH conditions in vitro. The results showed that BM-MSCs coculture modulated OxyHb-induced BV2 activation as well as polarization. We further implemented RNA-seq approaches to investigate differences in transcriptomes between OxyHb-stimulated BV2 cocultured with and without BM-MSCs. The RNA-seq results suggested that the levels of inflammatory genes were strongly altered when OxyHb-stimulated BV2 cells were cocultured with BM-MSCs. Moreover, we identified epigenetic regulators involved in the regulation of microglia-mediated inflammation by BM-MSCs. This study clarifies detailed transcriptomic mechanisms underlying the interaction between BM-MSCs and activated microglia and may lead to a new therapeutic strategy using stem cell therapy for SAH.

LRP1 activation attenuates white matter injury by modulating microglial polarization through Shc1/PI3K/Akt pathway after subarachnoid hemorrhage in rats.

White matter injury (WMI) is associated with motor deficits and cognitive dysfunctions in subarachnoid hemorrhage (SAH) patients. Therapeutic strategy targeting WMI would likely improve the neurological outcomes after SAH. Low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor of apolipoprotein E (apoE), is able to modulate microglia polarization towards anti-inflammatory M2 phenotypes during inflammatory and oxidative insult. In the present study, we investigated the effects of LRP1 activation on WMI and underlying mechanisms of M2 microglial polarization in a rat model of SAH. Two hundred and seventeen male Sprague Dawley rats (weight 280-330 g) were used. SAH was induced by endovascular perforation. LPR1 ligand, apoE-mimic peptide COG1410 was administered intraperitoneally. Microglial depletion kit liposomal clodronate (CLP), LPR1 siRNA or PI3K inhibitor were administered intracerebroventricularly. Post-SAH assessments included neurobehavioral tests, brain water content, immunohistochemistry, Golgi staining, western blot and co-immunoprecipitation. SAH induced WMI shown as the accumulation of amyloid precursor protein and neurofilament heavy polypeptide as well as myelin loss. Microglial depletion by CLP significantly suppressed WMI after SAH. COG1410 reduced brain water content, increased the anti-inflammatory M2 microglial phenotypes, attenuated WMI and improved neurological function after SAH. LRP1 was bound with endogenous apoE and intracellular adaptor protein Shc1. The benefits of COG1410 were reversed by LPR1 siRNA or PI3K inhibitor. LRP1 activation attenuated WMI and improved neurological function by modulating M2 microglial polarization at least in part through Shc1/PI3K/Akt signaling in a rat model of SAH. The apoE-mimic peptide COG1410 may serve as a promising treatment in the management of SAH patients.

Apolipoprotein E Exerts a Whole-Brain Protective Property by Promoting M1? Microglia Quiescence After Experimental Subarachnoid Hemorrhage in Mice.

Subarachnoid hemorrhage (SAH) is a neurologically destructive stroke in which early brain injury (EBI) plays a pivotal role in poor patient outcomes. Expanding upon our previous work, multiple techniques and methods were used in this preclinical study to further elucidate the mechanisms underlying the beneficial effects of apolipoprotein E (ApoE) against EBI after SAH in murine apolipoprotein E gene-knockout mice (Apoe-/-, KO) and wild-type mice (WT) on a C57BL/6J background. We reported that Apoe deficiency resulted in a more extensive EBI at 48 h after SAH in mice demonstrated by MRI scanning and immunohistochemical staining and exhibited more extensive white matter injury and neuronal apoptosis than WT mice. These changes were associated with an increase in NADPH oxidase 2 (NOX2) expression, an important regulator of both oxidative stress and inflammatory cytokines. Furthermore, immunohistochemical analysis revealed that NOX2 was abundantly expressed in activated M1 microglia. The JAK2/STAT3 signaling pathway, an upstream regulator of NOX2, was increased in WT mice and activated to an even greater extent in Apoe-/- mice; whereas, the JAK2-specific inhibitor, AG490, reduced NOX2 expression, oxidative stress, and inflammation in Apoe-deficient mice. Also, apoE-mimetic peptide COG1410 suppressed the JAK2/STAT3 signaling pathway and significantly reduced M1 microglia activation with subsequent attenuation of oxidative stress and inflammation after SAH. Taken together, apoE and apoE-mimetic peptide have whole-brain protective effects that may reduce EBI after SAH via M1 microglial quiescence through the attenuation of the JAK2/STAT3/NOX2 signaling pathway axis.

Apolipoprotein E Mimetic Peptide Increases Cerebral Glucose Uptake by Reducing Blood-Brain Barrier Disruption after Controlled Cortical Impact in Mice: An 18F-Fluorodeoxyglucose PET/CT Study.

Traumatic brain injury (TBI) disrupts the blood-brain barrier (BBB) and reduces cerebral glucose uptake. Vascular endothelial growth factor (VEGF) is believed to play a key role in TBI, and COG1410 has demonstrated neuroprotective activity in several models of TBI. However, the effects of COG1410 on VEGF and glucose metabolism following TBI are unknown. The current study aimed to investigate the expression of VEGF and glucose metabolism effects in C57BL/6J male mice subjected to experimental TBI. The results showed that controlled cortical impact (CCI)-induced vestibulomotor deficits were accompanied by increases in brain edema and the expression of VEGF, with a decrease in cerebral glucose uptake. COG1410 treatment significantly improved vestibulomotor deficits and glucose uptake and produced decreases in VEGF in the pericontusion and ipsilateral hemisphere of injury, as well as in brain edema and neuronal degeneration compared with the control group. These data support that COG1410 may have potential as an effective drug therapy for TBI.

An apoE-derived mimic peptide, COG1410, alleviates early brain injury via reducing apoptosis and neuroinflammation in a mouse model of subarachnoid hemorrhage.

This study investigated the neuroprotective effects of COG1410, an apoliporotein E (apoE)-derived mimic peptide, against early brain injury (EBI) after subarachnoid hemorrhage (SAH). SAH was induced in C57BL/6J mice (n=68) by endovascular perforation. Mice received intravenous injection of COG1410 (2mg/kg) or equal volume of vehicle (saline). The mortality rate, neurological score, rotarod latencies, cell apoptosis, microglial activation, pro-inflammatory cytokines production and protein levels of apoptotic and inflammatory markers were assessed at 24h after sham operation or SAH. Results showed that COG1410 alleviated the neurological deficits associated with SAH. Compared with vehicle treatment group, the number of apoptotic cells and activated microglia decreased significantly in the COG1410 treated group. COG1410 enhanced Akt activation and suppressed caspase-3 cleavage. The imbalance of Bax and Bcl-2 induced by SAH was regulated by COG1410. Additionally, COG1410 attenuated cytokines production of IL-1ß, IL-6 and TNF-α and suppressed the activation of JNK/c-Jun and NF-κB. Taken together, COG1410 protected against EBI via reducing apoptosis and neuroinflammation, through mechanisms that involve the regulation of apoptotic signaling and microglial activation. COG1410 is a potential neuroprotective agent for SAH treatment.