NEURAL PRECURSOR CELLS AS A POTENTIAL THERAPEUTIC APPROACH FOR RETT SYNDROME: IDENTIFICATION OF THE INVOLVED MOLECULAR MECHANISMS

Rett syndrome (RTT) is a rare, progressive, and profoundly debilitating neurodevelopmental disorder (NDD) and represents the leading cause of severe intellectual disability in females, with an estimated incidence of 1 in 10,000 live births. More than 95% of RTT cases result from sporadic mutations in the MECP2 gene, which is located on the X chromosome (Xq28) and encodes the methyl-CpG binding protein 2 (MeCP2), a nuclear protein that functions as an epigenetic regulator. Affected girls initially exhibit apparently normal development until 6 - 18 months of age, when they enter a severe regression phase characterized by the loss of previously acquired cognitive and motor skills. RTT is an extremely crippling disorder with significant phenotypic variability, influenced by the specific genetic variants causing the disease and by the degree of X chromosome inactivation (XCI). Common symptoms include communication impairments, stereotypical hand movements, respiratory abnormalities, and seizures. At the cellular level, RTT is associated with defects in neuronal morphology, activity, and synaptic transmission. Since the landmark discovery of RTT’s potential reversibility in 2007, significant progress has been made in elucidating the functional role of MECP2 and the consequences of its deficiency. Despite major advances over the past decades, the precise mechanisms underlying RTT pathophysiology remain poorly defined, and a definitive cure remains elusive. Indeed, currently, affected individuals receive symptomatic treatments. However, a significant milestone was reached in 2023 when the U.S. Food and Drug Administration (FDA) approved Trofinetide (DayBue) as the first treatment specifically designed for RTT. Despite this breakthrough, there remains an urgent need for additional therapeutic approaches to improve the severely compromised quality of life of affected individuals. This study was conducted within the broader context of identifying innovative therapeutic strategies for RTT and focused on investigating the potential of neural precursor cell (NPC) transplantation as a promising therapeutic approach for RTT. NPCs are multipotent stem cells that persist in the adult brain. Their therapeutic potential has been extensively explored in both preclinical and clinical studies of neurodegenerative diseases, where they have been shown to be safe and to exert beneficial effects primarily through “bystander” mechanisms. In the context of NDDs, we previously demonstrated that NPC transplantation in Mecp2 knockout (KO) mice rescued the characteristic cognitive and motor deficits in treated animals. When analyzing the transcriptomic profile of NPC-treated mice, we revealed an activation of the IFNγ pathway, that we further validated by observing symptoms improvements after direct IFNγ administrations. These preliminary results suggested a pivotal role for this cytokine in the therapeutic effects of NPCs. Building upon these findings, this study further explored the mechanisms underlying NPC-mediated benefits and leveraged NPCs as a platform to identify new molecular targets for RTT treatment. Using an in vitro transwell co-culture system, we demonstrated that NPC-secreted factors (NPC-SF) specifically rescued synaptic defects in Mecp2 KO neurons without affecting wild-type neurons. Furthermore, we characterized the temporal dynamics of NPC-neuron interactions. Notably, we found that NPCs respond specifically to the RTT pathological environment by releasing factors that promote synaptic repair in Mecp2 KO neurons. Remarkably, we demonstrated that Mecp2 KO NPCs exhibited therapeutic efficacy comparable to the one of wild-type NPCs, paving the way for patient-derived isogenic transplantation strategies. Furthermore, we investigated the role of IFNγ in our in vitro system and confirmed its involvement in NPC-mediated beneficial effects, particularly in the presynaptic compartment. To further elucidate the molecular basis of NPC-mediated effects, we performed transcriptomic analyses of Mecp2 KO neurons treated with NPCs, as well as of NPCs exposed to RTT neuronal media. RNA sequencing confirmed that NPC-SF promotes neuronal maturation, as evidenced by enrichment in synaptic and plasticity-related pathways, while also modulating IL-17 production in Mecp2 null treated neurons. Transcriptional profiling of NPCs highlighted genotype-specific adaptations and further implicated IFNγ in these processes. These findings suggest a multifaceted mechanism underlying NPC-mediated repair and underscore the dynamic interplay between NPCs and the RTT microenvironment. In conclusion, this study highlighted the therapeutic potential of NPCs in RTT and established the relevance of IFNγ in NPC-mediated beneficial effects in in vitro systems. Additionally, it demonstrated the capacity of NPCs to adapt to pathological environments and identifies novel pathways, such as IL-17 modulation, as promising targets for future research.