Abstract：
OBJECTIVE: The glymphatic system serves as a perivascular pathway that aids in clearing liquid and solute waste from the brain, thereby enhancing neurological function. Disorders in glymphatic drainage contribute to the development of vasogenic edema following cerebral ischemia, although the molecular mechanisms involved remain poorly understood. This study aims to determine whether a deficiency in dystrophin 71 (DP71) leads to aquaporin-4 (AQP4) depolarization, contributing to glymphatic dysfunction in cerebral ischemia and resulting in brain edema.
METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation.
RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level.
CONCLUSIONS: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.
METHODS: A mice model of middle cerebral artery occlusion and reperfusion was used. A fluorescence tracer was injected into the cortex and evaluated glymphatic clearance. To investigate the role of DP71 in maintaining AQP4 polarization, an adeno-associated virus with the astrocyte promoter was used to overexpress Dp71. The expression and distribution of DP71 and AQP4 were analyzed using immunoblotting, immunofluorescence, and co-immunoprecipitation techniques. The behavior ability of mice was evaluated by open field test. Open-access transcriptome sequencing data were used to analyze the functional changes of astrocytes after cerebral ischemia. MG132 was used to inhibit the ubiquitin-proteasome system. The ubiquitination of DP71 was detected by immunoblotting and co-immunoprecipitation.
RESULTS: During the vasogenic edema stage following cerebral ischemia, a decline in the efflux of interstitial fluid tracer was observed. DP71 and AQP4 were co-localized and interacted with each other in the perivascular astrocyte endfeet. After cerebral ischemia, there was a notable reduction in DP71 protein expression, accompanied by AQP4 depolarization and proliferation of reactive astrocytes. Increased DP71 expression restored glymphatic drainage and reduced brain edema. AQP4 depolarization, reactive astrocyte proliferation, and the behavior of mice were improved. After cerebral ischemia, DP71 was degraded by ubiquitination, and MG132 inhibited the decrease of DP71 protein level.
CONCLUSIONS: AQP4 depolarization after cerebral ischemia leads to glymphatic clearance disorder and aggravates cerebral edema. DP71 plays a pivotal role in regulating AQP4 polarization and consequently influences glymphatic function. Changes in DP71 expression are associated with the ubiquitin-proteasome system. This study offers a novel perspective on the pathogenesis of brain edema following cerebral ischemia.
摘要：
目的：淋巴系统作为血管周围通路，有助于清除大脑中的液体和溶质废物，从而增强神经功能。淋巴引流障碍有助于脑缺血后血管源性水肿的发展，尽管所涉及的分子机制仍然知之甚少。本研究旨在确定肌养蛋白71（DP71）的缺乏是否会导致水通道蛋白4（AQP4）去极化，导致脑缺血中的淋巴淋巴功能障碍并导致脑水肿。
方法：采用小鼠大脑中动脉闭塞再灌注模型。将荧光示踪剂注射到皮质中并评估淋巴清除。探讨DP71在维持AQP4极化中的作用,具有星形胶质细胞启动子的腺相关病毒用于过表达Dp71.采用免疫印迹法分析DP71和AQP4的表达和分布,免疫荧光，和免疫共沉淀技术。通过旷场试验评价小鼠的行为能力。采用开放式转录组测序数据分析脑缺血后星形胶质细胞的功能变化。MG132用于抑制泛素-蛋白酶体系统。通过免疫印迹和免疫共沉淀检测DP71的泛素化。
结果：在脑缺血后的血管源性水肿阶段，观察到间质液示踪剂的外排减少。DP71和AQP4在血管周围星形胶质细胞末端足中共同定位并相互作用。脑缺血后，DP71蛋白表达显着降低，伴有AQP4去极化和反应性星形胶质细胞增殖。DP71表达增加可恢复淋巴引流并减轻脑水肿。AQP4去极化，反应性星形胶质细胞增殖，小鼠的行为得到改善。脑缺血后，DP71被泛素化降解，MG132抑制DP71蛋白水平的降低。
结论：脑缺血后AQP4去极化导致脑淋巴清除障碍，加重脑水肿。DP71在调节AQP4极化中起关键作用，从而影响淋巴功能。DP71表达的变化与泛素-蛋白酶体系统相关。本研究为脑缺血后脑水肿的发病机制提供了新的视角。
方法：采用小鼠大脑中动脉闭塞再灌注模型。将荧光示踪剂注射到皮质中并评估淋巴清除。探讨DP71在维持AQP4极化中的作用,具有星形胶质细胞启动子的腺相关病毒用于过表达Dp71.采用免疫印迹法分析DP71和AQP4的表达和分布,免疫荧光，和免疫共沉淀技术。通过旷场试验评价小鼠的行为能力。采用开放式转录组测序数据分析脑缺血后星形胶质细胞的功能变化。MG132用于抑制泛素-蛋白酶体系统。通过免疫印迹和免疫共沉淀检测DP71的泛素化。
结果：在脑缺血后的血管源性水肿阶段，观察到间质液示踪剂的外排减少。DP71和AQP4在血管周围星形胶质细胞末端足中共同定位并相互作用。脑缺血后，DP71蛋白表达显着降低，伴有AQP4去极化和反应性星形胶质细胞增殖。DP71表达增加可恢复淋巴引流并减轻脑水肿。AQP4去极化，反应性星形胶质细胞增殖，小鼠的行为得到改善。脑缺血后，DP71被泛素化降解，MG132抑制DP71蛋白水平的降低。
结论：脑缺血后AQP4去极化导致脑淋巴清除障碍，加重脑水肿。DP71在调节AQP4极化中起关键作用，从而影响淋巴功能。DP71表达的变化与泛素-蛋白酶体系统相关。本研究为脑缺血后脑水肿的发病机制提供了新的视角。
