Abstract：
BACKGROUND: Post-stroke cognitive impairment is one of the major causes of disability due to cerebral ischemia. MAD2B is an inhibitor of Cdh1/APC, and loss of Cdh1/APC function in mature neurons increases ROCK2 activity, leading to changes in synaptic plasticity and memory loss in mouse neurons. Whether MAD2B regulates learning memory capacity through ROCK2 in cerebral ischemia is not known.
OBJECTIVE: We investigated the role and mechanism of MAD2B in cerebral ischemia-induced cognitive dysfunction.
METHODS: The expression of MAD2B and its downstream related molecules was detected by immunoblotting and intervened with neuroprotectants after middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R). We constructed MAD2B-cKO-specific knockout mice, knocked down and overexpressed MAD2B in mouse hippocampus by lentiviral injection in brain stereotaxis, modeled cerebral ischemia by using MCAO, and explored the role of MAD2B in post-stroke cognitive impairment (PSCI) by animal behaviors such as Y-maze and Novel object recognition test. Then the expression of MAD2B/ROCK2, downstream molecules and apoptosis-related molecules was detected. Finally, ROCK2 expression was intervened using its inhibitor and shRNA-ROCK2 lentivirus.
RESULTS: The expression of MAD2B and its downstream molecules increased after MCAO and OGD/R. Nonetheless, this expression underwent a decline post-therapy with neuroprotective agents. Deletion of MAD2B in the hippocampus ameliorated memory and learning deficits and improved motor coordination in MCAO mice. Conversely, the overexpression of MAD2B in the hippocampus exacerbated learning and memory deficits. Deletion of MAD2B resulted in the downregulation of ROCK2/LIMK1/cofilin. It effectively reduced ischemia-induced upregulation of BAX and cleaved caspase-3, which could be reversed by MAD2B overexpression. Inhibition or knockdown of ROCK2 expression in primary cultured neurons led to the downregulation of LIMK1/cofilin expression and reduced the expression of apoptosis-associated molecules induced by ischemia.
CONCLUSIONS: Our findings suggest that MAD2B affects neuronal apoptosis via Rock2, which affects neurological function and cerebral infarction.
OBJECTIVE: We investigated the role and mechanism of MAD2B in cerebral ischemia-induced cognitive dysfunction.
METHODS: The expression of MAD2B and its downstream related molecules was detected by immunoblotting and intervened with neuroprotectants after middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R). We constructed MAD2B-cKO-specific knockout mice, knocked down and overexpressed MAD2B in mouse hippocampus by lentiviral injection in brain stereotaxis, modeled cerebral ischemia by using MCAO, and explored the role of MAD2B in post-stroke cognitive impairment (PSCI) by animal behaviors such as Y-maze and Novel object recognition test. Then the expression of MAD2B/ROCK2, downstream molecules and apoptosis-related molecules was detected. Finally, ROCK2 expression was intervened using its inhibitor and shRNA-ROCK2 lentivirus.
RESULTS: The expression of MAD2B and its downstream molecules increased after MCAO and OGD/R. Nonetheless, this expression underwent a decline post-therapy with neuroprotective agents. Deletion of MAD2B in the hippocampus ameliorated memory and learning deficits and improved motor coordination in MCAO mice. Conversely, the overexpression of MAD2B in the hippocampus exacerbated learning and memory deficits. Deletion of MAD2B resulted in the downregulation of ROCK2/LIMK1/cofilin. It effectively reduced ischemia-induced upregulation of BAX and cleaved caspase-3, which could be reversed by MAD2B overexpression. Inhibition or knockdown of ROCK2 expression in primary cultured neurons led to the downregulation of LIMK1/cofilin expression and reduced the expression of apoptosis-associated molecules induced by ischemia.
CONCLUSIONS: Our findings suggest that MAD2B affects neuronal apoptosis via Rock2, which affects neurological function and cerebral infarction.
摘要：
背景：卒中后认知障碍是脑缺血致残的主要原因之一。MAD2B是Cdh1/APC的抑制剂，成熟神经元中Cdh1/APC功能的丧失会增加ROCK2活性，导致小鼠神经元突触可塑性和记忆丧失的变化。MAD2B是否通过ROCK2调节脑缺血中的学习记忆能力尚不清楚。
目的：研究MAD2B在脑缺血后认知功能障碍中的作用及机制。
方法：采用免疫印迹法检测MAD2B及其下游相关分子的表达，并在大脑中动脉闭塞（MCAO）和氧糖剥夺/复氧（OGD/R）后给予神经保护剂干预。我们构建了MAD2B-cKO特异性基因敲除小鼠，通过在脑立体定向中注射慢病毒在小鼠海马中击倒并过表达MAD2B，用MCAO模拟脑缺血，并通过Y迷宫和新颖物体识别试验等动物行为探讨了MAD2B在卒中后认知障碍（PSCI）中的作用。然后检测MAD2B/ROCK2、下游分子和凋亡相关分子的表达。最后,使用其抑制剂和shRNA-ROCK2慢病毒干预ROCK2表达。
结果：MCAO和OGD/R后，MAD2B及其下游分子的表达增加。尽管如此，使用神经保护剂治疗后,该表达出现下降.海马中MAD2B的缺失改善了MCAO小鼠的记忆和学习缺陷并改善了运动协调。相反，海马中MAD2B的过度表达加剧了学习和记忆缺陷.MAD2B的缺失导致ROCK2/LIMK1/cofilin的下调。它有效地减少了缺血诱导的BAX和caspase-3的上调，这可以通过MAD2B过表达来逆转。在原代培养的神经元中抑制或敲低ROCK2表达导致LIMK1/cofilin表达下调，并降低缺血诱导的凋亡相关分子的表达。
结论：我们的发现表明MAD2B通过Rock2影响神经元凋亡，从而影响神经功能和脑梗死。
目的：研究MAD2B在脑缺血后认知功能障碍中的作用及机制。
方法：采用免疫印迹法检测MAD2B及其下游相关分子的表达，并在大脑中动脉闭塞（MCAO）和氧糖剥夺/复氧（OGD/R）后给予神经保护剂干预。我们构建了MAD2B-cKO特异性基因敲除小鼠，通过在脑立体定向中注射慢病毒在小鼠海马中击倒并过表达MAD2B，用MCAO模拟脑缺血，并通过Y迷宫和新颖物体识别试验等动物行为探讨了MAD2B在卒中后认知障碍（PSCI）中的作用。然后检测MAD2B/ROCK2、下游分子和凋亡相关分子的表达。最后,使用其抑制剂和shRNA-ROCK2慢病毒干预ROCK2表达。
结果：MCAO和OGD/R后，MAD2B及其下游分子的表达增加。尽管如此，使用神经保护剂治疗后,该表达出现下降.海马中MAD2B的缺失改善了MCAO小鼠的记忆和学习缺陷并改善了运动协调。相反，海马中MAD2B的过度表达加剧了学习和记忆缺陷.MAD2B的缺失导致ROCK2/LIMK1/cofilin的下调。它有效地减少了缺血诱导的BAX和caspase-3的上调，这可以通过MAD2B过表达来逆转。在原代培养的神经元中抑制或敲低ROCK2表达导致LIMK1/cofilin表达下调，并降低缺血诱导的凋亡相关分子的表达。
结论：我们的发现表明MAD2B通过Rock2影响神经元凋亡，从而影响神经功能和脑梗死。
