Optimization and evaluation of surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry for protein profiling of cerebrospinal fluid

Cerebrospinal fluid (CSF) potentially carries an archive of peptides and small proteins relevant to pathological processes in the central nervous system (CNS) and surrounding brain tissue. Proteomics is especially well suited for the discovery of biomarkers of diagnostic potential in CSF for early diagnosis and discrimination of several neurodegenerative diseases. ProteinChip surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is one such approach which offers a unique platform for high throughput profiling of peptides and small proteins in CSF. In this study, we evaluated methodologies for the retention of CSF proteins < 20 kDa in size, and identify a strategy for screening small proteins and peptides in CSF. ProteinChip array types, along with sample and binding buffer conditions, and matrices were investigated. By coupling the processing of arrays to a liquid handler reproducible and reliable profiles, with mean peak coefficients of variation < 20%, were achieved for intra- and inter-assays under selected conditions. Based on peak m/z we found a high degree of overlap between the tested array surfaces. The combination of CM10 and IMAC30 arrays was sufficient to represent between 80–90% of all assigned peaks when using either sinapinic acid or α-Cyano-4-hydroxycinnamic acid as the energy absorbing matrices. Moreover, arrays processed with SPA consistently showed better peak resolution and higher peak number across all surfaces within the measured mass range. We intend to use CM10 and IMAC30 arrays prepared in sinapinic acid as a fast and cost-effective approach to drive decisions on sample selection prior to more in-depth discovery of diagnostic biomarkers in CSF using alternative but complementary proteomic strategies

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status. RESULTS: We have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity. CONCLUSIONS: We identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification

CGG Repeat-Induced FMR1 Silencing Depends on the Expansion Size in Human iPSCs and Neurons Carrying Unmethylated Full Mutations

In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in&nbsp;neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population

Autism-Tweaking the social network

A Focus article for Science’s sister journal Science Translational Medicine on a paper entitled “Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism” by Silverman et al

Molecular basis for prospective pharmacological treatment strategies in intellectual disability syndromes

A number of mutated genes that code for proteins concerned with brain synapse function and circuit formation have been identified in patients affected by intellectual disability syndromes over the last fifteen years. These genes are functionally involved in synapse formation and plasticity, the regulation of dendritic spine morphology, the regulation of the synaptic cytoskeleton, the synthesis and degradation of specific synapse proteins and the control of correct balance between excitatory and inhibitory synapses. In most of the cases even mild alterations in synapse morphology, function and balance give rise to mild or severe intellectual disabilities. These studies provided the rationale for the development of pharmacological agents that are able to counteract functional synaptic anomalies and potentially improve the symptoms of some of these conditions. This review summarizes recent findings on the functions of some of the genes responsible for intellectual disability syndromes and some of the new potential pharmacological treatments for these diseases

Patient perspectives on current and potential therapies and clinical trial approaches for cocaine use disorder

BackgroundCocaine use disorder (CUD) is characterized by the continued use of cocaine despite serious impacts on life. This study focused on understanding the perspective of individuals with current CUD, individuals in CUD remission, and their supporters regarding current therapies, future therapies, and views on clinical trials for CUD.MethodsThe online bulletin board (OBB) is a qualitative tool where participants engage in an interactive discussion on a virtual forum. Following completion of a screening questionnaire to determine eligibility, individuals in CUD remission and their supporters logged in to the OBB and responded to questions posed by the moderator. Individuals with current CUD participated in a one-time virtual focus group.ResultsAll individuals with current CUD and 94% of those in CUD remission reported a diagnosis consistent with CUD or substance use disorder during screening. Individuals with current CUD and their supporters were recruited from the United States (US). Individuals in CUD remission were recruited from five countries, including the US. Individuals with current CUD reported hesitation about seeking treatment due to stigma, a lack of privacy, and being labeled as a drug seeker; barriers to therapy included time, cost, and a lack of privacy. Participants wanted a safe therapy to stop cravings and withdrawal symptoms. Seven clinical trial outcomes, including long-term abstinence and craving control, were suggested based on collected insights.ConclusionThis study can help inform the design of clinical trials and emphasize the need for effective, safe, and accessible therapies. Recruiting participants will require significant trust building