The discovery that mutations in MECP2 are responsible for most cases of RTT has led researchers to search for transcriptional targets and misregulated expression of genes. A profile of aberrantly expressed genes or classes of genes has slowly emerged, and subsequent downstream functional analyses of these genes are shedding light on the impact that their abnormal expression has on the pathology of RTT. Various mRNA microarray or differential display studies in human postmortem tissue , Mecp2-mutant mice [9, 19–22, 30], patient fibroblast , lymphoblastoid [16, 18] and lymphocyte cells , and neuroblastoma (SH-SY5Y) cells in which MECP2 has been knocked down , have revealed that MeCP2 transcriptionally controls a wide range of functionally distinct genes.
Our aim was to expand the repertoire of genes that are aberrantly expressed in the RTT brain. To achieve this, we took the novel approach of evaluating differential mRNA expression between an affected and an unaffected brain region in MECP2 mutation-positive RTT patient postmortem tissue samples while also comparing mRNA expression in diseased and healthy tissue. We acknowledge that a potential limitation of our study is that at the time of this study we were unable to obtain equivalent age-matched control samples. It could be argued that the observed differences are a consequence of the developmental and aging processes or could be an epiphenomenon as a result of the disease process. However, we feel that the conclusions drawn in this exploratory phase of the study were valid for several reasons. Firstly, we applied very stringent criteria in selecting genes for further study, including the requirement that for a gene to be flagged as potentially significant it had to be differentially expressed in at least 5 out of the 6 relevant microarray data sets. Secondly, comparison of microarray data sets of different regions in the same subject yielded results that were consistent with our central hypothesis, namely that brain region-specific differences in gene expression would allow us to identify genes of potential biological relevance to the RTT neurodevelopmental phenotype. Thirdly, the subset of genes we chose for further analysis all showed differential expression in our RNAi-mediated knockdown system in the same direction as was seen in the microarray experiments. Finally, enzymatic studies of cytochrome c oxidase in the RNAi treated SH-SY5Y cells confirmed that the abnormalities seen at the RNA level were of functional significance at the protein level.
Only one other microarray study has used RTT patient brain samples as the source material . There were, however, a number of limitations of this study, including difficulties in comparing expression data obtained by using different microarray procedures, which differed significantly in four ways - substrates (eg nylon filters versus glass slides), types of labels (radioactivity versus fluorophores), numbers of cDNA clones (eg 597 clones for one type of array versus ~7000 clones for another), and the threshold for delineating differential expression. For our studies we used only one array platform, containing around 19,000 transcripts, and employed very stringent filtering criteria with the aim of having a low false discovery rate of less than 0.6%. Using this approach we identified only a small number of genes showing altered expression in the frontal cortex of RTT patients, and our in vitro studies of a subset of these genes recapitulated our microarray results.
Recognized, but little studied, findings in RTT patients are morphological abnormalities of mitochondria [24, 25, 27], functional defects of the mitochondrial respiratory chain [26, 29], and evidence of increased oxidative stress . In addition, some of the non-neurological manifestations of RTT have been seen in patients with primary respiratory chain disorders, including short stature, myopathy, cardiac arrhythmias, and intestinal pseudo-obstruction . However, the contribution of these functional abnormalities of the respiratory chain to the pathogenesis of RTT remains unclear. At a molecular level, an association between MeCP2 and the promoter of ubiquinol-cytochrome c reductase core protein 1 has been demonstrated (Uqcrc1) with subsequent up-regulation of this gene and mitochondrial respiratory chain dysfunction in Mecp2-null mice , as noted above. Here we show that cytochrome c oxidase subunit 1 expression was down-regulated exclusively in the frontal cortex of the RTT brain. This down-regulation was a consequence of MeCP2 deficiency (by RNAi analysis) and manifested as a decrease in COX enzyme activity. These two findings suggest that a defect in MeCP2 activity is likely to result in significant dysfunction of the mitochondrial respiratory chain in the brain and possibly other organs, which could contribute to the clinical abnormalities seen in RTT patients. It should be noted however that CpG methylation is absent in mitochondrially-encoded genes. Therefore, it is not likely that cytochrome c oxidase subunit 1 is under the direct transcriptional control of MeCP2, but may be affected as a consequence of a direct interaction of MeCP2 and a nuclear-encoded component of the processes responsible for mitochondrial DNA replication, transcription or translation, or direct interaction of MeCP2 with a nuclear-encoded protein that plays a role in maintaining the structure and stability of the COX protein complex.
Dynamin I was upregulated in the frontal cortex of the RTT brain, and we have demonstrated in vitro that it was upregulated as a result of MeCP2 knockdown, and that the promoter region of dynamin I associated with MeCP2. Dynamin I is a large GTPase that is involved synaptic vesicle endocytosis. Specifically, it assembles around the invaginated clathrin-coated pit and pinches the vesicle from the plasma membrane . Other indications of abnormalities in synaptic transmission in RTT have come from studies investigating the aberrant expression of glutamate receptors such as NMDA and AMPA receptors at excitatory synapses [33, 34], and associated impairment of both long term potentiation (LTP) and depression (LTD) , and by the correction of abnormalities in LTP when Mecp2 deficiency is reversed in a mouse model of RTT . NMDA receptor expression is decreased in RTT and interestingly, the internalization of this receptor is clathrin-mediated and is facilitated by a postsynaptic member of the dynamin family, dynamin 2 . Additionally, it has been demonstrated that synaptic activity can induce phosphorylation of MeCP2 which subsequently leads to gene transcription . The increase in the expression of dynamin I in the frontal cortex of RTT patients may manifest as abnormal rates of synaptic vesicle cycling which adds another dimension to the idea that anomalous synaptic transmission is contributing to the severe cognitive impairment in RTT.
