Rett syndrome mutation MeCP2 T158A disrupts DNA binding, protein stability and ERP responses.

Abstract

Mutations in the MECP2 gene cause the autism spectrum disorder Rett syndrome (RTT). One of the most common MeCP2 mutations associated with RTT occurs at threonine 158, converting it to methionine (T158M) or alanine (T158A). To understand the role of T158 mutations in the pathogenesis of RTT, we generated knockin mice that recapitulate the MeCP2 T158A mutation. We found a causal role for T158A mutation in the development of RTT-like phenotypes, including developmental regression, motor dysfunction, and learning and memory deficits. These phenotypes resemble those present in Mecp2 null mice and manifest through a reduction in MeCP2 binding to methylated DNA and a decrease in MeCP2 protein stability. The age-dependent development of event-related neuronal responses was disrupted by MeCP2 mutation, suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment of event-related potentials (ERPs) may serve as a biomarker for RTT and treatment evaluation.

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Rett Syndrome Mutation MeCP2 T158A Disrupts DNA Binding,
Protein Stability and ERP Responses
Darren Goffin1, Megan Allen1,5, Le Zhang1,5, Maria Amorim1,5, I-Ting Judy Wang1,5, Arith-
Ruth S. Reyes1, Amy Mercado-Berton2, Caroline Ong4, Sonia Cohen4, Linda Hu4, Julie A.
Blendy2, Gregory C. Carlson3, Steve J. Siegel3, Michael E. Greenberg4, and Zhaolan (Joe)
Zhou1,*
1Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
2Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA
19104
3Department of Psychiatry, University of Pennsylvania School of Medicine, Philadelphia, PA
19104
4Department of Neurobiology, Harvard Medical School, Boston, MA 02115
Abstract
Mutations in the MECP2 gene cause the autism spectrum disorder Rett Syndrome (RTT). One of
the most common mutations associated with RTT occurs at MeCP2 Threonine 158 converting it to
Methionine (T158M) or Alanine (T158A). To understand the role of T158 mutation in the
pathogenesis of RTT, we generated knockin mice recapitulating MeCP2 T158A mutation. Here
we show a causal role for T158A mutation in the development of RTT-like phenotypes including
developmental regression, motor dysfunction, and learning and memory deficits. These
phenotypes resemble those in Mecp2-null mice and manifest through a reduction in MeCP2
binding to methylated DNA and a decrease in MeCP2 protein stability. Importantly, the age-
dependent development of event-related neuronal responses are disrupted by MeCP2 mutation,
suggesting that impaired neuronal circuitry underlies the pathogenesis of RTT and that assessment
of event-related potentials may serve as a biomarker for RTT and treatment evaluation.
INTRODUCTION
RTT is an autism spectrum disorder caused by mutations in the X-linked gene encoding
methyl-CpG binding protein 2 (MeCP2)1. RTT is associated with different types of
mutations within MECP2, including missense, nonsense, deletions and insertions2. Classical
RTT patients, irrespective of the type of mutation, develop normally for the first 6–18
months of age, after which they enter a period of regression characterized by deceleration of
head growth and a loss of acquired motor and language skills. Frequently, patients develop
stereotypic hand wringing, abnormal breathing, seizures and autistic behaviors3. The
*Correspondence: Zhaolan@mail.med.upenn.edu.
5These authors contributed equally to this work.
Author Contributions
D.G. designed and performed EEG/ERP studies, analyzed protein stability, and was involved in most aspects of the project except
generation of mice. M. Allen and I-T.J.W. characterized mouse phenotypes. L.Z. analyzed protein expression and interaction. M.
Amorim analyzed DNA binding and gene expression. A.S.R and C.O. provided technical assistance. S.C. assisted with targeting
construct. L.H. assisted with generation of T158 antibody. A.M. and J.A.B. contributed to behavioral tests. G.C.C. and S.J.S.
contributed to EEG/ERP study. Z.Z. generated the knockin mice with supervision from M.E.G., designed the experiments and
supervised the project. D.G. and Z.Z. wrote the paper.
