“Celts” up and down the Alps. Insights on mobility patterns in the pre‐Roman /Celtic population from Verona ( NE Italy, 3rd–1st c. BCE ): A multi‐isotopic approach

OBJECTIVES: The Late Iron Age in continental Europe featured complex demographic processes including, among others, the establishment of transalpine “Celtic” communities on the Italian peninsula between the 4th and 1st centuries BCE. To date, only few data are available about mobility and migration in these populations. Here we explore these topics among the Cenomani of Seminario Vescovile (SV‐Verona, Italy, 3rd–1st c. BCE) through a multi‐isotopic approach and test the possible associations with sex, age and funerary treatment. MATERIALS AND METHODS: We analyzed isotopic ratios of oxygen (δ (18)O) and carbon (δ (13)C) from bone phosphate and collagen, respectively, of 49 individuals (23 males, 17 females, and 9 nonadults). In addition, we explored possible intraindividual lifetime changes by comparing collagen δ (13)C from bone and dentine of 26 individuals. We assessed nonlocality based on individual deviation of isotopic values from the population mean plus three times the median absolute deviation from the median (±3MAD). We then checked for isotopic differences between sexes and type of funerary treatment using Mann–Whitney tests. RESULTS: One individual shows isotopic values consistent with a nonlocal origin. Five more individuals may have originated from a different locality. No statistical differences separate sexes and types of funerary treatment. DISCUSSION: Results suggest a local origin of most of the individuals of SV with the few exceptions pointing especially to an Alpine origin. The low frequency of nonlocals at SV suggest a reduced mobility in this population, or the preeminence of short distance movements undetected by our analyses

"Celts" up and down the Alps. A multi-isotopic exploration of mobility among the pre-Roman population of Verona (NE Italy, 3rd -1st c. BCE)

During the Late Iron Age (4th-1st centuries BCE) Europe was interested by intense migratory processes across the Alps. In this period, several "Celtic" populations settled in the Italian peninsula. So far, only scarce data are available about mobility in these communities. In this work, we explore mobility patterns among the Cenomani population of Seminario Vescovile (SV-Verona, Italy, 3rd-1st c. BCE) through a multi-isotopic perspective and test the possible correlation between mobility, sex, age and funerary treatment. We analyzed isotopic ratios of oxygen (δ18O) and carbon (δ13C) from bone phosphate and collagen, respectively, of 49 individuals (23 males, 17 females and 9 nonadults). We also compared collagen δ13C from bone and dentine of 26 individuals. We assessed nonlocality based on individual deviation of isotopic values from the population mean plus three times the median absolute deviation from the median (±3MAD). We then checked for isotopic differences between sexes and type of funerary treatment using Mann-Whitney tests. A nonlocal origin can be proposed for one individual and cautiously suggested for five more individuals. No statistical difference separates sex nor funerary treatment based on isotopic values. Results highlight a local origin of most of the individuals of SV with few individuals that may point to an Alpine origin. The degree of mobility at SV is lower compared with other contemporaneous sites of Europe, a result that can be attributed to chronological and social factors but also to methodological differences with previous studies

Circadian cycle-dependent MeCP2 and brain chromatin changes

Abstract 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

Whole exome sequencing of Rett syndrome-like patients reveals the mutational diversity of the clinical phenotype

Classical Rett syndrome (RTT) is a neurodevelopmental disorder where most of cases carry MECP2 mutations. Atypical RTT variants involve mutations in CDKL5 and FOXG1. However, a subset of RTT patients remains that do not carry any mutation in the described genes. Whole exome sequencing was carried out in a cohort of 21 female probands with clinical features overlapping with those of RTT, but without mutations in the customarily studied genes. Candidates were functionally validated by assessing the appearance of a neurological phenotype in Caenorhabditis elegans upon disruption of the corresponding ortholog gene. We detected pathogenic variants that accounted for the RTT-like phenotype in 14 (66.6 %) patients. Five patients were carriers of mutations in genes already known to be associated with other syndromic neurodevelopmental disorders. We determined that the other patients harbored mutations in genes that have not previously been linked to RTT or other neurodevelopmental syndromes, such as the ankyrin repeat containing protein ANKRD31 or the neuronal acetylcholine receptor subunit alpha-5 (CHRNA5). Furthermore, worm assays demonstrated that mutations in the studied candidate genes caused locomotion defects. Our findings indicate that mutations in a variety of genes contribute to the development of RTT-like phenotypes

Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro

Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG- binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763

The multi-KH domain protein of Saccharomyces cerevisiae Scp160p contributes to the regulation of telomeric silencing

Multi-KH domain proteins are highly evolutionarily conserved proteins that associate to polyribosomes and participate in RNA metabolism. Recent evidence indicates that multi-KH domain proteins also contribute to the structural organization of heterochromatin both in mammals and Drosophila. Here, we show that the multi-KH domain protein of Saccharomyces cerevisiae, Scp160p, contributes to silencing at telomeres and at the mating-type locus, but not to ribosomal silencing. The contribution of Scp160p to silencing is independent of its binding to the ribosome as deletion of the last two KH domains, which mediate ribosomal binding, has no effect on silencing. Disruption of SCP160 increases cell ploidy but this effect is also independent of the contribution of Scp160p to telomeric silencing as strong relief of silencing is observed in Δscp160 cells with normal ploidy and, vice versa, Δscp160 cells with highly increased ploidy show no significant silencing defects. The TPE phenotype of Δscp160 cells associates to a decreased Sir3p deposition at telomeres and, in good agreement, silencing is rescued by SIR3 overexpression and in a Δrif1 Δrif2 mutant. Scp160p shows a distinct perinuclear localization that is independent of its ability to bind ribosomes. Moreover, telomere clustering at the nuclear envelope is perturbed in Δscp160 cells and disruption of the histone deacetylase RPD3, which is known to improve telomere clustering, rescues telomeric silencing in Δscp160 cells. These results are discussed in the context of a model in which Scp160p contributes to silencing by helping telomere clustering. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by Grants from the MCyT (BMC2003-243), the CIRIT (2001SGR00344), and the EU (LSHB-CT-2004-511965)Peer Reviewe

