Summary

GABRB3, in chromosome 15q11.2-q12, an important neurodevelopmental gene, is regulated by non-Mendelian processes, epigenetic modulation, and sex-specific transcription with deviation of parental gene expression. GABRB3 encodes the β3 subunit of GABA_A receptor. It is highly expressed in embryonic brain where repressor-element-1-silencing transcription factor (REST) regulates neuronal genes. GABRB3 is expressed at lower levels in adult brain, except in hippocampus where it remains high. Homozygous disruption of Gabrb3 in mice leads to myoclonic and atypical absence seizures, and impaired cognition, motor coordination, and somatosensory processes. Heterozygous disruption produces increased epileptiform EEG activity and elevated seizure susceptibility. In human, three different point mutations in exon 1A, coding the signal peptide and exon 2 of GABRB3, segregate with childhood absence epilepsy (CAE), and result in decreased neuronal GABA currents, Three neurological disorders, Rett syndrome, (a deficiency of MeCP2), Angelman syndrome, and autism, each exhibit reduced expression of GABRB3 and UBE3A, along with mental retardation and epilepsy. GABRB3 is highly associated with epilepsy and when a deficiency of UBE3A is also present, more severe symptoms result. UBE3A modulation of REST, which controls GABRB3 expression, and MeCP2 modification of UBE3A link Rett, Angelman, and autism syndromes with epilepsy, and invoke epigenetic mechanisms in epileptogenesis.

Introduction

Three neurodevelopmental disorders, Angelman syndrome (AS), Rett syndrome (RS), and Autism spectrum disorders (ASD), share several clinical features in common, most notably neurodevelopmental delay and epilepsy. Here, we ask--what common mechanisms do these three neurodevelopmental disorders share that lead to a decline in cognitive development and epilepsy? Based on our observations on the genetic regulation of GABRB3 in childhood absence epilepsy (CAE), we posit that the genetic mutations in these 3 neurodevelopmental disorders converge on a common disease mechanism involving genetic and epigenetic regulation of GABRB3. We first explain the data on human CAE, supporting this hypothesis and show that three different point mutations in alternative signal sequence (exon 1A) and N-terminus (exon 2) in GABRB3 result in hyperglycosylation and decreased GABA currents, all of which segregate with childhood absence epilepsy (CAE). One of the variants of exon 1A, P11S, which is maternally transmitted, links GABRB3 dysfunction with ASD, and provides a possible cause of seizures in this syndrome. In Angelman syndrome, GABRB3 deletion contributes to their severe seizure phenotype. Rett syndrome (RS) is caused by mutations in MeCp2. Because MeCp2 epigenetically regulates GABRB3, reduced expression of GABRB3 and epilepsy are considered consequences of MeCp2 mutations. We then review the significance of GABRB3 in embryonic and adult neurogenesis and neuronal development in mammalian brain. We point to a master regulator of neurogenesis, REST or RE1 silencing transcription factor, which binds to repressor element 1 (RE1) in intron3 and the 5′ region of GABRB3. REST epigenetically regulates tissue and developmental expression of GABRB3. Finally, we chart the future challenges and experiments that could prove or disprove our hypothesis that REST and epigenetic regulation are involved in neurogenesis and epileptogenesis of absence seizures.

Background

Genomics of GABRB3

GABRB3 generates two alternative signal peptide sequences, which are derived from alternative mRNA transcripts (exon 1A and exon 1), along with 8 additional exons (exon 2-9). From this, two mature polypeptides (NCBI accession number: NM_021912, NM_000814) are produced, each exhibiting a distinct signal peptide sequence with slight variations in residues at the N-terminus due to differing cleavage sites.¹