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Nat Neurosci. ; 15(2): 274–283. doi:10.1038/nn.2997.
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molecular mechanisms through which different types of MECP2 mutations lead to
disruptions in proper brain function are not fully understood.
Mice engineered with different Mecp2 alterations present phenotypes that are both similar
and distinct from those observed in Mecp2-null mice4–11. These similarities and differences
in mouse models suggest that different MECP2 mutations are likely to have both shared and
distinct biochemical and physiological correlates. Intriguingly, reintroduction of MeCP2 into
behaviorally affected Mecp2-null mice is sufficient to rescue RTT-like phenotypes12 and
restoration of MeCP2 function in astrocytes alone significantly improve the developmental
outcome of Mecp2-null mice13, suggesting that RTT is reversible upon restoration of
MeCP2 function. Thus, understanding the mechanisms by which different MeCP2 mutations
lead to RTT may reveal effective strategies tailored to the particular mutation to restore
MeCP2 function.
Mutation of the Threonine 158 (T158) residue, located at the C-terminus of the methyl-CpG
binding domain (MBD) of MeCP2, represents one of the most common mutations observed
in RTT. Approximately 10% of all RTT cases carry a single nucleotide mutation converting
T158 to Methionine (T158M) or in rare cases to Alanine (T158A)2. The T158 residue has
been suggested to play an important structural role in the stabilization of the MBD and the
binding of MeCP2 to methylated DNA14. Whether mutation of T158 leads to MeCP2 gain-
of-function or loss-of-function, however, is not clear.
The onset of RTT symptoms occurs during the establishment and refinement of neural
networks in early postnatal development. Studies in Mecp2-null mice have suggested that
reductions in connectivity between excitatory pyramidal neurons are associated with RTT-
like phenotypes15–17. How reductions in neuronal connectivity lead to the manifestation of
the age-dependent cognitive and behavioral deficits in RTT is currently unknown. Cognitive
dysfunctions are frequently assessed by measuring the neurophysiological responses that
occur during passive processes or during the performance of cognitive, sensory or motor
tasks. Brain activations that occur during these tasks manifest as event-related potentials
(ERPs). Disruptions in ERPs and the oscillations that underlie them are associated with a
number of cognitive disorders such as schizophrenia18,19 and autism20,21. EEG recordings in
Mecp2-null mice22,23 and ERP recordings in RTT patients24,25 also suggest that alterations
in brain activity are associated with behavioral and cognitive deficits. How ERPs are
affected by MeCP2 dysfunction and how changes in EEG and ERPs correlate with the age-
dependent progression of RTT-like symptoms, however, remains to be determined.
Given the high frequency of T158 mutations in RTT and its role in methyl-DNA binding,
we sought to model this mutation in vivo and developed knockin mice containing MeCP2
T158A mutation. We found that these mice recapitulate a number of RTT-like symptoms
observed in Mecp2-null mice including late onset of hypoactivity, poor motor control,
irregular breathing, altered anxiety, impaired learning and memory, and shortened lifespan.
We demonstrated that T158A mutation decreases the binding of MeCP2 to methylated DNA
in vitro and in vivo, and reduces MeCP2 protein stability. Moreover, the amplitude and
latency of ERPs in both MeCP2 T158A and Mecp2-null mice are significantly altered.
Time-frequency analysis of these ERPs revealed that MeCP2 T158A mice failed to show a
developmental increase in event-related power and phase locking in contrast to wild-type
(WT) mice, demonstrating that MeCP2 is required for the development of functional
neuronal circuits. Our studies suggest that stabilization of MeCP2 protein and enhancement
of its affinity for methylated DNA may provide a potential therapeutic approach to treat
patients with MeCP2 T158 mutation. Furthermore, assessment of ERPs may serve as a
biomarker for RTT and the evaluation of therapeutic efficacy in RTT treatment.
