Understanding how neuronal glucose metabolism changes in AD due to ApoE4

了解 AD 中 ApoE4 导致的神经元葡萄糖代谢如何变化

• 批准号: 10680020
• 负责人: Yoshi Sei
• 金额: $ 7.38万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-03 至 2026-05-02
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680020
• 关键词: Acceleration; Affect; Age; Age Months; Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease diagnosis; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease risk; Alzheimer' s disease therapy; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Apolipoprotein E; Arctic Regions; Arctic mutation; Astrocytes; Bioenergetics; Bioinformatics; Biological Markers; Biology; Biosensor; Brain; Brain region; C57BL/6 Mouse; CRISPR interference; CRISPR-mediated transcriptional activation; Carbohydrates; Catabolism; Catalogs; Cell Count; Cell Survival; Central Nervous System; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Consumption; Data; Dementia; Development; Disease; Disease Progression; Elderly; Exhibits; Fluorescence Resonance Energy Transfer; Future; GLUT-3 protein; Gene Expression; Genes; Genomics; Glucose; Glucose Transporter; Goals; Healthcare; Hippocampus; Human; Image; Immunofluorescence Immunologic; Impaired cognition; Impairment; In Situ; In Vitro; Individual; Induced pluripotent stem cell derived neurons; Insulin Receptor; Knock-in; Label; Link; Manufacturer; Measures; Memory impairment; Mentors; Mentorship; Messenger RNA; Metabolic; Metabolic dysfunction; Metabolism; Mitochondria; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Output; Pathology; Pathway interactions; Patients; Positron-Emission Tomography; Protein Isoforms; Proteins; Recovery; Research; Risk; Risk Factors; Role; Science; Senile Plaques; Symptoms; Testing; Time; Training; Transgenic Mice; Universities; Validation; Work; age related; apolipoprotein E-3; apolipoprotein E-4; behavior test; conditional knockout; differential expression; emerging adult; fluorodeoxyglucose positron emission tomography; glucose metabolism; glucose uptake; improved; in vivo; in vivo Model; induced pluripotent stem cell; insight; insulin signaling; interest; metabolic phenotype; metabolome; metabolomics; middle age; mouse model; mutant; neuronal metabolism; novel therapeutics; programs; stem cells; symposium; targeted biomarker; tau Proteins; therapeutic candidate; therapeutic target; trafficking; transcriptomics; translational potential

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.

项目概要 随着我们医疗保健质量的迅速提高，迫切需要更好地了解年龄 阿尔茨海默病 (AD) 等相关疾病的进展与血糖之间存在明确的联系。 大脑中依赖的生物能量缺陷促使研究人员发现 AD 诊断中的关键生物标志物； 载脂蛋白 E4 (ApoE4) 已被证明是患有 AD 风险的患者的标志性指标。 是大脑中 ApoE4 的主要制造者，促使大多数 AD 相关研究集中于定义 尽管有证据表明神经元代谢功能障碍与神经胶质细胞和 ApoE4 之间的关系有关。 我们主要感兴趣的是 ApoE4 的表达，即代谢变化的潜在生物学原理。 该项目的目的是确定 ApoE4 表达和降低神经葡萄糖的机制 我们努力纠正表达 ApoE4 的神经元中受损的机制。 将导致代谢表型恢复到更接近表达 ApoE3 的神经元。 我们的假设是，我们开发了一种范式，结合了人类的靶向代谢组学分析 通过对小鼠的空间转录组分析，体外诱导多能干细胞（iPSC）衍生的神经元 体内模型将是全身敲入人 ApoE3 或 ApoE4 的 C57BL/6 小鼠。 淀粉样前体蛋白 (APP) 背景具有瑞典、伊比利亚和北极突变 (APPNL-G-F)。 对海马体（受 AD 进展影响的首批区域之一）进行空间转录组学，以 纵向追踪由于 ApoE4 表达差异最大的基因。 ApoE 的 E3 或 E4 亚型可实现近乎纯的 (>99%) 神经培养物 整个神经代谢组。 用统一 13C 标记的葡萄糖（[U-13C] 葡萄糖）进行探测，以量化神经元中的哪些代谢物 来自葡萄糖的 ApoE3 和 ApoE4 表达神经元的代谢组将与 根据 ApoE 亚型定义神经元之间葡萄糖代谢的主要差异。 将使用 CRISPR 抑制或激活 (CRISPRi/a) 来改变目标基因中我们感兴趣的基因的表达 代谢组学研究评估与神经元葡萄糖代谢相关的每个基因的功能。 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也将为未来关注神经元代谢恢复的研究提供信息 该研究将提供针对神经退行性疾病的候选治疗靶点。 在 Ken Nakamura 博士的指导下以及主要设施的指导下，在格拉德斯通研究所和加州大学旧金山分校进行 例如干细胞、基因组学和生物信息学核心来完成拟议的工作培训计划。 该计划为期 3 年，主要侧重于研究，每个目标花费 1.5 年。 对于通过会议、指导和大学/社区项目培育学术科学至关重要。