Inducible pluripotent stem (iPS) cell-derived human astrocytes as a new disease model for the megalencephalic leukoencephalopathy with subcortical cysts (MLC): searching for shared pathological mechanisms of myelin degeneration in astrocytopathies

Inducible pluripotent stem (iPS) cell-derived human astrocytes as a new disease model for the megalencephalic leukoencephalopathy with subcortical cysts (MLC): searching for shared pathological mechanisms of myelin degeneration in astrocytopathies. S. Sposito1, M.S. Brignone1, E.S. Caprini1, C. Veroni1, C. Meloni1, C. De Nuccio2, S. Camerini3, M. Belfiore4, F. Nicita5, E. Bertini5, E. Ambrosini 1 and A. Lanciotti1 1 Dept. of Neuroscience, Istituto Superiore di Sanità, Rome, Italy 2 Research Coordination and Support Service, Istituto Superiore di Sanità, Rome, Italy 3 Core Facilities, Istituto Superiore di Sanità, 00161, Rome, Italy. 4 National Centre for Preclinical and Clinical Evaluation and Research of Drugs (FARVA), Istituto Superiore di Sanità, Rome, Italy. 5 Dept. Neuromuscular and neurodegenerative diseases, Pediatric Hospital Bambino Gesù (OPBG), Rome, Italy Abstract MLC is a rare neurodegenerative and incurable leukodystrophy characterized by brain edema, subcortical cysts and myelin vacuolation causing macrocephaly, motor/cognitive disabilities and epilepsy. In the majority of patients, MLC is caused by mutations in MLC1 gene. In the central nervous system, MLC1 protein expression is restricted to astrocytes, the glial cells with a primary role in the maintenance of brain homeostasis. Studies in MLC mouse models and in cellular heterologous expression systems showed that MLC1 plays a primary role in the activation of volume regulatory decrease (RVD) after astrocyte swelling in response to osmotic stress. In addition, MLC1 was found to inhibit intracellular signaling pathways responsible for astrocyte proliferation and activation following inflammatory, oxidative and osmotic brain insults, leading to hypothesize that in response to brain injury/stress an abnormal astrocyte activation/swelling occurs in MLC, being the mutated MLC1 unable to regulate cell signaling needed to restore normal cell volume. Although this experimental evidence disclose MLC1 involvement in molecular processes controlling astrocyte reactivity and ion/fluid exchanges, its proper function and the MLC molecular pathogenesis remain to be fully elucidated. This lack of knowledge strongly hamper the development of possible pharmacological treatments to arrest the progression of the clinical symptoms that were found reversible in some patients. To fill this gap, we generated iPSC from healthy and MLC donors and differentiated them into astrocytes. By performing electrophysiology, biochemical and imaging analysis and proteomics characterization of control and mutated astrocytes, we found that astrocytes derived from MLC patients recapitulated the key pathological features described in humans and rodent models (RVD and intracellular signaling defects). We also revealed new MLC associated molecular alterations responsible for oxidative stress and mitochondrial dysfunctions there were already described in other astrocytopathies. These observations suggest a common molecular base for the astrocyte-mediated myelin damage whose understanding is mandatory for therapy development and that deserve further investigation. Overall, our data provide evidence that iPSC-derived astrocytes can be used as valid human disease model for the identification of molecular targets and development of drug screening tests for therapeutic purposes.