Mutation of MeCP2 T158 to M or in rare cases to A represents one of the most common mutations observed in RTT patients2
. Previous in vitro
experiments established a critical role for this residue in the binding of MeCP2 to methylated DNA. To address the causal role of T158A mutation in the pathogenesis of RTT and the role of methyl-DNA binding in the proper functions of MeCP2, we developed and characterized MeCP2 T158A knockin mice. We found that MeCP2 T158A mice develop normally for the first 4–5 weeks of life after which they present RTT-like symptoms including decreased motor performance, altered anxiety, aberrant gait, hindlimb clasping, breathing abnormalities, and impaired learning and memory. The similarity in the identity and severity of symptoms with those observed in Mecp2
-null mice indicates that MeCP2 T158A mutation is a partial loss-of-function mutation.
The development of this mouse line allowed us to investigate the biochemical consequences of MeCP2 T158A mutation in vivo. In agreement with previous in vitro studies, we find that MeCP2 T158A mutation leads to a reduction in the affinity of MeCP2 for methylated DNA in vivo. Surprisingly, we also observed that T158A mutation concomitantly decreases MeCP2 protein expression in vivo. Consistent with these data, we find that fibroblasts obtained from a female RTT patient carrying MeCP2 T158M mutation express decreased levels of MeCP2 protein. These findings reveal two consequences of T158 mutation: impaired MeCP2 binding to DNA and decreased MeCP2 protein stability of MeCP2.
Previous studies have demonstrated that MeCP2 protein levels must be tightly regulated to ensure its proper function. A 50% reduction in MeCP2 protein levels leads to progressive neurological symptoms9,10
, although symptoms appeared later and do not fully recapitulate RTT-like symptoms as in Mecp2
-null mice. Therefore, the destabilization of MeCP2 protein alone, as observed in our T158A mice, may not be sufficient to cause the RTT-like symptoms. We propose that the combined reduction in MeCP2 protein levels and the decreased binding to methylated DNA contribute to the loss-of-function phenotype in T158A knockin mice. The development of knockin mice carrying other mutations that disrupt DNA binding will provide further insights into this hypothesis. Given that the reintroduction of MeCP2 protein into Mecp2
-null mice is sufficient to rescue RTT-like phenotypes12
we suggest a dual approach to restore MeCP2 function in patients carrying MeCP2 T158 mutations: increasing MeCP2 affinity for methylated DNA and enhancing MeCP2 protein stability. Indeed, the feasibility of increasing affinity for DNA has been shown for other DNA-binding proteins such as p5341
. It is conceivable that increasing MeCP2 affinity to methylated DNA may help stabilize MeCP2 protein expression. Targeting one or both of these possibilities may lead to the amelioration of RTT-like phenotypes. Our study also suggests that different therapeutic strategies should be considered for treating patients with different MeCP2 mutations.
Given their neurological origin, many of the symptoms associated with RTT have been hypothesized to result from imbalanced neural networks3
. Evidence to support this arises from observed alterations in synaptic connectivity and plasticity12,15–17,42
and hyperexcitability in the EEG22,23
-null mice. Furthermore, ERP analysis in RTT females suggests alterations in sensory processing of information24,25
. Given the delayed onset of symptoms in RTT patients, MeCP2 T158A mice and Mecp2
-null mice, we sought to examine whether neurophysiological responses as measured by EEG were altered during development in MeCP2 T158A mice. Indeed, we found that the power of high-gamma EEG signals is significantly increased in MeCP2 T158A mice when these mice exhibit RTT-like symptoms, suggesting hyperexcitability in the brain. Furthermore, assessment of auditory-evoked ERPs revealed a significant and marked reduction in the amplitude and increased latency of ERPs in MeCP2 T158A and Mecp2
-null mice suggesting deficits in information processing in the brain similar to that observed in RTT females24,25
and other disorders including schizophrenia18
. Further studies are needed to address the neuronal mechanisms that underlie these deficits in ERP response.
Our data show that disturbances in event-related power and phase locking also occur in MeCP2 mouse models and may play a role in the etiology of RTT. In humans and animal models, changes in the power and phase locking of neuronal responses coordinate neuronal activity across different brain regions. These changes are involved in the development and efficacy of motor, perceptual and memory tasks and deficits in neuronal oscillations are consistently observed in neurological disorders in which these functions are impaired18,43
. Our findings that both low- and high-frequency event-related oscillations are disrupted lead us to hypothesize that deficits in local and long-distance neuronal circuitry occur following MeCP2 dysfunction. The neurophysiological mechanisms that lead to disturbances in these oscillations are not known, but may involve the reduced neuronal connectivity that leads to a redistribution of neuronal activity away from excitation and towards inhibition as observed in Mecp2
. Furthermore, given the important role that event-related neuronal responses play in the development of the nervous system44
, their disruption prior to symptom presentation may augment the deficits in neuronal activity caused by MeCP2 dysfunction. Indeed, MeCP2 T158A mice do not exhibit developmental increases in event-related power or phase locking suggestive of stagnation in the developmental of neuronal circuits. Moreover, our findings that event-related changes in power and phase locking occur in MeCP2 T158A mice with no behavioural symptoms suggest that disruptions in neuronal networks may precede the behavioral RTT-like phenotypes. The identification of the mechanisms that lead to these disturbances will provide valuable insights into the pathogenesis of RTT and the neuronal networks underlying manifestation of behavioral phenotypes in RTT.
In summary, the development of MeCP2 T158A mice has uncovered a novel role for T158 in the pathogenesis of RTT and revealed an alternative strategy to restore MeCP2 function. These mice provide an in vivo animal model for assessing therapeutic efficacy in pre-clinical trials. Moreover, given that ERP studies can be readily performed in humans, assessment of ERP and the changes in oscillation and phase locking may serve as a valuable biomarker for evaluating RTT phenotypes.