Transcriptional disruptions and functional correlates in a human induced pluripotent stem cell – derived motor neuron model of Spinocerebellar ataxia type 1

Abstract

It is hypothesized that degeneration of the motor neurons (MNs) in the brain stem and spinal cord contributes to premature lethality in spinocerebellar ataxia type 1 (SCA1) by affecting the strength of swallowing and respiratory drive. While we can recreate some aspects of MN pathology in mouse models, loss of MNs has only been seen in SCA1 patient populations. This, in addition to other species differences that exist between rodents and humans, accentuates the need for translational human models of disease which can be used to uncover therapeutically relevant cellular and molecular mechanisms underlying dysfunction in vulnerable cell types. To investigate potential mechanisms underlying SCA1 pathology in human MNs, I developed a human induced pluripotent stem cell (iPSC)-derived MN model of SCA1. This is, to our knowledge, the first cell-type specific iPSC-derived model made to study SCA1. Previous work in mouse models has demonstrated both that ATXN1 regulates cellular transcription (Ingram et al 2016, Rousseaux et al 2018, Friedrich et al 2019) and that its entry into the nuclei of cells is important for the longevity of mouse models of SCA1 (Handler et al 2022). Thus, I predicted that the presence of mutant ATXN1 in hiPSC-derived MNs would drive measurable transcriptomic changes in SCA1 samples compared to unaffected controls. Furthermore, transcriptomic disruptions might indicate functional pathways of interest for future studies into the lethality of SCA1. I performed bulk RNA sequencing on MN-enriched cultures to assess underlying transcriptional changes that may be affecting SCA1 human MNs, and subsequently identified disruptions in key cellular processes including extracellular matrix (ECM) regulation, calcium ion binding, and mitotic cell cycle regulation. Proper regulation of ECM is key to many aspects of neuronal development and function, including extension of neurites, proper placement and presentation of receptors and ion channels, cell-cell communication, synapse formation, and intracellular transport. As such, I predicted that broad changes in ECM regulation might result in measurable changes in some of these capabilities in SCA1 MN enriched cultures. Neurite outgrowth was measured in motor neuron progenitors (pMNs) and immature motor neurons (iMNs). I determined that SCA1 samples do not exhibit remarkable disruptions in neurite outgrowth at either timepoint. Assessment of spontaneous calcium activity exhibited a similar degree of physiological maturity between SCA1 and unaffected control MN enriched cultures. Additionally, measurement of miniature excitatory postsynaptic currents (mEPSCs) demonstrated formation of synapses but no functional differences in SCA1 samples compared to controls. However, glutamate evoked calcium activity demonstrated a reduced amplitude of calcium response in SCA1 cultures. This occurred despite any measurable transcriptomic changes in glutamate receptor expression. This indicates potential underlying disruptions in receptor activity and calcium dynamics in SCA1 MN-enriched cultures and provides a potential avenue of interest for future work investigating disruptions in communication in the SCA1 spinal cord.