Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
Department of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
J Alzheimers Dis. 2022;85(1):207-221. doi: 10.3233/JAD-215305.
DNA damage accumulation and mitochondrial abnormalities are elevated in neurons during aging and may contribute to neurodegenerative pathologic conditions such as Alzheimer's disease. BRCA1 interacting protein 1 or BRIP1 is a 5' to 3' DNA helicase that catalyzes many abnormal DNA structures during DNA replication, gene transcription, and recombination, and contribute to genomic integrity.
BRIP1 functions were reasonably well studied in DNA repair; however, there is limited data on its role and regulation during aging and neurodegenerative diseases.
We used immunohistochemistry, western blot, and qRT-PCR assays to analyze the expression of BRIP1. Immunofluorescence studies were performed to study the formation of R-loops, reactive oxygen species (ROS) generation, and mitochondrial morphology. Flow cytometry and transmission electron microscopy were used to evaluate mitochondrial ROS and mitochondrial structures, respectively. Oxygen consumption rate was measured using Seahorse, and the Presto Blue™ assays were used to evaluate cell viability.
Our results demonstrate the expression of BRIP1 in mouse and human brain tissues and in neuronal cell lines. BRIP1 levels were elevated in the hippocampal regions of the brains, specifically in the dentate gyrus. BRIP1 downregulation in neuronal cells caused increased R-loop formation basally and in response to H2O2 treatment. Furthermore, BRIP1 deficient cells exhibited elevated levels of excitotoxicity induced by L-Glutamic acid exposure as evidenced by (mitochondrial) ROS levels, deteriorated mitochondrial health, and cell death compared to BRIP1 proficient neuronal cells.
Overall, our results indicate an important role for BRIP1 in maintaining neuronal cell health and homeostasis by suppressing cellular oxidative stress.
在衰老过程中，神经元中的 DNA 损伤积累和线粒体异常升高，可能导致阿尔茨海默病等神经退行性病理状况。BRCA1 相互作用蛋白 1 或 BRIP1 是一种 5' 到 3' DNA 解旋酶，在 DNA 复制、基因转录和重组过程中催化许多异常 DNA 结构，并有助于基因组完整性。
BRIP1 的功能在 DNA 修复中得到了很好的研究；然而，关于其在衰老和神经退行性疾病中的作用和调节的研究数据有限。
我们使用免疫组织化学、western blot 和 qRT-PCR 分析来分析 BRIP1 的表达。进行免疫荧光研究以研究 R-环的形成、活性氧 (ROS) 的产生和线粒体形态。流式细胞术和透射电子显微镜分别用于评估线粒体 ROS 和线粒体结构。使用 Seahorse 测量耗氧率，并使用 Presto Blue™ 测定法评估细胞活力。
我们的结果表明 BRIP1 在小鼠和人类脑组织以及神经元细胞系中表达。BRIP1 水平在大脑海马区升高，特别是在齿状回。神经元细胞中 BRIP1 的下调导致基础和 H2O2 处理后 R-环形成增加。此外，与 BRIP1 功能正常的神经元细胞相比，BRIP1 缺陷细胞表现出更高水平的谷氨酸暴露引起的兴奋性毒性，这表现为（线粒体）ROS 水平升高、线粒体健康恶化和细胞死亡。
总的来说，我们的结果表明 BRIP1 通过抑制细胞氧化应激在维持神经元细胞健康和体内平衡方面发挥重要作用。
