Cultured Mesenchymal Cells from Nasal Turbinate as a Cellular Model of the Neurodevelopmental Component of Schizophrenia Etiology.

The study of neurodevelopmental molecular mechanisms in schizophrenia requires the development of adequate biological models such as patient-derived cells and their derivatives. We previously utilized cell lines with neural progenitor properties (CNON) derived from the superior or middle turbinates of patients with schizophrenia and control groups to study schizophrenia-specific gene expression. In this study, we analyzed single-cell RNA seq data from two CNON cell lines (one derived from an individual with schizophrenia (SCZ) and the other from a control group) and two biopsy samples from the middle turbinate (MT) (also from an individual with SCZ and a control). We compared our data with previously published data regarding the olfactory neuroepithelium and demonstrated that CNON originated from a single cell type present both in middle turbinate and the olfactory neuroepithelium and expressed in multiple markers of mesenchymal cells. To define the relatedness of CNON to the developing human brain, we also compared CNON datasets with scRNA-seq data derived from an embryonic brain and found that the expression profile of the CNON closely matched the expression profile one of the cell types in the embryonic brain. Finally, we evaluated the differences between SCZ and control samples to assess the utility and potential benefits of using CNON single-cell RNA seq to study the etiology of schizophrenia.

Contributions of common genetic variants to risk of schizophrenia among individuals of African and Latino ancestry.

Schizophrenia is a common, chronic and debilitating neuropsychiatric syndrome affecting tens of millions of individuals worldwide. While rare genetic variants play a role in the etiology of schizophrenia, most of the currently explained liability is within common variation, suggesting that variation predating the human diaspora out of Africa harbors a large fraction of the common variant attributable heritability. However, common variant association studies in schizophrenia have concentrated mainly on cohorts of European descent. We describe genome-wide association studies of 6152 cases and 3918 controls of admixed African ancestry, and of 1234 cases and 3090 controls of Latino ancestry, representing the largest such study in these populations to date. Combining results from the samples with African ancestry with summary statistics from the Psychiatric Genomics Consortium (PGC) study of schizophrenia yielded seven newly genome-wide significant loci, and we identified an additional eight loci by incorporating the results from samples with Latino ancestry. Leveraging population differences in patterns of linkage disequilibrium, we achieve improved fine-mapping resolution at 22 previously reported and 4 newly significant loci. Polygenic risk score profiling revealed improved prediction based on trans-ancestry meta-analysis results for admixed African (Nagelkerke's R2 = 0.032; liability R2 = 0.017; P < 10-52), Latino (Nagelkerke's R2 = 0.089; liability R2 = 0.021; P < 10-58), and European individuals (Nagelkerke's R2 = 0.089; liability R2 = 0.037; P < 10-113), further highlighting the advantages of incorporating data from diverse human populations.

Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Transcriptional Gene Silencing of the Autism-Associated Long Noncoding RNA MSNP1AS in Human Neural Progenitor Cells.

The long noncoding RNA MSNP1AS (moesin pseudogene 1, antisense) is a functional element that was previously associated with autism spectrum disorder (ASD) with genome-wide significance. Expression of MSNP1AS was increased 12-fold in the cerebral cortex of individuals with ASD and 22-fold in individuals with a genome-wide significantly associated ASD genetic marker on chromosome 5p14.1. Overexpression of MSNP1AS in human neuronal cells caused decreased expression of moesin protein, which is involved in neuronal process stability. In this study, we hypothesize that MSNP1AS knockdown impacts global transcriptome levels. We transfected the human neural progenitor cell line SK- N-SH with constructs that caused a 50% suppression of MSNP1AS expression. After 24 h, cells were harvested for total RNA isolation. Strand-specific RNA sequencing analysis indicated altered expression of 1,352 genes, including altered expression of 318 genes following correction for multiple comparisons. Expression of the OAS2 gene was increased >150-fold, a result that was validated by quantitative PCR. Gene ontology analysis of the 318 genes with altered expression following correction for multiple comparisons indicated that upregulated genes were significantly enriched for genes involved in immune response, and downregulated genes were significantly enriched for genes involved in chromatin remodeling. These data indicate multiple transcriptional and translational functions of MSNP1AS that impact ASD-relevant biological processes. Chromatin remodeling and immune response are biological processes implicated by genes with rare mutations associated with ASD. Our data suggest that the functional elements implicated by association of common genetic variants impact the same biological processes, suggesting a possible shared common molecular pathway of ASD.

