Pathologies of MeCP2 Loss-of-Function Mutation in Rett Syndrome
Jack Raskin
Abstract
Rett Syndrome (RTT) is a rare, postnatal neurological disorder caused loss-of-function mutations in the methyl-CpG binding protein 2 (MeCP2), an X-linked epigenetic transcriptional regulator. RTT is associated with a myriad of neurological and developmental deficits in females, but to date no clear mechanism of etiology has been discovered. MeCP2 loss-of-function has a wide range of consequences, including “long-gene” expression on the genetic level, dendritic spine dysgenesis on the cellular level, and GABAergic transmission dysfunction on the synaptic level. These phenomena explain some of the many pathologies associated with RTT, but there are limitations to studies of MeCP2 dysfunction and its associated consequences.
Introduction
Rett Syndrome (RTT) is a postnatal neurological disorder that primarily affects females in their early development (Banerjee 2017). The syndrome is rare, with an incidence of 1 in 10,000-15,000 live births, and is associated with a variety of devastating neurological and developmental consequences, including but not limited to ataxia, respiratory abnormalities, and mild to severe cognitive impairment (Banerjee 2017). The disease is linked to loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (MeCP2), and these mutations have been found to be the causative genetic component of RTT in 90% of patients (Banerjee 2017).
MeCP2 is an epigenetic transcriptional regulator that has high binding affinity for a symmetrically methylated CpG site, and has been found to inhibit transcription via its association with Sin3A, a transcriptional repressor, and histone deacetylases (HDACs), which are linked to chromatin condensing (Christopher 2017). MeCP2 has two clinically important structural domains, the methyl-binding domain (MBD) and the transcriptional repressor domain (TRD) (Christopher 2017). Genetic mutations coding for these domains are associated with many of the consequences of Rett Syndrome (Banerjee 2017). Despite decades of research, there is still no clear mechanism linking the loss-of-function mutations of MeCP2 to the myriad of neurological and developmental impairments associated with Rett Syndrome.
Despite the lack of clarity in etiology of Rett Syndrome, there have been several studies exploring distinct pathologies of dysfunctional MeCP2 expression on both the genetic level, the cellular level, and the synaptic level. On the genetic level, there have been studies showing that there is a genome-wide length-dependent increase in models with MeCP2 loss-of-function, and that the expressed “long genes” may be the cause of neurological dysfunction (Gabel 2015). On the cellular level, studies have shown that MeCP2 loss-of-function negatively impacts neuronal maturation and synaptogenesis, and this has been observed morphologically in the irregular development of dendritic spines in cortical and hippocampal pyramidal neurons (Xu 2014). Lastly, on the synaptic level, there is emerging evidence that MeCP2 deficiency is linked to dysfunctional respiratory control mediated by “reduced phasic synaptic GABAergic transmission” (Chen 2017). These studies show the wide array of effects of MeCP2 deficiency and potential targets for therapy.
MeCP2 Loss-of-Function leads to long gene expression
In attempting to find commonality between genes affected by RTT, researchers found that MeCP2 loss-of-function mutations lead to the expression of longer genes than in models with functional MeCP2 (Fig 1) (Gabel 2015). This difference was found to be correlated only with RTT models and not with other models of neurological syndromes (Gabel 2015). Regulated expression of these long genes in control models have been found to modulate and maintain cellular adhesion and signaling, two hallmark characteristics of proper neuronal function (Sugino 2014). In the absence of functional MeCP2, these long genes are upregulated indiscriminately, and the researchers found that the magnitude of gene length was correlated with the severity of the RTT symptoms (Gabel 2015).
The mechanism by which these long genes are normally regulated involves the MeCP2 proteins binding affinity to methylated DNA on long genes, particularly methylated cytosine followed by adenine (mCA) (Gabel 2015). Here, the MeCP2 will bind to the mCA dinucleotide and recruit other transcription factors to repress transcription. The researchers found that in a representative set of genes misregulated by RTT, the gene length was longer than average and that they were enriched for mCA, implying that the genes that are down-regulated by functional MeCP2 activity are these long genes in question (Gabel 2015). The researchers concluded that the misregulation of these long genes implicated in normal neuronal function and communication may be an underlying cause to the neurological impairment associated with RTT.
MeCP2 loss-of-function confers dendritic spine dysgenesis
Dendritic spines are cytoplasmic processes that are involved in synaptic communication. While their morphology is greatly varied between different regions of the brain, proper dendritic spine development and maintenance is critical for normal brain function (Xu 2014). Dendritic spine dysgenesis refers to dendritic spine abnormalities in morphology or density (Chapleau 2009). Dendritic spine dysgenesis has been found to be a cause of neuronal dysfunction in many neurological or developmental disorders, and seems to be a part of the pathophysiology of the mental/cognitive impairment seen in RTT (Xu 2014). In both postmortem tissue analyses and RTT animal models, MeCP2 loss-of-function was found to greatly reduce the density of dendritic spines on pyramidal neurons in the hippocampus (Figure 2) (Chapleau 2009). Expression of a mutant MeCP2 or MeCP2 knockout was able to confer decreased dendritic spine density in the hippocampus, and similar affects have been seen in postmortem RTT patients in the cerebral cortex (Chapleau 2009). These results suggest that MeCP2 loss-of-function has a negative effect on dendritic spine density, which confers the cognitive and mental impairments associated with RTT.
