MULTI-OMICS APPROACHES TO CHARACTERIZE PATIENT-DERIVED SPINAL CORD ORGANOIDS AND ASSESS NOVEL GENES ASSOCIATED WITH C9ORF72-AMYOTROPHIC LATERAL SCLEROSIS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons (MNs) in brain and spinal cord triggering death mechanisms that are largely unknown. To date, a diagnostic test nor an effective therapy exist, making it crucial to better define ALS pathophysiology. The primary goal of this project was to unravel the role of genes, proteins, and pathways of interest by applying integrated multi-omics approaches to spinal cord organoids (SCOs) generated from patient-derived induced pluripotent stem cells (iPSCs) and isogenic lines obtained with CRISPR/Cas9 technology. Specifically, we used iPSCs derived from 3 patients carrying mutations in the C9ORF72 gene. The G4C2 hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic form of ALS and causes a complex and multifactorial pathological phenotype that is not properly understood. In fact, C9ORF72 protein participates in a variety of cellular functions including trafficking, lysosome homeostasis, and autophagy, but it has also been associated with inflammation, lipid synthesis, and axonal growth. C9ORF72-ALS (C9-ALS) and isogenic cell lines were employed to generate SCOs that were collected at 3 time points, specifically 30, 55, 80 days in vitro (DIV), to evaluate their proper development with immunocytochemistry (ICC) and qPCR. At the endpoint, SCOs underwent proteomic and transcriptomic analyses by performing nano-liquid chromatography–high-resolution mass spectrometry (nLC-HRMS), western blot (WB), qPCR, and single-cell RNA-sequencing (scRNA-seq). ICC and qPCR revealed the expression of neural, pan-neuronal, and MN markers in cryosections of DIV80-SCOs, particularly DCX, MAP2, PAX6, OLIG2, GFAP, and HOXB4. Time course quantification of precursors (SOX2 and ISL1), as well as the progressive increase of post-mitotic MNs (TUJ1 and SMI32) supported proper organoids maturation. This outcome highlights SCOs potential as a tool to recapitulate SC features and properties in vitro. Differential expression analysis performed on the proteomic and transcriptomic profiles of C9-ALS and isogenic SCOs depicted substantial dysregulations in many cellular functions typically associated with ALS, including cytoskeletal and differentiation defects, RNA metabolism and protein quality control, oxidative stress and mitochondrial impairment, cholesterol biosynthesis and regulation, and protective astrocyte reactivity. Taken together, these findings support that patient-derived SCOs are a good and predictive in vitro 3D model, able to recapitulate the main pathological features associated with C9-ALS. Moreover, the reliability of this model in recapitulating the disease neuropathological features makes it a promising platform both to elucidate the pathomechanisms underlying the disease, in the context of preclinical studies and in the development of new effective drug tests.