Subcellular mechanisms of subtype-specific neuron vulnerability in ALS and FTD: dysregulation of synapse-localized RNA, protein, and translation in mouse models and human cortico-spinal assembloids

ALS 和 FTD 中亚型特异性神经元脆弱性的亚细胞机制：小鼠模型和人类皮质脊髓组合体中突触定位 RNA、蛋白质和翻译的失调

• 批准号: 10716562
• 负责人: JEFFREY D MACKLIS
• 金额: $ 200.19万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716562
• 关键词: 3-Dimensional; Affect; Age; Amyotrophic Lateral Sclerosis; Area; Awareness; Axon; Biological Process; Brain; C9ORF72; Cells; Cerebral cortex; Cognition; Degenerative Disorder; Development; Elements; Emotions; Exhibits; Foundations; Frontotemporal Dementia; Functional disorder; Future; Genes; Genetic; Genetic Risk; Growth Cones; Human; Investigation; Knowledge; Label; Measures; Modeling; Molecular; Motor Neurons; Movement; Mus; Mutation; Neurons; Organoids; Output; Parents; Pathology; Pathway interactions; Patients; Population; Proteins; Proteome; Proteomics; RNA; RNA Processing; RNA Splicing; RNA-Binding Proteins; Sorting; Specificity; Spinal; Spinal Cord; Subcellular structure; Synapses; Synaptosomes; Testing; Therapeutic; Transcript; Transgenic Organisms; Translational Regulation; Translations; Untranslated RNA; Vertebral column; Wild Type Mouse; Work; comparative; familial amyotrophic lateral sclerosis; fluorophore; frontotemporal lobar dementia amyotrophic lateral sclerosis; genetic variant; induced pluripotent stem cell; insight; mosaic; motor deficit; mouse model; mutant; neuronal cell body; novel; novel strategies; particle; presynaptic neurons; prevent; protein TDP-43; ribosome profiling; risk variant; superoxide dismutase 1; synaptogenesis; trafficking; transcriptome; transcriptome sequencing

We propose to apply to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) newly developed subcellular approaches to investigate subtype-specific RNA, protein, and translational mechanisms of selective vulnerability to closely molecularly and developmentally related cerebral cortex neurons. ALS and FTD share genetics, and centrally involve degeneration of function-specific subtypes of subcerebral projection neruons (SCPN): in ALS, corticospinal neurons (CSN) controlling voluntary movement; in FTD, closely related von Economo neurons (VENs) and fork cells regulating emotion and cognition. Why these closely related neuronal subtypes are especially vulnerable in ALS and FTD is unknown and likely key to therapy, since in both patients and mice carrying variant genes of familial ALS or FTD (e.g. SOD1, TDP43, FUS, C9orf72), most neurons in the brain and most body cells express the variant risk gene, including many known RNA processing molecules, but only specific neuronal subtypes degenerate. Molecular differences between affected neuronal subtypes and other types of even closely related neurons might render affected neurons more vulnerable to dysfunction from mis-expression/mutation. By investigating vulnerable cortical populations and closely related unaffected populations, we aim to identify novel subcellular molecular specificities, abnormalities, or distinctly utilized pathways that render specific circuitry vulnerable to degeneration in ALS and FTD. This work will set a foundation toward future therapeutics to prevent/limit degeneration of specific cortical circuitry in ALS/FTD. We will quantitatively investigate subcellular RNA and protein localization (Aims 1, 3), and local translational regulation (Aims 2, 3), in subtype-specific synapses and somata of vulnerable CSN/SCPN, both in two complementary mouse models of ALS-FTD (hSOD1G93A Aims 1, 2; TDP-43Q331K Aim 3), and in refined human iPS cell-derived “assembloid” fusions of a cortical-like (Co-l) and a ventral spinal cord-like (vSC-l) organoid that exhibit remarkable neuron subset-specific projections and synapse formation from Co-l to vSC-l, modeling SCPN circuitry in ALS-FTD (Aim 4). We apply subtype-specific soma and synaptosome purification by FACS/novel small particle sorting with subtype-specific RNA-seq and new, ultra-low-input proteomics and ribosome profiling to interrogate ALS/FTD at the intersection of genetic risk, RNA processing, and subcellular specificity. We aim to identify subcellular molecular alterations in the affected circuitry, and to investigate whether these molecular alterations are specific to the ALS/FTD-affected subtypes by comparison with typically unaffected, but closely related cortical associative callosal projection neurons. This comprehensive “subtype-aware” analysis has promise to uniquely identify potential distinctions in subcellular (synapse- and soma-focused) RNA, protein, and/or translation as potential mechanisms of selective vulnerability. Together, this venturesome work will provide foundational molecular and subcellular insight into selective vulnerability of CSN/SCPN in ALS and FTD, merging new approaches across fields toward future therapy.

