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.

我们建议将其应用于肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的新 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。ALS和 FTD具有相同的遗传学特征，主要涉及脑下投射功能特异性亚型的变性 神经元（SCPN）：在ALS中，控制自主运动的皮质脊髓神经元（CSN）;在FTD中，密切相关 von Economo神经元（VENs）和叉细胞调节情绪和认知。为什么这些密切相关的 神经元亚型在ALS和FTD中特别脆弱，这是未知的，可能是治疗的关键，因为在这两种疾病中， 携带家族性ALS或FTD变异基因（例如SOD 1、TDP 43、FUS、C9 orf 72）的患者和小鼠， 大脑中的神经元和大多数体细胞表达变异风险基因，包括许多已知的RNA加工 分子，但只有特定的神经元亚型退化。受影响神经元之间的分子差异 亚型和其他类型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 由错误表达/突变引起的功能障碍。通过调查脆弱的皮层人群和与之密切相关的 未受影响的人群，我们的目标是确定新的亚细胞分子特异性，异常，或明显 利用使特定回路易受ALS和FTD退化影响的通路。这项工作将设置一个 为未来治疗奠定基础，以预防/限制ALS/FTD中特定皮质回路的退化。 我们将定量研究亚细胞RNA和蛋白质定位（目的1，3），和局部 翻译调控（目的2，3），在亚型特异性突触和脆弱的CSN/SCPN的胞体， 在ALS-FTD的两种互补小鼠模型（hSOD 1G 93 A目的1、2; TDP-43 Q331 K目的3）和精制小鼠模型中， 人iPS细胞衍生的皮质样（Co-1）和腹侧脊髓样（vSC-1）的“类胶质”融合体 从Co-1到vSC-1显示出显著的神经元亚群特异性投射和突触形成的类器官， 在ALS-FTD中建模SCPN电路（目标4）。我们应用亚型特异性索马和突触体纯化 通过FACS/新型小颗粒分选与亚型特异性RNA-seq和新的超低输入蛋白质组学， 核糖体分析在遗传风险、RNA加工和亚细胞的交叉点上询问ALS/FTD 的特异性我们的目标是确定亚细胞分子的改变，在受影响的电路，并调查 这些分子改变是否特异于ALS/FTD影响的亚型，通过与 通常不受影响，但密切相关的皮质联合胼胝体投射神经元。这一全面 “子类型感知”分析有希望唯一地识别亚细胞（突触和突触）中的潜在区别。 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。在一起， 这项冒险的工作将提供基础的分子和亚细胞的见解， CSN/SCPN在ALS和FTD中的脆弱性，将跨领域的新方法融合到未来的治疗中。