Spatially Resolved Transcriptomic Signatures of Hippocampal Subregions and Arc-Expressing Ensembles in Active Place Avoidance Memory.

The rodent hippocampus is a spatially organized neuronal network that supports the formation of spatial and episodic memories. We conducted bulk RNA sequencing and spatial transcriptomics experiments to measure gene expression changes in the dorsal hippocampus following the recall of active place avoidance (APA) memory. Through bulk RNA sequencing, we examined the gene expression changes following memory recall across the functionally distinct subregions of the dorsal hippocampus. We found that recall induced differentially expressed genes (DEGs) in the CA1 and CA3 hippocampal subregions were enriched with genes involved in synaptic transmission and synaptic plasticity, while DEGs in the dentate gyrus (DG) were enriched with genes involved in energy balance and ribosomal function. Through spatial transcriptomics, we examined gene expression changes following memory recall across an array of spots encompassing putative memory-associated neuronal ensembles marked by the expression of the IEGs Arc, Egr1, and c-Jun. Within samples from both trained and untrained mice, the subpopulations of spatial transcriptomic spots marked by these IEGs were transcriptomically and spatially distinct from one another. DEGs detected between Arc+ and Arc- spots exclusively in the trained mouse were enriched in several memory-related gene ontology terms, including "regulation of synaptic plasticity" and "memory." Our results suggest that APA memory recall is supported by regionalized transcriptomic profiles separating the CA1 and CA3 from the DG, transcriptionally and spatially distinct IEG expressing spatial transcriptomic spots, and biological processes related to synaptic plasticity as a defining the difference between Arc+ and Arc- spatial transcriptomic spots.

Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11-q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20-alpha-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.

Cell Type Catalog of Middle Turbinate Epithelium (Cell Catalog of Turbinate Epithelium).

Objective: Single-cell RNA-sequencing of middle turbinate mucosa was performed to create the first single-cell transcriptome catalog of this part of the human body. Study Design: Basic science research. Setting: Single center, tertiary care center. Methods: Samples were obtained from the head of the middle turbinate from a healthy volunteer. After the specimen was prepared per lab protocol, cells were dissociated, resuspended, and counted. Single-cell libraries were then prepared according to the 10x Genomics protocol and sequenced using NovaSeq 6000 (Illumina). Sequencing data were processed using Cell Ranger, and clustering and gene expression analysis was performed using Seurat. Cell types were annotated through expression profiling of single cells using known markers and data from other single-cell studies. Results: Fourteen unique cell types were identified, including serous, goblet, club, basal, ciliated, endothelial, and mesenchymal cells, as well as multiple types of blood cells. Conclusion: This catalog provides a comprehensive depiction of the cellular composition of middle turbinate mucosa. By uncovering the cellular stratification of gene expression profiles in the healthy middle turbinate epithelium, the groundwork has been laid for further investigation into the molecular pathogenesis and targeted therapy of sinonasal disease.

Cultured Mesenchymal Cells from Nasal Turbinate as a Cellular Model of the Neurodevelopmental Component of Schizophrenia Etiology.