Interestingly, a potential therapy to the neurological impairments was identified in exploring the mechanism by which MeCP2 loss-of-function confers cognitive impairment. MeCP2 regulated the expression of brain-derived neurotrophic factor (BDNF), which is implicated in neuronal development and synaptogenesis (Xu 2014). MeCP2 binds to the BDNF promoter and activates it in when neuronal activity is high, and therefore in the absence of correctly function MeCP2, BDNF levels remain low, which may give rise to the decrease in dendritic density seen in RTT models (Xu 2014). When exogenous BDNF is applied to hippocampal pyramidal neurons in situ, there is a marked increase in dendritic spine density (Xu 2014). This has also been observed in genetically engineered models where BDNF is overexpressed in vivo (Xu 2014). However, harnessing BDNF or BDNF-like neurotrophic factors poses challenges as a pharmacotherapy, due to its poor permeability through the blood brain barrier (Xu 2014).
MeCP2-knock out mice display defective GABAergic neurotransmission
Respiratory control involves the actions of two major neurotransmitters in the central nervous system; Glutamate and GABA (Chen 2017). Glutamate, an excitatory neurotransmitter, is implicated in generating the respiratory rhythm, while GABA, an inhibitory neurotransmitter, is implicated in providing phasic inhibitory waves to modulate the pattern of respiratory output (Chen 2017). Blocking the action of GABA with a receptor antagonist was found to increase inspiratory period and frequently resulted in apnea (Chen 2017). MeCP2 knock out mice seem to also have this effect on GABAergic regions in the nucleus tractus solitarius (NTS), and this has been causally linked to MeCP2 loss-of-function by animal models (Chen 2017).
In Mecp2-null mouse models, spontaneous inhibitory post synaptic currents (sIPSCs) were measured and compared to wild type mice in the NTS (Figure 3) (Chen 2017). In the MeCP2-null organisms, there was a decrease in both the frequency and amplitude of these pISPCs, suggesting that MeCP2 mice neurons have a hindered capacity for GABAergic neurotransmission in the NTS (Chen 2017). Given the role of GABA on respiratory control and the effects of MeCP2-knock out on GABAergic neurotransmission, it is concluded that MeCP2 loss-of-function mutations are a major cause of respiratory dysfunction commonly seen in RTT and could provide a target for future therapies. Indeed, researchers found that selectively recovering MeCP2 function in the Hox4A domain, which later develops into the NTS among other regions associated with respiration, effectively rescues mice from respiratory dysregulation seen in RTT models (Huang 2016).
Limitations
Although MeCP2 loss-of-function mutations are related to the neurological and developmental deficits associated with RTT, there is no clear mechanism connecting MeCP2 dysfunction and onset of RTT. As MeCP2 is a trans-acting transcriptional regulator, it has a host of hundreds of effector genes, each of which could be a mechanism for the pathology and severity of RTT (Pacheco 2017). Additionally, the actions of MeCP2 in wild-type, otherwise normal individuals are still largely unknown, and much of our understanding of the function of MeCP2 has been derived from studies of RTT (Banerjee 2017). Also, emerging evidence suggesting that MeCP2 can also function as an activator for specific targets has further confounded research efforts attempting to identify an underlying mechanism of RTT (Banerjee 2017).
An important distinction must be made between human RTT patients and RTT animal models induced by MeCP2 mutation or knock out. As MeCP2 is X-linked, most RTT patients in the natural world are female. Due to random X-chromosome inactivation, RTT in patients has a spectrum of severity, with some RTT cases being more severe due to genomic mosaicism (Banerjee 2017). This variability, while fortunate for some RTT patients, would be unreasonable in the lab, and therefore most animal models use male mice that are hemizygous for MeCP2 dysfunction. This makes research more generalizable to MeCP2 function, but could present future problems in developing therapies for the mostly female, randomly inactivated X-chromosome human patients.
Conclusion
Rett Syndrome is a devastating neurological and developmental disorder that is largely tied to MeCP2 loss-of-function mutations. MeCP2, as an epigenetic transcriptional regulator, has a myriad of gene targets, and any combination of them could give rise to the deficits observed in RTT patients. In this review, three consequences of MeCP2 loss-of-function mutations were explored on the genetic, cellular, and synaptic level, and they each contribute to the overall pathology of RTT syndrome. Future research should aim on fully understanding the role of MeCP2 on the development, maturation, and maintenance of neurons, as this information would likely elucidate potential therapies for RTT and other neurodevelopmental syndromes.
Figures
Figure 1
Compared to the genome-wide baseline (grey), genes that are repressed by MeCP2 normal function (red) are typically longer than those that are induced by MeCP2 normal function (blue). These genes are found to be upregulated in all parts of the brain and are statistically significant in all regions but LVR. This supports the claim that loss-of-function mutations of MeCP2 results in the expression of long genes. (Adapted from Gabel et al., 2015).
Figure 2
Compared to the unaffected (non-MR) control group, individuals with RTT show reduced dendritic spine density. In (A) and (B), apical dendrite density can be observed visually by noting the diameter and relative atrophy in the RTT vs. non-MR stains. These differences are quantified in (C) and are found to be statistically significant. (Adapted from Chapleau et al. 2009)
Figure 3
The sIPSCs are given as a function of time in (A). The probability of a given amplitude and interval between sIPSC is given in (B). In (C), the amplitude of current and frequency of sIPSC is given. The amplitude did decrease significantly, but the decrease in frequency was not significant. Overall, these results suggest MeCP2 plays a role in GABAergic neurotransmission. (Adapted from Chen et al. 2017).
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