Our results indicate that RTT-like symptoms, which are caused normally by germline mutations in Mecp2, can be recapitulated if MeCP2 is lost during the postnatal stage that coincides with the onset period of classic RTT (late juvenile stage). The immediate onset of symptoms in males, as soon as MeCP2 is mutated and the severity of symptoms, including death, indicates that MeCP2 function is critical at this juncture. Interestingly, although MeCP2 loss in heterozygous female mice was induced by tamoxifen at a similar postnatal stage as in males, the onset of symptoms was delayed until they were 20-week-old, similar to the onset of symptoms in female mice with germline mutations in Mecp2. Furthermore, the type of symptoms, and the kinetics of symptom progression and stabilization were similar to heterozygous female mice with germline mutation in Mecp2. These data further support a critical role for MeCP2 at the specific postnatal time window where RTT symptoms normally appear in males and females and further indicate that the absence of MeCP2 at earlier stages is not a prerequisite for manifestation of a severe RTT-like phenotype at later stages. Remarkably, a direct comparison of the loss of MeCP2 at late juvenile and adult stages showed parallel onset and progression of RTT symptoms, including the rate of lethality, and suggested an equal and critical requirement for MeCP2 function at these two different developmental stages.
Different mechanisms could potentially cause similar RTT-like symptoms at late juvenile and adult stages or upon the loss of MeCP2 in germ cells and postnatal stages. However, our detailed analysis of the brains that lost MeCP2 at postnatal stages suggests that although the state of brain maturation is different at late juvenile and adult stages, the loss of MeCP2 mediated similar brain abnormalities (). The brain abnormalities caused by the loss of MeCP2 at these postnatal stages are comparable in nature to the abnormalities caused by germline mutations in MeCP2 however they are clearly more robust and unequivocal. These results are surprising because manifestation of similar brain phenotypes upon loss of MeCP2 in adult brain requires regression from a more mature state of the brain, as opposed to developmental stagnation, which is thought to occur in RTT patients and mouse models with germline mutation in mecp2
. Indeed, our analysis shows that while the reduced brain size upon the loss of MeCP2 at a late juvenile stage was due partly to developmental stagnation and partly to shrinkage, at the adult stage, a time at which the brain already reached its full size, the reduced brain size was mediated solely by shrinkage of the brain (). The increase in neuronal cell packing, which is reciprocal to the reduced brain size, suggests that shrinkage of the brain is likely not due to neuronal degeneration. Interestingly, the loss of MeCP2 in heterozygous female mice elicited shrinkage of their brain similar to hemizygous male mice, suggesting that even mosaic depletion of MeCP2 in 50% of the cells in the brain is sufficient to affect the brain globally. Our detailed analysis of the pyramidal neurons in the hippocampus of symptomatic mice that lost MeCP2 either at late juvenile or adult stage clearly shows that dendritic complexity was significantly reduced, mainly due to retraction of dendritic arbors from a more advanced state of neuronal maturation. Our analysis further shows that dendritic spine density is dramatically reduced, suggesting that the loss of MeCP2 in the maturing or mature brain mediates alterations in synaptic structural plasticity. It is likely that in human RTT patients and RTT mouse models, in addition to developmental stagnation, shrinking of the maturing brain and retraction of dendritic arbor structures also take place. This scenario could explain the regression in RTT girls which manifests in loss of the already acquired developmental milestones, such as speech and motor coordination, which may not be simply a consequence of halting developmental processes, but rather, a result of actual regression of fundamental anatomical and functional neuronal features in the brain. Previously, it was shown that RTT symptoms can be reversed upon global reactivation of MeCP2 (Guy et al., 2007
) and attenuated by reactivation of MeCP2 in glia (Lioy et al., 2011
) (). Our present studies, which demonstrate that RTT-like brain abnormalities could be elicited similarly in normal adult brain, provide further support for the idea that the function of MeCP2 in maintaining mature neuronal networks maybe dynamic and that RTT has the potential to be fully reversed.