Study of the neurodevelopmental molecular mechanisms of schizophrenia requires the development of adequate biological models such as patient-derived cells and their derivatives. We previously used cell lines with neural progenitor properties (CNON) derived from superior or middle turbinates of patients with schizophrenia and control groups to study gene expression specific to schizophrenia. In this study, we compared single cell-RNA seq data from two CNON cell lines, one derived from an individual with schizophrenia (SCZ) and the other from a control group, with two biopsy samples from the middle turbinate (MT), also from an individual with SCZ and a control. In addition, we compared our data with previously published data from olfactory neuroepithelium (1). Our data demonstrated that CNON originated from a single cell type which is present both in middle turbinate and olfactory neuroepithelium. CNON express multiple markers of mesenchymal cells. In order to define relatedness of CNON to the developing human brain, we also compared CNON datasets with scRNA-seq data of embryonic brain (2) and found that the expression profile of CNON very closely matched one of the cell types in the embryonic brain. Finally, we evaluated differences between SCZ and control samples to assess usability and potential benefits of using single cell RNA-seq of CNON to study etiology of schizophrenia.

RseqFlow: workflows for RNA-Seq data analysis.

SUMMARY: We have developed an RNA-Seq analysis workflow for single-ended Illumina reads, termed RseqFlow. This workflow includes a set of analytic functions, such as quality control for sequencing data, signal tracks of mapped reads, calculation of expression levels, identification of differentially expressed genes and coding SNPs calling. This workflow is formalized and managed by the Pegasus Workflow Management System, which maps the analysis modules onto available computational resources, automatically executes the steps in the appropriate order and supervises the whole running process. RseqFlow is available as a Virtual Machine with all the necessary software, which eliminates any complex configuration and installation steps. AVAILABILITY AND IMPLEMENTATION: http://genomics.isi.edu/rnaseq CONTACT: wangying@xmu.edu.cn; knowles@med.usc.edu; deelman@isi.edu; tingchen@usc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

YPK9 and WHI2 Negatively Interact during Oxidative Stress.

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson's disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741COM). In contrast, deleting YPK9 from BY4741COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741COM and BY4741COMypk9∆ contain a novel premature stop codon near the 3' end of WHI2 (WHI2G1324T), whereas the collection's YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2pE442*) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2G1324T strain's genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

Transcriptome data of temporal and cingulate cortex in the Rett syndrome brain.

Rett syndrome is an X-linked neurodevelopmental disorder caused by mutation in the methyl-CpG-binding protein 2 gene (MECP2) in the majority of cases. We describe an RNA sequencing dataset of postmortem brain tissue samples from four females clinically diagnosed with Rett syndrome and four age-matched female donors. The dataset contains 16 transcriptomes, including two brain regions, temporal and cingulate cortex, for each individual. We compared our dataset with published transcriptomic analyses of postmortem brain tissue from Rett syndrome and found consistent gene expression alterations among regions of the cerebral cortex. Our data provide a valuable resource to explore the biology of the human brain in Rett syndrome.

Robust RNA-Seq of aRNA-amplified single cell material collected by patch clamp.

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq ("Patch-Seq"). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries - especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.

Gene Expression in Patient-Derived Neural Progenitors Implicates WNT5A Signaling in the Etiology of Schizophrenia.

BACKGROUND: Genome-wide association studies of schizophrenia have demonstrated that variations in noncoding regions are responsible for most of the common variation heritability of the disease. It is hypothesized that these risk variants alter gene expression. Therefore, studying alterations in gene expression in schizophrenia may provide a direct approach to understanding the etiology of the disease. In this study we use cultured neural progenitor cells derived from olfactory neuroepithelium (CNON cells) as a genetically unaltered cellular model to elucidate the neurodevelopmental aspects of schizophrenia. METHODS: We performed a gene expression study using RNA sequencing of CNON cells from 111 control subjects and 144 individuals with schizophrenia. Differentially expressed genes were identified with DESeq2 software, using covariates to correct for sex, age, library batches, and 1 surrogate variable component. RESULTS: A total of 80 genes were differentially expressed (false discovery rate < 10%), showing enrichment in cell migration, cell adhesion, developmental process, synapse assembly, cell proliferation, and related Gene Ontology categories. Cadherin and Wnt signaling pathways were positive in overrepresentation test, and, in addition, many genes were specifically involved in WNT5A signaling. The differentially expressed genes were modestly, but significantly, enriched in the genes overlapping single nucleotide polymorphisms with genome-wide significant association from the Psychiatric Genomics Consortium genome-wide association study of schizophrenia. We also found substantial overlap with genes associated with other psychiatric disorders or brain development, enrichment in the same Gene Ontology categories as genes with mutations de novo in schizophrenia, and studies of induced pluripotent stem cell-derived neural progenitor cells. CONCLUSIONS: CNON cells are a good model of the neurodevelopmental aspects of schizophrenia and can be used to elucidate the etiology of the disorder.

