- Email: [email protected]
Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities
european journal of paediatric neurology xxx (xxxx) xxx
Official Journal of the European Paediatric Neurology Society
Review article
Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities David C. Henshall a,b,* a b
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland FutureNeuro Research Centre, RCSI, Dublin, Ireland
abstract Keywords:
Acquired and genetic forms of epilepsy are associated with dysregulation of gene expression
DNA methylation
within the brain. Identifying mechanisms controlling gene expression may provide novel
Histone post-translational modifi-
opportunities for the development of disease-modifying therapies. Epigenetic processes in-
cation
fluence the medium-to long-term readability and accessibility of the genome to transcription.
Noncoding RNA
The mediators include biochemical modifications to DNA and the histones around which
Epileptogenesis
DNA is wrapped, as well as non-coding RNAs. Here, the main epigenetic processes are briefly
Hippocampal sclerosis
reviewed. Examples are provided of altered epigenetic processes and mutations in genes with
Antisense oligonucleotides
epigenetic functions in experimental models and human epilepsy. The outcome of recent
Gene therapy
studies manipulating epigenetics to protect the brain or reduce seizures in preclinical models is considered, with specific focus on RNA therapies. Last, future applications of epigenetics research are appraised, including opportunities to map and change epigenetic marks and the prospects for therapies based on manipulation of epigenetics and noncoding RNAs. In summary, epigenetics and noncoding RNAs are important mechanisms and targets to modulate brain excitability which together provides novel insight into patho-mechanisms, biomarkers and novel therapies for epilepsy. © 2019 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4.
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Epigenetic genes and epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manipulating epigenetics in epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What does the future hold for epigenetic therapies in epilepsy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
00 00 00 00 00 00
* Corresponding author. Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin, D02 YN77, Ireland. E-mail address: [email protected]. https://doi.org/10.1016/j.ejpn.2019.06.002 1090-3798/© 2019 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
2
european journal of paediatric neurology xxx (xxxx) xxx
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.
Background
The brain derives its structure and function from the temporally-controlled, cell-specific expression of over 15,000 genes.1 Many genes are uniquely expressed in neurons and glia (as well as other cells of the neurovascular unit - endothelial cells of the bloodebrain barrier (BBB), myocytes and pericytes). Their products bestow a myriad of properties, such as the ability to generate action potentials (neurons) or perform support roles such as uptake of released neurotransmitter (e.g. astrocytes). Gene expression begins with recruitment of factors to gene promoters followed by actions of RNA polymerases to generate a messenger RNA (mRNA). The transcripts then undergo splicing, editing and are eventually translated to protein (Fig. 1). Epigenetics and certain noncoding RNAs perform critical roles in shaping the gene expression landscape in the brain and important roles have emerged in both acquired and genetic forms of epilepsy.2,3 Manipulating epigenetic processes
represents, therefore, a novel approach to the treatment of epilepsy. A modern, operational definition of epigenetics covers the biochemical changes (“epigenetic marks”) that influence chromatin structure in a way that determines the readability (accessibility) of the genome to transcription.4,5 DNA is normally highly condensed within the nucleus, wrapped around octamers of histone proteins (forming nucleosomes). Highly condensed chromatin (“heterochromatin”) is not, however, accessible for transcription. Thus, in order for a gene to be transcribed, the chromatin must be in an “open” state (termed “euchromatin”). Epigenetic processes drive the transitions between these two states, thereby serving to control the gene expression landscape typically over the medium to long-term.4,6
2.
