Chemical Induction Of Aging In IPSC-Motor Nerve Organoids To Model Amyotrophic Lateral Sclerosis

Objectives Aging per se causes a variety of molecular and cellular alterations, which serve as a trigger in ALS etiology by making motor neurons more susceptible to disease-specific pathomechanisms. The establishment of ALS in vitro models recapitulating aging and more faithfully the neuropathological processes experienced by patients remains an ongoing challenge. Patient-derived iPSC-motoneurons and iPSC-motor nerve organoids (MN-ORGs) represent valuable 2D and 3D in vitro models, but their maturation stage is critical to study ALS-associated hallmarks. Aim of this work was to chemically induce senescence in iPSC-derived MN-ORGs to model TDP-43 proteinopathy in ALS and to evaluate the sensitivity to aging of MN-ORGs from C9ORF72 patients. Material and methods MN-ORGs were generated from 3 C9ORF72 and 3 healthy control (CTRL) iPSC lines by a 21-days differentiation protocol. Senescence was chemically induced for 4 days with the recently published SLOcocktail. Immunofluorescence and ImageJ and Scholl analyses were used to evalute axonal degeneration. Neurofilament light chain (NfL) in the culture medium was quantified by Simoa technology. Results MN-ORGs from C9ORF72 and CTRL iPSCs showed a similar size and differentiation efficiency as assessed by the expression of neuronal (pan-axonal SMI-312, III-Tubulin) and motoneuronal (ChAT, HB9) markers. Exposure to SLO cocktail confirmed aging of the MN-ORGs by Q-PCR analysis of LMNB1 and LAP2genes. Axonal branching and area were reduced in aged MN-ORGs from both CTRL and C9ORF72 individuals, while the release of NfL in the medium was significantly higher in C9ORF72 MN-ORGs, suggesting a greater axonal damage upon SLO treatment. SLO-driven chemical aging also induced features of TDP-43 proteinopathy with increased TDP-43 axonal mislocalization and C-terminal fragmentation in both CTRL and C9ORF72 MN-ORGs. Conclusion Our results show that iPSC-derived MN-ORGs treated with SLO represent suitable models of senescence in ALS and unveil differences in sensitivity to aging between C9ORF72 and CTRL.