genes associated with Rett syndrome
studies of Rett syndrome
Vonhoff, F., Williams, A., Ryglewski, S. and Duch, C. (2012). Drosophila as a model for MECP2 gain of function in neurons. PLoS One 7: e31835. PubMed ID: 22363746
The MECP2 protein contains at least five distinct functional domains (NTD, ID, MBD, TRD, and CTDα) which either bind DNA autonomously or regulate MBD (methyl-CpG binding) function. Historically, MECP2 is viewed as a transcriptional repressor that localizes to chromatin by binding to CpG dinucleotides to regulate gene expression through interactions with histone deacetylases and other cofactors. However, MECP2 can also activate transcription, associate also with un-methylated DNA, has chromatin compaction and RNA splicing functions, and several MECP2 interacting proteins are known. Therefore, multiple MECP2 functions might be mediated by interactions with diverse co-factors and by binding to both methylated and non-methylated DNA, consistent with the wide range of phenotypes observed in patients with RTT (Vonhoff, 2012).
Although Mecp2 mouse models recapitulate RTT phenotypes and provide valuable mechanistic insight into neuronal defects caused by Mecp2 mis-regulation, such as axon targeting, synaptic, and dendritic defects, the identification of MECP2 functions and target genes in this system is time intensive and complicated (Vonhoff, 2012).
The Drosophila genetic model system is increasingly being used as a tool to analyze specific genetic and cellular aspects of neurodevelopmental disorders. Short generation times, high fecundity, high throughput screening techniques, facile genetic tools, and relatively low costs have provided valuable mechanistic insights into inherited diseases like Fragile-X, Angelman syndrome, and neurofibromatosis. However, despite considerable conservation in fundamental cell biological pathways, the Drosophila genome encodes only about 75 percent of human disease associated genes, and mecp2 is not among these genes. Therefore, Drosophila can not be used to study the pathophysiology resulting from loss of endogenous mecp2. Instead, the Drosophila model relies on heterologous expression of human MECP2 allele and consequential gain of MECP2 function. Although classic Rett is mostly caused by loss-of-function of MECP2, this is likely not an artificial approach since in humans and in mouse models increased levels of MECP2 also cause disease. Genetic and behavioral proof of principle for the use of the Drosophila model to address MECP2 gain-of-function has been provided earlier. In MECP2 transgenic flies, the MECP2 protein associates with chromatin, interacts with homologs of known human MECP2 interactors, modifies the transcription of multiple genes, and is phosphorylated at serine 423, as in mammals. Most importantly, reported consequences are developmental dysfunctions and motor defects, suggesting parallels with RTT phenotypes. However, previous work on MECP2 in the Drosophila CNS has not tested for cellular phenotypes resulting from MECP2 over-expression in neurons, although mouse models demonstrate that disease phenotypes result from Mecp2 mis-regulation in postmitotic neurons. This study presents the first data on cellular defects as resulting from MECP2 gain-of-function in developing postmitotic Drosophila neurons (Vonhoff, 2012).
