MUSCLE-SPECIFIC MECP2 MISEXPRESSION INDUCES SKELETAL AND VISCERAL MUSCLE DEFECTS RESCUED BY BUTYRATE SUPPLEMENTATION IN DROSOPHILA

Summary
Background
MeCP2 is a chromatin-associated protein whose dosage alterations cause two severe neurodevelopmental disorders: Rett syndrome (RTT), linked to loss-of-function mutations, and MECP2 duplication syndrome (MDS), linked to overexpression. Although widely studied for their neurological symptoms, these disorders also display prominent non-exclusively neurological features, including gastrointestinal dysmotility and muscle hypotonia - suggesting that MeCP2 dysfunction may also impact peripheral tissues. Since MECP2 is expressed in muscle, yet its role outside the nervous system remains poorly defined, this work explores the tissue-autonomous effects of MECP2 misexpression in muscle.
Aims
This project aims to establish a Drosophila model to investigate the impact of MECP2 misexpression in muscle tissue and its role in muscle development, assess whether the resulting phenotypes are autonomous or secondary to neuronal dysfunction, and test the therapeutic potential of short-chain fatty acids (SCFAs) - specifically butyrate - as chromatin-targeting interventions.
Results
Because Drosophila lacks an endogenous MECP2 gene, we used transgenic lines overexpressing either wild-type or mutant human MECP2. Using the Gal4/UAS system, tissue-specific expression revealed that muscle-targeted MECP2 overexpression led to the strongest phenotypes, such as reduced viability, disorganized skeletal and visceral muscle, impaired locomotion, and delayed gut transit. In addition, mitochondrial abnormalities - increased organelle number and altered cristae structure - were observed by electron microscopy in muscle fibers. Visceral muscle defects were most pronounced when MECP2 was expressed during gut development, suggesting interference with tissue maturation.
We also employed the model to functionally classify MECP2 variants. Alleles were scored based on their ability to enhance or suppress wild-type MECP2 phenotypes. R106W suppressed lethality and caused mild muscle disruption, consistent with loss of function. Δ166 enhanced lethality but caused minimal pathology, suggesting a benign effect. R294X produced strong muscle phenotypes and variable lethality across tissues, indicating partial function.
To determine whether these effects were secondary to neural dysfunction, we expressed MECP2 in neurons but observed no impact on muscle structure or gut function. Similarly, MECP2 expression in muscle did not disrupt synaptic architecture. These findings support a tissue-autonomous mechanism.
With a clear muscle-specific phenotype and considering their energetic and epigenetic functions, we next tested whether SCFAs supplementation could reverse these defects. High-dose propionate reduced survival. In contrast, acetate, sodium butyrate, and the butyrate-rich postbiotic Lalbaay® improved viability and development, with Lalbaay® showing the strongest effects.
Motor function was also enhanced, supporting the beneficial role of butyrate molecule. Indeed, supplementation with sodium butyrate, an HDAC inhibitor with energetic role, specifically restored muscle structure and gut motility. Valproic acid, a pan-HDAC inhibitor without metabolic properties, produced similar improvements, suggesting an epigenetic mechanism of action.
Finally, we began dissecting genetic interactions. Since Drosophila lacks endogenous MECP2, it provides a clean background to identify functional modifiers. Recent work implicated PHF14 - a MeCP2 interactor mutated in a Rett-like syndrome - as a candidate modifier. Given its conservation in flies, we initiated a mutagenesis screen via transposon excision. To date, 22 candidate mutant lines have been isolated and are being validated, paving the way for future genetic interaction studies.
Conclusions
This thesi