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

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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.