Loss of function MECP2 mutations cause RTT in females, and duplication of the MECP2 locus leads to a progressive neurological syndrome in males. In this study we reveal how loss or doubling of MeCP2 levels impact neuronal function. First, we demonstrate that MeCP2 function is critical at a single neuron level. Second, we provide direct evidence that MeCP2 levels are critical for synapse formation in autaptic neurons and in vivo. Third, we uncover altered synapse number as the earliest morphological abnormality observed in mouse models of RTT and MECP2 duplication syndrome. Our discovery that the in vivo changes in synapse numbers appear early in postnatal development but disappear over time, suggests that homeostatic compensatory changes occur in response to the early perturbation of synapse numbers. This finding highlights the importance of probing the pathogenesis of Rett syndrome and MECP2 duplication disorders during the “pre-symptomatic” stage.
Synaptogenesis is a critical neuronal developmental process in the first two weeks of postnatal development in vitro
(Rao et al., 1998
) and in vivo
(Harris et al., 1992
). Our findings that glutamatergic synapses are altered in autaptic neurons and in vivo
during the first two weeks of development indicate that MeCP2 regulates genes required for the initial formation of synaptic contacts. Whether MeCP2 also functions in regulating the maintenance of these synapses in association with neuronal activity remains to be elucidated. The finding that MeCP2 plays a critical function in regulating neuronal developmental processes during the first two postnatal weeks is consistent with recent postnatal restoration studies. These studies showed that neurological impairments associated with loss of MeCP2 can be reversed by postnatal restoration of MeCP2 levels (Giacometti et al., 2007
; Guy et al., 2007
). Interestingly, however, Giacometti et al reported that MeCP2 restoration between one to two weeks of age gave the best rescue (Giacometti et al., 2007
), which is consistent with our finding that MeCP2 plays a critical role in regulating early neuronal development.
We find that alterations of synapse numbers occur prior to appreciable changes in dendritic complexity of hippocampal pyramidal neurons. This is consistent with neuropathological studies from postmortem Rett brains that did not find significant differences in dendrites of hippocampal CA1 pyramidal neurons (Armstrong et al., 1995
) but found reduced dendritic arborization only in the pyramidal neurons of layer III and V in frontal, motor and inferior temporal regions (Armstrong et al., 1995
; Belichenko et al., 1997
). In another study using rat hippocampal slice cultures, simplification of the dendritic complexity in the hippocampus was reported when using either shRNA-induced knockdown of MeCP2 or knockdown of MeCP2 concomitant with overexpression of wild-type MeCP2 (Zhou et al., 2006
). These approaches lead to rapid postnatal changes in MeCP2 levels, raising the possibility that the acute changes in MeCP2 levels might contribute to the observed dendritic abnormalities. Recently, a study by Guy et al demonstrated that rapid restoration of MeCP2 function is detrimental to mice, highlighting the fact that neurons are very sensitive to acute changes in MeCP2 levels (Guy et al., 2007
In examining mass cultured hippocampal neurons from Mecp2Null/y
, Nelson and colleagues used synaptophysin-1 (Syn1) as a synaptic marker and found no significant changes in synapse density (Nelson et al., 2006
). Because Syn1 marks the synapses in both excitatory and inhibitory neurons, their study does not address whether the number of only excitatory glutamatergic synapses are altered. We used the synaptic markers VGLUT1 and PSD95, which are specific to glutamatergic synapses, in order to specifically examine excitatory glutamatergic synapse numbers. Importantly, we observed correlated reduction in evoked EPSC response, RRP charge, mEPSC frequency, and glutamatergic synaptic densities with loss of MeCP2. Our results are consistent with the general view that mEPSC events are derived from the same quanta composing the RRP and that these parameters correlate with synaptic densities (Groemer and Klingauf, 2007
Although the functional analysis in the autaptic neuron showed no changes in release probability, short-term plasticity or postsynaptic strength, we cannot exclude the possibility that changes in these parameters occur during development in vivo
and contribute to pathogenesis. Release probability and postsynaptic strength in neuronal networks can change in response to perturbations in excitatory synaptic strength (Davis, 2006
; Turrigiano and Nelson, 2004
). Such compensatory changes may be responsible for the observed changes in short-term plasticity in hippocampal slices from MeCP2 deficient mice that showed paired-pulse facilitation, an indicator of decreased presynaptic release probability, occurred only in symptomatic six to ten week old mice, but not in presymptomatic mice (Asaka et al., 2006
). Asaka and colleagues’ discovery that presynaptic release properties appear unaltered in slices from presymptomatic Mecp2
null mice is consistent with our data in the autapse and support our conclusion that this is not an initial contributor to disease pathophysiology.
In sum, we provide direct evidence that MeCP2 levels are central and critical for synapse formation, that MeCP2 plays an important role at the level of a single neuron, and evidence for compensatory effects in response to the alterations in synapse number in vivo
. There are two additional clinically relevant conclusions that derive from the findings in this study. First, both a decrease and an increase in synaptic number are associated with neurological disorders that share several features including autism spectrum phenotypes. Second, given the emerging evidence that several synaptic proteins are implicated in autism (Durand et al., 2007
; Feng et al., 2006
; Jamain et al., 2003
; Zoghbi, 2003
), it is possible that the MeCP2 targets regulating synapse numbers may be involved in autism pathogenesis. Altogether, these findings provide an important framework for investigating the pathogenesis of MECP2
and autism spectrum disorders and a new avenue for exploring potential therapeutic interventions.