Autophagy-lysosomal defect in human CADASIL vascular smooth muscle cells
Autophagy-lysosomal defect in human CADASIL vascular smooth muscle cells
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DOI：
10.1016/j.ejcb.2018.10.001
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发表时间：
2018-11-01
影响因子：
6.6
通讯作者：
Behbahani, Homira
中科院分区：
文献类型：
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作者：
Hanemaaijer, Evelyn S.;Panahi, Mahmod;Behbahani, Homira
常染色体显性遗传性脑动脉病合并皮质下梗塞和白质脑病(CADASIL)是由NOTCH3基因突变引起的家族性进行性退行性疾病。以前的研究报道，突变体NOTCH3比野生型NOTCH3更容易形成聚集体，并且突变体聚集体具有抗降解能力。我们假设NOTCH3的聚集或聚集可能是由于VSMC的溶酶体-自噬机制受损所致。在这里，我们使用来自正常对照和携带NOTCH3(RI33C)突变的CADASIL患者的脑VSMCs来研究NOTCH3聚集/聚集的可能原因。硫代黄素-S染色证实，与VSMCWT相比，VSMCR133C中聚集的NOTCH3积累增加。在VSMCR133C中检测到溶酶体标记物Lamp2的水平升高，双重免疫组织化学显示其与NOTCH3的定位相同。在VSMCR133C中观察到Lc3-II/Lc3-I比值升高，表明自噬小体积累。与VSMCWT相比，VSMCR133C中NOTCH3与LC3和Lamp2共定位的减少，以及p62/SQSTM1水平的进一步增加。此外，Western印迹分析显示p-ERK、p-S6RP和p-P70 S6K被磷酸化。总之，这些结果表明VSMCR133C自噬-溶酶体途径存在功能障碍。本研究为研究CADASIL的分子机制提供了一条有趣的途径。
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC.Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3(RI33C) mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMCR133C compared to VSMCWT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMCR133C, which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMCR133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMCR133C compared to the VSMCWT. In addition, Western blot analysis indicated phosphorylation of p-ERK, p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMCR133C.The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.