Differential Sensitivity of the Protein Translation Initiation Machinery and mTOR Signaling to Gain- and Loss-of-Function Involves MeCP2 Isoform-Specific Homeostasis in the Brain.

Eukaryotic gene expression is controlled at multiple levels, including gene transcription and protein translation initiation. One molecule with key roles in both regulatory mechanisms is methyl CpG binding protein 2 (MeCP2). gain- and loss-of-function mutations lead to Rett Syndrome and Duplication Syndrome, respectively. To study gain-of-function, we generated stably transduced human brain cells using lentiviral vectors for both and isoforms. Stable overexpression was confirmed by Western blot and immunofluorescence. We assessed the impact of MeCP2E1-E2 gain-of-function on the MeCP2 homeostasis regulatory network (/BDNF-), mTOR-AKT signaling, ribosome biogenesis, markers of chromatin structure, and protein translation initiation. We observed that combined co-transduction of MeCP2 isoforms led to protein degradation of MeCP2E1. Proteosome inhibition by MG132 treatment recovered MeCP2E1 protein within an hour, suggesting its induced degradation through the proteosome pathway. No significant change was detected for translation initiation factors as a result of MeCP2E1, MeCP2E2, or combined overexpression of both isoforms. In contrast, analysis of human Rett Syndrome brains tissues compared with controls indicated impaired protein translation initiation, suggesting that such mechanisms may have differential sensitivity to gain- and loss-of-function. Collectively, our results provide further insight towards the dose-dependent functional role of MeCP2 isoforms in the human brain.