Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome–like phenotypes

Mutations in the X-linked gene encoding methyl-CpG–binding protein 2 (MeCP2) cause Rett syndrome (RTT), a neurological disorder affecting cognitive development, respiration, and motor function. Genetic restoration of MeCP2 expression reverses RTT-like phenotypes in mice, highlighting the need to search for therapeutic approaches. Here, we have developed knockin mice recapitulating the most common RTT-associated missense mutation, MeCP2 T158M. We found that the T158M mutation impaired MECP2 binding to methylated DNA and destabilized MeCP2 protein in an age-dependent manner, leading to the development of RTT-like phenotypes in these mice. Genetic elevation of MeCP2 T158M expression ameliorated multiple RTT-like features, including motor dysfunction and breathing irregularities, in both male and female mice. These improvements were accompanied by increased binding of MeCP2 T158M to DNA. Further, we found that the ubiquitin/proteasome pathway was responsible for MeCP2 T158M degradation and that proteasome inhibition increased MeCP2 T158M levels. Together, these findings demonstrate that increasing MeCP2 T158M protein expression is sufficient to mitigate RTT-like phenotypes and support the targeting of MeCP2 T158M expression or stability as an alternative therapeutic approach

Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome

Mutations in MECP2 cause Rett syndrome (RTT), an X-linked neurological disorder characterized by regressive loss of neurodevelopmental milestones and acquired psychomotor deficits. However, the cellular heterogeneity of the brain impedes an understanding of how MECP2 mutations contribute to RTT. Here we developed a Cre-inducible method for cell-type-specific biotin tagging of MeCP2 in mice. Combining this approach with an allelic series of knock-in mice carrying frequent RTT-associated mutations (encoding T158M and R106W) enabled the selective profiling of RTT-associated nuclear transcriptomes in excitatory and inhibitory cortical neurons. We found that most gene-expression changes were largely specific to each RTT-associated mutation and cell type. Lowly expressed cell-type-enriched genes were preferentially disrupted by MeCP2 mutations, with upregulated and downregulated genes reflecting distinct functional categories. Subcellular RNA analysis in MeCP2-mutant neurons further revealed reductions in the nascent transcription of long genes and uncovered widespread post-transcriptional compensation at the cellular level. Finally, we overcame X-linked cellular mosaicism in female RTT models and identified distinct gene-expression changes between neighboring wild-type and mutant neurons, providing contextual insights into RTT etiology that support personalized therapeutic interventions

Dopamine receptor signalling to GABA subscription A) receptors:bRegulation of cell-surface expression levels and GABAergic synapse formation

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Dopamine receptor signalling to GABAa receptors: Regulation of cell-surface expression levels and gabaergic synapse formation

GABAa receptors are GABA-gated, Cl' permeable ion channels that play an important role in regulating neuronal excitability and development. There is increasing evidence that dopamine receptors can regulate the activity and function of GABAa receptors; yet very little is known about this during development. We have investigated this regulation using the embryonic striatum, an area of the brain that receives considerable dopaminergic input from the substantia nigra. Since the function of GABAa receptors can be modulated through changes in their cell-surface expression we have investigated the ability of dopamine receptors to regulate GABAa receptor surface levels in cultured embryonic striatal neurones. Changes in the surface expression of GABAa P2/3 subunits were assessed using a cell-surface ELISA procedure that utilises an antibody directed against the extracellular epitopes of these subunits. Activation of D1- or D2-like dopamine receptors caused a prolonged, dose-dependent decrease in the surface expression of (32/3 subunits. These decreases were due to an increase in dynamin-dependent endocytosis, as assessed by visualising receptor internalisation via immunocytochemistry and the use of dynamin inhibitory peptides in cell-surface ELISA experiments. DiR modulation involved PKA and PP2A pathways: the effect of DiRs was occluded PKA activation; and attenuated by inhibitors of PP2A. Additionally, DiR activation increased the phosphorylation of the MAP kinases, p42 and p44; and inhibition of the MAP kinase kinase, MEK, significantly attenuated D1R- mediated decreases in cell-surface expression. The D2R-mediated regulation was prevented by the presence of a PPl inhibitor. To assess whether the prolonged attenuation in GABAa receptor levels at the cell-surface is associated with changes in the development of GABAergic synapses, embryonic striatal cultures were treated with D1R or D2R agonists and the number, as well as the size, of presynaptic vesicular inhibitory amino acid transporter-1 (VIAAT-l)-positive terminals and postsynaptic P2/3-positive clusters of GABAa receptors were determined using immunocytochemistry and confocal microscopy. We demonstrate that both DiR and D2R activation decreased the size of (32/3 clusters without affecting the total number at the cell surface, leading to a concomitant decrease in their colocalization with VIAAT-1, suggesting a decrease in the number of GABAergic synapses. Furthermore, using FM1-43FX, which is only incorporated into active synapses, we found that there was a significant reduction in the number of functional GABAergic synapses following these treatments. We have also investigated the regulation of GABAa receptors by dopamine receptors in cultures of neural progenitor (NP) cells from the postnatal mouse hippocampus. These NP cells are capable of differentiating into new neurones that incorporate into existing neural networks. We have demonstrated that these cells express functional GABAa receptors that upon activation leads to an increase of intracellular Ca2+ levels via opening of L-type Ca2+ channels, as measured by changes in fluo-4 fluorescence. Activation of these receptors also caused a significant decrease in proliferation, as assessed by a decrease in BrdU incorporation; an effect that requires the entry of Ca through L-type calcium channels. Furthermore, while activation of D1Rs had no effect on proliferation when added alone, their activation abrogated the effects of GABAa receptor activation on proliferation. The effects of D1Rs occur by decreasing the ability of GABAa receptors to increase intracellular calcium levels and, as revealed by cell surface ELISA experiments, by decreasing the surface expression of GABAa receptors. D2R activation on the other hand showed no effect on the proliferation of these NP cells in the absence or presence of GABAa receptor activation. In summary, these results lead us to propose a novel role for D1Rs and D2RS in fine-tuning of GABAergic synaptogenesis in the developing striatum potentially through regulation of GABAa receptor cell-surface levels. Additionally, we have also shown that DiR activation can increase the proliferation of NP cells by preventing GABAa receptor-mediated inhibition of proliferation