S920 collected. We followed the convention quality control and analytical processing on the raw data. We collected 543 “putative” marker genes for ten...

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S920 collected. We followed the convention quality control and analytical processing on the raw data. We collected 543 “putative” marker genes for ten cell types that have been commonly used from literature, In Situ Hybridization (ISH) databases and antibody companies. We defined general marker gene as their expressions in claimed target cells is the highest across all cell types tested. The expression difference for the rigorous marker gene defined as dividing the second highest expression level across all the other cell types by the expression level in target cell type. We set the fold change threshold for rigorous marker gene as at least 2. Parietal cortex tissue specimens from the Stanley Medical Research Institute (SMRI) Neuropathology Consortium and Array collections included schizophrenia, bipolar disorder and control samples were used for Weighted Gene CoExpression Network Analysis (WGCNA). Results: With the datasets collected and the criteria provided, we found that 44 general marker genes showed stable specificity across all data collected. That included 29 neuron markers, eight astrocyte markers, seven oligodendrocyte markers. The averaged correlation values of specific marker genes between human and mouse, transcriptome and proteome, RNA-Seq and microarray, acutely isolation and primary culture were 0.50, 0.58, 0.51 and 0.43, respectively. According to the criterion for rigorous marker genes, 23 of the 44 marker genes showed more than two-fold changes in at least seven data sets evaluated. The most specific marker gene (fold change=710.31) is RELN, a neuron marker gene. It is not clear whether the non-specificity of rest of marker genes is related to data quality or technical artifacts in the data we can use for the assessment. In the WGCNA analysis, we found general marker genes of astrocyte were significantly enriched (po2.2e-16) in disease associated module. The enriched six astrocyte marker genes (ALDH1L1, ALDOC, CLU, GJA1, SLC1A3, SLC4A4) showed co-expression with significant GWAS loci of schizophrenia and bipolar disorder in previous studies. Discussion: Based on transcriptome and proteomics of data of isolated cells, we confirmed a small set of 44 genes to be cell-type-specific in the brain while many other commonly-used marker genes will require additional studies to verify their specificity. Studies using these marker genes to tag cell types should exercise caution. Moreover, astrocyte marker genes enriched in disease-associated module co-expressed with known GWAS loci for schizophrenia and bipolar disorder. Further study and more data is need for evaluation of the rest non-specific marker genes and the coexpression of brain cell marker genes with psychiatric disorders.

Disclosure: Nothing to disclose.


Abstracts Jacob Garza2, Xiaoli Qi2, Klaudio Gjeluci2, Melanie Leussis3, n Gerard Martens4, Geert Poelmans4, Tracey Petryshen ,1 1

Massachusetts General Hospital, Harvard Medical School Massachusetts General Hospital 3 Emmanuel College 4 Radbound University Nijmegen Medical Centre 2

Background: Ankyrin3 has been identified as a risk gene for Bipolar Disorder (BD) and autism spectrum disorders by multiple genome-wide and targeted association studies as well as sequencing studies. Carriers of the BD risk alleles have reduced ankyrin3 expression in the brain, suggesting that ankyrin3 suppression contributes to disease. However, the mechanism through which ankyrin3 confers risk is unknown. Ankyrin3 encodes the ankyrinG protein that tethers integral membrane proteins to the cytoskeleton. We previously reported that Ank3 + /- heterozygous mice, which have 50% reduced ankyrinG in the brain, exhibit behavioral changes reminiscent of bipolar mania (impulsivity, increased motivation for reward), and altered expression of proteins involved in neuron axonal transport. Both the behavioral and protein expression changes in Ank3 + /mice are normalized by lithium treatment, supporting the disease relevance of these findings. This study sought to further examine the molecular mechanism underlying the behavioral and neuronal changes induced by ankyrin3 suppression in brain. Methods: We performed RNA sequencing of hippocampus from Ank3+ /- heterozygous and Ank3 + / + wildtype mice, followed by identification of differentially expressed genes using the Tuxedo package. Biological pathways implicated by RNAseq analysis were verified by Western blot of hippocampal protein and live cell imaging of forebrain primary neurons. CRISPR/dCas9 technology was used to repress ankyrin3 transcription in a mouse neuronal system in order to conduct in-depth biochemical analysis. Neurons were treated with lithium and CHIR-99021, a selective inhibitor of the lithium target GSK3B, as well as an inhibitor of CRMP2, an axon-specific substrate of GSK3B. Results: RNAseq identified 283 differentially expressed genes between Ank3 + /- and Ank3 +/ + mouse hippocampus (fold change41.2, po10-3). There was enrichment of genes involved in microtubule-mediated processes (e.g., cytoskeleton, cargo trafficking), implicating altered microtubule functions in Ank3 + /- mice. Western blot confirmed increased expression of the microtubule interacting protein EB3 in the hippocampus of Ank3 + /- compared to Ank3+ / + mice (40% increase, po0.01), supporting microtubule instability. Live cell imaging of EB3 movement in primary neurons verified microtubule instability in Ank3 +/- mice (p =0.002). This was further confirmed in ankyrin3 repressed neurons (i.e., 420% increased EB3, po0.01; 25% lower polymerized tubulin, po0.01), and was reversed by treatment with lithium and CHIR-99021. Increased GSK3B-specific phosphorylation of CRMP2-Thr514 (430%, po0.003) indicated impaired CRMP2 activity in ankyrin3 repressed neurons. Treatment with a CRMP2 inhibitor blocked lithium and CHIR-99021 reversal of elevated EB3, indicating that CRMP2 dependent signaling is involved in the microtubule defects of Ank3 + /- mice.



