Overexpression of REST Represses the Epithelial-Mesenchymal Transition Process and Decreases the Aggressiveness of Prostate Cancer Cells.

The RE-1 silencing transcription factor (REST) is a repressor factor related to neuroendocrine prostate cancer (PCa) (NEPC), a poor prognostic stage mainly associated with castration-resistant PCa (CRPC). NEPC is associated with cell transdifferentiation and the epithelial-mesenchymal transition (EMT) in cells undergoing androgen deprivation therapy (ADT) and enzalutamide (ENZ). The effect of REST overexpression in the 22rv1 cell line (xenograft-derived prostate cancer) on EMT, migration, invasion, and the viability for ENZ was evaluated. EMT genes, Twist and Zeb1, and the androgen receptor (AR) were evaluated through an RT-qPCR and Western blot in nuclear and cytosolic fractions of REST-overexpressing 22rv1 cells (22rv1-REST). The migratory and invasive capacities of 22rv1-REST cells were evaluated via Transwell® assays with and without Matrigel, respectively, and their viability for enzalutamide via MTT assays. The 22rv1-REST cells showed decreased nuclear levels of Twist, Zeb1, and AR, and a decreased migration and invasion and a lower viability for ENZ compared to the control. Results were expressed as the mean + SD of three independent experiments (Mann-Whitney U test, Kruskal-Wallis, Tukey test). REST behaves like a tumor suppressor, decreasing the aggressiveness of 22rv1 cells, probably through the repression of EMT and the neuroendocrine phenotype. Furthermore, REST could represent a response marker to ENZ in PCa patients.

RCOR1 improves neurobehaviors and neuron injury in rat cerebral palsy by Endothelin-1 targeting-induced Akt/GSK-3ß pathway upregulation.

BACKGROUND: RE1 Silencing Transcription factor (REST) corepressor 1 (RCOR1) has been reported to orchestrate neurogenesis, while its role in cerebral palsy (CP) remains elusive. Besides, RCOR1 can interact with Endothelin-1 (EDN1), and EDN1 expression is related to brain damage. Therefore, this study aimed to explore the effects of RCOR1/EDN1 on brain damage during the progression of CP. METHODS: CP rats were established via hypoxia-ischemia insult, and injected with lentivirus-RCOR1, followed by examination of brain pathological conditions. The RCOR1 and EDN1 interaction was recognized using hTFtarget. Healthy rat cortical neuron cells received interference of RCOR1/EDN1 expression, and underwent oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, after which phenotypic and molecular assays were conducted through the biochemical method, qRT-PCR and/or western blot. RESULTS: RCOR1 was low-expressed but EDN1 was high-expressed in CP model rats and OGD/R-treated neurons. RCOR1 overexpression ameliorated rat neurobehaviors, alleviated brain pathological conditions, reduced TUNEL-positive cells, decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), increased superoxide dismutase (SOD) level and repressed EDN1 expression in the brains of CP model rats. In neurons, RCOR1 overexpression counteracted OGD/R-induced viability decrease, reduction of the levels of RCOR1, SOD, Bcl-2, caspase-3, p-Akt/Akt and p-GSK-3ß/GSK-3ß, and elevation of the levels of EDN1, ROS, Bax, and cleaved caspase-3, while EDN1 overexpression did contrarily on these events. Moreover, there was a negative interplay between RCOR1 overexpression and EDN1 overexpression in OGD/R-induced neurons. CONCLUSION: RCOR1 ameliorates neurobehaviors and suppresses neuronal apoptosis and oxidative stress in CP through EDN1 targeting-mediated upregulation of Akt/GSK-3ß.

Ablation of placental REST deregulates fetal brain metabolism and impacts gene expression of the offspring brain at the postnatal and adult stages.

