Chemical induction of aging in iPSC-spinal cord organoids to model amyotrophic lateral sclerosis

The scarcity of experimental models of TDP-43 proteinopathy has hindered the discovery of novel therapeutic approaches for amyotrophic lateral sclerosis (ALS). Indeed, the establishment of ALS in vitro models more faithfully recapitulating the neuropathological processes experienced by patients remains an ongoing challenge. In ALS pathogenesis, aging per se causes a variety of molecular and cellular alterations, which serve as a trigger for ALS by making motor neurons more susceptible to disease-specific pathomechanisms. In this work, we chemically induced a senescent phenotype in iPSC-derived spinal cord organoids (SP-ORGs) to compare the sensitivity to aging of organoids from C9ORF72 ALS patients and from healthy controls (CTRL). We generated SP-ORGs from 3 CTRL and 3 C9ORF72 iPSC lines and characterized them for the expression of neuronal (pan-axonal SMI-312, β3-Tubulin) and motoneuronal (ChAT, HB9) markers. During their growth, we monitored their dimensions and preliminarily observed a reduced size of C9ORF72 SP-ORGs compared to CTRL ones. To trigger a senescent phenotype, we treated them with SLO, a cocktail of three molecules (SBI, Lopinavir, O151), recently described to induce aging in cultured neurons, and confirmed the senescent phenotype of the SP-ORGs by Q-PCR. We therefore investigated whether SLO treatment could induce TDP-43 proteinopathy, and demonstrated increased C-terminal TDP-43 fragmentation in both CTRL and C9ORF72 SP-ORGs. However, no alteration of TDP-43 nuclear activity as splicing regulator was observed. Furthermore, we measured the release of neurofilament light chain (NFL), a marker of axonal damage, in the culture medium using Simoa® technology, and preliminarily found that NFL levels were increased after aging induction by SLO in both CTRL and C9ORF72 SP-ORGs. These preliminary results show that iPSC-derived SP-ORGs treated with SLO might represent suitable models of senescence to be employed as drug screening platforms for ALS and to unveil possible differences in sensitivity to aging between C9ORF72 and CTRL in vitro models.