Generación de un modelo de Células de Purkinje de cerebelo para el estudio de dos enfermedades raras: PLAN y ARSACS

Human patient-derived induced pluripotent stem cells (hiPSCs) provide an unprecedented opportunity to recapitulate disease pathogenicity without the need for genetic manipulation and creation of gene targeted animal models. New technologies with hiPSCs are being developed to make human neurons and have the potential to improve our understanding underlying mechanisms of neurological diseases and development. Cerebellar atrophy is a condition associated with neurodegenerative diseases such as Charlevoix-Saguenay's Recessive Spastic Ataxia (ARSACS) and PLA2G6-Associated Neurodegeneration (PLAN). ARSACS is an early onset neurodegenerative disorder caused by mutations in the SACS gene, characterized with atrophy in superior cerebellum. PLAN, a NBIA (Neurodegeneration with Brain Iron Accumulation) form due to mutations in PLA2G6, is a neuroaxonal dystrophy, which presents cerebellar ataxia. Our aim was to generate a cerebellar Purkinje cell (CPC) model that will be used as models to investigate the cerebellar neurodegeneration to achieve a rationale therapy. We have developed a differentiation protocol using fibroblasts reprogrammed using Sendai virus to hiPSCs, from ARSACS and PLAN patients. We have successfully generated a functional CPC model from hiPSCs according to the protocol described by Shahin’s group [Mol Psychiatry 2018; 23: 2167-83] for each patient’s line. We have characterized this new models, CPC PLAN and CPC ARSACS by investigating the expression of genes and proteins involved in iron metabolism, lipid peroxidation, ferroptosis, and autophagy in PLAN patients and cytoskeleton, proteasome, and autophagy markers in ARSACS patients. In PLAN patients the findings showed an impaired ferritinophagy due to the ferritin and NCOA alterations among other altered markers, as well as an increased of lipid peroxidation levels, and altered gene antioxidant and ferroptosis gene expression altered, so that ferroptosis, iron metabolism and autophagy may be impaired and could contribute to neuronal death. In ARSACS patients the findings showed an autophagy pathway dysfunction and regulatory assembly and disassembly gene expression altered. Moreover, in mature ARSACS and healthy carriers CPCs we observed neurofilaments accumulations genotype-dependent, as patient and animal’s models showed previously as a principal characteristic of ARSACS disease. We also analyzed mitochondrial respiration and bioenergetics using a Seahorse XFe96 analyzer to confirm that the mitochondrial function was altered in both diseases. Finally, we examined the electrophysiological properties by whole cell patch clamp recordings on patients and control mature CPCs and neuronal morphology. The observed mitochondrial dysfunction and the abnormal electrophysiological properties of CPC’s patients carrying PLA2G6 and SACS mutations in heterozygosis and homozygosis indicate that normal expression of PLA2G6 and SACS is important for the development and functionality of the human CPCs. These results show that the CPC PLAN and CPC ARSACS model faithfully reproduces the underlying pathophysiology and, therefore, is suitable for drug screening and the achievement of rational therapy for this disease in the near future

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