In this study, the transcriptional repressor REST (Repressor Element 1 Silencing Transcription factor) was ablated in the mouse placenta to investigate molecular and cellular impacts on the offspring brain at different life stages. Ablation of placental REST deregulated several brain metabolites, including glucose and lactate that fuel brain energy, vitamin C (ascorbic acid) that functions in the epigenetic programming of the brain during postnatal development, and glutamate and creatine that help the brain to respond to stress conditions during adult life. Bulk RNA-seq analysis showed that a lack of placental REST persistently altered multiple transport genes, including those related to oxygen transportation in the offspring brain. While metabolic genes were impacted in the postnatal brain, different stress response genes were activated in the adult brain. DNA methylation was also impacted in the adult brain due to the loss of placental REST, but in a sex-biased manner. Single-nuclei RNA-seq analysis showed that specific cell types of the brain, particularly those of the choroid plexus and ependyma, which play critical roles in producing cerebrospinal fluid and maintaining metabolic homeostasis, were significantly impacted due to the loss of placental REST. These cells showed significant differential expression of genes associated with the metabotropic (G coupled protein) and ionotropic (ligand-gated ion channel) glutamate receptors, suggesting an impact of ablation of placental REST on the glutamatergic signaling of the offspring brain. The study expands our understanding of placental influences on the offspring brain.

Conditional knockout of REST/NRSF in excitatory neurons reduces seizure susceptibility to chemical kindling.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is an epigenetic master regulator that plays a crucial role during nervous system development and maturation. REST function was originally described during development, where it determines neuronal phenotype. However, recent studies showed that REST participates in several processes in the adult brain, including neuronal plasticity and epileptogenesis. In this regard, the relationships between REST and epilepsy are still controversial and need further investigation. As forebrain excitatory neurons are the common final pathway of seizure susceptibility, we investigated the role of REST in epilepsy by inducing REST conditional knockout (REST-cKO) specifically in excitatory neurons of the hippocampus. To target the excitatory neuronal population, we cloned the calcium/calmodulin-dependent protein kinase IIα minimal promoter upstream of Cre recombinase. After assessing the specificity of the promoter's expression, the transgenes were packaged in an engineered adeno-associated virus able to cross the blood-brain and blood-cerebrospinal fluid barriers and delivered in the lateral ventricles of 2-month-old RESTflox/flox mice to characterize, after 1 month, the cognitive phenotype and the seizure propensity. We show that REST-cKO mice display lower levels of anxiety in the light-dark test with respect to control mice but have unaltered motor, social, and cognitive profiles. The evaluation of the susceptibility to epileptic seizures showed that REST-cKO mice are more resistant to pentylenetetrazole-induced kindling but not to seizures induced by a single administration of the convulsant and show higher survival rates. Overall, these data suggest that the absence of REST in forebrain excitatory neurons decreases seizure susceptibility, pointing to a pro-epileptogenic role of the transcriptional repressor under conditions of pathological excitation/inhibition imbalance.

REST-restrained lncRNA EPB41L4A-AS2 Modulates Laryngeal Squamous Cell Carcinoma Development via Regulating miR-1254/HIPK2 Pathway.

BACKGROUND: LncRNAs have been corroborated to exert crucial effects in malignancies, including laryngeal squamous cell carcinoma (LSCC). Nevertheless, the role and mechanism of EPB41L4A- AS2 in LSCC are inadequately investigated and warrant further exploration. METHODS: Relevant database was adopted to analyze the relationship between EPB41L4A-AS2 expression level and tumors. The expressions and relationships of EPB41L4A-AS2, RE-1 silencing transcription factor (REST), miR-1254, and homeodomain interacting protein kinase 2 (HIPK2) in LSCC cells were evaluated by qRT-PCR, Pearson's correlation tests, RNA immunoprecipitation, RNA pull-down assay, chromatin immunoprecipitation, database, and dual-luciferase reporter assay. Following the required transfection, the biological behaviors of LSCC cells were examined using cell function experiments. Meanwhile, the levels of Ki-67 and apoptosis-, and epithelial-mesenchymal transition (EMT) pathway-related proteins were quantified with Western blot. Moreover, xenografts in nude mice were constructed, and the tumor volume and weight were measured. Ki-67 positivity was determined by immunohistochemical staining. RESULTS: EPB41L4A-AS2 and HIPK2 were lower-expressed, yet miR-1254 and REST were higher- expressed in LSCC cells. Pearson's correlation assay results exhibited a positive correlation between HIPK2 and EPB41L4A-AS2 and a negative correlation between HIPK2 and miR-1254. Overexpressed EPB41L4A-AS2 diminished the biological behavior, and repressed the levels of Ki-67 and EMT-related markers in LSCC cells whilst enhancing those of apoptosis-related markers. These aforementioned effects were counteracted by miR-1254 mimic. Moreover, EPB41L4A- AS2 overexpression suppressed the growth of tumors and reduced the positive expression of Ki-67 in nude mice. Besides, miR-1254 aggravated the biological behaviors and elevated the levels of Ki-67 and EMT-related proteins in LSCC cells while reducing the levels of apoptosis-related markers via targeting HIPK2. CONCLUSION: REST-restrained EPB41L4A-AS2 modulates LSCC development via regulating miR-1254/HIPK2 pathway.