Exon 1 and exon 1A each can elicit tissue- and temporal-specific expression with the unique signal peptide and N-terminus sequences influencing functions and/or subcellular localization. Both core promoters contain GC-rich regions and are characterized by TATA-less SP1 binding motifs.^{1, 2, 3} The abundant expression of exon 1A in fetal brain but not adult brain (Figure 1) suggests a role in embryonic neurogenesis, with exon 1 likely contributing to adult brain function. The Human Genome Feb. 2009 (GRCh37/hg19) assembly at the UCSC Genome Browser suggests additional GABRB3 sequences are expressed. One isoform found in brain has the first three exons (Exon 1A, 1, 2 and 3) truncated, with transcription beginning in intron 3 and extending through exon 9; the other, found in retina, consists of a long mRNA which is transcribed from intron 8 of GABRA5 and spliced to the short region at the interval between GABRB3 - GABRA5 (42 kb upstream region from exon 1A) and extending to exon 2 of GABRB3,. GABRB3 and GABRA5 are located in a ~94 kb interval and are transcribed in a head-to-head orientation.⁴ Recent NCBI reference sequence described four mRNAs of GABRB3 (variants NM_021912.4, NM_000814.5, NM_001191320.1, NM_001191321.1). Two are truncated mRNAs of GABRB3 without the first three exons (Exon 1A or, 1, 2, 3) which have two different lengths of intron 3 added to original variant 1 and 2. However, at the moment only two protein sequences of 473 amino acids derived from GABRB3 have consensus agreement (NP_068712, NP_000805.1).

Figure 1

Alternative transcripts (GABRB3 variant2) of the human GABA_A receptor beta3 subunit detected by RNase protection (from Kirkness & Fraser, 1993). The relative RNA content of different sample preparations was assessed by including an antisense riboprobe (more...)

Thus GABRB3 exhibits differing transcription start sites, chromatin accessibility, and histone modification patterns, and/or various splice patterns with stop codons, to produce these variants. Although some evidence suggests GABRB3 is not imprinted, the paternal GABRB3 gene region has been demonstrated to replicate earlier than maternal replication in T-cell lymphocytes,⁵ corresponding with distinct chromatin structure differences between paternal and maternal alleles.⁶ This observation suggests paternal expression bias or tissue-specific partial imprinting. In addition, there is evidence that Gabrb3 expression in brain is not always equivalent between male and female heterozygous mice.^{7, 8} In a mouse model of AS (Jiang et al., 2010⁹), maternal-derived heterozygotes for a deletion of the chromosome encompassing Ube3a and Gabrb3 show greater phenotypic correspondence with AS than a paternal-derived heterozygote, consistent with the known imprinting of the disorder; these mice show imprinting in some brain regions including cerebellum for Ube3a but biallelic expression of Gabrb3 in the brain regions and ages examined.

GABRB3 in Mammalian Brain Embryogenesis & Adult Neurogenesis

GABA and GABA_A receptors (GABA_AR) are the earliest neurotransmitter systems to emerge during development, even prior to glutamatergic synapses. GABA_AR’s mediate excitatory signaling during development and play a significant role in neuronal growth and differentiation.^{10, 11} The GABA_AR β3 subunit mRNA/protein (encoded by GABRB3) emerges at embryonic days 14–17 in rat whole brain, and reaches its strongest expression at the perinatal stage, which is 150% of the level expressed in the adult (Figure 2).¹² After birth, the β3 subunit protein decreases to moderate levels in adult cortex, while falling rapidly in most thalamic nuclei, except in the reticular thalamic nucleus (NRT), where it remains one of the main components of the GABA_AR.^{12, 13, 14} GABRB3 therefore plays a significant role in neurodevelopment as well as adult brain function. By virtue of its location in NRT and cortex, GABRB3 plays an important role in thalamocortical circuits. As these circuits are essential to sensory processing it is not surprising that heterozygous disruption of Gabrb3 in mice would elicit somatosensory disturbances.⁸ With GABRB3 being highly implicated in autism, it is interesting to note that somatosensory disturbances are common to autism.¹⁵

Figure 2

Expression level of the GABA_A receptor beta subunit mRNAs in selected regions of embryonic and postnatal rat brain (from Laurie et al., 1992). Black represents strong signal, dark gray represents moderate signal, light gray represents weak signal, and (more...)

