Bioenergetic and Proteolytic Impact of Ubiquitin-like Pathways in Metabolically Stressed Neurons

项目摘要 衰老过程和许多与衰老相关的疾病，如帕金森病（PD），被假设为 由细胞能量和线粒体功能下降引起。细胞能量对许多人来说至关重要 细胞过程，包括蛋白质降解，这是一个在神经退行性疾病中被破坏的过程。 识别调节ATP水平的基因，ATP是所有细胞中主要的能量携带分子， 对减缓或逆转衰老相关的病理，保护神经元功能以应对压力至关重要， 从而深入了解能量衰竭如何导致疾病。然而，这一分析受到了一个 缺乏以高通量筛选基因组中ATP水平修饰剂的工具，导致了 对导致能量衰竭的遗传因素的了解。我们开发了一种独特的筛选模式， 通过将基因编码的ATP传感器与基于CRISPR的全基因组相结合， 在暴露于急性代谢应激的细胞内进行筛选技术。通过这种方法，我们确定了 三个知之甚少的基因通路，对ATP水平有显着影响，特别是当细胞 代谢仅限于呼吸。这些通路是在细胞应激时触发的， 而且还在神经变性疾病的病理生理学中观察到。虽然这些途径都 在正常代谢条件下调节蛋白质代谢和应激反应的关键方面，目前尚不清楚 受损的代谢和低ATP如何影响这些途径对蛋白质消除的贡献 压力通常与神经退行性疾病有关。我们假设在代谢应激的情况下 神经元，这些应激反应途径加剧能量衰竭和蛋白质积累。我们将 通过研究这些过程是否以及如何影响神经元中的ATP水平，以及 这些途径对蛋白质降解和代谢应激的存活的功能后果 神经元这些目标的成功实现将为能源与环境之间的关系提供新的见解 内稳态和蛋白质稳态，以及代谢应激下神经变性的进展。

PROJECT SUMMARY The aging process and many aging-associated diseases like Parkinson's disease (PD) are hypothesized to be caused by a decline in cellular energy and mitochondrial function. Cellular energy is presumably critical to many cellular processes including protein degradation, a process that is disrupted in neurodegenerative disease. Identifying genes that modulate levels of ATP, the main energy-carrying molecule in all cells, could then be critical to slowing or reversing aging-associated pathology, preserving neuron function in response to stress, and providing insight into how energy failure contributes to disease. However, that analysis has been limited by a lack of tools to screen the genome at high throughput for modifiers of ATP levels, causing a critical gap in knowledge of genetic contributors to energy failure. We have developed a unique screening paradigm to address this knowledge gap by combining genetically encoded ATP sensors with CRISPR-based whole-genome screening technology within cells exposed to acute metabolic stress. With this approach, we have identified three poorly-understood gene pathways that have a prominent impact on ATP levels specifically when cells are metabolically restricted to using only respiration. These pathways are triggered in response to cellular stress, but have also been observed in the pathophysiology of neurodegenerative diseases. While these pathways all regulate key facets of protein metabolism and stress response under normal metabolic conditions, it is unclear how compromised metabolism and low ATP affect these pathways' contributions to the elimination of protein stresses commonly associated with neurodegenerative diseases. We hypothesize that in metabolically-stressed neurons, these stress-responsive pathways exacerbate energy failure and protein accumulation. We will address this hypothesis by investigating if and how these processes affect ATP levels in neurons, as well as the functional consequences of these pathways on protein degradation and survival of metabolically stressed neurons. Successful completion of these aims will provide new insight into the relation between energy homeostasis and proteostasis, as well as the progression of neurodegeneration under metabolic stress.