Altered circadian rhythms and oscillation of clock genes and sirtuin 1 in a model of sudden unexpected death in epilepsy.

OBJECTIVES: Circadian rhythms are affected in many neurological disorders. Although sleep disturbances are known in epilepsy, data on circadian rhythm disturbances in epilepsy are sparse. Here, we examined diurnal and circadian rest-activity and sleep-wake patterns in Kcna1-null mice, which exhibit spontaneous recurrent seizures and are a model of sudden unexpected death in epilepsy. Furthermore, we sought to determine whether seizures or aberrant oscillation of core clock genes and a regulator, sirtuin 1 (Sirt1), is associated with disrupted rhythms. METHODS: We used passive infrared actigraphy to assess rest-activity patterns, electroencephalography for seizure and sleep analysis, and reverse transcription polymerase chain reaction and Western blotting to evaluate expression of clock genes and Sirt1 in Kcna1-null and wild-type mice. RESULTS: Epileptic Kcna1-null animals have disrupted diurnal and circadian rest-activity patterns, tending to exhibit prolonged circadian periods. Electroencephalographic analysis confirmed disturbances in sleep architecture, with more time spent awake and less asleep. Although all epileptic mice manifested disrupted diurnal and circadian rest-activity patterns, we found no correlation between actual seizure burden and degree of sleep disruption. However, we found attenuated oscillations of several clock genes (ie, Clock, Bmal1, Per1, and Per2) and diurnal Sirt1 mRNA in the anterior hypothalamus. SIGNIFICANCE: Attenuated oscillation of several core clock genes correlates with, and may underlie, aberrant diurnal and circadian rest-activity and sleep-wake patterns observed in Kcna1-null mice. This could contribute to late complications in epilepsy, such as sudden unexpected death in epilepsy. Sirt1 may represent a useful therapeutic target for rescuing circadian clock gene rhythmicity and sleep patterns in epilepsy.

The transcription factor REST is lost in aggressive breast cancer.

The function of the tumor suppressor RE1 silencing transcription factor (REST) is lost in colon and small cell lung cancers and is known to induce anchorage-independent growth in human mammary epithelial cells. However, nothing is currently known about the role of this tumor suppressor in breast cancer. Here, we test the hypothesis that loss of REST function plays a role in breast cancer. To assay breast tumors for REST function, we developed a 24-gene signature composed of direct targets of the transcriptional repressor. Using the 24- gene signature, we identified a previously undefined RESTless breast tumor subtype. Using gene set enrichment analysis, we confirmed the aberrant expression of REST target genes in the REST-less tumors, including neuronal gene targets of REST that are normally not expressed outside the nervous system. Examination of REST mRNA identified a truncated splice variant of REST present in the REST-less tumor population, but not other tumors. Histological analysis of 182 outcome-associated breast tumor tissues also identified a subpopulation of tumors that lack full-length, functional REST and over-express the neuroendocrine marker and REST target gene Chromogranin A. Importantly, patients whose tumors were found to be REST-less using either the 24-gene signature or histology had significantly poorer prognosis and were more than twice as likely to undergo disease recurrence within the first 3 years after diagnosis. We show here that REST function is lost in breast cancer, at least in part via an alternative splicing mechanism. Patients with REST-less breast cancer undergo significantly more early disease recurrence than those with fully functional REST, regardless of estrogen receptor or HER2 status. Importantly, REST status may serve as a predictor of poor prognosis, helping to untangle the heterogeneity inherent in disease course and response to treatment. Additionally, the alternative splicing observed in REST-less breast cancer is an attractive therapeutic target.

Profound seizure suppression and disease modification by targeting JAK1, a key driver of a pro-epileptogenic gene network.

