"""Investigating autophagy enhancement as a therapeutic approach for the treatment of Rett syndrome."""

Periodic Reporting for period 1 - RTTOPHAGY ("Investigating autophagy enhancement as a therapeutic approach for the treatment of Rett syndrome.")

Période du rapport: 2019-06-03 au 2021-06-02

Mutations in the X-linked MECP2 gene cause Rett syndrome (RTT), a devastating neurological disorder which represents a first cause of severe intellectual disability in 1 out 10.000 girls worldwide. Affected females have an apparently normal development until 6-18 months of life when a regression phase begins, the purposeful use of hands is substituted by involuntary stereotyped movement, and all acquired motor and cognitive skills are lost. To date, there is no cure for Rett syndrome and current treatments are usually directed to alleviate secondary symptoms such as epilepsy, irritability and insomnia.
However, experimental evidence obtained in a mouse model of the disease, in which Mecp2 is missing, suggested that the disease is reversible. In particular, by gene therapy approach it was demonstrated that re-activation of Mecp2 gene reverted the majority of RTT symptoms. Unfortunately, this approach is not applicable to human being for many reasons but set it up the proof of principle that a cure is possible.
From there, several researchers exploited novel therapeutic strategy for RTT especially based on pharmacological approach aimed at modulating MeCP2-downstream pathways.
Autophagy is a catabolic process that is crucial for maintaining cellular homeostasis, energy balancing and ensuring quality control inside the cells, especially in neurons that are post-mitotic cell and metabolically very active.
Interestingly, defective autophagy has been recently recognized in fibroblasts of RTT patients and in the cerebellum of Mecp2-null mice, however it was not further investigated in the central nervous system.
Thus, this project aimed at the pinpointing the molecular deficits responsible for altered autophagy in RTT and determining a strategy could alleviate the developmental defects in cellular and animal models of the disease.
- Specifically, the characterization of the autophagy signal in brain cortices of RTT mouse model led to the identification an impairment of autophagy flux and a downregulation of a key regulator of autophagy biogenesis (objective 1).
- Given that autophagy flux is blocked and since that autophagy enhancement has been demonstrated to be effective for several neurological disorders, modulation of autophagy flux in cultured cortical neurons revealed an amelioration of RTT neuronal phenotypes (objective 2).
- Finally, it was demonstrated in a pre-clinical study the pharmacological potential of promoting autophagy. Mice model of the disease were treated with an autophagy enhancer which improved spontaneous and non-spontaneous motor skills, positively acted on explorative behaviour and more importantly ameliorated the general conditions and the survival of mutant mice (objective 3).
Overall all these data reinforced the hypothesis that autophagy dysfunction could contribute to RTT manifestations and that its modulation could be a good strategy to treat other neurodevelopmental disorders.
The social value of this project is twofold:
1) From a clinical perspective it will lead the way for further validation of a novel therapeutic option for RTT syndrome, which can currently benefit from very few treatments that are only
symptomatic. Importantly, several modulators of autophagy are already FDA-approved drugs for other diseases, thus making future repurposing easily feasible.
2) Defects in autophagic flux have been only scarcely investigated in neurodevelopmental disorders in general and RTT in particular. Thus, from the scientific perspective, this proposal led to a better comprehension of the basic pathological mechanisms underlying the disease.

Precise control of autophagic flux is necessary for maintenance of cellular balance between protein production and degradation. In contrast, alterations in autophagy have been implicated in many disorders, including metabolic and neurodegenerative diseases. However, there are only few studies investigating autophagy dysfunction in neurodevelopmental disorders, even if this cellular process regulates physiological functions of the nervous system.
Mutations in the X-linked MECP2 gene cause a broad spectrum of neuropsychiatric disorders including Rett syndrome (RTT). Many mouse models of RTT have been created to study the molecular pathogenesis of the disease and to define the role of MeCP2 in neurons. Although these studies suggest that MeCP2 has an essential function in brain development, it remains largely unclear which molecular pathways, directly or indirectly, lead to defects in neuronal maturation during the embryonic and postnatal stage.
A link between autophagy and RTT has been recently suggested in fibroblasts from RTT patients and Mecp2-null cerebellum, however no further studies were performed.
Here, investigation of autophagy pathway in brain of RTT animal model suggested a block of autophagy flux probably due to a deregulation of a key protein involved in the formation and maturation of autophagosomes, the major players in this process.
Mecp2 deficiency causes several morphological phenotypes in neurons, including reduced dendritic arborization, soma size and spine density. To assess the therapeutic potential of autophagy modulation, a short treatment on Mecp2-null neurons with an autophagy enhancer was performed which resulted in the dendritic branching rescue. Similarly, in vivo treatment on WT and Mecp2-null animals revealed an amelioration of overall general condition and life span of mutant mice.
In conclusion, these findings strengthen the hypothesis that dysfunctions of autophagy and related signaling pathways might represent a convergent molecular mechanism responsible for RTT pathogenesis. Moreover, this may represent a solid starting point for the development of novel therapies based on the proof of principle of autophagy modulation.

While previous report only exploited the autophagy function in fibroblasts of Rett patients, in this proposal a better characterization of this pathway was achieved. Key deregulated proteins of the signal were identified in brain cortices of Mecp2-null animals.
Moreover, in the pre-clinical study it was demonstrated the beneficial effect of using an autophagy modulator for the recovery of motor coordination and healthy conditions of mutant mice; thus setting up a proof of principle of autophagy modulation as a therapeutic strategy for the treatment of Rett syndrome. In the near future, it will be very interesting to test other molecules able to activate autophagy on RTT and other neurodevelopmental disorders.
Indeed, extending our fundamental scientific knowledge about neurological diseases and intellectual disabilities (ID), this project has the potential to make a valuable contribution to efforts for the identification of new therapeutic strategies that can be exploited in clinical setting.
Over 4.2 million people have ID in Europe with a total estimated cost of €43.3 billion each year. There is thus an unmet clinical need for a rationally-designed suite of therapeutic measures, ideally targeting early stages in the relevant pathogenetic pathways, that can ameliorate, fix or prevent a range of ID disorders that are still underrepresented in health care and health research.

Graphical image of RTTOPHAGY project

Periodic Reporting for period 1 - RTTOPHAGY ("Investigating autophagy enhancement as a therapeutic approach for the treatment of Rett syndrome.")

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