Epigenetic processes
There are at least five distinct epigenetic process,4,7 but for the purposes of this review, we focus on three of the major sub-
Fig. 1 e Overview of how epigenetic mechanisms affect the gene expression process and potential approaches to therapeutic targeting in epilepsy. Cartoon shows the gene expression pathway that begins with promoter elements driving transcription of a protein-coding gene. This results in production of messenger RNA (mRNA) copies which then transit to the cytoplasm for translation within the ribosome resulting in protein production. Among various epigenetic mechanisms affecting this pathway, methylation of DNA (top left, dark green), particularly around gene promoter elements, reduces transcription. Approaches to manipulation (e.g. to reactivate a silenced gene) could include use of DNA methylation inhibitors or a CRISPR-based approach could deliver a de-methylating enzyme to a specific promoter. Histones (bottom left, dark blue) comprise an octamer of proteins around which DNA is wrapped. Posttranslational modifications alter how closely packed these units are, promoting or repressing a transcriptionally permissive state by altering the open or closed state of chromatin. A variety of small molecule histone deacetylase inhibitors have been developed that could be used to activate gene expression. Long noncoding RNAs (middle, dark brown) are a diverse class of transcripts which can affect transcription, for example by acting as natural antisense transcripts (NAT) that block the protein-coding transcript. These NATs can be targeted (and thus their target gene upregulated) using oligonucleotides. MicroRNAs (miRNAs; top right, orange) are small noncoding RNAs that mainly work by directly binding the 3′ untranslated region (UTR) of protein-coding transcripts in the cytoplasm and promoting mRNA degradation or translation inhibition. Blocking certain miRNAs can upregulate their targets and have anti-seizure effects. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
european journal of paediatric neurology xxx (xxxx) xxx
categories: DNA methylation, post-translational modifications of histones and the functions of certain noncoding RNAs (and see Fig. 1). DNA methylation refers to the addition of a methyl group to a cytosine base, a reaction catalyzed by enzymes called DNA methyltransferases.8 Heavily methylated DNA invariably promotes condensation of chromatin and thus is normally associated with transcriptional silencing (although DNA methylation has been associated with other effects including promoting transcription).6 Histones undergo various posttranslational modifications, including phosphorylation, which serve to increase or decrease the permissiveness of a locus for transcription.9 One of the best understood modifications is acetylation, which, due to the addition of electrostatic charges, tends to cause histones to move apart and thus favour transcription. Finally, noncoding RNAs, of which two major sub-groups (long and short), are recognized. Numerous long noncoding RNAs have been linked to structuring chromatin and regulating transcription.10 These include enhancer RNAs and natural antisense transcripts (NATs).11 The best understood short RNAs are microRNAs, which target mRNAs to lower (buffer) protein levels.12 MostmicroRNAs do not, however, function in an epigenetic manner (i.e. acting to modify the biochemical and structural properties of chromatin). Rather, the role of microRNAs is within the cytoplasm at a step immediately prior to translation as part of the post-transcriptional machinery that regulates protein levels (Fig. 1). Some of their targets, however, may have direct epigenetic functions.
2.1.
Epigenetic genes and epilepsy
Since the discovery, in 1999, that mutations in the MECP2 gene caused Rett syndrome,13 a neurodevelopment disorder characterized by seizures, numerous genes whose products are involved in epigenetics have been linked to epilepsy. This includes BRD2 (a reader of histone marks),14 and CHD2 (a chromatin remodeler).15 This should not be altogether surprising. Any insult that disrupts the gene expression landscape by leading to genes being inappropriately on or off might be expected to disrupt neuronal network stability. The brain expresses an extensive complement of genes with epigenetic functions. In situ hybridization (see resources such as the Allen brain atlas) and single cell sequencing approaches,16 reveal particular enrichment in neurons compared to glia, including the molecular machinery for adding and removing DNA methylation and dozens of histones and histoneregulatory factors. There is strong evidence that all three epigenetic processes are altered in experimental and human epilepsy. DNA methylation patterns have been mapped at a global level in resected brain tissue from patients with focal epilepsy as well as in animal models.17e21 This has shown epilepsy to be associated with hypermethylation of numerous genes, indicating a restriction in transcription. For some genes that displayed hyper-methylation the tissue had correspondingly lower transcript levels. This would be consistent with a transcriptional-silencing influence of the epigenetic mark. For many other genes tested, however, methylation status was weakly associated with abundance of the corresponding transcript, meaning hyper-methylation was not necessarily
3
causing gene silencing. This raises questions of the importance of DNA methylation marks in regulating genes that control hyperexcitability and the need to individually validate whether a particular pattern of methylation affects transcription of a given gene.17 Another notable finding in relation to DNA methylation was the relative lack of overlap in DNA methylation patterns between epileptic animals which had epilepsy triggered by different methods (e.g. when comparing pilocarpine to traumatic brain injury).22 This may have diagnostic applications, enabling the cause of epilepsy to be deduced by an analysis of the DNA methylation profile in brain. Histone posttranslational modifications have been reported by several studies after experimental seizures, although there has been little reported for human epilepsy to date.23,24 Assessments of histone marks have been quite limited in scale, and the application of genome-wide histone mark-based sequencing should be undertaken to properly assess the role of these marks.25 Finally, changes to microRNA expression in experimental and human epilepsy have been extensively documented and, increasingly, there are reports of changes to long noncoding RNAs in experimental and human epilepsy.26,27
3.