Epilepsy is the 4th most prevalent neurological disorder with over 50 million cases worldwide. While a number of drugs exist to suppress seizures, approximately 1/3 of patients remain drug resistant, and no current treatments are disease modifying. Using network and systems-based approaches, we find that the histone methylase EZH2 suppresses epileptogenesis and slows disease progression, via repression of JAK1 and STAT3 signaling in hippocampal neurons. Pharmacological inhibition of JAK1 with the orally available, FDA-approved drug CP690550 (Tofacitinib) profoundly suppresses behavioral and electrographic seizures after the onset of epilepsy across preclinical rodent models of acquired epilepsy. This seizure suppression persists for weeks after drug withdrawal. Identification of an endogenous protective response to status epilepticus in the form of EZH2 induction has highlighted a critical role for the JAK1 kinase and STAT3 in both the initiation and propagation of epilepsy across preclinical rodent models and human disease. Overall, we find that STAT3 is transiently activated after insult, reactivates with spontaneous seizures, and remains targetable for disease modification in chronic epilepsy.

2-Deoxy-D-glucose reduces epilepsy progression by NRSF-CtBP-dependent metabolic regulation of chromatin structure.

Temporal lobe epilepsy is a common form of drug-resistant epilepsy that sometimes responds to dietary manipulation such as the 'ketogenic diet'. Here we have investigated the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in the rat kindling model of temporal lobe epilepsy. We show that 2DG potently reduces the progression of kindling and blocks seizure-induced increases in the expression of brain-derived neurotrophic factor and its receptor, TrkB. This reduced expression is mediated by the transcription factor NRSF, which recruits the NADH-binding co-repressor CtBP to generate a repressive chromatin environment around the BDNF promoter. Our results show that 2DG has anticonvulsant and antiepileptic properties, suggesting that anti-glycolytic compounds may represent a new class of drugs for treating epilepsy. The metabolic regulation of neuronal genes by CtBP will open avenues of therapy for neurological disorders and cancer.

Histone deacetylase inhibitors restore normal hippocampal synaptic plasticity and seizure threshold in a mouse model of Tuberous Sclerosis Complex.

Abnormal synaptic plasticity has been implicated in several neurological disorders including epilepsy, dementia and Autism Spectrum Disorder (ASD). Tuberous Sclerosis Complex (TSC) is an autosomal dominant genetic disorder that manifests with seizures, autism, and cognitive deficits. The abnormal intracellular signaling underlying TSC has been the focus of many studies. However, nothing is known about the role of histone modifications in contributing to the neurological manifestations in TSC. Dynamic regulation of chromatin structure via post translational modification of histone tails has been implicated in learning, memory and synaptic plasticity. Histone acetylation and associated gene activation plays a key role in plasticity and so we asked whether histone acetylation might be dysregulated in TSC. In this study, we report a general reduction in hippocampal histone H3 acetylation levels in a mouse model of TSC2. Pharmacological inhibition of Histone Deacetylase (HDAC) activity restores histone H3 acetylation levels and ameliorates the aberrant plasticity in TSC2+/- mice. We describe a novel seizure phenotype in TSC2+/- mice that is also normalized with HDAC inhibitors (HDACis). The results from this study suggest an unanticipated role for chromatin modification in TSC and may inform novel therapeutic strategies for TSC patients.

A systems approach identifies Enhancer of Zeste Homolog 2 (EZH2) as a protective factor in epilepsy.

Complex neurological conditions can give rise to large scale transcriptomic changes that drive disease progression. It is likely that alterations in one or a few transcription factors or cofactors underlie these transcriptomic alterations. Identifying the driving transcription factors/cofactors is a non-trivial problem and a limiting step in the understanding of neurological disorders. Epilepsy has a prevalence of 1% and is the fourth most common neurological disorder. While a number of anti-seizure drugs exist to treat seizures symptomatically, none is curative or preventive. This reflects a lack of understanding of disease progression. We used a novel systems approach to mine transcriptome profiles of rodent and human epileptic brain samples to identify regulators of transcriptional networks in the epileptic brain. We find that Enhancer of Zeste Homolog 2 (EZH2) regulates differentially expressed genes in epilepsy across multiple rodent models of acquired epilepsy. EZH2 undergoes a prolonged upregulation in the epileptic brain. A transient inhibition of EZH2 immediately after status epilepticus (SE) robustly increases spontaneous seizure burden weeks later. This suggests that EZH2 upregulation is a protective. These findings are the first to characterize a role for EZH2 in opposing epileptogenesis and debut a bioinformatic approach to identify nuclear drivers of complex transcriptional changes in disease.

Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST.

Loss of repressor element 1 silencing transcription factor (REST) occurs in 20% of breast cancers and correlates with a poor patient prognosis. However, the molecular basis for enhanced malignancy in tumors lacking REST (RESTless) is only partially understood. We used multiplatform array data from the Cancer Genome Atlas to identify consistent changes in key signaling pathways. Of the proteins screened in the reverse-phase protein array, we found that insulin receptor substrate 1 (IRS1) is the most highly upregulated protein in RESTless breast tumors. Analysis of breast tumor cell lines showed that REST directly represses IRS1, and cells lacking REST have increased levels of IRS1 mRNA and protein. We find that the upregulation of IRS1 function is both necessary and sufficient for enhanced signaling and growth in breast cancer cells lacking REST. IRS1 overexpression is sufficient to phenocopy the enhanced activation of the signaling hubs AKT and mitogen-activated protein kinase (MAPK) of MCF7 cells lacking REST. Loss of REST renders MCF7 and MDA-MB-231 breast tumor cells dependent on IRS1 activity for colony formation in soft agar. Inhibition of the type 1 insulin-like growth factor receptor (IGF1R) reduces the enhanced signaling, growth, and migration in breast tumor cells that occur upon REST loss. We show that loss of REST induces a pathogenic program that works through the IGF1R/IRS1 pathway.

Induction of the RNA regulator LIN28A is required for the growth and pathogenesis of RESTless breast tumors.

The transcription factor RE1 silencing transcription factor (REST) is lost in approximately 20% of breast cancers. Although it is known that these RESTless tumors are highly aggressive and include all tumor subtypes, the underlying tumorigenic mechanisms remain unknown. In this study, we show that loss of REST results in upregulation of LIN28A, a known promoter of tumor development, in breast cancer cell lines and human breast tumors. We found that LIN28A was a direct transcriptional target of REST in cancer cells and that loss of REST resulted in increased LIN28A expression and enhanced tumor growth both in vitro and in vivo, effects that were dependent on heightened LIN28A expression. Tumors lacking REST expression were locally invasive, consistent with the increased lymph node involvement observed in human RESTless tumors. Clinically, human RESTless breast tumors also displayed significantly enhanced LIN28A expression when compared with non-RESTless tumors. Our findings therefore show a critical role for the REST-LIN28A axis in tumor aggression and suggest a causative relationship between REST loss and tumorigenicity in vivo.

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of Apc(Min) (/+) ROSA11 Gene Trap Mice.

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in Apc(Min) (/+) mice. The insertion site of the ß-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors.

Localized domains of G9a-mediated histone methylation are required for silencing of neuronal genes.

Negative regulation of transcription is an important strategy in establishing and maintaining cell-specific gene expression patterns. Many neuronal genes are subject to active transcriptional repression outside the nervous system to establish neuronal specificity. NRSF/REST has been demonstrated to regulate at least 30 genes and contribute to their neuronal targeting by repressing transcription outside the nervous system. Further, human genome database searches reveal that over 800 genes contain an NRSE. Here we report that NRSF recruits the histone methylase G9a to silence NRSF target genes in nonneuronal cells. We show that G9a generates a highly localized domain of dimethylated histone H3-K9 around NRSEs, but H3-K27 remains unmethylated. The NRSEs are also associated with HP1. Finally, we demonstrate that dominant-negative G9a abrogates silencing of chromosomal neuronal genes. These findings implicate a role for histone methylation in targeting neuronal gene expression to the nervous system.