Schematic model for the function of MeCP2 during critical stage of postnatal brain development and in the mature brain
Importantly, our structural analysis of the hippocampal astrocytes shows that, like neurons, they acquired altered morphology upon the loss of MeCP2 (). Given that astrocytes not only provide metabolic support to neurons, but are in fact an integral part of synapses, with their processes ensheathing synapses (Bolton and Eroglu, 2009
), it is not surprising that such alteration in complexity of astrocytic processes could negatively affect the structure/function of the entire neuronal networks in the RTT brain.
Interestingly, our analysis of synaptic protein expression showed that, of the array of proteins analyzed, few were unaltered, more were robustly down regulated, and none were up regulated. The selective rather than global down-regulation of synaptic protein expression may indicate that MeCP2 regulates specific synaptic proteins, directly or indirectly, to up-regulate their expression in the normal maturing or mature brain (). Importantly, none of the genes whose protein expression was altered showed significant changes in their mRNA expression, suggesting that either MeCP2 directly regulates these genes post-transcriptionally, or indirectly by as yet unidentified primary target genes. These data could also explain why only subtle changes were mostly observed in mRNA levels in brains of conventional RTT mouse models (Tudor et al., 2002
, Chahrour et al., 2008
). Recent studies show aberrant expression of microRNA genes in a mouse model of RTT (Urdinguio et al., 2010
, Wu et al., 2010
). It is likely that specific microRNAs bind to the mRNAs of these specific synaptic proteins and inhibit their translation. Among the synaptic proteins that were down regulated, we identified pre- and post-synaptic proteins, both inhibitory and excitatory, supporting previous studies demonstrating that specific loss of MeCP2 in excitatory or inhibitory neurons could initiate RTT-like phenotypes (Chen et al., 2001
, Chao et al., 2010
). In normal brain, dendritic arbor growth is highly dynamic, and accumulating evidence suggests that synaptic strength and branch stability are concurrent (Cline and Haas, 2008
). Interestingly, several synaptic proteins, which were dramatically reduced upon postnatal loss of MeCP2 such as CaMKIIα, CaMKIIβ, AMPA receptors (GluR2/3 subunits), and NMDAR2A are involved in excitatory synapse maturation as well as in increasing and stabilizing dendritic arbor structures (Fink et al., 2003
, Haas et al., 2006
). It is likely that the reduction in the levels of these synaptic proteins upon loss of MeCP2 mediates the retraction of dendritic arbors as well as affecting synaptic function. However, we cannot exclude the possibility that the loss of dendritic arbors and synaptic contacts may trigger the degradation of proteins localized within these compartments. The effect of MeCP2 loss on the level of specific synaptic proteins in classic RTT mouse models remains ambiguous and partially controversial, however, several synaptic proteins that we found down regulated upon postnatal loss of MeCP2 are also down regulated in RTT mice with germline mutations in Mecp2
. Examples are, NMDAR2A (Asaka et al., 2006
) and Vglut1 (Chao et al., 2007
, Lioy et al., 2011
), which we also recently showed reduced in a RTT mouse model and rescued by reactivation of MeCP2 in astrocytes (Lioy et al., 2011
). Similarly, GABAA
receptor subunits are down regulated in classic RTT (Samaco et al., 2005
, Medrihan et al., 2008
) and our data showed that GABAB
receptor subunit (GABABR2) is also down regulated as it is in postmortem brain of autistic patients (Fatemi et al., 2009
). It is likely that reduced levels of both ionotropic and metabotropic GABA receptor subunits are also involved in manifestation of RTT symptoms. The reduction in the levels of synaptic proteins together with the reduction in dendritic complexity and spine density support the view that MeCP2 is required during late stages of brain maturation to establish and maintain mature neuronal circuitry throughout life (). Although studies using mouse models with germline mutations in MeCP2 helped to advance our knowledge about RTT development, a mouse model presented here, which allows to induce loss of MeCP2 at desired timing in otherwise healthy brain, provides a powerful tool for analyzing the direct effect of MeCP2 loss on brain structure/function. Further identification of key synaptic proteins and the mechanism, by which MeCP2 regulates these proteins, could provide important insights into the molecular pathways underlying the manifestation of brain abnormalities and identify new potential targets for therapeutic intervention of RTT.