Poster Session Iituesday, December 09, 2014
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Neuropsychopharmacology (2014) 39, S291–S472 & 2014 American College of Neuropsychopharmacology. All rights reserved 0893-133X/14 www.neuropsychopharmacology.org Tuesday, December 09, 2014 whether D-serine administration, which reverses these abnormalities at the RNA and protein level, also normalizes the epigenetic perturbations. T1. Altered CREB Binding to Activity-dependent Genes Keywords: CREB, Arc, BDNF, miR132. in Serine Racemase Deficient Mice, a Mouse Model of Disclosure: JTC served as a consultant for EnVivo and Schizophrenia Abbvie in the last 2 years. A patent owned by MGH for the use of D-serine as a treatment for serious mental illness Darrick Balu*, Joseph Coyle could yield royalties for Dr. Coyle. Harvard University, McLean Hospital, Belmont, Massachusetts T2. Wnt Signaling, Neurodevelopmental, and Behavioral Background: cAMP-response-element-binding protein Phenotypes in a Dixdc1 Knock-out Mouse Model of (CREB) is a ubiquitously expressed transcription factor in Psychiatric Illness the brain that regulates neuroplasticity by modulating gene expression. There are numerous signaling pathways by Benjamin Cheyette*, Robert Stanley, Pierre-Marie Martin which information is transmitted from the cell membrane to University of California at San Francisco, San Francisco, the nucleus that in turn affects CREB binding to DNA. The California influx of calcium through N-methyl-D-aspartate receptors (NMDARs) is a well-defined mechanism that leads to the Background: DIXDC1 encodes a scaffold protein enriched increased expression of CREB-dependent genes, including in neurons that physically interacts with the DISC1 protein brain derived neurotrophic factor (BDNF), microRNA-132 implicated in major mental illness, as well as with the (miR132), and activity-regulated cytoskeleton-associated Dishevelled protein central to Wnt/beta-catenin intercellu- protein (Arc). We have previously demonstrated that serine lar signaling. The Wnt/beta-catenin signaling pathway is racemase knockout (SR-/-) mice, which exhibit NMDAR broadly involved in neural development and for this reason hypofunction, also have reduced mRNA and protein levels has been considered a pontential pathogenic neurodevelop- of the aforementioned CREB-dependent genes in the mental pathway in psychiatric illness. Moreover, GSK3, the hippocampus. In addition, these molecules are reduced in central kinase in the Wnt/beta-catenin pathway, is a schizophrenia. pharmacological target of lithium at therapeutic doses. Methods: Wild-type (WT; n ¼ 5-6) and SR-/- (n ¼ 5) mice Recent human genetic studies have buttressed interest in were killed and their hippocampi were flash frozen on dry this pathway by showing that loss-of-function mutations in ice. Tissue samples were dissociated and then fixed with the CHD8 gene, which up-regulate Wnt/beta-catenin path- 1.5% paraformaldehyde. Chromatin was digested using the way target genes, are a susceptibility factor in the autism SimpleChIP Plus Enzymatic Chromatin IP kit (Cell Signal- spectrum disorders. ing Technologies). The chromatin was incubated with a Methods: We have generated a Dixdc1 knock-out (KO) rabbit anti-CREB antibody (1:50; Cell Signaling Technolo- mouse line using traditional (homologous recombina- gies) overnight at 41C on a rotisserie. The crosslinks were tion) gene-targeting techniques. We have analyzed and reversed and the DNA was purified for analysis using qPCR compared behavior in littermates homozygous for the (Sybr Green). To determine the levels of CREB bound at wild type Dixdc1 locus, heterozygous, or homozygous each gene of interest, PCR primers were directed near the for the Dixdc1 KO allele. We have tested the response CRE sequence of each promoter. All of the primers were of behavioral differences in these animals to psycho- validated in previous publications. Melting temperature pharmacological agents, including lithium. We have also analysis was performed after every PCR to ensure accuracy. analyzed neurodevelopmental phenotypes in the brains Results: We first validated the ChIP kit for brain tissue of these animals and in cultured forebrain (hippo- using a positive control antibody and primers to ensure campal) neurons. Separately, we have been probing for proper chromatin shearing. SR-/- mice have approximately SNVs at the DIXDC1 locus in human psychiatric patients 50% less CREB binding at the miR-132 gene than WT mice. (both in our own samples and in publicly-available Using BDNF exon-specific primers, SR-/- mice have sequence databases) and are testing these for functional significantly less CREB binding at the BDNF I promoter, differences in assays of Wnt/beta-catenin signaling and in but not at the BDNF IV promoter. Finally, SR-/- mice