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

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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.