The regulation of expression and function of the glycoprotein clusterin remains incompletely defined. It is associated with apoptosis, and whilst it is thought to promote tumor progression in cancer through Bax interaction , it possibly suppresses beta-amyloid deposition in Alzheimer disease . Upregulation of clusterin expression is not only associated with amyloid deposits in Alzheimer disease  but also with various other neurological disorders. Increased expression is associated with deposition of prion protein in transmissible spongiform encephalopathies , with status epilepticus  and following cerebral ischemia where it is associated with tissue remodeling . In the brain it is expressed in specific neuronal populations of cells in the hippocampus and cerebellum. These are regions of the brain that have been implicated in dysfunction in RTT at a histological level, and increasing evidence now also suggests that these regions are also dysfunctional at a molecular level. In this study, clusterin was upregulated in the frontal cortex of RTT brains and this upregulation was the result of MeCP2 deficiency as demonstrated by in vitro RNAi-mediated knockdown of MeCP2. In addition, the CpG-rich promoter region of clusterin associates with MeCP2, and it is interesting to note that it has previously been shown that expression of clusterin is induced by histone deacetylase inhibitors , raising the possibility that one mechanism by which MeCP2 mutations may exert their effect could be by failing to repress transcription through the interaction of MeCP2 with histone deacetylase. Interestingly, in the Delgado study, clusterin was underexpressed in mutant T-lymphocyte clones from RTT patients . Whether abnormal expression of clusterin is related to a pro- or anti-apoptotic function is unknown, but the fact that it is upregulated in a region that displays a greater level of cellular pathology makes it seem likely that its role in RTT is not of a neuroprotective nature.
CRMP1 is involved in semaphorin-induced growth cone collapse during neural development. It was upregulated in the RTT frontal cortex and we showed that MeCP2 associated with its promoter region in differentiated neuronal cell cultures. In adult mice, CRMP1 is localized to dendrites of cortical and hippocampal (CA1) pyramidal neurons , where it is possibly involved in neurite outgrowth and LTP . CRMP1 knockout mice display abnormal MAP2 staining . Abnormalities in CRMP1 expression in this study may explain in part the morphological abnormalities observed in neurons in the RTT brain whereby there is a decrease in dendritic arborization.
This study utilized stringent inclusion criteria for differentially expressed genes. It can be noted however (Table 1) that there was a large number of transcripts that were differentially expressed in some comparisons, but not in others. It is interesting to note that a number of the genes that were up- or downregulated in the frontal cortex were also up- or downregulated in other studies. Another component of the respiratory chain succinate dehydrogenase (subunit B), EH-domain containing 1 (endocytosis of IGF1 receptors) and amyloid precursor protein were all differentially expressed in some of the analyses in this study as well as in the study conducted by Colantuoni et al. . Genes that were statistically upregulated in this study (yet didn't have a significant fold change value) were also upregulated in the Delgardo study including eukaryotic translation initiation factor 2 subunit 1 alpha, thioredoxin, autism susceptibility candidate 2 and COX 15 homolog. MIR (Membrane-associated ring finger) is a protein linking membrane proteins to the actin cytoskeleton, PAM (Peptidylglycine alpha-amidating monooxygenase) catalyzes neuroendocrine peptides and FHL1 (four and a half LIM domains) involved in cell growth and differentiation. These transcripts were all differentially expressed in the frontal cortex in this study (SAM analysis) and in RTT-derived lymphoblastoid cell lines . Additionally, creatine kinase was upregulated by SAM analysis in the frontal cortex in this study and in the olfactory proteomic study .
This study has looked at the differential expression of genes in an area of the RTT brain that displays abnormal neuronal development. We have found that this differential expression was reproduced in an in vitro MeCP2 RNAi system. The promoter regions of two of these genes (DNMI and GNBI) associated with MeCP2 in differentiated and undifferentiated neuron-like cells, whilst two other genes (CRMP1 and CLU) associated with MeCP2 in differentiated cells only. In addition, downregulation of CO1 expression manifested as a downregulation of COX activity of the mitochondrial respiratory chain.
In summary, this study has further contributed to our understanding of how MeCP2-mediated misregulation of transcriptional repression of subsets of genes may influence the pathology in RTT. Of particular importance is the emerging role of mitochondrial dysfunction as well as the possibility that specific abnormalities in synaptic transmission contribute to the disease.
In considering the role of MeCP2-regulated genes highlighted in this study, further investigations of the effect of MeCP2 on apoptosis and/or neuroprotection, respiratory chain function, vesicle-mediated endocytosis, and secondary messenger/cell signaling in the RTT brain may provide further insight in to the pathophysiology of RTT.