The HDAC inhibitor CI-994 acts as a molecular memory aid by facilitating synaptic and intracellular communication after learning.

Long-term memory formation relies on synaptic plasticity, neuronal activity-dependent gene transcription, and epigenetic modifications. Multiple studies have shown that HDAC inhibitor (HDACi) treatments can enhance individual aspects of these processes and thereby act as putative cognitive enhancers. However, their mode of action is not fully understood. In particular, it is unclear how systemic application of HDACis, which are devoid of substrate specificity, can target pathways that promote memory formation. In this study, we explore the electrophysiological, transcriptional, and epigenetic responses that are induced by CI-994, a class I HDACi, combined with contextual fear conditioning (CFC) in mice. We show that CI-994­mediated improvement of memory formation is accompanied by enhanced long-term potentiation in the hippocampus, a brain region recruited by CFC, but not in the striatum, a brain region not primarily implicated in fear learning. Furthermore, using a combination of bulk and single-cell RNA-sequencing, we find that, when paired with CFC, HDACi treatment engages synaptic plasticity-promoting gene expression more strongly in the hippocampus, specifically in the dentate gyrus (DG). Finally, using chromatin immunoprecipitation-sequencing (ChIP-seq) of DG neurons, we show that the combined action of HDACi application and conditioning is required to elicit enhancer histone acetylation in pathways that underlie improved memory performance. Together, these results indicate that systemic HDACi administration amplifies brain region-specific processes that are naturally induced by learning.

Membrane activity detection in cultured cells using phase-sensitive plasmonics.

Despite the existence of various neural recording and mapping techniques, there is an open territory for the emergence of novel techniques. The current neural imaging and recording techniques suffer from invasiveness, a time-consuming labeling process, poor spatial/ temporal resolution, and noisy signals. Among others, neuroplasmonics is a label-free and nontoxic recording technique with no issue of photo-bleaching or signal-averaging. We introduced an integrated plasmonic-ellipsometry platform for membrane activity detection with cost-effective and high-quality grating extracted from commercial DVDs. With ellipsometry technique, one can measure both amplitude (intensity) and phase difference of reflected light simultaneously with high signal to noise ratio close to surface plasmon resonances, which leads to the enhancement of sensitivity in plasmonic techniques. We cultured three different types of cells (primary hippocampal neurons, neuroblastoma SH-SY5Y cells, and human embryonic kidney 293 (HEK293) cells) on the grating surface. By introducing KCl solution as a chemical stimulus, we can differentiate the neural activity of distinct cell types and observe the signaling event in a label-free, optical recording platform. This method has potential applications in recording neural signal activity without labeling and stimulation artifacts.

Comprehensive analysis of PM20D1 QTL in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a complex disorder caused by a combination of genetic and non-genetic risk factors. In addition, an increasing evidence suggests that epigenetic mechanisms also accompany AD. Genetic and epigenetic factors are not independent, but multiple loci show genetic-epigenetic interactions, the so-called quantitative trait loci (QTLs). Recently, we identified the first QTL association with AD, namely Peptidase M20 Domain Containing 1 (PM20D1). We observed that PM20D1 DNA methylation, RNA expression, and genetic background are correlated and, in turn, associated with AD. We provided mechanistic insights for these correlations and had shown that by genetically increasing and decreasing PM20D1 levels, AD-related pathologies were decreased and accelerated, respectively. However, since the PM20D1 QTL region encompasses also other genes, namely Nuclear Casein Kinase and Cyclin Dependent Kinase Substrate 1 (NUCKS1); RAB7, member RAS oncogene family-like 1 (RAB7L1); and Solute Carrier Family 41 Member 1 (SLC41A1), we investigated whether these genes might also contribute to the described AD association. RESULTS: Here, we report a comprehensive analysis of these QTL genes using a repertoire of in silico methods as well as in vivo and in vitro experimental approaches. First, we analyzed publicly available databases to pinpoint the major QTL correlations. Then, we validated these correlations using a well-characterized set of samples and locus-specific approaches-i.e., Sanger sequencing for the genotype, cloning/sequencing and pyrosequencing for the DNA methylation, and allele-specific and real-time PCR for the RNA expression. Finally, we defined the functional relevance of the observed alterations in the context of AD in vitro. Using this approach, we show that only PM20D1 DNA methylation and expression are significantly correlated with the AD-risk associated background. We find that the expression of SLC41A1 and PM20D1-but not NUCKS1 and RAB7L1-is increased in mouse models and human samples of AD, respectively. However, SLC41A1 and PM20D1 are differentially regulated by AD-related stressors, with only PM20D1 being upregulated by amyloid-ß and reactive oxygen species, and with only PM20D1 being neuroprotective when overexpressed in cell and primary cultures. CONCLUSIONS: Our findings reinforce PM20D1 as the most likely gene responsible of the previously reported PM20D1 QTL association with AD.

