Yan-Ying Fan

Ischemic stroke is a leading cause of mortality and long-term disability worldwide, with complex pathophysiological mechanisms and limited therapeutic options. The identification of key molecular targets that can effectively modulate the multifaceted pathological and reparative processes underlying ischemic stroke is urgently needed. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor widely expressed in the central nervous system (CNS), where it regulates lipid and bioenergetic metabolism, preserves blood‒brain barrier (BBB) function, modulates synaptic signaling, and supports neural stem/progenitor cell homeostasis. During cerebral ischemia, LRP1 undergoes a stage-dependent functional shift. In the acute phase, LRP1 promotes BBB disruption, tissue-type plasminogen activator (tPA)-associated hemorrhagic transformation, and early neuroinflammation, while also conferring neuroprotection by restraining inflammasome-mediated inflammatory responses and facilitating astrocyte-to-neuron mitochondrial transfer. In the recovery phase, LRP1 is involved in white matter injury and remodeling, including demyelination, remyelination, and white matter restoration. These effects are highly cell type–specific, reflecting distinct functions of LRP1 in endothelial cells, astrocytes, microglia, neutrophils, and oligodendrocyte-lineage cells. This context-dependent complexity underscores the need for a systematic review of the role of LRP1 in stroke. Here, we summarize the structural features and physiological functions of LRP1 in the CNS, discuss its roles and underlying mechanisms in ischemic stroke, and highlight the therapeutic potential and challenges of LRP1-targeted strategies for stroke intervention.

Lipid droplet (LD) accumulation in microglia results in a dysfunctional and proinflammatory state after ischemic stroke and worsens neurological outcomes; yet how this accumulation is regulated remains unclear. Interferon regulatory factor 7 (IRF7) is an immune regulatory factor whose role in lipid metabolism and autophagy has been increasingly studied in peripheral tissues. However, the role of IRF7 in microglial lipophagy (a selective autophagic process that targets LDs) and poststroke functional recovery remains unexplored. In this study, using a mouse photothrombotic ischemia (PTI) model, we observed that microglia in the peri-infarct region displayed persistent lipophagy impairment and LD accumulation for up to 21 days. Reanalysis of the single-cell RNA sequencing (scRNA-seq) dataset revealed that an Irf7 ^high microglial MG1 subcluster (disease-associated microglia) was significantly associated with autophagy and lipid metabolism poststroke. Furthermore, microglial Irf7 conditional knockout ( Irf7 cKO) mice exhibited a significant rescue of lipophagy impairment and an alleviation of the ensuing LD accumulation in microglia, accompanied by enhanced synaptic plasticity and motor functional recovery during the subacute phase poststroke. Consistently, in the 15-month-old distal middle cerebral artery occlusion (dMCAO) model, Irf7 cKO mice also displayed similar improvements. Similar results were also observed in vitro. Mechanistically, Gnai2 was identified as a positively regulated transcriptional target of IRF7. In BV2 cells and primary microglia, Gnai2 knockdown mitigated lipopolysaccharide (LPS)-induced lipophagy impairment, thereby reducing LD accumulation. This treatment also increased the level of phosphatidylcholine (PC), a key lipid for stabilizing small LDs as well as promoting autophagosome formation and autophagic flux. Consistently, microglial Irf7 deletion or knockdown attenuated stroke- or LPS-induced PC reduction both in vivo and in vitro. Furthermore, exogenous supplementation with CDP-choline, an intermediate in PC synthesis, alleviated LD accumulation and lipophagy impairment, thereby improving motor function. Additionally, delayed administration of an inhibitor of stimulator of interferon genes (STING, an upstream target of IRF7) replicated the beneficial effects observed in Irf7 cKO mice, and its effects were not further enhanced by microglial Irf7 deletion. Taken together, these novel findings reveal that persistent impairment of microglial lipophagy is a key contributor to poststroke LD accumulation, and that IRF7 is involved in this process through direct transcriptional activation of Gnai2 , which reduces the PC levels. Suppressing IRF7 with a STING inhibitor is a potential strategy for modulating microglial lipid metabolism and promoting functional recovery following stroke.

BACKGROUND: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown. METHODS: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65 ^−/− ) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65 ^−/− and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting. RESULTS: BTB09089 significantly promoted motor outcomes in WT but not in GPR65 ^−/− mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65 ^−/− mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65 ^−/− mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65 ^−/− mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice. CONCLUSIONS: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.

Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors nitric oxide and prostaglandins. The voltage-dependent K⁺ channel (K_V) blocker (4-aminopyridine) and selective K_Ca blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that K_V and K_Ca channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K⁺ channel (K_ir) blocker (barium chloride) and the K_ATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by K_ir channels. The vasodilation effect and expression of K_ir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca²⁺ levels in the isolated thoracic aorta. These results suggest that K_ir channels play a more important role than K_ATP channels in regulating vascular tone in larger arteries and that the activity of the K_ir channel is enhanced by the PKC pathway.

Orexin and its receptors are closely related to the pathogenesis of Alzheimer’s disease (AD). Although the expression of orexin system genes under physiological condition has circadian rhythm, the diurnal characteristics of orexin system genes, and its potential role in the pathogenesis in AD are unknown. In the present study, we hope to elucidate the diurnal characteristics of orexin system genes at the early stage of AD, and to investigate its potential role in the development of AD neuropathology. We firstly detected the mRNA levels of orexin system genes, AD risk genes and core clock genes (CCGs) in hypothalamus and hippocampus in 6-month-old male 3xTg-AD mice and C57BL/6J (wild type, WT) control mice, then analyzed diurnal expression profiles of all genes using JTK_CYCLE algorithm, and did the correlation analysis between expression of orexin system genes and AD risk genes or CCGs. In addition, the expression of β-amyloid protein (Aβ) and phosphorylated tau (p-tau) protein were measured. The results showed that the diurnal mRNA expression profiles of PPO, OX1R, OX2R, Bace2, Bmal1, Per1, Per2 and Cry1 in the hypothalamus, and gene expression of OX1R, OX2R, Bace1, Bmal1, Per1 and Cry2 in the hippocampus in 3xTg-AD mice were different from that in WT mice. Furthermore, there is positive correlation between orexin system genes and AD risk genes or CCGs in the brain in 3xTg-AD mice. In addition, the expression of Aβ and p-tau in hippocampus in 3xTg-AD mice were significantly increased, and the expression of p-tau is higher in night than in day. These results indicate that the abnormal expression profiles of orexin system genes and its interaction with AD risk genes or CCGs might exert important role in the pathogenesis of AD, which will increase the expression of Aβ and p-tau, and accelerate the development of AD.

Aims: Few treatments are available in the subacute phase of traumatic brain injury (TBI) except rehabilitation training. We previously reported that transient CO₂ inhalation applied within minutes after reperfusion has neuroprotective effects against cerebral ischemia/reperfusion injury. In this study, it was hypothesized that delayed CO₂ postconditioning (DCPC) starting at the subacute phase may promote neurological recovery of TBI.Methods: Using a cryogenic TBI (cTBI) model, mice received DCPC daily by inhaling 5%/10%/20% CO₂ for various time‐courses (one/two/three cycles of 10‐min inhalation/10‐min break) at Days 3–7, 3–14 or 7–18 after cTBI. Beam walking and gait tests were used to assess the effect of DCPC. Lesion size, expression of GAP‐43 and synaptophysin, amoeboid microglia number and glia scar area were detected. Transcriptome and recombinant interferon regulatory factor 7 (Irf7) adeno‐associated virus were applied to investigate the molecular mechanisms.Results: DCPC significantly promoted recovery of motor function in a concentration and time‐course dependent manner with a wide therapeutic time window of at least 7 days after cTBI. The beneficial effects of DCPC were blocked by intracerebroventricular injection of NaHCO₃. DCPC also increased puncta density of GAP‐43 and synaptophysin, and reduced amoeboid microglia number and glial scar formation in the cortex surrounding the lesion. Transcriptome analysis showed many inflammation‐related genes and pathways were altered by DCPC, and Irf7 was a hub gene, while overexpression of IRF7 blocked the motor function improvement of DCPC.Conclusions: We first showed that DCPC promoted functional recovery and brain tissue repair, which opens a new therapeutic time window of postconditioning for TBI. Inhibition of IRF7 is a key molecular mechanism for the beneficial effects of DCPC, and IRF7 may be a potential therapeutic target for rehabilitation after TBI.

Acidic postconditioning by transient CO₂ inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO₂ for various durations (three cycles of 10- or 20-min CO₂ inhalation/10-min break) at days 3–7, 7–21, or 3–21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO₂ concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3–7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.