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

The short time window from ALS symptoms onset to death, usually less than 3 years, has led to the hypothesis of a role of cerebral spinal fluid (CSF) in disease spreading, triggering a degenerative cascade in not-yet-degenerating motor neurons. We investigated the possible toxic effects of CSF from ALS patients (ALS-CSF) on healthy integrated, inducible and isogenic lower motor neurons (i3LMNs) differentiated from NIL-modified induced pluripotent stem cells (NIL-iPSCs). CSFs were obtained from 5 sporadic ALS patients carrying the risk alleles (CC) at the rs12608932 of UNC13A gene, 5 ALS patients carrying the corresponding wild-type allele (AA) and 5 familial patients with an hexanucleotide repeat expansion in C9ORF72. Treatment of i3LMNs with mild and chronic sodium arsenite (ARS) was used as a positive control of oxidative stress, while CSF from 5 individuals affected by a non-degenerative condition was used as a negative control. After 48h-treatment with 10% CSF, none of the tested conditions was sufficient to trigger significant alterations in cell viability, autophagic flux, neurite degeneration, DNA damage and Golgi apparatus integrity. Only UNC13A CC-CSF treatment induced a significant increase in generalized protein aggregation. On the other hand, chronic ARS significantly altered all the analyzed parameters. We also performed differential gene expression analyses by RNA-seq and observed that chronic ARS was able to alter 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. In conclusion, we have demonstrated that chronic oxidative stress by ARS is a powerful stressor for healthy motor neurons, triggering several pathological features and massive gene expression deregulation. On the other hand, ALS-CSF treatment, independently on patients’ CSF genotype, was not sufficient to induce typical ALS-like pathological features in human i3LMNs in our experimental settings and only had a mild effect in triggering gene expression changes.