In adult brain, GABA activates synaptic and extrasynaptic GABA_AR’s, producing phasic and tonic inhibitory chloride currents.¹⁶ Opening of chloride conductance channels stabilizes the mature neuron near the resting potential, serving to inhibit neuronal action potentials whether or not the current is hyperpolarizing. Furthermore, GABA_AR-mediated chloride conductance can depolarize neural progenitor cells in immature neurons in the adult brain, similar to what occurs in the embryonic nervous system.^{10, 17} Phasic and tonic GABA_AR activity both play an important role in proper regulation of adult neurogenesis within the subventricular zone of the lateral ventricles as well as in the dentate gyrus of the hippocampus.¹⁷

Recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice indicate that these cells receive direct GABAergic inputs, but not glutamatergic inputs from the hippocampal circuitry. These GABAergic inputs depolarize neuronal progenitor cells, causing an increase in [Ca ²⁺]_I, resulting in the induction of NeuroD expression, a positive regulator of neuronal differentiation. Thus GABAergic inputs to hippocampal progenitor cells are likely promoting activity-dependent neuronal differentiation.¹⁸ Since GABRB3 is one of main components of GABA_AR found in adult hippocampus, GABRB3 likely contributes to these GABAergic inputs. Furthermore, other transcription factors of the basic helix-loop-helix (bHLH) family to which NeuroD belongs, may likewise be similarly regulated by GABA. GABRB3 contains NEUROD1 binding motifs within intron3, as well as a MASH1 binding motif in 5′UTR of exon1A and two NEUROG1 binding motifs in exon1A (Figure 3). One of the NEUROG1 motifs contains a SNP (rs 25409) which, as described above, we discovered in two CAE families,¹ and is also associated with maternal transmission in autism disorders.¹⁹ This suggests that exon1A may contain transcription binding sites for essential factors of neurogenesis in addition to its role of encoding a signal peptide. While NEUROD1 regulates neurogenesis,^{18, 20, 21}, MASH1 is required for generation of GABAergic neurons^{21, 22} and NEUROG1 is required to specify glutamatergic neurons, while simultaneously repressing both GABAergic differentiation^{23, 24} and astrocyte differentiation.²⁵ Therefore, the SNP rs25409 in the coding sequence may induce malfunction or unbalanced generation of GABAergic and glutamatergic neurons.

Figure 3

Sequence from 5′ region to intron 3 of human GABRB3 and predicted transcriptional factor binding motifs. GABRB3 contains a long intron 3, spanning almost 151 kb. This, along with the first four exons (exon 1A, exon 1, exon 2, and exon 3) represents (more...)

The nuclear-localized small modulatory double-stranded RNA coding the RE1/NRSE sequence was discovered in hippocampal stem cells and found to contribute to adult neurogenesis by changing neuron-restrictive silencer factor (NRSF) from a repressor to an activator for neuron-specific gene with RE1.²⁶ Thus the role of GABRB3 in adult neurogenesis is promoted by an epigenetic modulator, REST.

Epigenetic modulation of GABRB3

Two NRSF binding motifs are found in GABRB3, one about 400 bp upstream from exon 1A, and the other within intron 3 (Figure 4),²⁷ both locations corresponding to predicted RE1 sites. NRSF, also known as repressor element 1 (RE1)-silencing transcription factor (REST), was originally reported as a transcriptional repressor of neuronal differentiation genes in non-neuronal cells and embryonic stem cells.²⁸ We confirmed the suppression of luciferase promoter activity in a construct containing RE1 in the 5′ region of exon 1A expressed in non-neuronal HEK 293 cells.² In contrast, promoter activity was not suppressed in neuron-like NT2 cells.²⁹

Figure 4

Chr 15q11.2-q12: GABRB3 and UBE3A. The initial exons of GABRB3 and UBE3A contain SP1 binding motifs in the proximal promoter region which are GC rich regions and are indicated by light blue painted squares. Green squares indicate exons of GABRB3 with (more...)