Case Report: Two clinical cases of Wilms tumor comorbid to gingival fibromatosis in young children with constitutionally mutated REST.

Introduction: Nephroblastoma (Wilms tumor (WT)) is an embryonal tumor accounting for >90% of pediatric renal cancers. About 10% of WTs harbor pathogenic germline mutations. The REST gene, classified as a putative tumor suppressor, is affected in 2% of WTs. High-throughput molecular methods facilitate advanced diagnostics of cancer. In addition to this, germline mutations in REST are also associated with familial gingival fibromatosis (GFM). Reciprocally, none of the articles on RESTmut WT mentions GFM as a comorbid condition. This report provides unique evidence on the WT-GFM comorbidity in RESTmut carriers. Case presentation: Patient 1 (a 5-year-old boy with unilateral WT) is a proband, who has two healthy siblings. Patient 2 (a 4-year-old girl with bilateral WT) is a proband from in vitro fertilization (IVF) triplets, with a sister and brother without WT. We analyzed probands' DNA extracted from peripheral blood leucocytes with a custom-targeted next-generation sequencing (NGS)-198 gene panel. The detected variants were checked in family members by Sanger sequencing. Patient 1 had a pathogenic germline mutation in REST: c.1035_1036insTA, p.(E346*), as did his mother and both brothers. There were two other WT cases in this family (proband's maternal uncles). Patient 2 had a pathogenic germline variant in REST: c.2668_2671del, p.(E891Pfs*6), as well as her sister. The mutation was probably inherited from their deceased father, as he had gingival fibromatosis. Family members with REST mutations from both families had gingival fibromatosis. A somatic REST c.663C>A p.C221* mutation was identified in one patient with WT. At the moment both patients with WT are under dynamic observation without signs of the disease. Conclusion: Here, we describe two clinical cases of WT in nonrelated young children with germline-inactivating REST variants identified by next-generation sequencing. Both patients present with familial gingival fibromatosis, regarded as clinically useful comorbidity indicative of the tumor predisposition syndrome. The two cases illustrate Wilms tumor-gingival fibromatosis comorbidity in carriers of germline-inactivated REST alleles previously identified as a predisposition factor for both conditions.

DNA methylation alterations at RE1-silencing transcription factor binding sites and their flanking regions in cancer.

BACKGROUND: DNA methylation changes, frequent early events in cancer, can modulate the binding of transcription factors. RE1-silencing transcription factor (REST) plays a fundamental role in regulating the expression of neuronal genes, and in particular their silencing in non-neuronal tissues, by inducing chromatin modifications, including DNA methylation changes, not only in the proximity of its binding sites but also in the flanking regions. REST has been found aberrantly expressed in brain cancer and other cancer types. In this work, we investigated DNA methylation alterations at REST binding sites and their flanking regions in a brain cancer (pilocytic astrocytoma), two gastrointestinal tumours (colorectal cancer and biliary tract cancer) and a blood cancer (chronic lymphocytic leukemia). RESULTS: Differential methylation analyses focused on REST binding sites and their flanking regions were conducted between tumour and normal samples from our experimental datasets analysed by Illumina microarrays and the identified alterations were validated using publicly available datasets. We discovered distinct DNA methylation patterns between pilocytic astrocytoma and the other cancer types in agreement with the opposite oncogenic and tumour suppressive role of REST in glioma and non-brain tumours. CONCLUSIONS: Our results suggest that these DNA methylation alterations in cancer may be associated with REST dysfunction opening the enthusiastic possibility to develop novel therapeutic interventions based on the modulation of this master regulator in order to restore the aberrant methylation of its target regions into a normal status.

Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

Cross-Talk Between Histone Methyltransferases and Demethylases Regulate REST Transcription During Neurogenesis.

The RE1 Silencing Transcription Factor (REST) is a major regulator of neurogenesis and brain development. Medulloblastoma (MB) is a pediatric brain cancer characterized by a blockade of neuronal specification. REST gene expression is aberrantly elevated in a subset of MBs that are driven by constitutive activation of sonic hedgehog (SHH) signaling in cerebellar granular progenitor cells (CGNPs), the cells of origin of this subgroup of tumors. To understand its transcriptional deregulation in MBs, we first studied control of Rest gene expression during neuronal differentiation of normal mouse CGNPs. Higher Rest expression was observed in proliferating CGNPs compared to differentiating neurons. Interestingly, two Rest isoforms were expressed in CGNPs, of which only one showed a significant reduction in expression during neurogenesis. In proliferating CGNPs, higher MLL4 and KDM7A activities opposed by the repressive polycomb repressive complex 2 (PRC2) and the G9A/G9A-like protein (GLP) complex function allowed Rest homeostasis. During differentiation, reduction in MLL4 enrichment on chromatin, in conjunction with an increase in PRC2/G9A/GLP/KDM7A activities promoted a decline in Rest expression. These findings suggest a lineage-context specific paradoxical role for KDM7A in the regulation of Rest expression in CGNPs. In human SHH-MBs (SHH-α and SHH-ß) where elevated REST gene expression is associated with poor prognosis, up- or downregulation of KDM7A caused a significant worsening in patient survival. Our studies are the first to implicate KDM7A in REST regulation and in MB biology.

The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

Comprehensive Analysis of REST/NRSF Gene in Glioma and Its ceRNA Network Identification.

We sought to clarify the clinical relationship between REST/NRSF expression and the prognosis of glioma and explore the REST-associated competitive endogenous RNA (ceRNA) network in glioma. We downloaded RNA-seq, miRNA-seq and correlated clinical data of 670 glioma patients from The Cancer Genome Atlas and analyzed the correlation between REST expression, clinical characteristics and prognosis. Differentially expressed genes (DEGs) were identified with DESeq2 and analyzed with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) using the Profiler package. Starbase was used to explore the regulatory interaction between REST and miRNAs or LncRNAs. The lncRNA-miRNA-REST ceRNA network was constructed with Cytoscape. RT-qPCR, WB, CCK8, wound-healing, and luciferase assays were performed to validate the ceRNA network. Results showed that REST expression was significantly higher in glioma patients than normal samples. Higher REST expression was significantly associated with worse overall survival, progression-free interval, and worse disease-specific survival in glioma patients. The DEGs of mRNA, miRNA, and lncRNA were identified, and GO and KEGG enrichment analyses were performed. Finally, REST-associated ceRNA networks, including NR2F2-AS1-miR129-REST and HOTAIRM1-miR137-REST, were experimentally validated. Thus, REST may be a prognostic biomarker and therapeutic target in glioma, and its regulatory network validated in this study may provide insights into glioma's molecular regulatory mechanisms.

Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity.

Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte-neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein-binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte-neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.

A Monoallelic Variant in REST Is Associated with Non-Syndromic Autosomal Dominant Hearing Impairment in a South African Family.

Hearing impairment (HI) is a sensory disorder with a prevalence of 0.0055 live births in South Africa. DNA samples from a South African family presenting with progressive, autosomal dominant non-syndromic HI were subjected to whole-exome sequencing, and a novel monoallelic variant in REST [c.1244GC; p.(C415S)], was identified as the putative causative variant. The co-segregation of the variant was confirmed with Sanger Sequencing. The variant is absent from databases, 103 healthy South African controls, and 52 South African probands with isolated HI. In silico analysis indicates that the p.C415S variant in REST substitutes a conserved cysteine and results in changes to the surrounding secondary structure and the disulphide bonds, culminating in alteration of the tertiary structure of REST. Localization studies using ectopically expressed GFP-tagged Wild type (WT) and mutant REST in HEK-293 cells show that WT REST localizes exclusively to the nucleus; however, the mutant protein localizes throughout the cell. Additionally, mutant REST has an impaired ability to repress its known target AF1q. The data demonstrates that the identified mutation compromises the function of REST and support its implication in HI. This study is the second report, worldwide, to implicate REST in HI and suggests that it should be included in diagnostic HI panels.

