Understanding how neuronal glucose metabolism changes in AD due to ApoE4

项目总结 随着我们的医疗质量迅速提高，我们迫切需要更好地了解年龄- 与阿尔茨海默病(AD)等相关疾病。明确AD进展和血糖之间的联系- 大脑中依赖的生物能量缺陷促使研究发现AD诊断中的关键生物标记物； 载脂蛋白E4(ApoE4)已被证明是有患AD风险的患者的标志指标。星形胶质细胞 是大脑中载脂蛋白E4的主要制造商，促使大多数与AD相关的研究集中在定义 胶质细胞与载脂蛋白E4的关系。尽管有证据表明神经元代谢功能障碍与 ApoE4的表达是代谢变化的基础生物学，目前还不清楚。我们的主要利益 这个项目是为了确定载脂蛋白E4表达和降低神经元血糖的机制 新陈代谢是相互联系的。我们假设纠正表达载脂蛋白E4的神经元的破坏机制 将导致代谢表型的恢复，使其更接近于表达载脂蛋白E3的神经元。为了测试 我们的假设，我们已经开发出一种范例，它结合了对人类代谢的目标分析 小鼠体外诱导多能干细胞来源神经元的空间转录分析 活着。体内模型将是全身敲入人载脂蛋白E3或载脂蛋白E4的C57BL/6小鼠 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)。我们会 对海马体进行空间转录，海马体是AD进展最早影响的区域之一，以 纵向追踪由于载脂蛋白E4而差异表达最多的基因。人类IPSCs表达的 ApoE的E3或E4亚型允许进行几乎纯的(&gt；99%)神经元培养。整个神经元代谢组 用统一的13C标记葡萄糖([U-13C]葡萄糖)进行探测，以量化神经元中的哪些代谢物 从葡萄糖中提取的。ApoE3和ApoE4表达神经元的代谢组将与 根据不同的载脂蛋白E亚型确定神经元之间葡萄糖代谢的主要差异。我们 将使用CRISPR抑制或激活(CRISPRi/a)来改变我们的目标基因的表达 代谢组学研究，以评估每个基因的功能与神经元中葡萄糖代谢的关系。我们 然后将通过比较ApoE4神经元和ApoE3神经元来评估是否实现了代谢恢复 基于葡萄糖衍生代谢物、胞浆三磷酸腺苷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也将为未来专注于神经元代谢恢复的研究提供信息 为治疗神经退行性疾病提供候选靶点。这项研究将是 在Gladstone研究所和加州大学旧金山分校，在Ken Nakamura博士的指导下进行，以及主要设施 例如干细胞、基因组学和生物信息学核心，以完成拟议的工作。培训计划 为期3年，主要专注于研究，每个目标花费1.5年。该计划还预留了时间 对于通过会议、指导和大学/社区项目培养学术科学来说是必不可少的。

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.