Control of the programmed cell death machinery by the ubiquitin-proteasome system in neurons

了解成熟神经元如何不仅存活，而且作为复杂神经网络的一部分在整个生命中保持功能是生物学的一个基本问题。神经元稳态维持的缺陷是正常脑老化的关键因素，并能极大地影响神经退行性疾病的发展。然而，在正常衰老过程中支持大脑健康的分子途径尚未得到很好的表征。了解它们的本质是至关重要的，因为它们的功能障碍可能会增加神经元对与年龄相关的神经变性的易感性，并影响我们社会面临的最大挑战之一的心理健康。半胱天冬酶是一种对程序性细胞死亡至关重要的蛋白酶。一旦被激活，效应半胱天冬酶负责数百种细胞蛋白质的蛋白水解裂解和细胞结构的拆除。半胱天冬酶也被认为在多种神经退行性疾病中发挥重要作用。因此，我们的目标是了解半胱天冬酶如何在神经元中受到限制，并确定负责控制神经元中半胱天冬酶活性的新分子。由于半胱天冬酶是通过蛋白水解裂解激活的，这是不可逆的，限制半胱天冬酶活性和随后对神经元细胞损伤的最有效机制是介导其降解。泛素-蛋白酶体系统是一个高度保守的途径，是靶向蛋白降解的主要途径。泛素连接酶的功能是将泛素共价转移到蛋白质底物上，它依赖于蛋白质对蛋白酶体降解的特异性靶向。重要的是，许多神经退行性疾病都表现出泛素-蛋白酶体通路的异常。本项目的具体目标是：a-确定神经元中关键凋亡调节因子和效应因子的调节机制。确定这些机制中的缺陷是否会改变神经元的结构和活动。利用生化技术，我们将表征小鼠初级神经元中泛素介导的半胱天冬酶活性调节的模式。CRISPRi技术将用于筛选泛素连接酶及其调控神经元凋亡机制。此外，将使用最先进的共聚焦成像技术评估泛素介导的凋亡机制调节的影响，以确定无限制的caspase活性如何影响神经元结构和活性。

Understanding how mature neurons not only survive but also remain functional throughout life as part of complex neuronal networks is a fundamental question in biology. Defects in the maintenance of neuronal homeostasis is a critical factor in normal brain aging and can drastically affect the development of neurodegenerative disorders. However, the molecular pathways that support brain health during normal aging are not well characterized. Understanding their nature is critical as their dysfunction could increase the susceptibility of neurons to age-related neurodegeneration and affect mental health which are among the greatest challenges of our society.Caspases are proteases which are essential for the execution of programmed cell death. Once activated, effector caspases are responsible for the proteolytic cleavage of hundreds of cellular proteins and the dismantlement of cellular structures. Caspases are also known to play a major role in multiple neurodegenerative diseases. Our goal is therefore to understand how caspases are restricted in neurons and identify new molecules responsible for the control of caspase activity in neurons. Because caspases are activated by proteolytic cleavage, which is non-reversible, the most efficient mechanism to limit caspase activity and subsequent damage to neuronal cells is to mediate their degradation. The ubiquitin-proteasome system is a highly conserved pathway and the main way to target proteins for degradation. It relies on the specific targeting of proteins for proteasomal degradation by enzymes called ubiquitin ligases whose function is to covalently transfer ubiquitin onto protein substrates. Importantly, many neurodegenerative disorders exhibit abnormalities in ubiquitin-proteasome pathway.The specific objectives of this project are to:a- Identify the mechanisms regulating critical apoptotic regulators and effectors in neurons.b- Determine whether defects in these mechanisms alter neuronal structure and activity.Using biochemical techniques, we will characterize the modalities of ubiquitin-mediated regulation of caspase activity in primary mouse neurons. CRISPRi technology will be employed to screen for ubiquitin ligases and their regulators controlling the apoptotic machinery in neurons. Furthermore, the impact of ubiquitin-mediated regulation of the apoptotic machinery will be assessed using state of the art confocal imaging to determine how unrestricted caspase activity affects neuronal structure and activity.