Manipulating epigenetics in epilepsy
All three epigenetic processes are targetable using pharmacologic, genetic and oligonucleotide-based approaches (see Fig. 1).2,28 DNA methylation is a potentially attractive target because the marks are medium-to-long lasting, providing a means to impose a lasting change on gene expression. Studies to date, however, have been limited by the rather blunt instruments available; broad-spectrum DNA methyltransferase inhibitors such as 5-azacytidine. There have been reports of anti-seizure effects of this class of DNA methylation inhibitor in animal models of epilepsy,19 which is promising for the future. However, contrasting anti-seizure actions of enhancing DNA methylation by other groups suggest hypermethylation of DNA may also serve anti-excitability effects.20 Ultimately, more specific tools which can target DNA methylation at specific gene loci are needed. Targeting histone modifications may also enable the up- or down-regulation of a specific gene. The expected time course of action would be in the short-to medium-term. Similar to DNA methylation, however, there are few tools to selectively induce or remove histone marks. The main category of pharmacologic tool that has been used is histone deacetylase inhibitors (HDAC inhibitors).28 Treatment with a HDAC inhibitor would tend to promote transcription of genes since removal of acetylation favours silencing. A number of HDAC inhibitors have been tested in clinical trials and, notably, the common anti-epileptic drug sodium valproate has HDAC inhibitory activity. However, studies suggest the anti-seizure effects of valproate are unrelated to HDAC inhibition.29 Moreover, mixed results have emerged from studies of HDAC inhibitors on epileptogenesis, with some reporting disease-modifying actions,30 whereas others finding no effect of histone deacetylase inhibition during epileptogenesis.31 Progress on targeting histones requires a better understanding of the influence of these epigenetic marks and a more extensive tool-set for their specific manipulation.
Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
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Non-coding RNAs have the advantage over the other two categories of epigenetic process because highly selective targeting is possible through antisense-based oligonucleotide inhibitors.32 A sequence of oligonucleotides complementary to a unique stretch of the RNA molecule can be designed and deployed, producing suppression of the actions of the noncoding RNA. Knock-down of the target RNA can be longlasting (e.g. over one month following a single injection). Excitingly, oligonucleotide therapies are now approved (e.g. nusinersen for spino-muscular atrophy33). RNA therapeutics have been highly effective in preclinical studies targeting microRNAs, with over a dozen different microRNAs now having been inhibited using this approach and in several cases effects on evoked or spontaneous seizures have been reported.34 Long noncoding RNAs are also targetable using a similar approach. An exciting recent study used oligonucleotide inhibitors of a NAT long noncoding RNA to upregulate expression of the SCN1A mRNA.35 The human SCN1A gene locus expresses a NAT which functions to limit SCN1A proteins levels in cells. Introduction of an oligonucleotide targeting the SCN1A-NAT was reported to increase mRNA levels of SCN1A in cell models and protect against hyperthermic seizures in a Dravet mouse model.35 The oligonucleotide has entered clinical trials for Dravet syndrome. It is likely that other genes could be upregulated in diseases of haploinsufficiency. Given the extensive class of long-noncoding RNAs that are yet to be targeted in epilepsy, RNA therapeutic-based strategies are likely to be of strong interest in the treatment of epilepsy in the future.32,33 The challenge, however, is delivery. Large oligonucleotides do not pass across the blood brain barrier meaning that therapeutic delivery must circumvent this problem. For example, by intra-thecal injection. In our laboratory, we recently tested systemic delivery of an oligonucleotide inhibitor of a microRNA timed with disruption of the bloodebrain barrier after status epilepticus. We found that the large molecule did pass into brain tissue, particularly at sites of focal seizure activity, and produced similar anti-seizure effects to when it had been given intracerebroventricularly (unpublished data).