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RESULTS
Generation of MeCP2 T158A and loxP knockin mice
Although mutation of MeCP2 T158A occurs at a lower frequency than T158M in RTT
patients, the mechanism through which both mutations impair MeCP2 function is believed
to be the same: the lack of hydroxyl group in alanine and methionine destabilizes the tandem
Asx-ST motif in the MBD and thus reduces MeCP2 affinity for methylated DNA14. Indeed,
patients carrying MeCP2 T158A or T158M mutation are phenotypically similar in the
identity and severity of presented symptoms26,27. To examine the importance of the T158
residue in the functioning of MeCP2, we developed a knockin mouse with T158A mutation
to circumvent the potential steric interference brought about by the larger Methionine
residue. To facilitate screening of properly targeted ES cells, we incorporated a silent
mutation at codon 160 to create a new BstEII restriction site and a floxed Neomycin
expression cassette in intron III of the Mecp2 gene (Supplementary Fig. 1). Given that our
goal was to test a rather subtle mutation on MeCP2 – a single amino acid change of T158A
– we also engineered loxP knockin mice that contain the BstEII restriction site and loxP
insertion but that lack T158A mutation, to rule out the possibility that manipulation of the
Mecp2 locus may affect MeCP2 expression (Supplementary Fig. 1). Sequencing of MeCP2
mRNA extracted from brain tissues of WT, Mecp2T158A/y, and Mecp2loxP/y mice verified
that both Mecp2T158A/y and Mecp2loxP/y knockin mice contained the T to C mutation at
codon 160 for the generation of the BstEII restriction site, whereas only Mecp2T158A/y mice
contain the A to G mutation at codon 158 (Fig. 1a). Furthermore, an MeCP2 T158 site-
specific antibody, recognized MeCP2 protein from WT and Mecp2loxP/y mice, but not
Mecp2T158A/y mice confirming the successful generation of MeCP2 T158A knockin mice
(Fig. 1b).
MeCP2 T158A mice recapitulate RTT-like phenotypes
RTT is characterized by relatively normal development during the first 6–18 months of life,
followed by a period of developmental stagnation leading to motor impairments, breathing
abnormalities, and intellectual disability. We evaluated the presence, development and
progression of RTT-like phenotypes in MeCP2 T158A mice following a previously reported
scoring system12. We found that male Mecp2T158A/y mice present no overt symptoms
during the first 4 weeks of life, but become progressively symptomatic after 5 weeks of age
as indicated by significantly increasing phenotypic score (Fig. 1c). The hindlimb clasping
apparent in Mecp2-null mice is also observed in Mecp2T158A/y mice (Supplementary Fig.
2a). Mecp2T158A/y mice weighed significantly less than their WT littermates starting from 4
weeks of age but then gradually gained weight to WT levels after 8 weeks (Supplementary
Fig. 2b). In contrast, the body weights of Mecp2loxP/y mice are indistinguishable from their
WT littermates (data not shown). Occasionally, we also observed seizure behaviors in
Mecp2T158A/y mice after 5 weeks of age.
Given that MECP2 is an X-linked gene and that the majority of RTT patients are girls with
mosaic MeCP2 expression due to random X-chromosome inactivation, female
Mecp2T158A/+ mice represent a close genetic match to RTT patients. Phenotypic scoring
revealed no apparent symptoms in these mice until 17 weeks of age, after which they
become progressively and increasingly symptomatic (Fig. 1d). A significant increase in
body weight also occurred in female Mecp2T158A/+ mice at the time of symptom
presentation (Supplementary Fig. 2c) similar to that observed in heterozygous Mecp2−/+
female mice12.