Psychotropic drug-induced genetic-epigenetic modulation of CRTC1 gene is associated with early weight gain in a prospective study of psychiatric patients.

BACKGROUND: Metabolic side effects induced by psychotropic drugs represent a major health issue in psychiatry. CREB-regulated transcription coactivator 1 (CRTC1) gene plays a major role in the regulation of energy homeostasis and epigenetic mechanisms may explain its association with obesity features previously described in psychiatric patients. This prospective study included 78 patients receiving psychotropic drugs that induce metabolic disturbances, with weight and other metabolic parameters monitored regularly. Methylation levels in 76 CRTC1 probes were assessed before and after 1 month of psychotropic treatment in blood samples. RESULTS: Significant methylation changes were observed in three CRTC1 CpG sites (i.e., cg07015183, cg12034943, and cg 17006757) in patients with early and important weight gain (i.e., equal or higher than 5% after 1 month; FDR p value = 0.02). Multivariable models showed that methylation decrease in cg12034943 was more important in patients with early weight gain (≥ 5%) than in those who did not gain weight (p = 0.01). Further analyses combining genetic and methylation data showed that cg12034943 was significantly associated with early weight gain in patients carrying the G allele of rs4808844A>G (p = 0.03), a SNP associated with this methylation site (p = 0.03). CONCLUSIONS: These findings give new insights on psychotropic-induced weight gain and underline the need of future larger prospective epigenetic studies to better understand the complex pathways involved in psychotropic-induced metabolic side effects.

Correction: Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

[This corrects the article DOI: 10.1371/journal.pone.0132546.].

Whole genome grey and white matter DNA methylation profiles in dorsolateral prefrontal cortex.

The brain's neocortex is anatomically organized into grey and white matter, which are mainly composed by neuronal and glial cells, respectively. The neocortex can be further divided in different Brodmann areas according to their cytoarchitectural organization, which are associated with distinct cortical functions. There is increasing evidence that brain development and function are governed by epigenetic processes, yet their contribution to the functional organization of the neocortex remains incompletely understood. Herein, we determined the DNA methylation patterns of grey and white matter of dorsolateral prefrontal cortex (Brodmann area 9), an important region for higher cognitive skills that is particularly affected in various neurological diseases. For avoiding interindividual differences, we analyzed white and grey matter from the same donor using whole genome bisulfite sequencing, and for validating their biological significance, we used Infinium HumanMethylation450 BeadChip and pyrosequencing in ten and twenty independent samples, respectively. The combination of these analysis indicated robust grey-white matter differences in DNA methylation. What is more, cell type-specific markers were enriched among the most differentially methylated genes. Interestingly, we also found an outstanding number of grey-white matter differentially methylated genes that have previously been associated with Alzheimer's, Parkinson's, and Huntington's disease, as well as Multiple and Amyotrophic lateral sclerosis. The data presented here thus constitute an important resource for future studies not only to gain insight into brain regional as well as grey and white matter differences, but also to unmask epigenetic alterations that might underlie neurological and neurodegenerative diseases.