REST, along with its primary cofactor, CoREST, dynamically recruits cellular cofactors including MeCP2 and other silencing machinery to element-1/neuron restrictive silencer element (RE1/NRSE) sites to suppress the expression of the target gene, primarily neural genes, by chromatin remodeling.³⁰ REST not only prevents extraneural expression of target genes but also regulates by repressing the differentiation of neuronal subtypes required for proper tissue differentiation during embryonic development.^{28, 31, 32} REST-mediated regulation of its target genes is not an all-or-none function. It depends on the cellular environment e.g., intercellular signaling, Ca²⁺ dynamics, cell depolarization, etc., resulting in context-dependent gene repression.^{32, 33} REST and CoREST can mediate cell type and developmental stage–specific gene repression for protein-coding genes and for several classes of non-coding RNA (ncRNA), (e.g., micro RNA and long ncRNAs). The REST and CoREST network is highly integrated with ncRNA and mediates neural gene expression programs including bidirectional feedback.^{34, 35} REST is an essential epigenetic modulator for neuronal differentiation, homeostasis, and plasticity. Deregulation of REST and ncRNAs are implicated in cancer, neurodegenerative diseases, and neurodevelopmental diseases, including epilepsy.^{30, 36, 37}

Recent studies concluded that REST regulates neurogenesis by reciprocal actions of REST and non-coding double-stranded RNAs that exhibit RE1 sequence homology; this directly interacts with the REST transcriptional machinery in adult hippocampal neuronal stem cells.²⁶ The interaction transforms the REST complex into a transcriptional activator, inducing neuronal differentiation. The expression of GABRB3, which has two RE1/NRSE sites, is regulated by REST. In addition, Hogart et al.³⁸ found a MeCP2 binding site in intron 3 of GABRB3 close to the REST binding site (RE1). MeCP2, a methylated DNA binding protein, is an epigenetic regulator which is required for development and maintenance of neurons.³⁹ Hogart et al.³⁸ report that GABRB3 expression is biallelic but paternally biased in human prefrontal cortex (Brodmann field 9) via MeCP2 activation of GABRB3 expression. In other words, REST, which connects with various co-factors including MeCP2, could modulate GABRB3 expression in age-dependent, tissue-specific development, not only in stem cells, but also in mature neurons through environmental stimulation that could occur throughout the lifespan.

Neurodevelopmental disorders

I. GABRB3, Epilepsy, and Autism

Various clinical reports, animal studies, genetic association and basic molecular studies have provided evidence that GABRB3 is involved in developmental neurological disorders including epilepsy.^40–46 Three missense mutations (P11S, S15F, G32A) found in an alternative signal peptide and the N-terminus of GABRB3, segregated with remitting childhood absence epilepsy (rCAE) within four families. Two multiplex and multigeneration families containing eight affected members (Figure 5A) and one proband of a singleton have mutations in an alternative signal peptide. When expressed in vitro, each of the mutations in the signal peptide coding region, exon 1A of GABRB3, caused hyperglycosylation of the GABA_AR β3 subunit protein with concurrent reduction in GABA currents.¹ Furthermore, the promoter region of exon 1A of GABRB3 displayed a significant reduction in promoter activity for a common SNP in the promoter region.² We concluded that the functional abnormality resulting from missense mutations, as well as certain combinations of SNPs in multiple regulatory elements in the 5′ region of GABRB3, causes reduced expression of GABRB3, and a concurrent reduction in inhibition, leading to an increase in susceptibility to absence seizures. The CAE disease phenotype exhibits paternal transmission (Figure 5A) of the rare GABRB3 signal peptide variant (rs 25409: P11S), whereas maternal transmission of the same variant appears to be associated with autism (Figure 5B). The association of phenotype transmission with parental gender suggests that parent-of-origin gene expression is likely occurring in certain brain region(s) within some temporal range (partial imprinting). Further genetic studies are needed to determine conclusively the importance of these rare GABRB3 variants in CAE and autism.

