Understanding how neuronal glucose metabolism changes in AD due to ApoE4

项目摘要 随着医疗质量的迅速提高，我们迫切需要更好地了解年龄- 相关疾病如阿尔茨海默病（AD）。AD进展与血糖之间的明确联系- 大脑中的依赖性生物能量缺陷促使研究发现AD诊断中的关键生物标志物; 载脂蛋白E4（ApoE 4）已被证明是处于发展AD风险的患者的标志性指标。星形细胞 是大脑中ApoE 4的主要制造商，促使大多数AD相关研究专注于定义 胶质细胞与ApoE 4的关系尽管有证据表明神经元代谢功能障碍与 ApoE 4的表达，代谢变化的潜在生物学尚不清楚。我们的主要兴趣 该项目的目的是确定ApoE 4表达和降低神经元葡萄糖的机制， 新陈代谢是有联系的。我们假设，纠正表达ApoE 4的神经元中被破坏的机制， 将导致代谢表型恢复到更接近表达ApoE 3的神经元。测试 我们的假设，我们已经开发出一种范式，结合有针对性的代谢组学分析的人类 体外诱导多能干细胞（iPSC）衍生的神经元， vivo.体内模型将是C57 BL/6小鼠，其在小鼠体内具有人ApoE 3或ApoE 4的全身敲入。 淀粉样前体蛋白（APP）背景与瑞典，伊比利亚和北极突变（APPNL-G-F）。我们将 对海马进行空间转录组学，海马是受AD进展影响的第一个区域之一， 纵向追踪ApoE 4引起的最大差异表达基因。表达以下任一种的人iPSC ApoE的E3或E4同种型允许几乎纯的（>99%）神经元培养。整个神经元代谢组 用均匀的13 C标记的葡萄糖（[U-13 C]葡萄糖）探测，以定量神经元中的哪些代谢物是 由葡萄糖衍生而来。将表达ApoE 3和ApoE 4的神经元的代谢组与 根据ApoE亚型，确定神经元之间葡萄糖代谢方式的主要差异。我们 将使用CRISPR抑制或激活（CRISPRi/a）来改变我们感兴趣的基因在靶向细胞中的表达。 代谢组学研究，以评估每个基因在神经元中与葡萄糖代谢相关的功能。我们 然后将通过比较ApoE 4神经元与ApoE 3神经元来评估是否实现代谢恢复 基于葡萄糖衍生代谢物、胞质ATP和葡萄糖水平以及细胞存活的等效性。 这些目标的成功完成将为未来的研究提供信息，重点是神经元的代谢恢复 作为针对神经变性疾病的治疗靶点的候选物。这项研究将 在中村博士沿着的指导下，在格拉德斯通研究所和加州大学旧金山分校进行， 如干细胞、基因组学和生物信息学核心，以完成拟议的工作。培训计划 跨越3年的时间，主要集中在研究上，每个目标花费1.5年。该计划还留出时间 通过会议，指导和大学/社区计划培养学术科学必不可少。

PROJECT SUMMARY With rapid improvements to the quality of our healthcare, there is an urgency to better our understanding of age- related diseases like Alzheimer’s Disease (AD). Clear connections between AD progression and glucose- dependent bioenergetic deficits in the brain have motivated studies to uncover key biomarkers in AD diagnosis; apolipoprotein E4 (ApoE4) has proven to be a hallmark indicator for patients at risk of developing AD. Astrocytes are the primary manufacturer of ApoE4 in the brain, motivating most AD-relevant studies to focus on defining the relationship between glial cells and ApoE4. Despite evidence connecting neuronal metabolic dysfunction to the expression of ApoE4, the underlying biology of the metabolic changes is not understood. Our primary interest with this project is to define the mechanisms through which ApoE4 expression and reduced neuronal glucose metabolism are connected. We hypothesize that correcting disrupted mechanisms in ApoE4-expressing neurons will result in a recovery of metabolic phenotypes to more closely resemble neurons expressing ApoE3. To test our hypothesis, we have developed a paradigm that combines the targeted metabolomic analysis of human induced pluripotent stem cell (iPSC) derived neurons in vitro with the spatial transcriptomic analysis of mice in vivo. The in vivo model will be C57BL/6 mice with a whole-body knock in of human ApoE3 or ApoE4 on an amyloid precursor protein (APP) background with the Swedish, Iberian, and Arctic mutations (APPNL-G-F). We will perform spatial transcriptomics on the hippocampus, one of the first regions affected by AD progression, to longitudinally track the most differentially expressed genes due to ApoE4. The human iPSCs expressing either the E3 or E4 isoform of ApoE allows for a nearly pure (>99%) neuronal culture. The whole neuronal metabolome is probed with uniformly 13C labeled glucose ([U-13C] glucose) to quantify which metabolites in neurons are being derived from glucose. The metabolome of the ApoE3 and ApoE4 expressing neurons will be compared to define the primary differences in how glucose is metabolized between neurons based on their ApoE isoform. We will use CRISPR inhibition or activation (CRISPRi/a) to alter the expression of our genes of interest in a targeted metabolomics study to evaluate the functionality of each gene in relation to glucose metabolism in neurons. We will then evaluate whether metabolic recovery is achieved through comparing ApoE4 neurons to ApoE3 neurons based on equivalence of glucose-derived metabolites, cytosolic ATP and glucose levels, and cell survival. Successful completion of these aims will inform future studies focusing on metabolic recovery in neurons as well as providing candidates for therapeutic targets against neurodegenerative disease. The research will be conducted at Gladstone Institutes and UCSF under the mentorship of Dr. Ken Nakamura along with key facilities such as the Stem Cell, Genomics, and Bioinformatics Cores to complete the proposed work. The training plan spans a 3-year period focused primarily on research with 1.5 years spent per aim. The plan also sets aside time essential for nurturing academic science through conferences, mentoring, and university/community programs.