Total RNA Sequencing of Rett Syndrome Autopsy Samples Identifies the M₄ Muscarinic Receptor as a Novel Therapeutic Target

Abstract

Mutations in the gene are responsible for the neurodevelopmental disorder Rett syndrome (RTT). MeCP2 is a DNA-binding protein whose abundance and ability to complex with histone deacetylase 3 is linked to the regulation of chromatin structure. Consequently, loss-of-function mutations in MeCP2 are predicted to have broad effects on gene expression. However, to date, studies in mouse models of RTT have identified a limited number of gene or pathway-level disruptions, and even fewer genes have been identified that could be considered amenable to classic drug discovery approaches. Here, we performed RNA sequencing (RNA-seq) on nine motor cortex and six cerebellar autopsy samples from RTT patients and controls. This approach identified 1887 significantly affected genes in the motor cortex and 2110 genes in the cerebellum, with a global trend toward increased expression. Pathway-level analysis identified enrichment in genes associated with mitogen-activated protein kinase signaling, long-term potentiation, and axon guidance. A survey of our RNA-seq results also identified a significant decrease in expression of the gene, which encodes a receptor [muscarinic acetylcholine receptor 4 (M)] that is the subject of multiple large drug discovery efforts for schizophrenia and Alzheimer's disease. We confirmed that expression was decreased in RTT patients, and, excitingly, we demonstrated that M potentiation normalizes social and cognitive phenotypes in mice. This work provides an experimental paradigm in which translationally relevant targets can be identified using transcriptomics in RTT autopsy samples, back-modeled in mice, and assessed for preclinical efficacy using existing pharmacological tool compounds.

Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence

“… (B) RT-PCR on RNA isolated from Rett brain versus age-matched control brains for P53 targets. (C) A re-analysis of data published by Gogliotti et al. (2018) .…”

Pharmacological enhancement of KCC2 gene expression exerts therapeutic effects on human Rett syndrome neurons and Mecp2 mutant mice

“…A reduction in KCC2 protein expression has been detected in MECP2-deficient human RTT neurons and in the brains of Mecp2 mutant mouse model of RTT (27,28), in autopsy brain samples (48), and in the cerebrospinal fluid of patients with RTT (49). To assess whether the KEECs identified above would enhance KCC2 expression in RTT neurons, we screened MECP2-null RTT human KCC2 reporter neurons (isogenic to the WT reporter cells used in Fig.…”

A Coordinated Attack: Rett Syndrome Therapeutic Development

“…Studies in autopsy samples have demonstrated that the expression of ~2000 genes is compromised by pathogenic mutations in MeCP2 , most of which are only moderately shifted up or down [12]. This increase in transcriptional noise is postulated to result in a series of interactomes of affected proteins that may additively contribute to the manifestation of RTT phenotypes [12]. De novo discovery efforts for RTT seek to identify nodes on these interactomes, which can serve as potentially druggable access points to modify specific symptom domains.…”

“…For example, both glutamatergic and GABAergic signaling are compromised in MeCP2 -knockout mice. Based on our experience with modulating synaptic plasticity using allosteric modulators of metabotropic glutamate (mGlu) and muscarinic receptors, we profiled human RTT and control tissues for expression of these targets and found significant changes in two metabotropic glutamate (mGlu) receptors, mGlu 5 [13] and mGlu 7 [14], as well as in the M 4 muscarinic receptor [12]. Using positive allosteric modulators that were originally under development for other indications, such as schizophrenia, we have built preclinical datasets for these novel targets in RTT model mice.…”

“…Furthermore, there remains considerable debate regarding how distinct preclinical measures will translate to clinical trials. For example, RTT model mice exhibit reproducible impairments in learning and memory paradigms that are responsive to numerous pharmacological interventions [12,16,17]. However, it will likely be challenging to quantify changes in cognitive ability clinically in patients who are primarily nonverbal and have limited use of their extremities.…”