4. What does the future hold for epigenetic therapies in epilepsy? The development of epigenetic-based therapies remains in its infancy but already we can see exciting opportunities emerging. What might be possible in the near future? If we take DNA methylation, the key limitation currently is specificity. A solution may be at hand. Work by Jaenisch's group showed that locus-specific hyper- or de-methylation is possible. The team used a CRISPR-based system to bring either a methylating enzyme or de-methylating enzyme to a gene locus and showed they could enhance methylation or reduce methylation in a specific manner.36 The applications of this approach are clear. If a gene were epigenetically silenced by DNA methylation, you could increase its expression using this approach, wiping clean the methylation mark that suppressed expression. The RELN gene might be an example for proof-of-concept. Kobow and colleagues showed the RELN gene to be hyper-methylated in
hippocampus from patients with granule cell dispersion.37 The CRISPR-based approach could, in principal, bring about de-methylation of the promoter, switching the gene back on and restoring production of the Reelin protein. This could help reduce pathological granule cell dispersion in patients with intractable epilepsy. Applying de-methylation to reactivate otherwise silenced ion channels could also be of therapeutic benefit. While the study by Liu and colleagues reported high efficiency of their CRISPR-based DNA methylation editing technique in vitro and in vivo (including in mouse brain) and high specificity (with minimal changes to methylation in non-targeted regions), the duration of the effects were uncertain. More recent work by the same group has shown that re-activation of FMR1 (loss of which causes Fragile X syndrome/FXS) using CRISPR-based demethylation lasts for at least two weeks.38 Further optimisation may be needed if the longevity of CRISPR-based editing is limited by the endogenous machinery for applying and erasing DNA methylation marks. Finally, what might be possible in the future in the area of RNA therapeutics targeting noncoding RNAs? We and our collaborators recently showed that a potassium channel (encoded by Kv4.2) could be upregulated by targeting the microRNA (miR-324-5p) that was normally bound to the 30 UTR.39 Injection of an oligonucleotide targeting the microRNA increased surface expression of the potassium channel and reduced excitability. Seizures and hippocampal damage were also reduced in mice given an inhibitor of the microRNA. This approach could offer numerous other opportunities for therapies in epilepsy. In theory, any gene whose haploinsufficiency caused epilepsy could be treated in a similar way. A microRNA that strongly suppresses the transcript can be identified (for example using iCLIP data40). A microRNA inhibitor could be designed to block the microRNA and used to upregulate the previously repressed gene. And microRNAs are really just the tip of the iceberg. RNA-based therapeutics offer the ability to selectively manipulate many genes, including down-regulating gain-of-function mutated transcripts.32 As we continue to uncover the roles of noncoding RNAs in the future, further opportunities may arise for the development of novel and disease-modifying therapies for epilepsy.
Conflicts of interest This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number 16/RC/3948 and co-funded under the European Regional Development Fund and by FutureNeuro industry partners. Epigenetics and noncoding RNA studies in the author’s laboratory were also supported by SFI grants 11/ TIDA/B1988 and 13/IA/1891, the European Union’s ‘Seventh Framework’ Programme (FP7) under Grant Agreement No. 602130 (EpimiRNA), Health Research Board HRA-POR-2013325, Epilepsy Ireland/Medical Research Charities Group (20169, 2011/7) and Charitable Infirmary Charitable Trust (Ireland). The author holds US patent No. US 9,803,200 B2 “Inhibition of microRNA-134 for the treatment of seizure-related disorders and neurologic injuries”.
Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
european journal of paediatric neurology xxx (xxxx) xxx
Acknowledgments The author would like to thank Gary P. Brennan for helpful comments on the overview of epigenetics figure, to his colleagues who have contributed to the field, and apologises to those whose work could not be cited due to space limitations.
