Characterization of human healthy i3 lower motor neurons exposed to CSF from ALS patients stratified by UNC13A and C9ORF72 genotype
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motor neurons. Neurodegeneration in ALS might be driven by proteotoxicity or neuroinflammation, which have also been proposed to be promoted by toxic components of the cerebrospinal fluid (CSF). We investigated the possible toxicity of ALS CSF on healthy induced pluripotent stem cells (iPSC)-derived integrated, inducible, and isogenic lower motor neurons (i3LMNs). CSFs were obtained from ALS patients homozygous for the risk UNC13A rs12608932 single nucleotide polymorphism (CC) and for the corresponding major allele (AA), ALS patients with C9ORF72 hexanucleotide repeat expansion, and individuals affected by normal pressure hydrocephalus (NPH), a non-degenerative condition, as controls (ND). Chronic sodium arsenite (ARS) treatment was used as positive control of oxidative stress. We found that 10 % ALS CSF treatment for 48 h was not sufficient to induce significant alterations in viability, autophagic flux, axonal degeneration, DNA damage, and Golgi apparatus integrity in healthy i3LMNs, in contrast to ARS treatment. Only UNC13A CC CSF significantly increased protein aggregation and Golgi apparatus fragments dimension. RNA-sequencing revealed that all ALS and ND CSFs induced expression changes of few genes, while chronic ARS deregulated the expression of thousands of genes, mostly involved in inflammation and synapse biology. In this work, we demonstrated that in our experimental settings only UNC13A CC CSF induced some ALS-associated pathological features in healthy i3LMNs. Further studies will be required to elucidate the mechanistic link between the risk UNC13A genotype and CSF composition and toxicity. Overall design: RNA-seq profiling of i3 lower motor neurons treated with 10% CSF from UNC13A CC, UNC13 AA or C9ORF72 ALS patients, ND controls or chronic ARS, as well as their untreated counterparts.