In Vitro Characterization of Motor Neurons and Purkinje Cells Differentiated from Induced Pluripotent Stem Cells Generated from Patients with Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay

Abstract

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an early-onset neurodegenerative disease mainly characterized by spasticity in the lower limbs and poor muscle control. The disease is caused by mutations in the SACS gene leading in most cases to a loss of function of the sacsin protein, which is highly expressed in motor neurons and Purkinje cells. To investigate the impact of the mutated sacsin protein in these cells in vitro, induced pluripotent stem cell- (iPSC-) derived motor neurons and iPSC-derived Purkinje cells were generated from three ARSACS patients. Both types of iPSC-derived neurons expressed the characteristic neuronal markers β3-tubulin, neurofilaments M and H, as well as specific markers like Islet-1 for motor neurons, and parvalbumin or calbindin for Purkinje cells. Compared to controls, iPSC-derived mutated SACS neurons expressed lower amounts of sacsin. In addition, characteristic neurofilament aggregates were detected along the neurites of both iPSC-derived neurons. These results indicate that it is possible to recapitulate in vitro, at least in part, the ARSACS pathological signature in vitro using patient-derived motor neurons and Purkinje cells differentiated from iPSCs. Such an in vitro personalized model of the disease could be useful for the screening of new drugs for the treatment of ARSACS.

Proteomics and lipidomic analysis reveal dysregulated pathways associated with loss of sacsin

“…Cytoskeleton components are yet known to have an important role in ARSACS pathogenesis ( Duncan et al, 2017 ; Louit et al, 2023 ), but microfilaments structure and dynamics have never been reported in ARSACS models. Our analysis indicated an aberrant expression of actin and actin-binding proteins, unraveling a potential role of actin cytoskeleton in the disease.…”

“…ARSACS is caused by mutations in SACS ( Engert et al, 2000 ), a gene encoding sacsin whose “scaffold- like” and multidomain organization suggests involvement in protein quality control ( Romano et al, 2013 ). Recent findings point to loss-of-function mechanism in ARSACS ( Longo et al, 2021 ), and multiple in vitro and in vivo studies suggest that sacsin plays roles in mitochondrial dynamics ( Girard et al, 2012 ; Criscuolo et al, 2015 ), cytoskeletal filament assembly and dynamics ( Duncan et al, 2017 ; Louit et al, 2023 ), axonal development ( Ady et al, 2018 ; Romano et al, 2022 ), and Ca 2+ homeostasis ( Del Bondio et al, 2023 ). Yet, the question of how mutant sacsin leads to disease status in patients with ARSACS remains to be answered.…”