Metabolic Vulnerability and Effects of APOE in Human Neurons with Impaired Endocytic Recycling

项目总结 神经退行性痴呆，包括阿尔茨海默病，会造成毁灭性的认知衰退，因此 没有治愈的方法。代谢应激被认为是导致痴呆症的原因：葡萄糖 低代谢是阿尔茨海默病(AD)和帕金森病(PD)的早期特征，而糖尿病是一种 患痴呆症的重要危险因素。然而，新陈代谢压力如何与基因结合 神经退行性疾病导致神经元死亡的危险因素还不是很清楚，也没有已知的 提高易感神经元新陈代谢弹性的方法。为了解决这些知识差距，我们已经确定了 保持细胞能量水平的遗传靶点，使用一种独特的筛选范例，结合切割- EDGE，遗传编码的ATP传感器，细胞中主要的能量输送分子，全基因组 基于CRISPR的基因操作。通过这种方法，我们已经确定了具有 当细胞处于代谢应激状态时，对ATP水平的显著影响。特别是，我们的初步数据 提示与细胞内循环相关的AD/PD疾病危险基因的敲除会导致神经元死亡 尤其是当葡萄糖缺乏的时候。其中一个基因是SORL1，这是一个可能导致阿尔茨海默病的风险基因。 已知SORL1与APOE相互作用，而APOE4变异是AD最大的遗传风险因素。但是 APOE和SORL1之间的相互作用或细胞内循环途径还不是很清楚。在.之下 Ken Nakamura博士和Robert Mahley博士的指导，与Martin Kampmann博士和 Thomas Graeber，并在Gladstone研究所充满活力的研究社区和核心的支持下 和加州大学旧金山分校，我将检验这样一个假设，即内吞循环中的AD风险突变会导致能量衰竭和 增加神经元对脂质缺陷的易感性以及APOE表达和 载脂蛋白E4基因。我将通过以下目的来研究这一假说：1)确定 破坏细胞内循环对能量消耗和呼吸的影响，2)确定受损的影响 糖代谢对内体蛋白转运的影响，3)决定载脂蛋白E的表达和基因型 有助于内细胞性循环缺陷神经元的代谢脆弱性和内体蛋白的运输。 拟议中的研究还将确定是通过遗传操作还是通过处理 代谢缺陷具有治疗神经元脆弱性和功能衰退的潜力。这个 建议的研究和职业发展计划将建立在我之前的培训基础上，并提升我的职业发展轨迹 通过培养研究能量代谢和蛋白质平衡的技能，成为一名独立的研究者 在单个神经元中，掌握分析技术来研究代谢功能障碍和对代谢的反应 强调系统级别，获得实验设计、沟通和指导方面的经验，以及 建立对痴呆和神经退行性疾病病理生理学的生物学理解的基础。

PROJECT SUMMARY Neurodegenerative dementias, including Alzheimer’s disease, inflict devastating cognitive decline, for which there is no cure. Metabolic stress is hypothesized to contribute to the development of dementia: glucose hypometabolism is an early feature in Alzheimer’s (AD) and Parkinson’s disease (PD), and diabetes is a substantial risk factor for developing dementia. However, how metabolic stress combines with genetic neurodegenerative disease risk factors to lead to neuronal death is not well understood, and there are no known ways to boost metabolic resilience in susceptible neurons. To address these knowledge gaps, we have identified genetic targets that maintain cellular energy levels, using a unique screening paradigm that combines cutting- edge, genetically encoded sensors for ATP, the main energy-carrying molecule in cells, with whole-genome CRISPR-based gene manipulations. With this approach, we have identified gene pathways that have a prominent impact on ATP levels when cells are under metabolic stress. In particular, our preliminary data indicate that knockdown of AD/PD disease risk genes associated with endocytic recycling lead to neuronal death specifically when glucose is scarce. One of these genes is SORL1, a risk gene that is potentially causal for AD. SORL1 is known to interact with APOE, and the APOE4 variant is the largest genetic risk factor for AD. But the interaction between APOE and SORL1 or the endocytic recycling pathway is not well understood. Under the mentorship of Dr. Ken Nakamura and Dr. Robert Mahley, in collaboration with Drs. Martin Kampmann and Thomas Graeber, and with the support of the vibrant research community and cores at the Gladstone Institutes and UCSF, I will test the hypothesis that AD risk mutations in endocytic recycling create an energy failure and increases the susceptibility of neurons to lipid deficits and the deleterious effects of APOE expression and APOE4 genotype. I will investigate this hypothesis through the following Aims: 1) Determine the impact of disrupting endocytic recycling on energy consumption and respiration, 2) Determine the effect of impaired glucose metabolism on endosomal protein trafficking, 3) Determine how APOE expression and genotype contribute to metabolic vulnerability and endosomal protein trafficking in endocytic recycling deficient neurons. The proposed studies will also determine if maintaining ATP levels via genetic manipulations or by addressing metabolic deficits have therapeutic potential for treating neuron vulnerability and functional decline. The proposed research and career development plan will build on my previous training and enhance my trajectory toward becoming an independent investigator by developing skills to study energy metabolism and proteostasis in individual neurons, mastering analytical techniques to study metabolic dysfunction and response to metabolic stress on a systems-level, gaining experience with experimental design, communication, and mentorship, and building a foundation of biological understanding of dementia and neurodegenerative disease pathophysiology.