我们建议新适用于肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD） 开发了亚细胞方法来研究亚型特异性 RNA、蛋白质和翻译机制 对分子和发育密切相关的大脑皮层神经元的选择性脆弱性。肌萎缩侧索硬化症和 FTD 具有相同的遗传学特征，主要涉及大脑下投射功能特异性亚型的退化 神经元 (SCPN)：在 ALS 中，皮质脊髓神经元 (CSN) 控制随意运动；在 FTD 中，密切相关 von Economo 神经元 (VEN) 和叉细胞调节情绪和认知。为什么这些密切相关 神经元亚型在 ALS 中尤其脆弱，而 FTD 尚不清楚，但可能是治疗的关键，因为在这两种情况下 携带家族性 ALS 或 FTD 变异基因（例如 SOD1、TDP43、FUS、C9orf72）的患者和小鼠，大多数 大脑中的神经元和大多数身体细胞表达变异风险基因，包括许多已知的 RNA 处理 分子，但只有特定的神经元亚型会退化。受影响的神经元之间的分子差异 亚型和其他类型的甚至密切相关的神经元可能会使受影响的神经元更容易受到影响 错误表达/突变导致的功能障碍。通过调查脆弱的皮质群体和密切相关的 对于未受影响的人群，我们的目标是识别新的亚细胞分子特异性、异常或明显的亚细胞分子特异性。 利用使特定电路容易在 ALS 和 FTD 中退化的途径。这项工作将设定一个 为未来预防/限制 ALS/FTD 特定皮质回路退化的治疗奠定基础。 我们将定量研究亚细胞 RNA 和蛋白质定位（目标 1、3）以及局部 翻译调节（目标 2、3），在易受攻击的 CSN/SCPN 的亚型特异性突触和胞体中，两者 在 ALS-FTD 的两个互补小鼠模型（hSOD1G93A 目标 1、2；TDP-43Q331K 目标 3）中，以及在精制 人类 iPS 细胞衍生的皮质样 (Co-l) 和腹侧脊髓样 (vSC-l) 的“组合体”融合 类器官表现出显着的神经元子集特异性投射和从 Co-l 到 vSC-l 的突触形成， 在 ALS-FTD 中对 SCPN 电路进行建模（目标 4）。我们应用亚型特异性体细胞和突触体纯化 通过 FACS/新型小颗粒分选，结合亚型特异性 RNA 测序和新的超低输入蛋白质组学， 核糖体分析可在遗传风险、RNA 加工和亚细胞交叉点上检测 ALS/FTD 特异性。我们的目标是识别受影响电路中的亚细胞分子改变，并研究 通过与 通常不受影响，但密切相关的皮质关联胼胝体投射神经元。这个全面的 “亚型感知”分析有望独特地识别亚细胞（突触和 以体细胞为中心的）RNA、蛋白质和/或翻译作为选择性脆弱性的潜在机制。一起， 这项大胆的工作将为选择性的分子和亚细胞洞察提供基础 CSN/SCPN 在 ALS 和 FTD 中的脆弱性，融合跨领域的新方法以实现未来的治疗。