Figure 5

Figure 5-A. Paternally transmitted rs 25409 (P11S) in CAE (Tanaka et al., 2008) The rare variant (dbSNP ID, rs 25409 (P11S)) is segregated in affected persons in two families out of four families with rCAE probands as reported. Black circles (female) (more...)

IIA. GABRB3 in Chromosome 15Q11-13, Angelman Syndrome, and Prader-Willi Syndrome

GABRB3 is spatially clustered with GABRA5 and GABRG3 on chromosome 15q11-q13, with UBE3A (ubiquitin protein ligase E3A) lying about 1 cM upstream (centromeric) from GABRB3. This region contains a variety of imprinted genes (Figure 6) and exhibits genomic rearrangements at five common breakpoints (BP1-BP5) that often result in deletions and duplications of these genes.⁴⁷ Parent-of-origin phenotypes are characteristic of chr. 15q11-q13 abnormalities. For example, Angelman syndrome (AS),⁴⁸ caused by deficiencies of maternal genomic information on chr. 15q11-q13, confers a severe phenotype. In contrast, individuals with Prader-Willi syndrome (PWS), resulting from deficiency of paternal genes from the same region, exhibits a less severe phenotype.⁴⁹ The nature of the genomic defect also influences the severity of the phenotype, for instance, a de novo 15q11-q13 deletion on the maternal genome, occurring in 70% of AS cases, is characterized by severe mental retardation, motor dysfunction, and early onset intractable epilepsy (primarily absences and myoclonic seizures). In contrast, 10% of AS probands exhibit only a mutation in UBE3A also found on chr 15q11-13,⁴⁹ and are less severely affected than individuals with the full 15q11-q13 deletion. Such patients typically display only mild epilepsy.⁵⁰ Recurrence risk and severity of epilepsy also differ according to the genetic type of AS. Minassian et al., 1998,⁵⁰ suggested that the severe epilepsy in deletion cases of AS appears to be due to the lack of maternal GABRB3 in addition to the AS gene, UBE3A. This is consistent with the epilepsy present in human GABRB3¹ and mouse Gabrb3 mutations.⁴⁵ Epilepsy in most AS cases is refractory to medication, but shows improvement with age.⁵¹ PWS, which results from paternal deletion of the same chr. 15q11-q13 region, displays a very different phenotype than AS, with mild mental retardation, obesity, and a lower likelihood to exhibit epilepsy. Furthermore, Wang et al.⁵² reported that 8 of 38 PWS patients (21%) with large deletions of paternal 15q11-q13 genes, including GABRB3, had seizures. In contrast, all 12 PWS patients without a GABRB3 deletion had no seizures. These differences further support the involvement of GABRB3 in the epilepsy displayed by these probands.⁵² It is also important to note that duplications or micro-deletions of the maternal 15q11-q13 region are one of the more frequently reported observations (~2%) in autism spectrum disorder.^{53, 54} Interestingly, paternally-derived duplications of 15q11-q13 rarely display autistic symptoms.⁵⁵

Figure 6

Gene order and REST binding sites on chromosome 15q11-13 (Modified from Bittel et al. 2006). Chromosome 15 contains segmental duplications and repeated transcribed DNAsequences (i.e., HERC2 genes) located at the proximal and distal ends of the 15q11-q13 (more...)

