A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects.

The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25-92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10(-8)). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10(-30)). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.

Metabolic actions of kisspeptin signaling: Effects on body weight, energy expenditure, and feeding.

Kisspeptin (encoded by the Kiss1 gene) and its receptor, KISS1R (encoded by the Kiss1r gene), have well-established roles in stimulating reproduction via central actions on reproductive neural circuits, but recent evidence suggests that kisspeptin signaling also influences metabolism and energy balance. Indeed, both Kiss1 and Kiss1r are expressed in many metabolically-relevant peripheral tissues, including both white and brown adipose tissue, the liver, and the pancreas, suggesting possible actions on these tissues or involvement in their physiology. In addition, there may be central actions of kisspeptin signaling, or factors co-released from kisspeptin neurons, that modulate metabolic, feeding, or thermoregulatory processes. Accumulating data from animal models suggests that kisspeptin signaling regulates a wide variety of metabolic parameters, including body weight and energy expenditure, adiposity and adipose tissue function, food intake, glucose metabolism, respiratory rates, locomotor activity, and thermoregulation. Herein, the current evidence for the involvement of kisspeptin signaling in each of these physiological parameters is reviewed, gaps in knowledge identified, and future avenues of important research highlighted. Collectively, the discussed findings highlight emerging non-reproductive actions of kisspeptin signaling in metabolism and energy balance, in addition to previously documented roles in reproductive control, but also emphasize the need for more research to resolve current controversies and uncover underlying molecular and physiological mechanisms.

Conditional Deletion of KOR (Oprk1) in Kisspeptin Cells Does Not Alter LH Pulses, Puberty, or Fertility in Mice.

Classic pharmacological studies suggested that endogenous dynorphin-KOR signaling is important for reproductive neuroendocrine regulation. With the seminal discovery of an interconnected network of hypothalamic arcuate neurons co-expressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons), the KNDy hypothesis was developed to explain how gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) pulses are generated. Key to this hypothesis is dynorphin released from KNDy neurons acting in a paracrine manner on other KNDy neurons via kappa opioid receptor (KOR) signaling to terminate neural "pulse" events. While in vitro evidence supports this aspect of the KNDy hypothesis, a direct in vivo test of the necessity of KOR signaling in kisspeptin neurons for proper LH secretion has been lacking. We therefore conditionally knocked out KOR selectively from kisspeptin neurons of male and female mice and tested numerous reproductive measures, including in vivo LH pulse secretion. Surprisingly, despite validating successful knockout of KOR in kisspeptin neurons, we found no significant effect of kisspeptin cell-specific deletion of KOR on any measure of puberty, LH pulse parameters, LH surges, follicle-stimulating hormone (FSH) levels, estrous cycles, or fertility. These outcomes suggest that the KNDy hypothesis, while sufficient normally, may not be the only neural mechanism for sculpting GnRH and LH pulses, supported by recent findings in humans and mice. Thus, besides normally acting via KOR in KNDy neurons, endogenous dynorphin and other opioids may, under some conditions, regulate LH and FSH secretion via KOR in non-kisspeptin cells or perhaps via non-KOR pathways. The current models for GnRH and LH pulse generation should be expanded to consider such alternate mechanisms.

Nicotine preference and affective behavior of Cd81 knockout mice.

RATIONALE: Cd81 -/- (knockout) mice have previously been reported to have reduced cocaine preference and increased striatal dopamine content and dopamine turnover, but normal learning and memory in the Morris water maze. The effects of Cd81 on other behaviors and drugs of abuse have not been investigated. OBJECTIVES AND METHODS: We measured the effects of Cd81 -/- in a modified two-bottle choice test for nicotine, as well as in somatic signs of nicotine withdrawal, four tests of affective behavior, and tyrosine hydroxylase gene expression assays. RESULTS: We found that Cd81 loss-of-function significantly increased voluntary nicotine consumption and somatic signs of nicotine withdrawal. Nicotine consumption of Cd81 -/- female mice increased for 3 weeks and then remained relatively stable for the next 5 weeks, suggesting that their nicotine consumption continued to be limited by aversion to higher nicotine doses. Cd81 -/- also produced a dramatic and significant increase in struggling in the forced swim test and a significant increase in the time spent in the light chamber of the light/dark box. The elevated plus maze and the tail suspension test did not show a main effect of genotype. Therefore, we conclude that Cd81 did not have an overall effect on anxiety- or depression-like behavior. Tyrosine hydroxylase mRNA levels were unchanged. CONCLUSIONS: Cd81 knockouts have a strongly increased nicotine preference, plus a proactive response to specific stressful situations. Together with reports of increased striatal dopamine content and anecdotal reports of increased aggressiveness, these provide intriguing parallels to some aspects of post-traumatic stress disorder.

MBD-seq as a cost-effective approach for methylome-wide association studies: demonstration in 1500 case--control samples.

AIM: We studied the use of methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq) as a cost-effective screening tool for methylome-wide association studies (MWAS). MATERIALS & METHODS: Because MBD-seq has not yet been applied on a large scale, we first developed and tested a pipeline for data processing using 1500 schizophrenia cases and controls plus 75 technical replicates with an average of 68 million reads per sample. This involved the use of technical replicates to optimize quality control for multi- and duplicate-reads, an in silico experiment to identify CpGs in loci with alignment problems, CpG coverage calculations based on multiparametric estimates of the fragment size distribution, a two-stage adaptive algorithm to combine data from correlated adjacent CpG sites, principal component analyses to control for confounders and new software tailored to handle the large data set. RESULTS: We replicated MWAS findings in independent samples using a different technology that provided single base resolution. In an MWAS of age-related methylation changes, one of our top findings was a previously reported robust association involving GRIA2. Our results also suggested that owing to the many confounding effects, a considerable challenge in MWAS is to identify those effects that are informative about disease processes. CONCLUSION: This study showed the potential of MBD-seq as a cost-effective tool in large-scale disease studies.