目的:加重神经元丢失,主要由神经元凋亡引起,在阿尔茨海默病(AD)患者和AD动物模型的大脑中观察到。双特异性和酪氨酸磷酸化调节蛋白激酶1A(Dyrk1A)的截短形式在AD发病机理中起着至关重要的作用。抗凋亡Bcl-xL的下调与AD中的神经元丢失密切相关。然而,Dyrk1A在AD中对神经元凋亡和Bcl-x表达的分子调节仍然很难理解。这里,我们旨在探讨Dyrk1A在细胞凋亡中的作用及其分子机制。
方法:细胞计数套件-8(CCK8),流式细胞术,和TdT介导的dUTP尼克末端标记(TUNEL)用于检查细胞凋亡。细胞,用Dyrk1A或/和用Bcl-x小基因的ASF转染,通过RT-PCR和Western印迹分析Bcl-x的表达。免疫共沉淀,放射自显影,免疫荧光检测ASF与Dyrk1A的相互作用。在过表达Dyrk1A(TgDyrk1A)的小鼠和AD模型5xFAD小鼠中进行凋亡相关基因的基因集富集分析(GSEA)。
结果:Dyrk1A促进Bcl-xS表达和细胞凋亡。剪接因子ASF促进Bcl-x外显子2b包含,导致Bcl-xL表达增加。Dyrk1A通过磷酸化抑制ASF介导的Bcl-x外显子2b包涵。Dyrk1A的C端缺失促进了其与ASF的结合和激酶活性。此外,Dyrk1a1-483进一步抑制ASF介导的Bcl-x外显子2b包涵体并加重细胞凋亡。截断的Dyrk1A,增加Bcl-xS,在5xFAD小鼠脑中观察到凋亡相关基因的富集。
结论:我们推测增加的Dyrk1A和截短的Dyrk1A可能通过磷酸化ASF降低Bcl-xL/Bcl-xS的比例来加重AD的神经元凋亡。
Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer\'s disease (AD) and animal models of AD. A truncated form of Dual-specific and tyrosine phosphorylation-regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti-apoptotic Bcl-xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl-x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis.
Cell Counting Kit-8 (CCK8), flow cytometry, and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and
ASF with Bcl-x minigene, were used to assay Bcl-x expression by RT-PCR and Western blots. Co-immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis-related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice.
Dyrk1A promoted Bcl-xS expression and apoptosis. Splicing factor
ASF promoted Bcl-x exon 2b inclusion, leading to increased Bcl-xL expression. Dyrk1A suppressed
ASF-mediated Bcl-x exon 2b inclusion via phosphorylation. The C-terminus deletion of Dyrk1A facilitated its binding and kinase activity to
ASF. Moreover, Dyrk1a1-483 further suppressed the ASF-mediated Bcl-x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl-xS, and enrichment of apoptosis-related genes was observed in the brain of 5xFAD mice.
We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl-xL/Bcl-xS via phosphorylating
ASF in AD.