α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a late-onset adult neurodegenerative disease, although there is mounting electrophysiological and pathological evidence from patients and animal models for a protracted preclinical period of motor neuron susceptibility and dysfunction, long before clinical diagnosis. The key molecular mechanisms linked to motor neuron vulnerability in amyotrophic lateral sclerosis have been extensively studied using transcriptional profiling in motor neurons isolated from adult mutant superoxide dismutase 1 mice. However, neonatal and embryonic motor neurons from mutant superoxide dismutase 1 mice show abnormal morphology and hyperexcitability, suggesting preceding transcriptional dysregulation. Here, we used RNA sequencing on motor neurons isolated from embryonic superoxide dismutase 1 mice to determine the earliest molecular mechanisms conferring neuronal susceptibility and dysfunction known in a mouse model of amyotrophic lateral sclerosis. Transgenic superoxide dismutase 1 mice expressing the spinal motor neuron homeobox HB9:green fluorescent protein reporter allowed unambiguous identification and isolation of motor neurons using fluorescence-activated cell sorting. Gene expression profiling of isolated motor neurons revealed transcriptional dysregulation in superoxide dismutase 1 mice as early as embryonic Day 12.5 with the majority of differentially expressed genes involved in RNA processing and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-mediated glutamate receptor signalling. We confirmed dysregulation of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2, at transcript and protein levels, in embryonic superoxide dismutase 1 mouse motor neurons and human motor neurons derived from amyotrophic lateral sclerosis patient induced pluripotent stem cells harbouring pathogenic superoxide dismutase 1 mutations. Mutant superoxide dismutase 1 induced pluripotent stem cell-derived motor neurons showed greater vulnerability to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-mediated excitotoxicity, consistent with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2 downregulation. Thus, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor Subunit 2 deficiency leading to enhanced α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor signalling, calcium influx, hyperexcitability, and chronic excitotoxicity is a very early and intrinsic property of spinal motor neurons that may trigger amyotrophic lateral sclerosis pathogenesis later in life. This study reinforces the concept of therapeutic targeting of hyperexcitability and excitotoxicity as potential disease-modifying approaches for amyotrophic lateral sclerosis.