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

了解成熟神经元作为复杂神经网络的一部分如何存活并在整个生命过程中保持功能是生物学中的一个基本问题。神经元动态平衡的维持缺陷是正常脑老化的关键因素，并可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还没有得到很好的描述。了解它们的性质是至关重要的，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退化的敏感性，并影响精神健康，这是我们社会面临的最大挑战之一。半胱氨酸天冬氨酸酶是执行细胞程序性死亡所必需的蛋白水解酶。一旦激活，效应半胱氨酸酶负责数百种细胞蛋白的蛋白水解性切割和细胞结构的分解。半胱氨酸天冬氨酸氨基转移酶在多种神经退行性疾病中也发挥着重要作用。因此，我们的目标是了解caspase是如何在神经元中受到限制的，并识别负责控制神经元中caspase活性的新分子。由于半胱氨酸天冬氨酸酶是由蛋白水解性裂解激活的，这是不可逆转的，限制半胱氨酸氨基转移酶活性和随后对神经细胞损伤的最有效的机制是调节它们的降解。泛素-蛋白酶体系统是一种高度保守的途径，也是靶向蛋白质降解的主要途径。它依赖于被称为泛素连接酶的酶对蛋白质进行蛋白酶体降解的特定靶点，其功能是将泛素共价转移到蛋白质底物上。重要的是，许多神经退行性疾病都表现出泛素-蛋白酶体途径的异常。这个项目的具体目标是：a-确定神经元中关键的凋亡调节因子和效应因子的调节机制。b-确定这些机制中的缺陷是否会改变神经元的结构和活性。利用生化技术，我们将表征泛素介导的原代小鼠神经元中caspase活性的调节方式。CRISPRi技术将被用于筛选控制神经元凋亡机制的泛素连接酶及其调节器。此外，将使用最先进的共聚焦成像来评估泛素介导的对凋亡机制的调节的影响，以确定不受限制的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.