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Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
Official Journal of the European Paediatric Neurology Society
Review article
Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities David C. Henshall a,b,* a b
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland FutureNeuro Research Centre, RCSI, Dublin, Ireland
abstract Keywords:
Acquired and genetic forms of epilepsy are associated with dysregulation of gene expression
DNA methylation
within the brain. Identifying mechanisms controlling gene expression may provide novel
Histone post-translational modifi-
opportunities for the development of disease-modifying therapies. Epigenetic processes in-
cation
fluence the medium-to long-term readability and accessibility of the genome to transcription.
Noncoding RNA
The mediators include biochemical modifications to DNA and the histones around which
Epileptogenesis
DNA is wrapped, as well as non-coding RNAs. Here, the main epigenetic processes are briefly
Hippocampal sclerosis
reviewed. Examples are provided of altered epigenetic processes and mutations in genes with
Antisense oligonucleotides
epigenetic functions in experimental models and human epilepsy. The outcome of recent
Gene therapy
studies manipulating epigenetics to protect the brain or reduce seizures in preclinical models is considered, with specific focus on RNA therapies. Last, future applications of epigenetics research are appraised, including opportunities to map and change epigenetic marks and the prospects for therapies based on manipulation of epigenetics and noncoding RNAs. In summary, epigenetics and noncoding RNAs are important mechanisms and targets to modulate brain excitability which together provides novel insight into patho-mechanisms, biomarkers and novel therapies for epilepsy. © 2019 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4.
Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epigenetic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Epigenetic genes and epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manipulating epigenetics in epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . What does the future hold for epigenetic therapies in epilepsy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
00 00 00 00 00 00
* Corresponding author. Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin, D02 YN77, Ireland. E-mail address: [email protected]. https://doi.org/10.1016/j.ejpn.2019.06.002 1090-3798/© 2019 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
2
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.
Background
The brain derives its structure and function from the temporally-controlled, cell-specific expression of over 15,000 genes.1 Many genes are uniquely expressed in neurons and glia (as well as other cells of the neurovascular unit - endothelial cells of the bloodebrain barrier (BBB), myocytes and pericytes). Their products bestow a myriad of properties, such as the ability to generate action potentials (neurons) or perform support roles such as uptake of released neurotransmitter (e.g. astrocytes). Gene expression begins with recruitment of factors to gene promoters followed by actions of RNA polymerases to generate a messenger RNA (mRNA). The transcripts then undergo splicing, editing and are eventually translated to protein (Fig. 1). Epigenetics and certain noncoding RNAs perform critical roles in shaping the gene expression landscape in the brain and important roles have emerged in both acquired and genetic forms of epilepsy.2,3 Manipulating epigenetic processes
represents, therefore, a novel approach to the treatment of epilepsy. A modern, operational definition of epigenetics covers the biochemical changes (“epigenetic marks”) that influence chromatin structure in a way that determines the readability (accessibility) of the genome to transcription.4,5 DNA is normally highly condensed within the nucleus, wrapped around octamers of histone proteins (forming nucleosomes). Highly condensed chromatin (“heterochromatin”) is not, however, accessible for transcription. Thus, in order for a gene to be transcribed, the chromatin must be in an “open” state (termed “euchromatin”). Epigenetic processes drive the transitions between these two states, thereby serving to control the gene expression landscape typically over the medium to long-term.4,6
2.
