ABSTRACT

Stroke is a leading cause of death and disability. The pathophysiological mechanisms associated with stroke are very complex and not fully understood. Lysosomal function has a vital physiological function in the maintenance of cellular homeostasis. In neurons, CTSD (cathepsin D) is an essential protease involved in the regulation of proteolytic activity of the lysosomes. Loss of CTSD leads to lysosomal dysfunction and accumulation of different cellular proteins implicated in neurodegenerative diseases. In cerebral ischemia, the role of CTSD and lysosomal function is not clearly defined. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons and the middle cerebral artery occlusion (MCAO) model of stroke to assess the role of CTSD in stroke pathophysiology. Our results show a time-dependent decrease in CTSD protein levels and activity in the mouse brain after stroke and neurons following OGD, with concurrent defects in lysosomal function. We found that shRNA-mediated knockdown of CTSD in neurons is sufficient to cause lysosomal dysfunction. CTSD knockdown further aggravates lysosomal dysfunction and cell death in OGD-exposed neurons. Restoration of CTSD protein levels via lentiviral transduction increases CTSD activity in neurons and, thus, renders resistance to OGD-mediated defects in lysosomal function and cell death. This study indicates that CTSD-dependent lysosomal function is critical for maintaining neuronal survival in cerebral ischemia; thus, strategies focused on maintaining CTSD function in neurons are potentially novel therapeutic approaches to prevent neuronal death in stroke.

Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; CTSL: cathepsin L; FTD: frontotemporal dementia, HD: Huntington disease; LAMP1: lysosomal associated membrane protein 1; LSD: lysosomal storage disease; MCAO: middle cerebral artery occlusion; OGD: oxygen glucose deprivation; OGR: oxygen glucose resupply; PD: Parkinson disease; SQSMT1: sequestosome 1; TCA: trichloroacetic acid; TTC: triphenyl tetrazolium chloride.

摘要

中风是导致死亡和残疾的主要原因。与中风相关的病理生理机制非常复杂，尚未完全了解。溶酶体功能在维持细胞稳态中具有重要的生理功能。在神经元中，CTSD（组织蛋白酶 D）是一种必需的蛋白酶，参与调节溶酶体的蛋白水解活性。CTSD 的缺失导致溶酶体功能障碍和与神经退行性疾病有关的不同细胞蛋白的积累。在脑缺血中，CTSD 和溶酶体功能的作用尚不明确。我们在小鼠皮质神经元中使用氧-葡萄糖剥夺 (OGD) 和中风的大脑中动脉闭塞 (MCAO) 模型来评估 CTSD 在中风病理生理学中的作用。我们的研究结果显示，中风后小鼠大脑和 OGD 后神经元中 CTSD 蛋白水平和活性随时间下降，同时溶酶体功能缺陷。我们发现 shRNA 介导的神经元 CTSD 敲低足以导致溶酶体功能障碍。CTSD 敲低进一步加剧了 OGD 暴露神经元中的溶酶体功能障碍和细胞死亡。通过慢病毒转导恢复 CTSD 蛋白水平会增加神经元中的 CTSD 活性，从而对 OGD 介导的溶酶体功能缺陷和细胞死亡产生抗性。该研究表明 CTSD 依赖性溶酶体功能对于维持脑缺血中的神经元存活至关重要。因此，专注于维持神经元 CTSD 功能的策略是预防中风神经元死亡的潜在新治疗方法。

缩写： 3-MA：3-甲基腺嘌呤；ACTB：肌动蛋白β；AD：阿尔茨海默病；ALS：肌萎缩侧索硬化症；CQ：氯喹；CTSB：组织蛋白酶 B；CTSD：组织蛋白酶 D；CTSL：组织蛋白酶L；FTD：额颞叶​​痴呆，HD：亨廷顿病；LAMP1：溶酶体相关膜蛋白 1；LSD：溶酶体贮积病；MCAO：大脑中动脉闭塞；OGD：氧葡萄糖剥夺；OGR：氧葡萄糖补给；PD：帕金森病；SQSMT1：隔离体 1；TCA：三氯乙酸；TTC：三苯基四唑氯化物。