It was demonstrated that heterologous expression of human MECP2 in Drosophila motoneurons does not affect axonal pathfinding, dendritic territory boundaries, or the neurons' electrophysiology, but it causes a significant reduction in new dendritic branch formation during development. Similarly, in the mouse model Mecp2 mis-regulation results in pyramidal neuron dendritic defects. This study provides four lines of evidence that dendritic defects in Drosophila motoneurons are caused by specific cellular functions that result from MECP2 gain-of-function, and not from non-specific over-expression or sequestering effects. First, MECP2 protein specifically localizes to the nucleus of Drosophila neurons, so that interactions of MECP2 with molecules in the cytoplasm are unlikely. Second, targeted expression of MECP2 in Drosophila motoneurons causes significant dendritic branching defects but does not affect firing responses to current injections, voltage activated potassium current, or firing frequencies during motor behavior, indicating normal regulation of electrophysiological properties. Although it was earlier demonstrated that Drosophila motoneuron dendritic structure may undergo compensatory changes in response to altered neuronal activity, and a link between motoneuron activity and dendritic growth has clearly been established, any evidence for homeostatic changes in motoneuron excitability in response to developmental defects in dendritic structure was not found in this study. Third, MECP2-induced dendritic defects require intact MBD function of the MECP2 protein because dendritic architecture is not affected following expression of MECP2 alleles with non-functional MBD. This indicates that human MECP2 exerts specific action in Drosophila neurons via chromatin remodeling. Fourth, MECP2-induced dendritic phenotypes can be ameliorated by reducing the dose of osa, a member of the SWI/SNF complex. This genetic interaction is consistent with the hypothesis that human MECP2 may exert specific action in Drosophila motoneurons via chromatin remodeling. It also indicates that MECP2 gain-of-function activates specific cell signaling pathways in Drosophila, and may not cause unspecific over-expression effects. Therefore, the study concludes that Drosophila neurons can serve as a valuable model system to identify some cellular mechanisms by which MECP2 gain-of-function affects neuronal development (Vonhoff, 2012).
It was shown that dendritic defects, as induced by heterologous expression of MECP2 in Drosophila motoneurons, require an intact MBD domain, because expression of MECP2 with a point mutated or truncated MBD domain does not affect dendritic structure. However, each UAS-MECP2 transgene is likely inserted into a unique site in the Drosophila genome, and therefore, the possibility that different UAS-MECP2 transgenes may yield different expression levels or other genetic interactions can not be excluded. The finding that dendritic defects as caused by the expression of full length UAS-MECP2, but not by the expression of UAS-MECP2 transgenes with defective MBD domain, are a result of the unique insertion sites of the UAS-MECP2 constructs into the Drosophila genome, is unlikely for two reasons. First, both UAS-transgenes with defective MBD do not cause dendritic defects. Second, similar dendritic defects are observed following the expression of the full length MECP2 construct inserted in the second or in the third chromosome (Vonhoff, 2012).
MBD domains recognize two key mechanisms of chromatin regulation in eukaryotes, C5 methylations of DNA at cytosines and post-translational histone modifications. Although the existence of DNA methylation was demonstrated earlier in the fly genome, methylation levels are several orders of magnitude lower than in mammals. The fly genome contains only one methylated DNA binding protein (dMBD2/3) and only one DNA methyltransferase (dDNMT2), which shows highest affinity to t-RNA. Consequently, Drosophila DNA is only sparsely methylated, so that MECP2 interactions with modified histone tails seem the more parsimonious scenario. This is consistent with the finding in this study that MECP2-dependent dendritic defects are suppressed in an osa heterozygous mutant background. Osa is a member of the SWI/SNF complex (human homolog is BAF250), a class of trithorax proteins involved in chromatin remodeling which are highly conserved between flies and humans. This indicates that human MECP2 may exert specific action in Drosophila motoneurons via chromatin remodeling. In fact, it was previously suggested that MECP2 associates with human Brahma, a catalytic component of the SWI/SNF chromatin remodeling complex to regulate gene repression, although this finding is disputed. Nevertheless, the Drosophila system provides some unique advantages to study possible interactions of MECP2 and members of the SWI/SNF chromatin remodeling complex with genetic tools (Vonhoff, 2012).