Epigenetic processes
There are at least five distinct epigenetic process,4,7 but for the purposes of this review, we focus on three of the major sub-
Fig. 1 e Overview of how epigenetic mechanisms affect the gene expression process and potential approaches to therapeutic targeting in epilepsy. Cartoon shows the gene expression pathway that begins with promoter elements driving transcription of a protein-coding gene. This results in production of messenger RNA (mRNA) copies which then transit to the cytoplasm for translation within the ribosome resulting in protein production. Among various epigenetic mechanisms affecting this pathway, methylation of DNA (top left, dark green), particularly around gene promoter elements, reduces transcription. Approaches to manipulation (e.g. to reactivate a silenced gene) could include use of DNA methylation inhibitors or a CRISPR-based approach could deliver a de-methylating enzyme to a specific promoter. Histones (bottom left, dark blue) comprise an octamer of proteins around which DNA is wrapped. Posttranslational modifications alter how closely packed these units are, promoting or repressing a transcriptionally permissive state by altering the open or closed state of chromatin. A variety of small molecule histone deacetylase inhibitors have been developed that could be used to activate gene expression. Long noncoding RNAs (middle, dark brown) are a diverse class of transcripts which can affect transcription, for example by acting as natural antisense transcripts (NAT) that block the protein-coding transcript. These NATs can be targeted (and thus their target gene upregulated) using oligonucleotides. MicroRNAs (miRNAs; top right, orange) are small noncoding RNAs that mainly work by directly binding the 3′ untranslated region (UTR) of protein-coding transcripts in the cytoplasm and promoting mRNA degradation or translation inhibition. Blocking certain miRNAs can upregulate their targets and have anti-seizure effects. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
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categories: DNA methylation, post-translational modifications of histones and the functions of certain noncoding RNAs (and see Fig. 1). DNA methylation refers to the addition of a methyl group to a cytosine base, a reaction catalyzed by enzymes called DNA methyltransferases.8 Heavily methylated DNA invariably promotes condensation of chromatin and thus is normally associated with transcriptional silencing (although DNA methylation has been associated with other effects including promoting transcription).6 Histones undergo various posttranslational modifications, including phosphorylation, which serve to increase or decrease the permissiveness of a locus for transcription.9 One of the best understood modifications is acetylation, which, due to the addition of electrostatic charges, tends to cause histones to move apart and thus favour transcription. Finally, noncoding RNAs, of which two major sub-groups (long and short), are recognized. Numerous long noncoding RNAs have been linked to structuring chromatin and regulating transcription.10 These include enhancer RNAs and natural antisense transcripts (NATs).11 The best understood short RNAs are microRNAs, which target mRNAs to lower (buffer) protein levels.12 MostmicroRNAs do not, however, function in an epigenetic manner (i.e. acting to modify the biochemical and structural properties of chromatin). Rather, the role of microRNAs is within the cytoplasm at a step immediately prior to translation as part of the post-transcriptional machinery that regulates protein levels (Fig. 1). Some of their targets, however, may have direct epigenetic functions.
2.1.
Epigenetic genes and epilepsy
Since the discovery, in 1999, that mutations in the MECP2 gene caused Rett syndrome,13 a neurodevelopment disorder characterized by seizures, numerous genes whose products are involved in epigenetics have been linked to epilepsy. This includes BRD2 (a reader of histone marks),14 and CHD2 (a chromatin remodeler).15 This should not be altogether surprising. Any insult that disrupts the gene expression landscape by leading to genes being inappropriately on or off might be expected to disrupt neuronal network stability. The brain expresses an extensive complement of genes with epigenetic functions. In situ hybridization (see resources such as the Allen brain atlas) and single cell sequencing approaches,16 reveal particular enrichment in neurons compared to glia, including the molecular machinery for adding and removing DNA methylation and dozens of histones and histoneregulatory factors. There is strong evidence that all three epigenetic processes are altered in experimental and human epilepsy. DNA methylation patterns have been mapped at a global level in resected brain tissue from patients with focal epilepsy as well as in animal models.17e21 This has shown epilepsy to be associated with hypermethylation of numerous genes, indicating a restriction in transcription. For some genes that displayed hyper-methylation the tissue had correspondingly lower transcript levels. This would be consistent with a transcriptional-silencing influence of the epigenetic mark. For many other genes tested, however, methylation status was weakly associated with abundance of the corresponding transcript, meaning hyper-methylation was not necessarily
3
causing gene silencing. This raises questions of the importance of DNA methylation marks in regulating genes that control hyperexcitability and the need to individually validate whether a particular pattern of methylation affects transcription of a given gene.17 Another notable finding in relation to DNA methylation was the relative lack of overlap in DNA methylation patterns between epileptic animals which had epilepsy triggered by different methods (e.g. when comparing pilocarpine to traumatic brain injury).22 This may have diagnostic applications, enabling the cause of epilepsy to be deduced by an analysis of the DNA methylation profile in brain. Histone posttranslational modifications have been reported by several studies after experimental seizures, although there has been little reported for human epilepsy to date.23,24 Assessments of histone marks have been quite limited in scale, and the application of genome-wide histone mark-based sequencing should be undertaken to properly assess the role of these marks.25 Finally, changes to microRNA expression in experimental and human epilepsy have been extensively documented and, increasingly, there are reports of changes to long noncoding RNAs in experimental and human epilepsy.26,27
3.