Deconvolution of transcriptional networks identifies TCF4 as a master regulator in schizophrenia.

Applying tissue-specific deconvolution of transcriptional networks to identify their master regulators (MRs) in neuropsychiatric disorders has been largely unexplored. Here, using two schizophrenia (SCZ) case-control RNA-seq datasets, one on postmortem dorsolateral prefrontal cortex (DLPFC) and another on cultured olfactory neuroepithelium, we deconvolved the transcriptional networks and identified TCF4 as a top candidate MR that may be dysregulated in SCZ. We validated TCF4 as a MR through enrichment analysis of TCF4-binding sites in induced pluripotent stem cell (hiPSC)-derived neurons and in neuroblastoma cells. We further validated the predicted TCF4 targets by knocking down TCF4 in hiPSC-derived neural progenitor cells (NPCs) and glutamatergic neurons (Glut_Ns). The perturbed TCF4 gene network in NPCs was more enriched for pathways involved in neuronal activity and SCZ-associated risk genes, compared to Glut_Ns. Our results suggest that TCF4 may serve as a MR of a gene network dysregulated in SCZ at early stages of neurodevelopment.

Endogenous Cell Type-Specific Disrupted in Schizophrenia 1 Interactomes Reveal Protein Networks Associated With Neurodevelopmental Disorders.

BACKGROUND: Disrupted in schizophrenia 1 (DISC1) has been implicated in a number of psychiatric diseases along with neurodevelopmental phenotypes such as the proliferation and differentiation of neural progenitor cells. While there has been significant effort directed toward understanding the function of DISC1 through the determination of its protein-protein interactions within an in vitro setting, endogenous interactions involving DISC1 within a cell type-specific setting relevant to neural development remain unclear. METHODS: Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome engineering technology, we inserted an endogenous 3X-FLAG tag at the C-terminus of the canonical DISC1 gene in human induced pluripotent stem cells (iPSCs). We further differentiated these cells and used affinity purification to determine protein-protein interactions involving DISC1 in iPSC-derived neural progenitor cells and astrocytes. RESULTS: We were able to determine 151 novel cell type-specific proteins present in DISC1 endogenous interactomes. The DISC1 interactomes can be clustered into several subcomplexes that suggest novel DISC1 cell-specific functions. In addition, the DISC1 interactome in iPSC-derived neural progenitor cells associates in a connected network containing proteins found to harbor de novo mutations in patients affected by schizophrenia and contains a subset of novel interactions that are known to harbor syndromic mutations in neurodevelopmental disorders. CONCLUSIONS: Endogenous DISC1 interactomes within iPSC-derived human neural progenitor cells and astrocytes are able to provide context to DISC1 function in a cell type-specific setting relevant to neural development and enables the integration of psychiatric disease risk factors within a set of defined molecular functions.

Analysis of Gene Expression Variance in Schizophrenia Using Structural Equation Modeling.