Study of breast cancer incidence in patients of lymphangioleiomyomatosis.

Molecular evidence has linked the pathophysiology of lymphangioleiomyomatosis (LAM) to that of metastatic breast cancer. Following on this observation, we assessed the association between LAM and subsequent breast cancer. An epidemiological study was carried out using three LAM country cohorts, from Japan, Spain, and the United Kingdom. The number of incident breast cancer cases observed in these cohorts was compared with the number expected on the basis of the country-specific incidence rates for the period 2000-2014. Immunohistochemical studies and exome sequence analysis were performed in two and one tumors, respectively. All cohorts revealed breast cancer standardized incidence ratios (SIRs) ≥ 2.25. The combined analysis of all cases or restricted to pre-menopausal age groups revealed significantly higher incidence of breast cancer: SIR = 2.81, 95 % confidence interval (CI) = 1.32-5.57, P = 0.009; and SIR = 4.88, 95 % CI = 2.29-9.99, P = 0.0007, respectively. Immunohistochemical analyses showed positivity for known markers of lung metastatic potential. This study suggests the existence of increased breast cancer risk among LAM patients. Prospective studies may be warranted to corroborate this result, which may be particularly relevant for pre-menopausal women with LAM.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Circadian cycle-dependent MeCP2 and brain chromatin changes.

Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

Promoter hypermethylation of the phosphatase DUSP22 mediates PKA-dependent TAU phosphorylation and CREB activation in Alzheimer's disease.

Genetic screening in Alzheimer's disease (AD) has identified only a handful of genes that are mutated in the disorder. Thus, for a very large proportion of patients, the biology of their disease is poorly understood. Epigenetic alterations may provide an explanation in these cases. Using DNA methylation profiles of human hippocampus from controls and patients, we have identified the presence of promoter hypermethylation of the dual-specificity phosphatase 22 (DUSP22) gene in AD. DUSP22 is a likely candidate gene for involvement in the pathogenesis of the disorder since, as we demonstrate here, it inhibits PKA activity and thereby determines TAU phosphorylation status and CREB signaling.

Phylogenetic and in silico structural analysis of the Parkinson disease-related kinase PINK1.

Parkinson disease (PD) is the second most common neurodegenerative disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra. Mutations in PINK1 were shown to cause recessive familial PD, and today are proposed to be associated with the disease via mitochondrial dysfunction and oxidative damage. The PINK1 gene comprises eight exons, which encode a ubiquitously expressed 581 amino acid protein that contains an N-terminal mitochondrial targeting domain and a serine/threonine protein kinase. To better understand the relationship between PINK1 and PD we have first analyzed the evolutionary history of the gene showing its late emergence in evolution. In addition, we have modeled the three-dimensional structure of PINK1 and found some evidences that help to explain the effect of some PD-related mutations in this protein's function.

Parkinson's disease due to the R1441G mutation in Dardarin: a founder effect in the Basques.

The recent discovery of mutations in Dardarin (LRRK2) have been related to the appearance of Parkinson's disease in several families. Notably, one single mutation in this gene (R1441G) not only appeared in familial, but also in apparently sporadic Parkinson disease (PD) patients of Basque descent. A clinical population was ascertained, and subjects were classified into Basque and non-Basque descent according to their known ancestry. The R1441G mutation was assayed using an allele-specific polymerase chain reaction, and several single nucleotide polymorphisms surrounding this mutation were analyzed by direct sequencing. In addition to 22 members of the original Basque families where R1441G was identified, we observed 17 carriers of the mutation who were apparently related through a common ancestor. From a clinical perspective, the disease observed in mutation carriers is indistinguishable from that in noncarriers. The R1441G mutation causes a form of Parkinson's disease that is equivalent to that observed in idiopathic Parkinson's disease. This mutation appears in 16.4% and 4.0% of familial and sporadic PD in this Basque population, respectively.