Manipulating epigenetics in epilepsy
All three epigenetic processes are targetable using pharmacologic, genetic and oligonucleotide-based approaches (see Fig. 1).2,28 DNA methylation is a potentially attractive target because the marks are medium-to-long lasting, providing a means to impose a lasting change on gene expression. Studies to date, however, have been limited by the rather blunt instruments available; broad-spectrum DNA methyltransferase inhibitors such as 5-azacytidine. There have been reports of anti-seizure effects of this class of DNA methylation inhibitor in animal models of epilepsy,19 which is promising for the future. However, contrasting anti-seizure actions of enhancing DNA methylation by other groups suggest hypermethylation of DNA may also serve anti-excitability effects.20 Ultimately, more specific tools which can target DNA methylation at specific gene loci are needed. Targeting histone modifications may also enable the up- or down-regulation of a specific gene. The expected time course of action would be in the short-to medium-term. Similar to DNA methylation, however, there are few tools to selectively induce or remove histone marks. The main category of pharmacologic tool that has been used is histone deacetylase inhibitors (HDAC inhibitors).28 Treatment with a HDAC inhibitor would tend to promote transcription of genes since removal of acetylation favours silencing. A number of HDAC inhibitors have been tested in clinical trials and, notably, the common anti-epileptic drug sodium valproate has HDAC inhibitory activity. However, studies suggest the anti-seizure effects of valproate are unrelated to HDAC inhibition.29 Moreover, mixed results have emerged from studies of HDAC inhibitors on epileptogenesis, with some reporting disease-modifying actions,30 whereas others finding no effect of histone deacetylase inhibition during epileptogenesis.31 Progress on targeting histones requires a better understanding of the influence of these epigenetic marks and a more extensive tool-set for their specific manipulation.
Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
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Non-coding RNAs have the advantage over the other two categories of epigenetic process because highly selective targeting is possible through antisense-based oligonucleotide inhibitors.32 A sequence of oligonucleotides complementary to a unique stretch of the RNA molecule can be designed and deployed, producing suppression of the actions of the noncoding RNA. Knock-down of the target RNA can be longlasting (e.g. over one month following a single injection). Excitingly, oligonucleotide therapies are now approved (e.g. nusinersen for spino-muscular atrophy33). RNA therapeutics have been highly effective in preclinical studies targeting microRNAs, with over a dozen different microRNAs now having been inhibited using this approach and in several cases effects on evoked or spontaneous seizures have been reported.34 Long noncoding RNAs are also targetable using a similar approach. An exciting recent study used oligonucleotide inhibitors of a NAT long noncoding RNA to upregulate expression of the SCN1A mRNA.35 The human SCN1A gene locus expresses a NAT which functions to limit SCN1A proteins levels in cells. Introduction of an oligonucleotide targeting the SCN1A-NAT was reported to increase mRNA levels of SCN1A in cell models and protect against hyperthermic seizures in a Dravet mouse model.35 The oligonucleotide has entered clinical trials for Dravet syndrome. It is likely that other genes could be upregulated in diseases of haploinsufficiency. Given the extensive class of long-noncoding RNAs that are yet to be targeted in epilepsy, RNA therapeutic-based strategies are likely to be of strong interest in the treatment of epilepsy in the future.32,33 The challenge, however, is delivery. Large oligonucleotides do not pass across the blood brain barrier meaning that therapeutic delivery must circumvent this problem. For example, by intra-thecal injection. In our laboratory, we recently tested systemic delivery of an oligonucleotide inhibitor of a microRNA timed with disruption of the bloodebrain barrier after status epilepticus. We found that the large molecule did pass into brain tissue, particularly at sites of focal seizure activity, and produced similar anti-seizure effects to when it had been given intracerebroventricularly (unpublished data).
