In vitro studies of amyotrophic lateral sclerosis using human pluripotent stem cell-derived motor neurons

Among the disciplines of medicine, the study of neurological disorders such as amyotrophic lateral sclerosis (ALS) is particularly challenging. In ALS, both cortical and spinal motor neurons progressively degenerate, leading to paralysis and death. The fundamental inaccessibility and the postmitotic state of these cells prevent their isolation and culture for studies of degenerative mechanisms or for drug screening efforts. The studies presented here support the premise that human motor neurons (MNs) generated by directed differentiation of induced pluripotent (iPS) or embryonic stem cells (ES) represent a great research tool to address this challenge. We show that MNs derived from patient-specific iPS cells with known ALS-linked mutations can recapitulate molecular and functional phenotypes associated with the disease. The phenotypes observed included transcriptional and morphological changes in mitochondria, protein solubility, membrane hyperexcitability, and defects in cell survival and axonal transport. By utilizing gene-targeting technology we further validated the requirement, and in some cases the sufficiency, of the SOD1A4V mutant allele to drive some of these phenotypes. Additionally, by means of a human ES cell line with a stable MN-specific green fluorescence protein (GFP) reporter, we carried out transcriptome profiling of cultured GFP+ cells following dysregulation of the ALS-associated RNA-binding protein TDP-43. We uncovered novel molecular targets downstream of the activity of TDP-43, one of which is the gene encoding for the tubulin-binding protein STMN2, with functional roles in cytoskeletal dynamics and axonal regeneration. Furthermore, we confirmed STMN2 response to TDP-43 downregulation at the protein level and provide evidence supporting its altered expression in spinal cord tissues from ALS cases. We propose that depletion of this neuronal growth factor following TDP-43 dysregulation could be a molecular mechanism by which the loss of normal nuclear TDP-43, seen in most ALS cases, contributes to MN degeneration. Finally, we discuss how future experiments, by integrating our findings with recent technological breakthroughs in genome-editing, stem cell differentiation and single-cell analysis, will further our understanding of disease mechanisms and facilitate the identification of novel therapeutic interventions.