Department of Pharmacology, School of Basic Medical Science (R.C., M.-Q.Z., Y.-L.M., S.-H.Z., Y.C., J.Y., C.-H.Y., H.-H.Z., Y.L., Y.-Y.F.), Shanxi Medical University, Jinzhong, China.
Medicinal Basic Research Innovation Centre of Chronic Kidney Disease, Ministry of Education (Y.C., L.T., Y.-Y.F.), Shanxi Medical University, Jinzhong, China.
Stroke. 2024 Aug;55(8):2151-2162. doi: 10.1161/STROKEAHA.124.046954. Epub 2024 Jul 1.
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.
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.
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.
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.
GPR65（G 蛋白偶联受体 65）可以感知细胞外的酸性环境，从而调节生理病理过程。研究表明，GPR65 激动剂 BTB09089 预处理可在急性缺血性脑卒中发挥神经保护作用。然而，延迟给予 BTB09089 治疗和神经元 GPR65 的激活是否能促进神经修复仍不清楚。
光血栓性脑缺血诱导野生型（WT）或 GPR65 敲除（GPR65）小鼠发生缺血性脑卒中。雄性小鼠在脑卒中后第 3、7 或 14 天，每隔一天通过腹腔注射 BTB09089 进行治疗。AAV-Syn-GPR65（腺相关病毒-突触素-GPR65）用于过表达 GPR65 于 GPR65 和 WT 小鼠的梗死周边皮质神经元。通过网格行走和圆筒测试监测运动功能。通过免疫组化、高尔基染色和 Western blot 观察 BTB09089 的神经修复作用。
即使在脑卒中后 3 至 7 天给予 BTB09089 治疗，BTB09089 也显著改善了 WT 但不改善 GPR65 小鼠的运动功能。BTB09089 抑制了 WT 但不抑制 GPR65 小鼠小胶质细胞的激活和神经胶质瘢痕的进展。同时，BTB09089 减少了 WT 小鼠神经元密度的下降，但在 GPR65 小鼠中这种益处被消除，而过表达 GPR65 于梗死周边皮质神经元后又重新出现。此外，过表达 GPR65 于 GPR65 小鼠的梗死周边皮质神经元，可增加 BTB09089 诱导的 GAP43（生长相关蛋白-43）和突触小体蛋白的点状密度、树突棘密度、树突分支长度和树突复杂度，同时伴随着磷酸化 cAMP 反应元件结合蛋白（p-CREB）水平的升高。此外，过表达 GPR65 还将 BTB09089 的治疗窗口延长至 WT 小鼠脑卒中后第 14 天。
我们的研究结果表明，延迟给予 BTB09089 治疗通过激活神经元 GPR65 改善了脑卒中后的神经功能恢复和脑组织修复。过表达 GPR65 可能是扩大 GPR65 激动剂治疗缺血性脑卒中后神经康复治疗时间窗的一种潜在策略。
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