Schizophrenia (SCZ) is a psychiatric disorder of unknown etiology. There is evidence suggesting that aberrations in neurodevelopment are a significant attribute of schizophrenia pathogenesis and progression. To identify biologically relevant molecular abnormalities affecting neurodevelopment in SCZ we used cultured neural progenitor cells derived from olfactory neuroepithelium (CNON cells). Here, we tested the hypothesis that variance in gene expression differs between individuals from SCZ and control groups. In CNON cells, variance in gene expression was significantly higher in SCZ samples in comparison with control samples. Variance in gene expression was enriched in five molecular pathways: serine biosynthesis, PI3K-Akt, MAPK, neurotrophin and focal adhesion. More than 14% of variance in disease status was explained within the logistic regression model (C-value = 0.70) by predictors accounting for gene expression in 69 genes from these five pathways. Structural equation modeling (SEM) was applied to explore how the structure of these five pathways was altered between SCZ patients and controls. Four out of five pathways showed differences in the estimated relationships among genes: between KRAS and NF1, and KRAS and SOS1 in the MAPK pathway; between PSPH and SHMT2 in serine biosynthesis; between AKT3 and TSC2 in the PI3K-Akt signaling pathway; and between CRK and RAPGEF1 in the focal adhesion pathway. Our analysis provides evidence that variance in gene expression is an important characteristic of SCZ, and SEM is a promising method for uncovering altered relationships between specific genes thus suggesting affected gene regulation associated with the disease. We identified altered gene-gene interactions in pathways enriched for genes with increased variance in expression in SCZ. These pathways and loci were previously implicated in SCZ, providing further support for the hypothesis that gene expression variance plays important role in the etiology of SCZ.

Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation.

As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium.

Integrative functional genomic analysis of human brain development and neuropsychiatric risks.

To broaden our understanding of human neurodevelopment, we profiled transcriptomic and epigenomic landscapes across brain regions and/or cell types for the entire span of prenatal and postnatal development. Integrative analysis revealed temporal, regional, sex, and cell type-specific dynamics. We observed a global transcriptomic cup-shaped pattern, characterized by a late fetal transition associated with sharply decreased regional differences and changes in cellular composition and maturation, followed by a reversal in childhood-adolescence, and accompanied by epigenomic reorganizations. Analysis of gene coexpression modules revealed relationships with epigenomic regulation and neurodevelopmental processes. Genes with genetic associations to brain-based traits and neuropsychiatric disorders (including MEF2C, SATB2, SOX5, TCF4, and TSHZ3) converged in a small number of modules and distinct cell types, revealing insights into neurodevelopment and the genomic basis of neuropsychiatric risks.

Genetics of recurrent early-onset major depression (GenRED): significant linkage on chromosome 15q25-q26 after fine mapping with single nucleotide polymorphism markers.

OBJECTIVE: The authors studied a dense map of single nucleotide polymorphism (SNP) DNA markers on chromosome 15q25-q26 to maximize the informativeness of genetic linkage analyses in a region where they previously reported suggestive evidence for linkage of recurrent early-onset major depressive disorder. METHOD: In 631 European-ancestry families with multiple cases of recurrent early-onset major depressive disorder, 88 SNPs were genotyped, and multipoint allele-sharing linkage analyses were carried out. Marker-marker linkage disequilibrium was minimized, and a simulation study with founder haplotypes from these families suggested that linkage scores were not inflated by linkage disequilibrium. RESULTS: The dense SNP map increased the information content of the analysis from around 0.7 to over 0.9. The maximum evidence for linkage was the Z likelihood ratio score statistic of Kong and Cox (Z(LR))=4.69 at 109.8 cM. The exact p value was below the genomewide significance threshold. By contrast, in the genome scan with microsatellite markers at 9 cM spacing, the maximum Z(LR) for European-ancestry families was 3.43 (106.53 cM). It was estimated that the linked locus or loci in this region might account for a 20% or less populationwide increase in risk to siblings of cases. CONCLUSIONS: This region has produced modestly positive evidence for linkage to depression and related traits in other studies. These results suggest that DNA sequence variations in one or more genes in the 15q25-q26 region can increase susceptibility to major depression and that efforts are warranted to identify these genes.