Discussion: Ankyrin3 disruption in mice is associated with altered neuronal microtubule dynamics. Lithium reversal of both the microtubule and behavioral changes of Ank3 +/mice suggest that microtubule defects underlie manic-like behaviors observed in these mice. Our results support the investigation of risk genes using mouse and neuronal models to elucidate the neural mechanisms underlying psychiatric illness.

Disclosure: Biohaven Pharmaceuticals – Royalties, Self


the result of a secondary defect or suggest that the Dhcr7T93M/Δ3-5 model does not replicate the behavioral traits of patients. The hippocampus is the part of the brain responsible for learning, memory and spatial awareness. As such, the changes seen in the hippocampus could explain the decreased shredding observed in the Nestlet test and the potential delayed learning of the Dhcr7T93M/Δ3-5 mice, observed by the normalization of the behaviors in the EPM and open field tasks. These results are suggestive of subtle structural defects and justify further analysis, particularly using mice with an isogenic background where the differences may be more distinct. As SLOS does not have an optimal treatment pathway, if the results suggested by these tests are accurate, it could help aid the development of therapeutic interventions.

Disclosure: Nothing to disclose.

Joanna Cross ,1, Margaret Keil1, Forbes Porter1, Frances Platt2





National Institutes of Health University of Oxford

Background: Smith-Lemli-Opitz Syndrome (SLOS) is an autosomal recessive inborn error of cholesterol synthesis caused by mutation of the 7-dehydrocholesterol reductase (DHCR7) gene. This results in abnormal sterol levels, increased 7-dehydrocholesterol and typically decreased cholesterol. Although SLOS has a characteristic physical phenotype, with the most common finding being 2-3 toe syndactyly, there are also multiple behavioral abnormalities. These include cognitive deficits, anxiety, hyperactivity, sleep cycle disturbance, language impairment and autism spectrum behaviors. Methods: There are currently two main mouse models of SLOS; a homozygous null, Dhcr7Δ3-5/Δ3-5, and a model combining the null mutation and the common p.T93M missense mutation, Dhcr7Δ3-5/T93M. Unlike the null model, the hypomorphic Dhcr7T93M/Δ3-5 mice can live to adulthood and are therefore suitable for behavioral studies. Anxiety can be measured via multiple commonly used protocols; elevated plus maze (EPM), open field test and assessing burrowing and nesting behavior. Results: Although there was variation in all genders and genotypes, overall the Dhcr7T93M/Δ3-5 mice were marginally more likely to have increased burrowing compared to controls. However, this was only significant at 4 months in the overnight study. In contrast, the Nestlet test and EPM are both suggestive of decreased anxiety in the Dhcr7T93M/ Δ3-5 mice. Additional histological analysis showed that Dhcr7T93M/Δ3-5 mice tended to have a smaller hippocampus and anterior commissure than age and gender matched controls. Discussion: The increased burrowing could be showing the increased anxiety phenotype or hyperactivity, which is also noted in the increased gait speed of the Dhcr7T93M/ Δ3-5 mice in the open field test. However, the results of the Nestlet test and EPM are opposite to what is typically observed in patients, therefore these findings are either


Paola Giusti-Rodriguez ,1, Leina Lu2, Cheynna Crowley1, Julien Bryois3, Xiaoxiao Liu2, Amir Sariaslan3, Ivan Juric4, Joshua Martin1, NaEshia Ancalade1, Daniela DeCristo1, Craig Stockmeier5, Ming Hu4, Fulai Jin2, Yun Li1, Patrick Sullivan1 1

University of North Carolina at Chapel Hill Case Western Reserve University 3 Karolinska Institutet 4 Lerner Research Institute, Cleveland Clinic Foundation 5 University of Mississippi School of Medicine 2

Background: Schizophrenia is an often devastating psychiatric disorder with substantial morbidity, mortality, and personal and societal costs. Genome-wide association, Copy Number Variant (CNV), and exome studies have demonstrated that schizophrenia is a complex genetic disorder, with likely hundreds of genes contributing to its genetic architecture. For most schizophrenia GWAS loci, we are unable to pinpoint the specific genes that connect with these GWAS signals or the direction of association. This markedly limits the biological, clinical, and therapeutic utility of these findings. Prior studies have demonstrated the importance of chromatin looping as a regulatory mechanism and as a way to connect Single Nucleotide Polymorphisms (SNPs) to genes. We aim to facilitate the interpretation and prioritization of genomic findings from schizophrenia and other psychiatric disorders by generating a catalog of chromatin interactions in fetal and adult brain using easy Hi-C (eHi-C, a variation of Hi-C). Generating CNSspecific functional genomics data is critical for the development of a mechanistic understanding of genetic findings in psychiatric disorders. Methods: Chromosome conformation capture methods enable the identification of chromatin interactions in vivo. In