4. What does the future hold for epigenetic therapies in epilepsy? The development of epigenetic-based therapies remains in its infancy but already we can see exciting opportunities emerging. What might be possible in the near future? If we take DNA methylation, the key limitation currently is specificity. A solution may be at hand. Work by Jaenisch's group showed that locus-specific hyper- or de-methylation is possible. The team used a CRISPR-based system to bring either a methylating enzyme or de-methylating enzyme to a gene locus and showed they could enhance methylation or reduce methylation in a specific manner.36 The applications of this approach are clear. If a gene were epigenetically silenced by DNA methylation, you could increase its expression using this approach, wiping clean the methylation mark that suppressed expression. The RELN gene might be an example for proof-of-concept. Kobow and colleagues showed the RELN gene to be hyper-methylated in
hippocampus from patients with granule cell dispersion.37 The CRISPR-based approach could, in principal, bring about de-methylation of the promoter, switching the gene back on and restoring production of the Reelin protein. This could help reduce pathological granule cell dispersion in patients with intractable epilepsy. Applying de-methylation to reactivate otherwise silenced ion channels could also be of therapeutic benefit. While the study by Liu and colleagues reported high efficiency of their CRISPR-based DNA methylation editing technique in vitro and in vivo (including in mouse brain) and high specificity (with minimal changes to methylation in non-targeted regions), the duration of the effects were uncertain. More recent work by the same group has shown that re-activation of FMR1 (loss of which causes Fragile X syndrome/FXS) using CRISPR-based demethylation lasts for at least two weeks.38 Further optimisation may be needed if the longevity of CRISPR-based editing is limited by the endogenous machinery for applying and erasing DNA methylation marks. Finally, what might be possible in the future in the area of RNA therapeutics targeting noncoding RNAs? We and our collaborators recently showed that a potassium channel (encoded by Kv4.2) could be upregulated by targeting the microRNA (miR-324-5p) that was normally bound to the 30 UTR.39 Injection of an oligonucleotide targeting the microRNA increased surface expression of the potassium channel and reduced excitability. Seizures and hippocampal damage were also reduced in mice given an inhibitor of the microRNA. This approach could offer numerous other opportunities for therapies in epilepsy. In theory, any gene whose haploinsufficiency caused epilepsy could be treated in a similar way. A microRNA that strongly suppresses the transcript can be identified (for example using iCLIP data40). A microRNA inhibitor could be designed to block the microRNA and used to upregulate the previously repressed gene. And microRNAs are really just the tip of the iceberg. RNA-based therapeutics offer the ability to selectively manipulate many genes, including down-regulating gain-of-function mutated transcripts.32 As we continue to uncover the roles of noncoding RNAs in the future, further opportunities may arise for the development of novel and disease-modifying therapies for epilepsy.
Conflicts of interest This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number 16/RC/3948 and co-funded under the European Regional Development Fund and by FutureNeuro industry partners. Epigenetics and noncoding RNA studies in the author’s laboratory were also supported by SFI grants 11/ TIDA/B1988 and 13/IA/1891, the European Union’s ‘Seventh Framework’ Programme (FP7) under Grant Agreement No. 602130 (EpimiRNA), Health Research Board HRA-POR-2013325, Epilepsy Ireland/Medical Research Charities Group (20169, 2011/7) and Charitable Infirmary Charitable Trust (Ireland). The author holds US patent No. US 9,803,200 B2 “Inhibition of microRNA-134 for the treatment of seizure-related disorders and neurologic injuries”.
Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002
european journal of paediatric neurology xxx (xxxx) xxx
Acknowledgments The author would like to thank Gary P. Brennan for helpful comments on the overview of epigenetics figure, to his colleagues who have contributed to the field, and apologises to those whose work could not be cited due to space limitations.
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Please cite this article as: Henshall DC, Epigenetics and noncoding RNA: Recent developments and future therapeutic opportunities, European Journal of Paediatric Neurology, https://doi.org/10.1016/j.ejpn.2019.06.002