Multispectral Imaging of Neurons and Astrocytes: Revealing Spatiotemporal Organelle Phenotypes in Health and Neurodegeneration

神经元和星形胶质细胞的多光谱成像：揭示健康和神经退行性疾病中的时空细胞器表型

• 批准号: 10674346
• 负责人: Shannon Rhoads
• 金额: $ 3.91万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-15 至 2026-06-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674346
• 关键词: 3-Dimensional; ALS patients; Affect; Amyotrophic Lateral Sclerosis; Astrocytes; Automobile Driving; Binding; Brain; Cell physiology; Cells; Cessation of life; Characteristics; Clinical; Computational Biology; Confocal Microscopy; DNA-Binding Proteins; Data; Dementia; Disease; Disparate; Endoplasmic Reticulum; Etiology; Fibroblasts; Frontotemporal Dementia; Functional disorder; Genes; Genetic; Goals; Golgi Apparatus; Health; Human; Image; Induced Mutation; Induced pluripotent stem cell derived neurons; Label; Lead; Link; Lipids; Lysosomes; Membrane; Methods; Microscopy; Mitochondria; Morphology; Muscular Atrophy; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Organelles; Pathogenesis; Pathogenicity; Pathologic; Pathology; Phenotype; Physiological; Physiology; Population; Process; Series; System; Techniques; Testing; Time; Visualization; Work; analysis pipeline; brain cell; brain tissue; cell type; effective therapy; frontotemporal lobar dementia amyotrophic lateral sclerosis; imaging modality; improved; induced pluripotent stem cell; innovation; novel; novel strategies; peroxisome; pharmacologic; protein TDP-43; protein aggregation; response; sample fixation; spatiotemporal; spectrograph; stressor; success; trafficking; training opportunity

PROJECT SUMMARY/ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are now thought to represent a spectrum of rapidly progressing neurodegenerative diseases. There are currently no cures or effective treatments for them. Though separate entities, these diseases share common genetic and pathological hallmarks and can have overlapping clinical presentations resulting in FTD-ALS. The underlying genetics linked to FTD, ALS, and FTD-ALS span a broad range of functions, making it difficult to interpret their contribution to disease pathophysiology. Moreover, most cases (70% FTD, 95% ALS) are sporadic with unknown etiology. Therefore, new techniques are needed to evaluate cellular pathomechanisms in the FTD-ALS spectrum of diseases. Because many causative genes are integral to organelle functions or contacts, we propose to use Multispectral Imaging (MSI) as a novel approach to accomplish this goal. MSI is a cutting-edge confocal microscopy technique used to simultaneously visualize up to seven fluorescent labels in living cells. In the proposed work, we will label major membrane-bound organelles (endoplasmic reticulum, peroxisomes, Golgi apparatus, mitochondria, lysosomes, lipid droplets) to quantify organelle morphology, interactions, and dynamics that are integral to cell function and/or perturbed in disease. The resulting organelle metrics will be combined to produce an organelle signature for neurons and astrocytes, cell types relevant to FTD-ALS. To our knowledge, this is the first study to utilize MSI to assess cellular mechanisms of the brain. Therefore, Aim 1 will establish organelle signatures for healthy neurons and astrocytes, examine how the signatures change upon pharmacological perturbations, and create a platform to automate the MSI analysis process. We hypothesize that neurons and astrocytes will have disparate organelle signatures related to their differing physiology. Aim 2 will evaluate FTD-ALS phenotypes induced by a familial mutation in the TARDBP gene. TARDBP encodes the TDP-43 protein, which has been identified to aggregate in the majority of FTD and ALS cases (45% and 97%, respectively). We hypothesize that this neurodegenerative perturbation will result in aberrant organelle morphology and dynamics in both neurons and astrocytes. Our preliminary findings demonstrate the feasibility of our approach and a likelihood of success, as we have optimized MSI labels in both cell types and began to examine pharmacological and genetic perturbations that result in altered organelle morphology and interactions. To substantiate our findings, we will confirm and expand on the MSI data in human induce pluripotent stem cell-derived neurons and astrocytes and validate significant phenotypes in FTD-ALS brain tissue. Our study will establish MSI as an innovative approach to evaluate neurodegeneration and could reveal cellular phenotypes integral to FTD-ALS onset or progression that traditional techniques have thus far missed.

项目摘要/摘要 额颞叶痴呆（FTD）和肌萎缩侧索硬化症（ALS）现在被认为是 代表了一系列迅速发展的神经退行性疾病。目前还没有治愈方法 或有效的治疗方法。虽然这些疾病是独立的实体，但它们有着共同的遗传和 病理学特征，并可能具有重叠的临床表现，导致FTD-ALS。的 与FTD、ALS和FTD-ALS相关的潜在遗传学跨越了广泛的功能， 来解释它们对疾病病理生理学的贡献。此外，大多数病例（70% FTD，95% ALS） 散发，病因不明。因此，需要新的技术来评估细胞 FTD-ALS疾病谱中的病理机制。因为许多致病基因是 细胞器功能或接触，我们建议使用多光谱成像（MSI）作为一种新的方法， 实现这一目标。MSI是一种尖端的共聚焦显微镜技术，用于同时 在活细胞中可视化多达七个荧光标记。在拟议的工作中，我们将标记主要 膜结合细胞器（内质网，过氧化物酶体，高尔基体，线粒体， 溶酶体，脂滴）来量化细胞器形态，相互作用和动力学， 细胞功能和/或疾病中的干扰。由此产生的细胞器指标将被组合以产生 神经元和星形胶质细胞的细胞器特征，与FTD-ALS相关的细胞类型。据我们所知， 这是第一项利用MSI评估大脑细胞机制的研究。目标1将 建立健康神经元和星形胶质细胞的细胞器特征， 并创建一个平台来自动化MSI分析过程。我们 假设神经元和星形胶质细胞将具有不同的细胞器特征， physiology.目的2将评估由TARDBP家族性突变诱导的FTD-ALS表型 基因TARDBP编码TDP-43蛋白，其已被鉴定为在大多数人中聚集。 FTD和ALS病例（分别为45%和97%）。我们假设这种神经退行性疾病 扰动将导致神经元和星形胶质细胞中异常的细胞器形态和动力学。 我们的初步研究结果证明了我们的方法的可行性和成功的可能性，因为我们 已经优化了两种细胞类型的MSI标记，并开始研究药理学和遗传学 扰动导致细胞器形态和相互作用的改变。为了证实我们的发现， 我们将确认和扩展人类诱导多能干细胞衍生神经元的MSI数据， 星形胶质细胞和验证FTD-ALS脑组织中的显著表型。我们的研究将建立MSI， 一种创新的方法来评估神经变性，并可以揭示细胞表型组成， FTD-ALS的发病或进展，传统技术迄今错过。