The finding in this study that flies with MECP2 over-expression in motoneurons show normal take-off likelihoods as well as normal motoneuron firing and wing beat frequencies but can not sustain flight is in accord with specific MECP2 effects on dendrite development in otherwise normal motoneurons. In Drosophila, take-off can be mediated by the escape response neural circuitry. This circuitry bypasses flight motoneuron dendrites by synapsing directly on MN5 axon, but it relies on normal synaptic transmission and flight motoneuron physiology. Therefore, initial take-off and initial motoneuron firing are not affected by dendritic defects. In Drosophila motoneuron, firing frequencies are directly proportional to wing beat frequency, and thus, these are also not affected. By contrast, flight can not be sustained because the significantly reduced dendritic surface likely reduces the excitatory synaptic drive to motoneuron dendrites that is necessary to stay in flight. Therefore, flies with MECP2-caused motoneuron dendritic defects show a 30- to 60-fold reduction in flight duration. This behavioral phenotype is obvious, and thus, useful for screening. Although the quantification of flight durations and take-off likelihoods does not allow for rapid genetic screening, high throughput screening can easily be developed based on the observed reduction in flight duration by more than 30-fold. Moreover, high throughput assays which utilize Drosophila behavior for rapid screening have been developed by others. Such approaches may help the future identification of candidate MECP2 targets or interactors (Vonhoff, 2012).
Identification of genetic interactors and modifiers of MECP2 function in neurons will be imperative toward developing future treatment strategies. MECP2 itself is not a promising treatment target because the X-linked MECP2 gene is mosaic regulated in the human brain. Furthermore, both loss and gain of function cause disease phenotypes. The sparse methylation landscape in Drosophila may offer unique promise of identifying non-methylated DNA-dependent functions of MECP2 in neurons, the cell type that is most relevant to Rett syndrome. Since known binding partners of MECP2 are conserved in flies (e.g. YB-1, mSin3A etc.), it seems plausible that gain-of-function of human MECP2 may affect neural development via a cellular machinery that is partly conserved between flies and humans (Vonhoff, 2012).
MECP2-induced dendritic phenotypes in flight motoneurons cause a severe motor behavioral phenotype in that flight bout duration is reduced approximately 30- to 60-fold. Rapid screening assays for Drosophila behavioral phenotypes are available. Combined with the fast generation times, high fecundity and facile genetic tools available in Drosophila, this offers a powerful tool to identify molecules that interact with MECP2 in neurons. However, potential MECP2 candidate target genes or genetic modifiers of MECP2 function that can readily be identified in the Drosophila system will then have to be further evaluated in the existing mouse models of RTT (Vonhoff, 2012).
Hess-Homeier, D.L., Fan, C.Y., Gupta, T., Chiang, A.S and Certel, S.J. (2014). Astrocyte-specific regulation of hMeCP2 expression in Drosophila. Biol Open 3: 1011-1009. PubMed ID: 25305037
This study reports on the spatial and temporal post-transcriptional regulation of human MeCP2 levels in Drosophila astrocytes and on circuit functional changes due to glial expression. Until relatively recently, astrocytes, along with other glia cell types, were believed to be structural cells that function to hold neurons together. It is now appreciated that astrocytes serve many functions, including developmental roles during synaptogenesis, maintenance of the extracellular environment and stabilization of cell–cell communications in the CNS. In addition, astrocytes are increasingly recognized as active partners in synaptic function including regulating basal synaptic transmission and synaptic efficacy leading to the proposal that normal brain output arises from the coordinated activity of a network comprising both neurons and glia. A number of earlier studies highlight the role of astrocytes in the modulation of circuit concerned with sleep and sleep-related rhythmogenesis. Glial cells regulate slow oscillations, a specific thalamocortical activity that characterizes non-REM sleep, and sleep-associated behaviors (Hess-Homeier, 2014).
In this study, it was found that hMeCP2 expression in Drosophila astrocytes causes a significant decrease in sleep with the reduction occurring at a specific time point, immediately after the day:evening transition. How potential cell or non-cell autonomous morphological or functional defects cause the distinct deficit in sleep in the Drosophila males expressing hMeCP2 in astrocytes is presently unknown. However, a recent study shows that dendritic structure defects in motor neurons are caused by MeCP2 expression and as the neurons and circuits that regulate sleep duration, initiation, and maintenance are well studied in Drosophila, further analysis of these described sleep deficits should prove fruitful. Results from the sleep paradigm in this study can also be viewed as the endpoint behavioral representation of synaptic connectivity and dysfunction of circuits in general, which is a fundamental theme in neurodevelopmental syndromes including RTT (Hess-Homeier, 2014).