Serum repressor element-1 silencing transcription factor levels in alzheimer's patients from a national institute of health in mexico city, elderly and young controls

ABSTRACT Background: Decreased levels of repressor element-1 silencing transcription (REST) factor in the brain, plasma, and neuron-derived exosomes are associated with Alzheimer’s disease (AD). Objective: The objective of the study was to test the viability of serum REST as a possible blood-based biomarker for AD, comparing serum REST levels in AD patients from a National Institute of Health in Mexico City (with different levels of severity and comorbidities), with elderly controls (EC) and young controls (YC). Methods: We used an enzyme-linked immunosorbent assay to determine serum REST levels in AD patients (n = 28), EC (n = 19), and YC (n = 24); the AD patients were classified by dementia severity and comorbidities (depression and microangiopathy) using clinimetric tests and magnetic resonance imaging. Results: Mean serum REST levels did not differ between AD patients, EC, and YC. The severity of AD and the presence of depression or microangiopathy were not associated with serum REST levels. Conclusion: Our results differ from previously published patterns found for plasma and cerebral REST levels. Free serum REST levels may not be a viable AD blood-based biomarker. (REV INVEST CLIN. 2021;73(1):17-22)

Natural products as LSD1 inhibitors for cancer therapy.

Natural products generally fall into the biologically relevant chemical space and always possess novel biological activities, thus making them a rich source of lead compounds for new drug discovery. With the recent technological advances, natural product-based drug discovery is now reaching a new era. Natural products have also shown promise in epigenetic drug discovery, some of them have advanced into clinical trials or are presently being used in clinic. The histone lysine specific demethylase 1 (LSD1), an important class of histone demethylases, has fundamental roles in the development of various pathological conditions. Targeting LSD1 has been recognized as a promising therapeutic option for cancer treatment. Notably, some natural products with different chemotypes including protoberberine alkaloids, flavones, polyphenols, and cyclic peptides have shown effectiveness against LSD1. These natural products provide novel scaffolds for developing new LSD1 inhibitors. In this review, we mainly discuss the identification of natural LSD1 inhibitors, analysis of the co-crystal structures of LSD1/natural product complex, antitumor activity and their modes of action. We also briefly discuss the challenges faced in this field. We believe this review will provide a landscape of natural LSD1 inhibitors.

Homeostatic Plasticity in Epilepsy.

In the healthy brain, neuronal excitability and synaptic strength are homeostatically regulated to keep neuronal network activity within physiological boundaries. Epilepsy is characterized by episodes of highly synchronized firing across in widespread neuronal populations, due to a failure in regulation of network activity. Here we consider epilepsy as a failure of homeostatic plasticity or as a maladaptive response to perturbations in the activity. How homeostatic compensation is involved in epileptogenic processes or in the chronic phase of epilepsy, is still debated. Although several theories have been proposed, there is relatively little experimental evidence to evaluate them. In this perspective, we will discuss recent results that shed light on the potential role of homeostatic plasticity in epilepsy. First, we will present some recent insights on how homeostatic compensations are probably active before and during epileptogenesis and how their actions are temporally regulated and closely dependent on the progression of pathology. Then, we will consider the dual role of transcriptional regulation during epileptogenesis, and finally, we will underline the importance of homeostatic plasticity in the context of therapeutic interventions for epilepsy. While classic pharmacological interventions may be counteracted by the epileptic brain to maintain its potentially dysfunctional set point, novel therapeutic approaches may provide the neuronal network with the tools necessary to restore its physiological balance.

DNA methylation of the RE-1 silencing transcription factor in peripheral blood mononuclear cells and gene expression of antioxidant enzyme in patients with late-onset Alzheimer disease.

