Rett Syndrome (RTT) is a severe and progressive neurodevelopmental disorder that affects approximately 1 in 10,000 live female births, resulting in profound cognitive and physical disabilities. One of the most common mutations associated with RTT is the T158M missense mutation, found in approximately 12% of cases. T158M mice demonstrate a loss of localization to heterochromatic foci and a redistribution of mutant MeCP2 to the nucleolus. This mutation disrupts the methyl-CpG binding domain (MBD) of MeCP2, impairing its ability to bind to methylated DNA. Normally, MeCP2 binds to DNA in heterochromatin regions. These heterochromatin regions exhibit liquid-like properties, forming condensates, which compartmentalize and concentrate molecules to facilitate complex gene regulatory processes. This mislocalization impairs chromatin regulation and leads to significant cellular dysfunction, contributing to the neurological symptoms seen in RTT.

The T158M mutation causes cellular dysfunction by disrupting the localization of MeCP2, trapping it in the nucleolus rather than allowing it to bind to chromatin, where it regulates gene expression. Our hypothesis is that re-distributing the mutant MeCP2 out of the nucleolus and into the DNA-rich heterochromatin regions could improve its function, potentially restoring normal gene expression and neuronal function.

Currently, no approved therapies directly address the loss of MeCP2 function central to Rett Syndrome (RTT). The reduced levels of MeCP2 and phospho-riboprotein S6 observed in T158M mutants, compared to wild-type, further suggest that phospho-riboprotein S6 could serve as a direct readout of MeCP2 function due to its positive correlation with MeCP2 activity. C-mods may also help relocate mutant MeCP2 from nucleoli to chromatin, potentially mitigating the loss of function and restoring normal cellular processes. Biotin tagging of MeCP2 has proven to be a reliable method for assessing MeCP2 localization, and this research highlights the therapeutic potential of c-mods in treating RTT by improving MeCP2 chromatin localization and function.

Experimental Approach: To investigate this, we conducted immunohistochemistry (IHC), which allows us to visualize specific proteins in tissue samples using antibodies. We utilized several markers:

1. Rabbit anti-MeCP2: To detect and visualize MeCP2 levels and localization. We expect reduced MeCP2 in T158M samples.
2. Rabbit anti-nucleolin: To identify the nucleolus and determine whether MeCP2 is mislocalized.
3. Rabbit anti-phospho-S6: Used to analyze MeCP2 activity. Higher phospho-S6 levels correlate with increased MeCP2 activity.

Results: Biotin-tagged MeCP2 was used as a proxy for MeCP2 localization. In wild-type (WT) cells, MeCP2 is normally located outside the nucleolus's chromatin. However, in T158M mutants, the biotin signal was found inside the nucleolus, indicating mislocalized MeCP2. Furthermore, reduced phospho-S6 levels confirmed impaired MeCP2 function in T158M mutants.

Future Directions: Our next steps involve intracerebroventricular (ICV) injections and subsequent IHC with a larger sample size of mice to assess the efficacy of potential therapies. We will test various c-mods (chemical modifiers) to relocate mutant MeCP2 away from the nucleolus. We believe that improving MeCP2 chromatin localization may mitigate its loss of function and show the therapeutic potential of c-mods in treating Rett Syndrome.