Characterization of metabolism and mitochondrial dynamics in astrocytes derived from induced pluripotent stem cells of multiple sclerosis patients

Multiple sclerosis (MS) is an autoimmune disease characterized by a chronic and progressive inflammatory condition in the Central Nervous System (CNS), which results in an axonal neurodegeneration process, culminating in neurological deficiency in patients. Recently, it was established that astrocytes (glial cells) play a key role in the regulation of neuronal synapses, along with inflammatory and neurodegenerative processes in the CNS. It is also known that astrocytes are one of the major sources of reactive oxygen species and this is directly related to the regulation of mitochondrial function. Mitochondria constantly modify their morphology according to the bioenergetic needs of the cell and changes in these regulatory mechanisms can trigger neurodegenerative processes. MS is hardly represented in all its scope by animal models, and many therapeutic studies in experimental models have failed to be translated into humans due to interspecific differences. Still, the study of the role of CNS resident cells in MS patients is hampered by ethical issues. In this sense, induced pluripotent stem cells (hiPSC), which can be reprogrammed from patients\' blood or skin samples and differentiated into any somatic cell population, appear as a powerful approach to investigate molecular mechanisms that may be associated the development of complex diseases like MS.Therefore, in this work we formulated the hypothesis that hiPSC-derived astrocytes from patients with Multiple Sclerosis should have changes in mitochondrial dynamics and metabolism that may be associated with the MS phenotype. In a brief manner, hiPSC-derived astrocytes were successfully obtained and characterized. We observed baseline differences between the control and patients\' astrocytes, with enrichment of genes associated with mitophagy and neurodegenerative processes, as well as the transport of molecules through the mitochondria in MS astrocytes. Then, we observed in this same group an increase in superoxide production and a decrease in mitochondrial quality. Analyzing the cells functionally using the Seahorse technique, we observed an increase in oxidative and glycolytic metabolism in patients\' astrocytes, indicating a process of metabolic stress in these cells, in addition to a significant decrease in their mitochondrial bioenergetics efficiency. Using transmission electron microscopy, we analyzed the mitochondrial morphology in astrocytes and observed a significant increase in mitochondrial fission in the MS group, corroborating our gene expression results obtained by PCR array. Also, in line with the previous results, we observed a decrease in the ratio between mitochondrial DNA and nuclear DNA. As we observed a swelling of the endoplasmic reticulum in the astrocytes of patients by electron microscopy, we analyzed the expression of genes related to the UPR (Unfolded Protein Response) pathway as well as genes linked to the regulation of the response to mitochondrial stress and we observed that several of them are significantly decreased in patient astrocytes, indicating a possible defect in thecellular stress response machinery. Thus, our results suggest a phenotype of mitochondrial dysfunction in astrocytes derived from MS patients and open new perspectives for both disease modeling and future therapeutic approaches. (AU)