The above evidence suggests a significant role for imprinted genes in neurodevelopmental disorders like AS and PWS. Consequently, their control by the imprinting center on chr. 15q11-q13 is of importance. The imprinting center IC (Figure 6) was originally demonstrated and defined ^{56, 57} by the smallest microdeletion (currently 880 BP) that resulted in a block of resetting the imprint, stabilizing on that chromosome the parental imprint (epigenotype) on which the mutation arose. The IC for AS is located centromeric to the IC for PWS, which includes the first exon of the SNRPN gene (Figure 6). None of the paternally imprinted genes (ZNF127, SNRPN, PAR-5, IPW, and PAR-1), located in the vicinity of the IC, are expressed when the PWS IC is mutated. By contrast, these same genes are expressed biparentally in mutations of the AS IC. However, these AS probands lack expression of UBE3A (the AS gene), located 250–1000 kb distally to the IC. To quote Saitoh et al., 1996,⁵⁶ “the paternal chromosome of these PWS [IC mutation] patients carries an ancestral maternal epigenotype, and the maternal chromosome of these AS [IC mutation] patients carries an ancestral paternal epigenotype. The IC therefore functions to reset the maternal and paternal imprints throughout a 2 Mb imprinted domain [in cis] within human chromosome 15q11-q13 during gametogenesis”.⁵⁷ The two IC sequences presumably also interact in a DNA methylation-regulated manner with proteins and noncoding mRNAs that act in trans with target imprinted genes.^{58, 59} At this time, neurological defects in AS are considered to be due primarily to reduced UBE3A function leading to inactivation of crucial gene products by ubiquitin-dependent proteasome degradation.⁵⁹ The latter is involved in neuronal growth and development, possibly involving dendritic spine morphology⁶⁰ and/or excitatory AMPA-type glutamatergic receptor trafficking.⁶¹ Localization of GABA_A receptors including β3 subunit in postmortem AS brain would be interesting to examine.

IIB. GABRB3 and UBE3A in Hippocampus

In the hippocampus, GABRB3 expression levels remain constant throughout maturation and into adulthood. Exon 1 of GABRB3 is strongly expressed in many regions of adult human brain including the hippocampus (Figure 2), and initiates transcription by SP1 as well as other transcriptional factor(s).³ The AS gene UBE3A, located at 1.1 megabase upstream from GABRB3 in chromosome 15q11.2-q12, is a maternally imprinted gene. Although it has been accepted that UBE3A displays maternally imprinted expression in hippocampus, cerebellum, and several lobes of neocortex,^{62, 63} recent reports utilizing immunoblotting and immunohistochemistry methods, found that maternally predominant Ube3a protein expression is not limited to these regions but instead occurs throughout all brain regions.⁶⁴

Three variants of UBE3A are transcribed from the same exon in the 5′ region, but different N-termini are produced by various splice patterns including stop codons.⁶⁵ The common 5′ region contains a GC- rich region as well as a SP1 binding motif as predicted by MatInspector software. This suggests that the common ubiquitous transcriptional factor SP1 could transcribe both the UBE3A and GABRB3 genes on the same chromatin at the same time.

III. UBE3A and GABRB3 Deficiency with MeCP2 Dysregulation: Rett syndrome and Angelman syndrome

Angelman syndrome (AS), Rett syndrome (RS), and autism spectrum disorders (ASD), each exhibit mental retardation, and epilepsy, of varying severity, as part of their phenotypes. Reduced GABRB3 in prefrontal cortex is common to these three disorders.^{38, 46} RS, caused by a MeCP2 mutation, displays deficiency of GABRB3 and UBE3A even though the genes for GABRB3 and UBE3A are intact. Therefore MeCP2 deficiency alone can result in reduced UBE3A and GABRB3. In contrast, studies of MeCP2 deficient mice were not consistent,^{46, 66, 67} suggesting that epigenetic regulation that includes RE1 sites is not necessarily the same between human and mice. Makedonski et al.⁶⁸ found the PWS-IC (PWS-Imprinting Center) on the maternal allele is normally methylated. This allows MeCP2 to bind and recruit histone deacetylases 1 and 2 that deacetylate histone H3 and histone methyltransferase to methylate H3(K9). This chain of events leads to heterochromatinization and silencing of all paternally expressed genes. However, in the absence of MeCP2, heterochromatin cannot form, allowing an open chromatin structure which is sufficient to transcribe antisense UBE3A RNA, thereby, reducing UBE3A transcription. MeCP2 deficiency in RS can lead to epigenetic aberrations via the imprinting center (IC).⁶⁸ REST is degraded by the ubiquitin-proteasome system through SCFbeta-TRCP.⁶⁹ UBE3A is therefore hypothesized to degrade REST. Deficient UBE3A could then affect GABRB3 expression and other neuronal genes through elevated REST/NRSF.

