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

项目摘要 神经退行性痴呆，包括阿尔茨海默氏病，造成毁灭性的认知能力下降， 无法治愈代谢压力被假设为有助于痴呆症的发展：葡萄糖 代谢减退是阿尔茨海默氏病（AD）和帕金森氏病（PD）的早期特征，而糖尿病是阿尔茨海默氏病（AD）和帕金森氏病（PD）的早期特征。 是导致痴呆症的重要危险因素。然而，代谢压力如何结合遗传 导致神经元死亡的神经退行性疾病风险因素尚未得到很好的理解， 增强易受影响神经元代谢恢复能力的方法。为了解决这些知识差距，我们确定了 遗传目标，保持细胞能量水平，使用一种独特的筛选模式，结合切割， 细胞中主要的能量携带分子ATP的边缘基因编码传感器， 基于CRISPR的基因操作通过这种方法，我们已经确定了基因通路， 当细胞处于代谢压力下时，对ATP水平的显著影响。特别是，我们的初步数据 表明与内吞再循环相关AD/PD疾病风险基因的敲低导致神经元死亡 特别是当葡萄糖缺乏时。其中一个基因是SORL 1，这是一种可能导致AD的风险基因。 已知SORL 1与APOE相互作用，而APOE 4变体是AD的最大遗传风险因素。但 APOE和SORL 1之间的相互作用或内吞再循环途径还不清楚。下 Ken中村博士和Robert Mahley博士与Martin Kampmann博士合作， 托马斯格雷伯，并在充满活力的研究社区和核心的支持下，在格莱斯顿研究所 和加州大学旧金山分校，我将测试这一假设，即AD风险突变的内吞再循环创造了一个能源故障， 增加神经元对脂质缺乏的易感性和APOE表达的有害作用， APOE 4基因型。我将通过以下目的来研究这一假设：1）确定 破坏内吞再循环对能量消耗和呼吸的影响，2）确定受损的 葡萄糖代谢对内体蛋白运输的影响，3）确定APOE表达和基因型 导致内吞再循环缺陷神经元的代谢脆弱性和内体蛋白运输。 拟议的研究还将确定是否通过基因操作或通过解决 代谢缺陷具有治疗神经元脆弱性和功能衰退的治疗潜力。的 拟议的研究和职业发展计划将建立在我以前的培训和提高我的轨迹 通过发展研究能量代谢和蛋白质稳态的技能，成为一名独立的研究者 在单个神经元中，掌握分析技术，研究代谢功能障碍和对代谢的反应， 强调系统级，获得实验设计、沟通和指导方面的经验， 建立对痴呆症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.