Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder.

Most genetic risk for psychiatric disease lies in regulatory regions, implicating pathogenic dysregulation of gene expression and splicing. However, comprehensive assessments of transcriptomic organization in diseased brains are limited. In this work, we integrated genotypes and RNA sequencing in brain samples from 1695 individuals with autism spectrum disorder (ASD), schizophrenia, and bipolar disorder, as well as controls. More than 25% of the transcriptome exhibits differential splicing or expression, with isoform-level changes capturing the largest disease effects and genetic enrichments. Coexpression networks isolate disease-specific neuronal alterations, as well as microglial, astrocyte, and interferon-response modules defining previously unidentified neural-immune mechanisms. We integrated genetic and genomic data to perform a transcriptome-wide association study, prioritizing disease loci likely mediated by cis effects on brain expression. This transcriptome-wide characterization of the molecular pathology across three major psychiatric disorders provides a comprehensive resource for mechanistic insight and therapeutic development.

Neural-specific deletion of the focal adhesion adaptor protein paxillin slows migration speed and delays cortical layer formation.

Paxillin and Hic-5 are homologous focal adhesion adaptor proteins that coordinate cytoskeletal rearrangements in response to integrin signaling, but their role(s) in cortical development are unknown. Here, we find that Hic-5-deficient mice are postnatal viable with normal cortical layering. Mice with a neural-specific deletion of paxillin are also postnatal viable, but show evidence of a cortical neuron migration delay that is evident pre- and perinatally, but is not detected at postnatal day 35 (P35). This phenotype is not modified by Hic-5 deficiency (double knockout). Specific deletion of paxillin in postmitotic neurons using Nex-Cre-mediated recombination as well as in utero electroporation of a Cre-expression construct identified a cell-autonomous requirement for paxillin in migrating neurons. Paxillin-deficient neurons have shorter leading processes that exhibited multiple swellings in comparison with control. Multiphoton imaging revealed that paxillin-deficient neurons migrate ∼30% slower than control neurons. This phenotype is similar to that produced by deletion of focal adhesion kinase (FAK), a signaling partner of paxillin, and suggests that paxillin and FAK function cell-autonomously to control migrating neuron morphology and speed during cortical development.

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca2+ signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr192 phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr192 phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.

Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction.

Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50cm-long column packed with 1.9µm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of over 300% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results reveal the analytical power of optimized chromatography using sub 2µm particles for the analysis of the T cell phosphoproteome to reveal a vast landscape of significantly altered phosphorylation changes in response to T cell receptor stimulation.

Genetic variation and RNA binding proteins: tools and techniques to detect functional polymorphisms.

At its most fundamental level the goal of genetics is to connect genotype to phenotype. This question is asked at a basic level evaluating the role of genes and pathways in genetic model organism. Increasingly, this question is being asked in the clinic. Genomes of individuals and populations are being sequenced and compared. The challenge often comes at the stage of analysis. The variant positions are analyzed with the hope of understanding human disease. However after a genome or exome has been sequenced, the researcher is often deluged with hundreds of potentially relevant variations. Traditionally, amino-acid changing mutations were considered the tractable class of disease-causing mutations; however, mutations that disrupt noncoding elements are the subject of growing interest. These noncoding changes are a major avenue of disease (e.g., one in three hereditary disease alleles are predicted to affect splicing). Here, we review some current practices of medical genetics, the basic theory behind biochemical binding and functional assays, and then explore technical advances in how variations that alter RNA protein recognition events are detected and studied. These advances are advances in scale-high-throughput implementations of traditional biochemical assays that are feasible to perform in any molecular biology laboratory. This chapter utilizes a case study approach to illustrate some methods for analyzing polymorphisms. The first characterizes a functional intronic SNP that deletes a high affinity PTB site using traditional low-throughput biochemical and functional assays. From here we demonstrate the utility of high-throughput splicing and spliceosome assembly assays for screening large sets of SNPs and disease alleles for allelic differences in gene expression. Finally we perform three pilot drug screens with small molecules (G418, tetracycline, and valproic acid) that illustrate how compounds that rescue specific instances of differential pre-mRNA processing can be discovered.