De novo Mutation Enables NOTCH3^ECD Aggregation and Mitochondrial Dysfunction via Interactions with BAX and BCL-2

Article type: Research Article

Authors: Liu, Ruijie^{a; 1} | Gao, Chenhao^{b; c; 1} | Shang, Junkui^{a; b} | Sun, Ruihua^{b; c} | Wang, Wenjing^d | Li, Wei^b | Gao, Dandan^b | Huo, Xuejing^b | Shi, Yingying^{a; b} | Wang, Yanliang^e | Wang, Fengyu^{a; b; *} | Zhang, Jiewen^{a; b; c; d; *}

Affiliations: [a] Department of Neurology, Henan University People’s Hospital, Henan Provincial People’s Hospital, Zhengzhou, Henan, China | [b] Department of Neurology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Zhengzhou, Henan, China | [c] Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China | [d] Department of Neurology, Xinxiang Medical University, Henan Provincial People’s Hospital, Zhengzhou, Henan, China | [e] Henan Provincial Key Laboratory of Kidney Disease and Immunology, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Zhengzhou, Henan, China

Correspondence: [*] Corresponding authors: Jiewen Zhang, Department of Neu-rology, Henan University People’s Hospital, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Academy of Medical Sciences, Zhengzhou University, Xinxiang Medical University, Zhengzhou 450003, China. E-mail: zhangjiewen9900@126.com and Fengyu Wang, Department of Neurology, Henan University People’s Hospital, Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Zhengzhou 450003, China. E-mail: wangfengyu1979@163.com.

Note: [1] These authors contributed equally to this work.

Abstract: Background:Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most common monogenic hereditary pattern of cerebral small vessel disease. The aggregation of the mutant NOTCH3 may play a cytotoxic role in CADASIL. However, the main mechanism of this process remains unclear. Objective:We aimed to investigate the possible pathogenesis of the mutant NOTCH3 in CADASIL. Methods:The clinical information of two pedigrees were collected and analyzed. Furthermore, we constructed cell lines corresponding to this mutation in vitro. The degradation of the extracellular domain of NOTCH3 (NOTCH3ECD) was analyzed by Cycloheximide Pulse-Chase Experiment. Flow cytometry and cell counting kit-8 assay were performed to observe the effects of the NOTCH3 mutation on mitochondrial function and apoptosis. Results:We confirmed a de novo heterozygous missense NOTCH3 mutation (c.1690G > A, p. A564T) in two pedigrees. In vitro, the NOTCH3ECD aggregation of A564T mutant may be related to their more difficult to degrade. The mitochondrial membrane potential was attenuated, and cell viability was significant decreased in NOTCH3ECD A564T group. Interestingly, BAX and cytochrome c were significantly increased, which are closely related to the mitochondrial-mediated pathway to apoptosis. Conclusion:In our study, the aggregation of NOTCH3ECD A564T mutation may be associated with more difficult degradation of the mutant, and the aggregation may produce toxic effects to induce apoptosis through the mitochondrial-mediated pathway. Therefore, we speculated that mitochondrial dysfunction may hopefully become a new breakthrough point to explain the pathogenesis of cysteine-sparing NOTCH3 mutations.

Keywords: Apoptosis, CADASIL, cysteine-sparing NOTCH3 mutation, flow cytometry, mitochondrial dysfunction

DOI: 10.3233/JAD-215256

Journal: Journal of Alzheimer's Disease, vol. 86, no. 1, pp. 67-81, 2022