Distribution and evolution of methylation profiles in mouse brain cells.

The methylation profiles generated during the early stages of mouse brain development play a critical role in the gene expression regulation program; given the large number of different cellular population in murine brain, is the great importance the study of the cell to cell heterogeneity in terms of DNA methylation. In this work, I performed an in-depth single molecule methylation analysis in order to assess the cell to cell methylation heterogeneity analyzing the epialleles composition at the D-aspartate oxidase (ddo) putative regulatory region, that is a gene implicated in a correct neurodevelopment and that undergoes a strong methylation changes during the early stages of mouse development. I found that brain methylation heterogeneity is generated and develops in an extremely conserved fashion, giving rise to a deterministically regulated distribution of different epialleles, distinct for each stage and cell type, evoking the possible existence of a novel, cell population based, combinatorial code of CpG methylation. Importantly, rapid epialleles remodeling toward mature neuronal and glial patterns was observed in ES cells population upon neural differentiation. The high degree of epipolimorphism, detected also in pure cell populations, supports the existence of mechanisms oriented to maintain the epiallele patterns in a dynamic equilibrium involving continuously occurring methylation and demethylation events in each single cell. The interplay between contiguous CpGs differing in methylation susceptibility likely underlies specific epiallele frequency and dynamics in a spatial-specific manner. The present data on Ddo gene provide a proof of principle that employment of high coverage single molecule methylation analysis, may potentially reveal unprecedented mechanisms underlying methylation establishment, changes and alterations within cell populations in development and diseases, unpredictable by classical methylation analyses

D2R signaling in striatal spiny neurons modulates L-DOPA induced dyskinesia

Degeneration of dopaminergic neurons leads to Parkinson's disease (PD), characterized by reduced levels of striatal dopamine (DA) and impaired voluntary movements. DA replacement is achieved by levodopa treatment which in long-term causes involuntary movements or dyskinesia. Dyskinesia is linked to the pulsatile activation of D1 receptors of the striatal medium spiny neurons (MSNs) forming the direct output pathway (dMSNs). The contribution of DA stimulation of D2R in MSNs of the indirect pathway (iMSNs) is less clear. Using the 6-hydroxydopamine model of PD, here we show that loss of DA-mediated inhibition of these neurons intensifies levodopa-induced dyskinesia (LID) leading to reprogramming of striatal gene expression. We propose that the motor impairments characteristic of PD and of its therapy are critically dependent on D2R-mediated iMSNs activity. D2R signaling not only filters inputs to the striatum but also indirectly regulates dMSNs mediated responses

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles

The Brain’s Reward System in Health and Disease

Rhythmic gene expression is found throughout the central nervous system. This harmonized regulation can be dependent on- and independent of- the master regulator of biological clocks, the suprachiasmatic nucleus (SCN). Substantial oscillatory activity in the brain's reward system is regulated by dopamine. While light serves as a primary time-giver (zeitgeber) of physiological clocks and synchronizes biological rhythms in 24-h cycles, nonphotic stimuli have a profound influence over circadian biology. Indeed, reward-related activities (e.g., feeding, exercise, sex, substance use, and social interactions), which lead to an elevated level of dopamine, alters rhythms in the SCN and the brain's reward system. In this chapter, we will discuss the influence of the dopaminergic reward pathways on circadian system and the implication of this interplay on human health

The Interplay between Defensins and Microbiota in Crohn’s Disease

Crohn’s disease (CD) is a chronic inflammation of the intestinal mucosa, characterized by periods of acute recurrence and remission. Depending on the specific region affected, CD is classified as ileal CD or colonic CD. It is largely accepted that the intestinal microbiota is involved in the onset of the pathology. Indeed, a reduced immune tolerance to components of the intestinal commensal microbiota and inflammation of the intestinal barrier typifies patients with CD. Several studies have shown defective expression of intestinal antimicrobial peptides (AMPs) in patients with CD compared to controls, particularly defensins. A reduction in α-defensins is observed in ileal CD, while β-defensins are increased in colonic CD. In addition to an immunological basis, the disease is frequently associated with genetic alterations including mutations of NOD2 gene. Several therapeutic strategies to circumvent the dysfunction observed in CD are currently under investigation. These include the use of delivery systems to administer endogenous AMPs and the engineering of peptidomimetics that could ameliorate the severity of CD. In this review, the role defensins play in CD and the strategies aimed at overcoming bacterial resistance will be discussed

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons

Background CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Results Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. Conclusions ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net. Keyword

D2R signaling in striatal spiny neurons modulates L-DOPA induced dyskinesia

Degeneration of dopaminergic neurons leads to Parkinson's disease (PD), characterized by reduced levels of striatal dopamine (DA) and impaired voluntary movements. DA replacement is achieved by levodopa treatment which in long-term causes involuntary movements or dyskinesia. Dyskinesia is linked to the pulsatile activation of D1 receptors of the striatal medium spiny neurons (MSNs) forming the direct output pathway (dMSNs). The contribution of DA stimulation of D2R in MSNs of the indirect pathway (iMSNs) is less clear. Using the 6-hydroxydopamine model of PD, here we show that loss of DA-mediated inhibition of these neurons intensifies levodopa-induced dyskinesia (LID) leading to reprogramming of striatal gene expression. We propose that the motor impairments characteristic of PD and of its therapy are critically dependent on D2R-mediated iMSNs activity. D2R signaling not only filters inputs to the striatum but also indirectly regulates dMSNs mediated responses