Conclusions

GABRB3 plays an important role in embryonic/adult neurogenesis and neuronal development in mammalian brain. GABRB3 is expressed at the embryonic and perinatal stages as well as in childhood brain as a main component of GABA_AR’s in most brain regions. This includes many thalamic nuclei that mediate the EEG spike-wave complex of absence seizures. In adult brain, GABRB3 is dominantly expressed in hippocampus and cerebellum, but is very low in most parts of thalamus, the only exception being the nucleus reticularis where it remains relatively high. GABRB3 mutations expressed in embryonic and neonatal but not in adult thalamic regions of the brain, could explain the reduction of seizures in AS and the remission of CAE with age, since CAE remits during puberty at the time when expression of GABRB3 diminishes/ceases in most thalamic nuclei.

We suggest that GABRB3 variant1, and variant2, need to be examined in thalamus and hippocampus as a function of development, in comparison to UBE3A expression, in order to establish the significance of GABRB3 in neurodevelopment and epilepsy. In mouse, such assays of GABRB3 expression could be performed on animals engineered to produce a maternal-derived heterozygous deletion of the chromosomal region encompassing both the UBE3A gene and GABRB3, an animal model of Angelman syndrome (Jiang et al., 2010⁹).

Future experiments also should consider the epigenetic modulation of REST and MeCP2 in ASD, AS, RS and CAE. One study⁷⁰ has already broadened the view for REST and CoREST action by genome-wide analysis of the binding status of REST and CoREST to various promoters in embryonic mouse forebrain. Expression of REST and CoREST varies in different brain regions and is specific for neuronal subtypes of GABAergic, glutamatergic, and cholinergic neurons. REST and CoREST differential expression in brain regions also affects the generation of factors responsible for neuronal diversity, e.g., homeostasis, cell cycle dynamics, cell viability, stress responses, and epigenetic regulation. REST is likely to have multiple roles, not only in immature neurons, but also in the function of mature neurons.⁷⁰ In situ hybridization analysis in normal brains has already revealed that REST mRNA is highest in adult hippocampus⁷¹ and REST protein expression studies utilizing mature hippocampal neurons have replicated the in situ hybridization results.⁷² Epileptic insults and ischemic changes have also been observed to elevate mRNA of REST.^{32, 71, 73}

UBE3A is hypothesized to regulate REST by degradation through the ubiquitin pathway, with altered GABRB3 expression resulting from maladjustment of REST. When UBE3A is mutated, reduced degradation of REST would result in excessive REST. When REST acts as a repressor, excessive REST in a developing brain could hypothetically reduce GABRB3 expression, resulting in cognitive decline and epilepsy. Experiments that would demonstrate the above hypothesis are warranted. The deletion of UBE3A, thereby influencing REST could also lead to a severe phenotype due to inappropriate regulation of a number of other targets, including GABRB3, with which REST interacts. Future experiments should also consider whether epigenetic mal-regulation of GABRB3 by REST and/or MeCP2 produces the phenotypes of mental retardation and epilepsy even without a GABRB3 mutation.^{74, 75} Understanding these epigenetic mechanisms is likely to lead to novel interventions for neurodevelopmental diseases like ASD, AS, RS and CAE.

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Support: NIH grant NS035985 to RO, MH065393 to TD, and NIH Grant NS055057 and a VA Merit review grant to AVDE.

Disclosure: the authors declare no conflicts of interest.