Late-onset Alzheimer disease (LOAD) is the most frequent cause of dementia in elderly adults. However, the factors determining disease onset remain unclear. In the elderly, the activation and expression of the gene encoding RE-1 silencing transcription factor (REST) may be a determinant of neuroprotective mechanisms and good amyloidogenic pathway management. In the present study, the minimal promoter region of REST1 was genetically and epigenetically analyzed in blood samples from 21 subjects with LOAD and 20 cognitively healthy elderly subjects. Genomic DNA was isolated, treated with bisulfite and pyrosequenced, and gene expression was determined using real-time PCR. Notably, subjects with LOAD exhibited hypermethylation and significantly diminished expression of REST1 compared with healthy subjects (p = 0.001). In the LOAD group, the gene expression of CAT, SOD2 and GPX also showed a significant decrease and an increase in malondialdehyde. A docking analysis revealed that the first zinc finger protein Sp1 recognized and bound the methylated sequence in subjects with LOAD differently than the binding observed in control subjects. These results reveal that in patients with LOAD the methylation of specific sites in the promoter sequence of REST suppresses its expression and this could be regulating the decreased expression of CAT, SOD and GPX, besides interfering with the action of transcription factors as Sp1.

Disturbed Yin-Yang balance: stress increases the susceptibility to primary and recurrent infections of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1), a neurotropic herpes virus, is able to establish a lifelong latent infection in the human host. Following primary replication in mucosal epithelial cells, the virus can enter sensory neurons innervating peripheral tissues via nerve termini. The viral genome is then transported to the nucleus where it can be maintained without producing infectious progeny, and thus latency is established in the cell. Yin-Yang balance is an essential concept in traditional Chinese medicine (TCM) theory. Yin represents stable and inhibitory factors, and Yang represents the active and aggressive factors. When the organism is exposed to stress, especially psychological stress caused by emotional stimulation, the Yin-Yang balance is disturbed and the virus can re-engage in productive replication, resulting in recurrent diseases. Therefore, a better understanding of the stress-induced susceptibility to HSV-1 primary infection and reactivation is needed and will provide helpful insights into the effective control and treatment of HSV-1. Here we reviewed the recent advances in the studies of HSV-1 susceptibility, latency and reactivation. We included mechanisms involved in primary infection and the regulation of latency and described how stress-induced changes increase the susceptibility to primary and recurrent infections.

The transcription factor REST up-regulates tyrosine hydroxylase and antiapoptotic genes and protects dopaminergic neurons against manganese toxicity.

Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein-binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1ß (IL-1ß), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2-associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration.

The deficiency of NRSF/REST enhances the pro-inflammatory function of astrocytes in a model of Parkinson's disease.

Neuroinflammation, as an important pathological characteristic of Parkinson's disease (PD), is primarily mediated by activated astrocytes and microglia. Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) regulates many genes and signal pathways involved in the inflammatory process in astrocytes. In the present study, we established the GFAP-Cre:NRSFflox/flox conditional knockout (cKO) mice. The expression of inflammation-associated molecules were measured in primary astrocytes from wild type (WT) and cKO mice after stimulation by 1-Methyl-4-phenylpyridine (MPP+), LPS, and conditioned medium (CM) of LPS-treated BV-2 microglial cells. The inflammatory molecule expression in BV-2 microglial cells exposed to conditioned medium of MPP+-treated primary astrocytes were also analyzed. Moreover, a subacute regimen of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP) was used to establish mouse PD model and the damages to the nigrostriatal pathway were comprehensively evaluated in WT and cKO mice. We found that MPP+ induced a remarkable increase of NRSF expression in cultured astrocytes. Compared to WT astrocytes, the expression of inflammatory molecules IL-1ß, IL-6, COX-2, and iNOS increased dramatically in NRSF deficient astrocytes challenged with CM of LPS-treated BV-2 cells. COX-2 and IL-1ß transcripts were significantly elevated in BV-2 microglial cells exposed to CM of MPP+-treated NRSF deficient astrocytes compared to WT astrocytes. In cKO mice, the activation of astrocytes and microglial cells was more obvious, and the nigrostriatal dopaminergic system was more heavily injured compared to their WT counterparts after MPTP administration. Our results suggest that reactive NRSF deficient astrocytes orchestrated with microglial cells aggravate the pathophysiological progress in PD.