Combined cellomics and proteomics analysis reveals shared neuronal morphology and molecular pathway phenotypes for multiple schizophrenia risk genes.

An enigma in studies of neuropsychiatric disorders is how to translate polygenic risk into disease biology. For schizophrenia, where > 145 significant GWAS loci have been identified and only a few genes directly implicated, addressing this issue is a particular challenge. We used a combined cellomics and proteomics approach to show that polygenic risk can be disentangled by searching for shared neuronal morphology and cellular pathway phenotypes of candidate schizophrenia risk genes. We first performed an automated high-content cellular screen to characterize neuronal morphology phenotypes of 41 candidate schizophrenia risk genes. The transcription factors Tcf4 and Tbr1 and the RNA topoisomerase Top3b shared a neuronal phenotype marked by an early and progressive reduction in synapse numbers upon knockdown in mouse primary neuronal cultures. Proteomics analysis subsequently showed that these three genes converge onto the syntaxin-mediated neurotransmitter release pathway, which was previously implicated in schizophrenia, but for which genetic evidence was weak. We show that dysregulation of multiple proteins in this pathway may be due to the combined effects of schizophrenia risk genes Tcf4, Tbr1, and Top3b. Together, our data provide new biological functions for schizophrenia risk genes and support the idea that polygenic risk is the result of multiple small impacts on common neuronal signaling pathways.

Transcriptome analysis provides genome annotation and expression profiles in the central nervous system of Lymnaea stagnalis at different ages.

BACKGROUND: The pond snail, Lymnaea stagnalis (L. stagnalis), has served as a valuable model organism for neurobiology studies due to its simple and easily accessible central nervous system (CNS). L. stagnalis has been widely used to study neuronal networks and recently gained popularity for study of aging and neurodegenerative diseases. However, previous transcriptome studies of L. stagnalis CNS have been exclusively carried out on adult L. stagnalis only. As part of our ongoing effort studying L. stagnalis neuronal growth and connectivity at various developmental stages, we provide the first age-specific transcriptome analysis and gene annotation of young (3 months), adult (6 months), and old (18 months) L. stagnalis CNS. RESULTS: Using the above three age cohorts, our study generated 55-69 millions of 150 bp paired-end RNA sequencing reads using the Illumina NovaSeq 6000 platform. Of these reads, ~ 74% were successfully mapped to the reference genome of L. stagnalis. Our reference-based transcriptome assembly predicted 42,478 gene loci, of which 37,661 genes encode coding sequences (CDS) of at least 100 codons. In addition, we provide gene annotations using Blast2GO and functional annotations using Pfam for ~ 95% of these sequences, contributing to the largest number of annotated genes in L. stagnalis CNS so far. Moreover, among 242 previously cloned L. stagnalis genes, we were able to match ~ 87% of them in our transcriptome assembly, indicating a high percentage of gene coverage. The expressional differences for innexins, FMRFamide, and molluscan insulin peptide genes were validated by real-time qPCR. Lastly, our transcriptomic analyses revealed distinct, age-specific gene clusters, differentially expressed genes, and enriched pathways in young, adult, and old CNS. More specifically, our data show significant changes in expression of critical genes involved in transcription factors, metabolisms (e.g. cytochrome P450), extracellular matrix constituent, and signaling receptor and transduction (e.g. receptors for acetylcholine, N-Methyl-D-aspartic acid, and serotonin), as well as stress- and disease-related genes in young compared to either adult or old snails. CONCLUSIONS: Together, these datasets are the largest and most updated L. stagnalis CNS transcriptomes, which will serve as a resource for future molecular studies and functional annotation of transcripts and genes in L. stagnalis.

Differential Effects of the G-Protein-Coupled Estrogen Receptor (GPER) on Rat Embryonic (E18) Hippocampal and Cortical Neurons.

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERß. Studies related to the extranuclear, membrane-bound G-protein-coupled ER (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic day 18 (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and nonselective agonist, 17ß-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involves internal Ca2+ store release via activation of phospholipase C (PLC) and extracellular entry via Ca2+ channels. Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet real-time qPCR (RT-qPCR) and Western blotting showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen's action in early neuronal development.

Novel GPER Agonist, CITFA, Increases Neurite Growth in Rat Embryonic (E18) Hippocampal Neurons.

Numerous studies have reported neuroprotective and procognitive effects of estrogens. The estrogen 17ß-estradiol (E2) activates both the classical nuclear estrogen receptors ERα and ERß as well as the G protein-coupled estrogen receptor (GPER). The differential effects of targeting the classical estrogen receptors over GPER are not well-understood. A limited number of selective GPER compounds have been described. In this study, 10 novel compounds were synthesized and exhibited half-maximal effective concentration values greater than the known GPER agonist G-1 in calcium mobilization assays performed in nonadherent HL-60 cells. Of these compounds, 2-cyclohexyl-4-isopropyl-N-((5-(tetrahydro-2H-pyran-2-yl)furan-2-yl)methyl)aniline, referred to as CITFA, significantly increased axonal and dendritic growth in neurons extracted from embryonic day 18 (E18) fetal rat hippocampal neurons. Confirmation of the results was performed by treating E18 hippocampal neurons with known GPER-selective antagonist G-36 and challenging with either E2, G-1, or CITFA. Results from these studies revealed an indistinguishable difference in neurite outgrowth between the treatment and control groups, exhibiting that neurite outgrowth in response to G-1 and CITFA originates from GPER activation and can be abolished with pretreatment of an antagonist. Subsequent docking studies using a homology model of GPER showed unique docking poses between G-1 and CIFTA. While docking poses differed between the ligands, CIFTA exhibited more favorable distance, bond angle, and strain for hydrogen-bonding and hydrophobic interactions.