An early diagnostic criterion for RTT is a deceleration in head growth often leading to
microcephaly by the first two years of life3,28. We observed a significant reduction in the
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sizes and weights of brains of presymptomatic (P30) and postsymptomatic (P90)
Mecp2T158A/y mice relative to their WT littermates (Fig. 1e), similar to that observed in
Mecp2-null mice4,5. The gross brain anatomy of Mecp2T158A/y mice, however, was
indistinguishable from their WT littermates (Supplementary Fig. 2d). The decreased brain
size and weight in Mecp2-null mice are purported to occur, at least in part, through a
reduction in neuronal soma size4,28. To assess whether soma size is affected in
Mecp2T158A/y mice, we bred MeCP2 T158A mice to those expressing GFP under the
control of the Thy1 promoter29. Focusing on the hippocampal CA1 region, confocal imaging
of GFP-positive pyramidal neurons revealed a significant reduction in soma size in
Mecp2T158A/y mice compared to WT littermates at both P30 and P90 (Fig. 1f).
MeCP2 mutation in boys often leads to infantile lethality3 and male Mecp2-null mice show
shortened life span4,5,8. We find that male Mecp2T158A/y mice die prematurely, with 50%
dying by 16 weeks of age (Fig. 1g), which is approximately 3–4 weeks longer that germline
Mecp2-null mice4,5. No apparent changes in the survival profiles of female Mecp2T158A/+
mice were observed before 6 months of age (data not shown). Importantly, we have not
observed any significant difference in longevity, body weights or brain weights of
Mecp2loxP/y knockin mice, supporting the conclusion that these changes are the result of
T158A mutation (data not shown). Together, these data demonstrate that mice carrying
MeCP2 T158A mutation manifest RTT-like phenotypes.
MeCP2 T158A mice present similar phenotypes to Mecp2-null mice
Given the clinical relevance of MeCP2 T158A mutation, we sought to carry out a side-by-
side comparison of behavioral phenotypes with a well-studied Mecp2-null mouse 5. In light
of the locomotor deficits, aberrant gait and hindlimb clasping observed in these mice
(Supplementary Video 1), we assessed locomotor activity in a home cage environment with
a cohort of age-matched WT, Mecp2T158A/y and Mecp2−/y littermates on the same C57BL/6
background at approximately 9 weeks of age. We found a significant reduction in the
locomotor activity of both Mecp2T158A/y and Mecp2−/y mice compared to WT littermates
(Fig. 2a). However, the reduction in locomotor activity was significant higher in Mecp2−/y
mice compared to Mecp2T158A/y mice (Fig. 2a). We also found a significant reduction in
distance traveled by Mecp2T158A/y mice at 11 but not 3 weeks compared to WT littermates
using the open field assay (Supplementary Fig. 3a). Similarly, locomotor activity is also
significantly reduced in female Mecp2T158A/+ mice at 20 weeks, but not 12 weeks of age
(Supplementary Fig. 3b), consistent with the age-dependent hypoactivity observed with
phenotypic scoring (Fig. 1c,d).
To examine motor coordination and motor learning in these two mouse models, we
performed the accelerating rotarod test. WT mice show an increase in the latency to fall over
the course of 4 trials per day and over 4 consecutive days, indicating improvements in motor
coordination and learning over time (Fig. 2b). However, both Mecp2T158A/y and Mecp2−/y
mice spent significantly less time on the rotarod compared to WT littermates and failed to
improve significantly over the course of 4 days suggestive of deficits in motor coordination
and motor learning (Fig. 2b). The latency to fall from the rotarod was moderately but
significantly decreased in Mecp2−/y mice compared Mecp2T158A/y mice (Fig. 2b). We
conclude that MeCP2 T158A mutation impairs motor function similar to that seen in RTT
patients and to a lesser degree than Mecp2-null mice.
RTT patients experience anxiety episodes, particularly in response to distressing events3.
The role of MeCP2 in anxiety in mice, however, is less clear: Mecp2-null mice and mice
with a 50% reduction in MeCP2 protein expression both show a decreased anxiety
phenotype8, whereas those containing an early-truncating mutation show increased anxiety6.
