NEURAL PRECURSOR/STEM CELL-BASED THERAPY: A NOVEL THERAPEUTIC APPROACH FOR THE TREATMENT OF RETT SYNDROME

Rett syndrome (RTT) is a postnatal progressive neurodevelopmental disorder with an incidence of 1:10,000 females born alive. It represents the most common genetic cause of severe intellectual disability in girls worldwide and it is caused by severe loss of function mutations in the MECP2 gene encoding for the methyl-CpG binding protein 2 (MeCP2), an epigenetic factor involved in gene transcription regulation and chromatin remodeling, particularly abundant in the central nervous system. RTT features include cognitive and motor impairments, hand stereotypies, seizures, loss of acquired speech and breathing abnormalities. MECP2 aberrations result in a wide range of neuropsychiatric disorders characterized by neuronal defects such as reduction in soma size, dendritic arborization and synaptic plasticity together with an excitation/inhibition unbalance. Basic and preclinical studies on RTT have largely benefitted from the use of mouse models mutated in Mecp2. Among these, the most studied is the full knock-out mouse (Mecp2 KO), that recapitulates many RTT features. The same animal model has permitted to demonstrate that RTT is a reversible condition, at least in mice. This breaking through discovery encouraged preclinical studies that in general have tested the efficacy of pharmacological or augmentative gene therapies. However, a cure for RTT is still lacking and the several functions exerted by MeCP2 and its involvement in different molecular pathways complicate the finding of a therapeutic approach; indeed, patients are treated only to ameliorate secondary phenotypes. Among all the preclinical works, no study assessed the possible efficacy of neural precursor/stem cell (NPC) transplantation, that has already been proved beneficial and safe for many neurological disorders, such as Parkinson’s disease, spinal cord injury, ischemia and multiple sclerosis. Mechanisms of action of NPCs can rely not only on the replacement of damaged neurons/cells, but also on the release of molecules, including trophic and immunomodulator factors, exerting beneficial effects through a “bystander” mechanism. With this PhD project I tested the validity of NPC treatment as a novel therapeutic approach for RTT. Through an in vitro co-culture, I initially proved that, by sensing the surrounding pathological context, NPCs use a bystander mechanism to recover typical morphological and synaptic defects of Mecp2 deficient neurons. To investigate whether the treatment could be beneficial also in vivo, in RTT mouse models, I intrathecally transplanted NPCs in symptomatic Mecp2 KO mice. NPC-transplanted Mecp2 KO mice showed improvements in motor and cognitive functions compared to Mecp2 KO control animals. In order to find the molecular factors responsible for these behavioral improvements, which is a fundamental step for moving from the bench to bedside, RNA sequencing analyses were performed on brain of the same transplanted animals. Bioinformatics analyses revealed several deregulated pathways, mostly upregulated and associated with the immune system, in Mecp2 KO mice treated with NPCs compared to the control group. Among these pathways emerged the upregulation of that one related to Cytokine X (CX), whose identity is not revealed considering that the results related to this molecule are under evaluation for patent. Since CX regulates neural connectivity and social behavior, I proceeded to test the therapeutic validity of treating RTT mice directly with this cytokine. Importantly, recombinant CX showed in vitro its ability to rescue typical Mecp2 KO synaptic defects. Then, to test whether CX treatment could be efficacious also in vivo, I intrathecally injected CX in symptomatic Mecp2 KO mice. Notably, CX treatment reverted both Mecp2 KO motor and cognitive defects. Overall, the data presented in this thesis demonstrate the efficacy of NPC treatment both in in vitro and in vivo Mecp2 KO models and identified CX as one of the possible factors responsible for the behavioral improvements observed in Mecp2 KO mice transplanted with NPCs.