Although MeCP2 is ubiquitously transcribed, the expression of distinct MeCP2 isoforms is developmentally regulated and heterogeneous in neuronal subpopulations and may be impacted by DNA methylation patterns at MeCP2 regulatory elements may impact the differential expression of MeCP2/MeCP2 isoforms in brain regions. A series of separate studies suggest a role of RNAi-induced down-regulation of MeCP2 expression. In this study, human MeCP2e2 and RTT R106W transcript levels were found to significantly decrease in astrocyte subsets, while expression of the MeCP2Δ166 allele was not altered. The region absent in MeCP2Δ166 contains the MBD domain, a nuclear localizing signal, and a region designated HMG1, due to the amino acid composition similarity to high mobility group (HMG) proteins. What types of evolutionarily conserved cis-regulatory elements are located in this area of the hMeCP2-coding region? Several studies have identified microRNA target sites, AU-rich elements, and G-quadruplexes within the transcribed regions of hMeCP2. Many of these DNA regions influence DNA replication, transcription, and epigenetic mechanisms. For example expression of the microRNA, miR-483-5p, decreases MeCP2 mRNA levels through the human-specific binding site in the MeCP2 long 3′ UTR. At this point, results from this study suggest that an endogenous factor expressed in Drosophila glia targets a regulatory component or components located within the first 498 base pair region of hMeCP2. As wildtype MeCP2 levels in glial cells are essential for proper development and maturation of the brain, identifying cell-type specific mechanisms that activate or repress normal levels to achieve a controlled balance of MeCP2 expression would be useful in therapeutic considerations (Hess-Homeier, 2014).
Kim, S., Chahrour, M., Ben-Shachar, S. and Lim, J. (2013). Ube3a/E6AP is involved in a subset of MeCP2 functions. Biochem Biophys Res Commun 437: 67-73. PubMed ID: 23791832
Next, it was shown that MeCP2 and E6AP do not regulate each other’s expression. Furthermore, E6AP does not affect expression levels of some of MeCP2-associated proteins in mice in vivo, which are crucial for the regulation of MeCP2-mediated target gene expression. There is still the possibility of novel unidentified MeCP2 corepressors regulated by E6AP, however. Rather, MeCP2 and E6AP can physically associate with each other and modulate expression of some common target genesin HEK293T cells. MeCP2 and E6AP may co-regulate gene expression by acting directly at the promoter and/or regulatory elements of their target genes. Alternatively, E6AP may affect MeCP2 function in organization of chromatin structures and gene expression changes might be secondary to the altered chromatin structures. Although the gene expression changes may not directly explain specific behavioral and neurological phenotypes, the molecular data are in agreement with genetic interaction data in that loss of (or decrease in) Ube3a expression suppresses MECP2 overexpression-induced phenotypes. This co-regulation of shared molecular targets between MeCP2 and E6AP may explain the similarities in disease features of RTT and AS (Kim, 2013).