Using the elevated zero maze paradigm, we found that Mecp2T158A/y and Mecp2−/y mice
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spend significantly less time in the closed arm and significantly more time in the open arm
compared to their WT littermates (Fig. 2c). This suggests that T158A mice show reduced
anxiety similar to those observations in Mecp2-null mice and mice with decreased MeCP2
protein expression.
Because RTT is the primary cause of intellectual disability in females, we examined whether
MeCP2 mutant mice have learning and memory deficits using contextual and cued fear
conditioning paradigms. Mice were trained to associate a context (testing box) and a cue
(auditory tone) with a coterminating foot shock. Animals typically freeze in response to the
shock. We found no differences in freezing behaviors in WT and Mecp2T158A/y littermates
prior to or immediately following shocks, suggesting no differences in pain sensitivity.
However, Mecp2−/y mice exhibit significantly increased freezing even prior to the shock
(Supplementary Fig. 3c). This is likely due to their decreased locomotor activity and
akinesia, thus confounding interpretation of the fear-conditioning test. These mice were
therefore excluded from this study. 24 hours after training, Mecp2T158A/y mice demonstrate
significantly less context- and cue-dependent freezing compared to their WT littermates,
suggesting deficits in learning and memory (Fig. 2d).
Together, these data demonstrate that MeCP2 T158A knockin mice present phenotypes
similar to those observed in Mecp2-null mice but to a lesser extent overall. We therefore
infer that T158A is a partial loss-of-function mutation.
Decreased MeCP2 protein stability in MeCP2 T158A mice
Alterations in MeCP2 protein levels, such as a 50% reduction or a two-fold increase, leads
to the progressive development of neurological deficits in mice, albeit at much later time
points than those observed in Mecp2-null mice7,9,10. We therefore examined whether
T158A mutation alters the expression of MeCP2 protein during development. Quantitative
Western blotting on whole-cell lysates from the brains of male Mecp2T158A/y mice revealed
that MeCP2 protein expression is significantly decreased at P2, P30 and P90 compared to
their WT littermates (Fig. 3a). The down-regulation of MeCP2 expression is significantly
higher at P90, when Mecp2T158A/y mice present overt RTT-like phenotypes, than at either
P2 or P30, when symptoms are not present (Fig. 1c). Importantly, MeCP2 protein levels
were not affected in Mecp2loxP/y mice indicating that the down-regulation of MeCP2 is due
to the presence of the T158A mutation rather than genomic modification (Fig. 1b). These
changes in MeCP2 expression are likely to be independent of gene transcription since
quantitative RT-PCR found no significant differences in the level of MeCP2 mRNA
expression between Mecp2T158A/y and WT mice at P0, P30 or P90 (Supplementary Fig. 4).
Despite RTT being primarily a neurological disorder3, MeCP2 is ubiquitously expressed in
all mammalian tissues30. Indeed, we found that MeCP2 protein levels are decreased to a
similar extent in kidney, liver, lung and heart in Mecp2T158A/y mice (Fig. 3b).
MeCP2 protein levels are also significantly decreased in the brains of female Mecp2T158A/+
mice compared to their WT littermates (Fig. 3c). The magnitude of MeCP2 down-regulation
in females is less than that observed in male Mecp2T158A/y mice and is likely due to the
mosaic expression of MeCP2 T158A in heterozygous Mecp2T158A/+ mice. To investigate
whether mutation at T158 disrupts MeCP2 protein expression in human RTT patients, we
obtained fibroblast cultures derived from a female RTT patient with T158M mutation and an
age-matched female control. We found that MeCP2 expression is also significantly down-
regulated by about 40% in cells with MeCP2 T158M mutation compared to control cells
(Fig. 3c). These data demonstrate that T158 mutation, to either A or M, triggers the down-
regulation of MeCP2 protein expression in both humans and mice, indicating that the
reduction in MeCP2 protein expression may be a contributing factor to RTT.
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