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) 发展亚细胞方法研究特定亚型的RNA、蛋白质和翻译机制 对与分子和发育密切相关的大脑皮层神经元的选择性易感性。肌萎缩侧索硬化和 FTD具有相同的遗传学，并集中涉及脑下投射功能特定亚型的退变 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，与之密切相关 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 神经元亚型在ALS中尤其脆弱，FTD是未知的，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(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这种变异的风险基因，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 由错误表达/突变引起的功能障碍。通过调查脆弱的皮质人口和密切相关的 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 所利用的通路使ALS和FTD中的特定电路容易退化。这项工作将建立一个 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和蛋白质的定位(目标1、3)和局部 易损性CSN/SCPN亚型特异性突触和胞体的翻译调控(AIMS 2，3) 在ALS-FTD的两个互补小鼠模型中(hSOD1G93A目标1，2；TDP-43Q331K目标3)，以及在精制的 人诱导性多能干细胞来源的皮质样(co-L)和腹侧脊髓样(vsc-L)的“集合体”融合 显示出显著的神经元亚群特异性投射和突触形成的有机体，从共同的L到VSC-L， 在ALS-FTD中对SCPN电路进行建模(目标4)。我们应用亚型特异性胞体和突触小体纯化 用亚型特异性RNA-SEQ和新的超低投入蛋白质组学进行FACS/新颖的小颗粒分选 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 专一性。我们的目标是识别受影响电路中的亚细胞分子变化，并研究 这些分子改变是否是ALS/FTD影响的亚型特有的 通常不受影响，但密切相关的皮质联合胼胝体投射神经元。这一全面的 “亚型识别”分析有望唯一地识别亚细胞(突触和 以SOMA为重点)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，为未来的治疗合并了跨领域的新方法。