have assays based on phenotypic findings in this knock-out 80% less CREB binding to the synaptic activity response mouse model. element (SARE) region of the Arc gene. Results: Mice homozygous for loss-of-function at the Conclusions: These data demonstrate that in vivo, NMDAR Dixdc1 locus are viable, fertile, and grossly normal in hypofunction caused by the selective removal of the development. In extended (unpublished) behavioral assays NMDAR co-agonist, D-serine, leads to altered CREB bind- we have found that loss-of-function at the Dixdc1 locus ing on known activity-dependent genes. The ChIP results leads to a specific dose-sensitive behavioral deficit: reduced suggest that reduced CREB binding contributes to the lower motivation (Forced Swim and Tail Suspension tests). This levels of BDNF, miR-132, and Arc that we previously deficit is responsive to correction by acute administration of observed in SR-/- mice. Future studies will determine either lithium or a specific GSK3-inhibitor. We have ACNP 53rd Annual Meeting Abstracts S292 concurrently found that forebrain pyramidal neurons from mice expressing cre-recombinase under the control of the Dixdc1 knock-out animals have reduced dendritic spines PACAP promotor. The PACAP-cre mice facilitate real time and glutamatergic synapses - tests of the response of this manipulation of PACAP expressing neurons, through synapse phenotype to lithium and to pharmacological GSK3 neuroanatomically directed viral injections conferring inhibition are ongoing at the time of this abstract. Unlike expression of chemogenetic tools. These neurons are then some other Wnt signal scaffold proteins that we have stimulated or inhibited by intraparenchymal injection of the studied previously, loss of Dixdc1 does not lead to more otherwise inert molecule, clozapine-N-oxide (CNO). The generally reduced dendrite complexity as quantified by same animals injected with saline serve as controls, and data Sholl analysis, nor to changes in inhibitory synaptic is analyzed by paired t-test. In our second approach, we markers. Excitatory synapse phenotypes in the mouse KO created lox-PACAP mice that possess loxP sites flanking the can be rescued by expression of a wild type human DIXDC1 2nd exon of the PACAP gene, allowing for deletion of cDNA. functional PACAP in the presence of cre-recombinase. Conclusions: Our data support a role for the Dixdc1 protein The PACAP-lox mice were bred with Sim1-cre mice to in the generation and maintenance of excitatory synapses in induce the conditional knock-out of PACAP within the PVH the forebrain. Our data further suggest that this occurs in order to discern the specific effect of PACAP in the downstream of activity in the Wnt/beta-catenin pathway circuit. Mice were subjected to assays for energy home- within developing neurons. Behavioral consequences of ostasis and anxiety, and compared to littermate controls Dixdc1 inactivation in mice are relatively specific for using ANOVA. motivation and can be reversed by lithium administration Results: Acute chemogenetic stimulation of paraventri- or by pharmacologically-specific GSK3 inhibition. This cular hypothalamic PACAP neurons caused an 2.5x supports a role for this protein and for its associated increase in food intake during the light cycle, when pathway in major affective disorders and their pharmaco- animals do not normally eat, and a trend toward increa- logical treatment. Using this animal model, we are testing sed anxiety like behavior, evidenced by less time spent in for differences in the signaling and neurodevelopmental the open arms of the elevated plus maze. Conditional knock activities of DIXDC1 SNVs found in psychiatric patients. out animals lacking PACAP in Sim1-expressing neurons Our studies provide independent support for the contribu- show a small, but significant, increase in body weight tion of abnormal Wnt/beta-catenin signaling pathway (B10%), as well as a trend to decrease anxiety-like activation during neural development to psychiatric patho- behavior. genesis. Conclusions: Stimulation of PVH PACAP neurons leads to Keywords: Wnt signaling, Excitatory Synapses, Motivation, hyperphagia and increased anxiety, while knock out of Lithium. PACAP from the same neurons leads to weight gain and Disclosure: Nothing to Disclose. decreased anxiety-like behavior. The non-reciprocal differ- ence in behavioral effects of stimulating synaptic transmis- sion versus effects of genetic knock out of PACAP in the T3. Toward an Understanding of Eating Disorders: same neurons points to the complex role neuromodulators Are PVH PACAP Neurons an Interface Between Stress play in neuronal communication. This circuitry may be Response and Feeding Behavior? important in understanding the genesis of eating disorders, as well as phenotypic differences in feeding behavior