Transcriptional modulation unique to vulnerable motor neurons predicts ALS across species and SOD1 mutations

Abstract Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons that innervate skeletal muscles. However, certain motor neuron groups including ocular motor neurons, are relatively resilient. To reveal key drivers of resilience versus vulnerability in ALS, we investigate the transcriptional dynamics of four distinct motor neuron populations in SOD1G93A ALS mice using LCM-seq and single molecule fluorescent in situ hybridization. We find that resilient ocular motor neurons regulate few genes in response to disease. Instead, they exhibit high baseline gene expression of neuroprotective factors including En1, Pvalb, Cd63 and Gal, some of which vulnerable motor neurons upregulate during disease. Vulnerable motor neuron groups upregulate both detrimental and regenerative responses to ALS and share pathway activation, indicating that breakdown occurs through similar mechanisms across vulnerable neurons, albeit with distinct timing. Meta-analysis across four rodent mutant SOD1 motor neuron transcriptome datasets identify a shared vulnerability code of 39 genes including e.g Atf4, Nupr1, Ddit3 and Penk, involved in apoptosis, as well as a proregenerative and anti-apoptotic signature consisting of Atf3, Vgf, Ina, Sprr1a, Fgf21, Gap43, Adcyap1, and Mt1. Machine learning using genes upregulated in SOD1G93A spinal motor neuron predicts disease in human stem cell-derived SOD1E100G motor neurons, and shows that dysregulation of VGF, INA, PENK and NTS are strong disease-predictors across species and SOD1 mutations. Our study reveals motor neuron population-specific gene expression and temporal disease-induced regulation that together provide a basis to explain ALS selective vulnerability and resilience and that can be used to predict disease. Competing Interest Statement The authors have declared no competing interest. Footnotes Figure 2. We have added RNA scope analysis of Chl1 in CN3/4, CN10 and CN12 motor neurons. Figure 3. We have added a species cross-comparison (mouse and human) regarding differences in baseline gene expression between CN3/4 and spinal motor neurons (DEGs and pathways). We have also added analysis, showing that particular genes that are normally (in control) highly expressed in CN3/4 motor neurons compared to spinal motor neurons are induced in vulnerable motor neurons with disease. This pinpoints a protective response to disease and identifies a particular CN3/4 gene expression signature that may explain their intrinsic resilience. Figure 6. We have added a heatmap displaying 34 ALS-induced DEGs common to four mutant SOD1 motor neuron (MN) studies (Mei, Lobsiger, Shadrach, and Sun). Genes are categorized as upregulated or downregulated in ALS motor neurons. The presence of each gene in different datasets is indicated in columns: MEI (disease-induced DEGs shared between CN12 and SC motor neurons at P112 from Mei et al.), CRUSH (sciatic nerve crush model from Shadrach et al., 2021) and if the regulation is considered GOOD or BAD. We also included a summary of transcriptional differences in baseline gene expression between resilient and vulnerable neurons as well as their differential responses to disease, organized into functional categories, reveal key pathways including ER stress and apoptosis, axon integrity and regeneration, neuroprotection, synapse, complement activation and ECM remodeling. We have also included several New Supplemental Figures: In Figure S4 we detail how the RNA scope quantification was conducted and the number of neurons counted. In Figure S6 we have included a cross-comparison with three other transcriptome data sets on CN3/4 and spinal motor neurons in control tissues (mouse and human). In Figure S7 we included new RNA scope analysis for Atf3, Sprr1a, Timp1, Pvalb and Fgf21, across motor neuron populations. In Figure S8 we have included alternative polyadenylation (APA) analysis on CN3/4 and spinal motor neurons in SOD1-ALS.