Drosophila DDP1, a multi-KH-domain protein, contributes to centromeric silencing and chromosome segregation

Background: The Drosophila melanogaster DDP1 protein is a highly evolutionarily conserved protein that is characterised by the presence of 15 tandemly organized KH domains, known for mediating high-affinity binding to single-stranded nucleic acids (RNA and ssDNA). Consistent with its molecular organization, DDP1 binds single-stranded nucleic acids with high affinity, in vitro. It was shown earlier that, in polytene chromosomes, DDP1 is found in association with chromocenter heterochromatin, suggesting a contribution to heterochromatin formation and/or maintenance. Results: In this paper, the actual contribution of DDP1 to the structural and functional properties of heterochromatin was determined through the analysis of the phenotypes associated with the hypomorphic ddp115.1 mutation that was generated through the mobilization of a P element inserted in the second intron of ddp1. ddp1 15.1 behaves as a dominant suppressor of PEV in the variegated rearrangement In(1)wm4 as well as in several transgenic lines showing variegated expression of a hsp70-white+ transgene. In polytene chromosomes from homozygous ddp115.1 larvae, histone H3-K9 methylation and HP1 deposition at chromocentre heterochromatin are strongly reduced. Our results also show that, when the maternal contribution of DDP1 is reduced, chromosome condensation and segregation are compromised. Moreover, in a ddp115.1 mutant background, transmission of the nonessential Dp1187 minichromosome is reduced. Conclusions: We conclude that DDP1 contributes to the structural and functional properties of heterochromatin. These results are discussed in the context of current models for the formation and maintenance of heterochromatin; in these models, HP1 deposition depends on H3-K9 methylation that, in turn, requires the contribution of the RNAi pathway.This work was financed by grants from the Ministerio de Ciencia y Tecnologı́a (BMC2000-878; BMC2003-243) and the Comissió Interdepertamental d'Investigació i Innovació Tecnològica (2001SGR00344). D.H. acknowledges receipt of a Postdoctoral Contract from the Ministerio de Ciencia y Tecnologı́a. A.C. received a doctoral fellowship from the Comissió Interdepertamental d'Investigació i Innovació Tecnològica. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the Generalitat de CatalunyaPeer Reviewe

Drosophila vigilin, DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum

Functional characterisation of vigilin, a highly conserved multi-KH-domain protein that binds RNA and ssDNA, remains elusive and, to some extent, controversial. Studies performed in Saccharomyces cerevisiae and human cells indicate that vigilin localises to the cytoplasm, binds ribosomes, associates to RER and regulates mRNA translation. On the other hand, we and others reported a contribution to heterochromatin-mediated gene silencing (PEV) and chromosome segregation in S. cerevisiae, Drosophila and human cells. Whether this contribution is direct remains, however, unclear. Here, we report that Drosophila vigilin, DDP1, vastly localises to the cytoplasm, being largely excluded from the nucleus. We also show that DDP1 preferentially associates to RER and co-purifies with several ribosomal proteins, suggesting a contribution to mRNA translation. In light of these results, the contribution of DDP1 to PEV was re-examined. Here, we show that a newly generated null ddp1Δ mutation is only a weak suppressor of PEV, which is in contrast with our own previous results showing dominant suppression in the presence of a strong hypomorphic ddp115.1 mutation. Similar results were obtained in the fission yeast Schizosaccharomyces pombe, where vigilin (Vgl1) also associates to RER, having no significant contribution to PEV at centromeres, telomeres and the mating-type locus. Altogether, these results indicate that cytoplasmic localisation and association to RER, but not contribution to heterochromatin organisation, are evolutionarily conserved features of vigilin, favouring a model by which vigilin acts in the cytoplasm, regulating RNA metabolism, and affects nuclear functions only indirectly. © 2010 Elsevier B.V.Este trabajo fue financiado por las subvenciones del Ministerio de Ciencia e Innovación, España (BFU2006-1627, CSD2006-49 y BFU2009-07111) y la Generalitat de Catalunya (SGR2005-678 y SGR2009-1023)Peer Reviewe

An increase in MECP2 dosage impairs neural tube formation

Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development. © 2014 .This study was supported by the European Community's Seventh Framework Program (FP7/2007–2013), under grant agreement PITN-GA-2009-238242 and the project DISCHROM; ERC with grant agreement 268626; the project EPINORC; the E-RARE EuroRETT network (Carlos II Health Institute project PI071327); the Fondation Lejeune (France); MINECO projects SAF2011-22803, CSD2006-00049, BFU2009-11527 and BFU-2012-34261; Grant 090210 from Fundaciò La Marató de TV3; the Cellex Foundation; the Botín Foundation; the Catalan Association for Rett Syndrome; and the Health and Science Departments of the Catalan government (Generalitat de Catalunya). NA and CE received an I3P fellowship (I3P-BPD2005) and FPU fellowships respectively. ME is an ICREA Research ProfessorPeer Reviewe