Cukier, H.N., Perez, A.M., Collins, A.L., Zhou, Z., Zoghbi, H.Y. and Botas, J. (2008). Genetic modifiers of MeCP2 function in Drosophila. PLoS Genet 4: e1000179. PubMed ID: 18773074
A commonly accepted model of MeCP2 function postulates that MeCP2 binds to methylated CpG islands in promoters where it recruits histone deacetylases and other co-repressors to silence gene transcription. However, accumulating evidence suggests that this may be too simple a view of MeCP2 function. For example, MeCP2 binds to unmethylated DNA with affinity only 3 times weaker than to methylated DNA, and MeCP2 also binds or requires AT sequences for binding. Moreover, MeCP2 interacts with both methylated and unmethylated chromatin and leads to alterations in the secondary structure of both types of chromatin. In addition, large-scale mapping of MeCP2 binding sites in chromosomal regions containing candidate MeCP2 target genes reveals that: 1) MeCP2 is absent from highly methylated promoters, 2) only ∼6% of MeCP2 binding sites are in CpG islands, and 3) many MeCP2-bound promoters are actively expressed. Furthermore, gene expression patterns in mice that either lack or overexpress MeCP2 suggest that many genes are activated by MeCP2. This study shows that the methyl-CpG-binding domain is not necessary for association of the MeCP2 protein with chromatin in polytene chromosomes, nor is it required to produce an eye phenotype in Drosophila. In this context, it is interesting to note that unlike mammals, bacteria, plants, and other insects, the levels of DNA methylation are very low in Drosophila. Together, these data suggest that MeCP2 function may be more complex than previously thought. MeCP2 may regulate both methylated and unmethylated target genes in vivo, possibly as part of large protein complex(es) of chromatin remodelling proteins regulating gene expression both positively and negatively (Cukier, 2008).
Using a candidate gene approach, this study provides proof of principle that modulating the activity of modifier genes can amend MeCP2 function in vivo. Among this group of genes is the kinase trc, a member of the NDR (nuclear Dbf-related) family. Alterations in the phosphorylation of MeCP2 in trc mutants are not detected. However, there is evidence that both trc and one of its mammalian homologs, NDR2, are involved in dendritic formation, a feature also found to be affected by mutations in MeCP2. Also, modification of the MeCP2 phenotype by the E3 ligase UBE3A target pbl is noteworthy due to the similarities between Rett and Angelman syndromes. Patients with Angelman-like features have been identified with MeCP2 mutations and, while controversial, some studies show a decrease of UBE3A in Rett patients and Mecp2 null mice. The data presented in this study suggests that shared pathways may be involved in Rett and Angelman syndromes (Cukier, 2008).
Misregulation of neuronal genes caused by alterations in MeCP2 activity is thought to cause Rett and Rett-like syndromes. One possible avenue for therapy is to identify the MeCP2 target genes misregulated during disease and to restore their normal regulation. This approach may prove impractical if the targets are numerous or difficult to identify due to subtle variations in expression levels in response to MeCP2 activity. A possible future treatment based on gene therapy to restore normal levels of MeCP2 also seems improbable. The nervous systems of Rett patients are mosaic due to random X-chromosome inactivation causing some neurons expressing the normal while others expressing the mutant allele. Therefore, in the context of neurons expressing the wild-type allele, gene therapy is not possible because doubling of MeCP2 also leads to disease. An alternative approach is to identify molecular mechanisms capable of compensating for the misregulation of target genes caused by MeCP2 altered levels. This study provides support for the validity of this approach and identifies specific chromatin remodeling genes of the Pc-G and Trx-G (i.e., Asx, corto, osa, and Scm) that suppress the phenotypes caused by MeCP2 overexpression in Drosophila. Interestingly, both in Drosophila and mammals, mutations in genes of either Pc-G or Trx-G also suppress the body patterning abnormalities caused by mutations in members of the other group (Cukier, 2008).
In conclusion, human MeCP2 protein expressed in Drosophila maintains important features observed in mammals such as phosphorylation and association with the chromatin. The novel modifiers identified in this model system point to potential therapeutic targets that might be more amenable to manipulation than MeCP2, and thus they provide new opportunities to develop therapies for Rett syndrome and related neurological disorders (Cukier, 2008).
Gatto, C.L. and Broadie, K. (2011). Drosophila modeling of heritable neurodevelopmental disorders. Curr Opin Neurobiol 21: 834-841. PubMed ID: 21596554
Discussion on role of glia in neurological diseases including Rett syndrome
Discussion on epigenetic
changes in neurological diseases including Rett syndrome
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Date revised: 20 June 2015
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