SummaryRett syndrome (RTT) is a neurodevelopmental disorder that is characterized by developmental regression, loss of communicative ability, stereotyped hand wringing, cognitive impairment, and central apneas, among many other symptoms. RTT is caused by loss-of-function mutations in a methyl-reader known as methyl-CpG-binding protein 2 (MeCP2), a protein that links epigenetic changes on DNA to larger chromatin structure. Historically, target identification for RTT has relied heavily on Mecp2 knockout mice; however, we recently adopted the alternative approach of performing transcriptional profiling in autopsy samples from RTT patients. Through this mechanism, we identified muscarinic acetylcholine receptors (mAChRs) as potential therapeutic targets. Here, we characterized a cohort of 40 temporal cortex samples from individuals with RTT and quantified significantly decreased levels of the M1, M2, M3, and M5 mAChRs subtypes relative to neurotypical controls. Of these four subtypes, M1 expression demonstrated a linear relationship with MeCP2 expression, such that M1 levels were only diminished in contexts where MeCP2 was also significantly decreased. Further, we show that M1 potentiation with the positive allosteric modulator (PAM) VU0453595 (VU595) rescued social preference, spatial memory, and associative memory deficits, as well as decreased apneas in Mecp2+/- mice. VU595’s efficacy on apneas in Mecp2+/- mice was mediated by the facilitation of the transition from inspiration to expiration. Molecular analysis correlated rescue with normalized global gene expression patterns in the brainstem and hippocampus, as well as increased Gsk3β inhibition and NMDA receptor trafficking. Together, these data suggest that M1 PAMs could represent a new class of RTT therapeutics.

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Hypofunction of cholinergic circuits and diminished cholinergic tone have been associated with the neurodevelopmental disorder Rett syndrome (RTT). Specifically, deletion of Mecp2 in cholinergic neurons evokes the same social and cognitive phenotypes in mice seen with global Mecp2 knockout, and decreased choline acetyltransferase activity and vesamicol binding have been reported in RTT autopsy samples. Further, we recently identified significant decreases in muscarinic acetylcholine receptor subtype 4 (M 4 ) expression in both the motor cortex and cerebellum of RTT patient autopsies and established proof of concept that an acute dose of the positive allosteric modulator (PAM) VU0467154 (VU154) rescued phenotypes in Mecp2 +/− mice. Here, we expand the assessment of M 4 PAMs in RTT to address clinically relevant questions of tolerance, scope of benefit, dose response, chronic treatment, and mechanism. We show that VU154 has efficacy on anxiety, social preference, cognitive, and respiratory phenotypes in Mecp2 +/− mice; however, the therapeutic range is narrow, with benefits seen at 3 mg/kg concentrations, but not 1 or 10 mg/kg. Further, sociability was diminished in VU154treated Mecp2 +/− mice, suggestive of a potential adverse effect. Compound efficacy on social, cognitive, and respiratory phenotypes was conserved with a 44-day treatment paradigm, with the caveat that breath rate was moderately decreased with chronic treatment in Mecp2 +/+ and Mecp2 +/− mice. VU154 effects on respiratory function correlated with an increase in Gsk3β inhibition in the brainstem. These results identify the core symptom domains where efficacy and adverse effects may present with M 4 administration in RTT model mice and advocate for the continued evaluation as potential RTT therapeutics.