Characterization of human i3 motor neurons exposed to CSF from ALS patients stratified by C9ORF72 and UNC13A genotype

It has been hypothesized that in ALS the cerebral spinal fluid (CSF) might contribute to disease spreading, triggering a degenerative cascade in not-yet-degenerating motor neurons. We investigated the possible effects of CSF from ALS patients on healthy integrated, inducible and isogenic lower motor neurons (i3LMNs) differentiated from induced pluripotent stem cells. CSFs were obtained from 5 ALS patients with the UNC13A rs12608932 risk genotype (CC) (a known ALS genetic risk factor), 5 ALS patients with the corresponding low-risk genotype (AA) and 5 patients with an hexanucleotide repeat expansion in the C9ORF72 gene. Treatment of i3LMNs with mild chronic sodium arsenite (ARS) was used as positive control of oxidative stress, while CSF from 5 individuals with non-degenerative conditions was used as negative control. After 48h-treatment with 10% CSF, none of the tested CSF triggered significant alterations in viability, autophagy, neurite degeneration, DNA damage and Golgi apparatus integrity. Only UNC13A CC-CSF induced a significant increase in generalized protein aggregation. On the other hand, chronic ARS altered all the analyzed parameters. Accordingly, differential gene expression analyses by RNA-seq revealed that chronic ARS altered the expression of thousands of genes, while ALS-CSF treatments only exerted a mild effect. Curiously, few genes were found to be commonly upregulated upon treatments with each of the different CSFs. We have demonstrated that ALS-CSF treatment, independently on patients’ CSF genotype, was not sufficient to induce ALS-like pathological features in human i3LMNs in our experimental settings and only had a mild effect in triggering gene expression changes. Conversely, chronic oxidative stress by ARS is a powerful stressor for healthy motor neurons, triggering pathological features and massive gene expression deregulation.