Poster Session II Cntnap2a and 2B Knockouts

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Poster Session II Cntnap2a and 2B Knockouts Neuropsychopharmacology (2011) 36, S198–S323 & 2011 American College of Neuropsychopharmacology. All rights reserved 0893-133X/11 S198 www.neuropsychopharmacology.org Poster Session II CNTNAP2a and 2b knockouts. These mutations are predicted to be Tuesday, December 6, 2011 5:30 PM – 7:30 PM damaging as they occur early in the coding regions of each gene and produce a frameshift, resulting in a premature stop codon and truncation of the protein in or immediately after the 1. A Zebrafish Model for the Functional Analysis of Genes in N-terminal discoidin domain. In total, we have successfully Autism generated zebrafish founders with deleterious germline mutations Ellen J. Hoffman*, Antonio Giraldez, Matthew State in both CNTNAP2 paralogs. We are currently conducting a battery of morphological and behavioral assays to identify quantifiable Yale University, New Haven, USA phenotypes in CNS structure and larval neural circuits in mutant fish. Discussion: Our experiments lay the foundation for the use of Background: A critical challenge in the genetics of neuropsychia- zebrafish as a model system for elucidating the function of tric disorders is distinguishing deleterious rare mutations from susceptibility genes in ASD. We have identified the zebrafish neutral variants, as rare sequence and structural variants have orthologs of the ASD susceptibility gene, CNTNAP2, demonstrated been identified throughout the genomes of both affected and its expression in the zebrafish CNS, and successfully generated unaffected individuals, including at candidate gene loci. The ability the first ZFN-induced deletions in each CNTNAP2 paralog. This to distinguish rare functional from rare neutral variation is critical model has tremendous promise for illuminating common path- for confirming the association of risk genes carrying rare alleles. ways involving ASD susceptibility genes and rapidly assessing the For this reason, we propose to develop an in vivo model that will functional consequences of rare sequence variation in a risk gene. allow us rapidly to differentiate mutations that alter the function Future applications of this research include large-scale pharma- of susceptibility genes from neutral rare variants. This novel cological screens to identify novel therapeutic targets aimed at the approach capitalizes on critical advantages of zebrafish, including mechanisms underlying the core deficits of ASD. visualization of the developing nervous system in transparent Disclosure: E. Hoffman: none. A. Giraldez: none. M. State: none. embryos, ease of genetic manipulation, and large progenies that facilitate the conduct of large-scale pharmacologic screens. Therefore, we generated zebrafish knockouts of the ASD risk gene, CNTNAP2, using the emerging technology of zinc finger nucleases (ZFN). We anticipate that CNTNAP2 will be particularly 2. Histidine Decarboxylase Deficiency produces Tourette informative in this regard, as homozygous disruption of CNTNAP2 Syndrome Phenomenology and Dopamine Dysregulation in by a single base pair deletion in the Old Order Amish population Humans and Mice Lissandra C. Baldan Ramsey, Kyle Williams, Jean-Dominique causes a monogenic syndrome that is highly associated with ASD. Gallezot, Michael Crowley, George Anderson, Bennett L. Leventhal, In addition, the State laboratory identified a de novo chromosome Hiroshi Ohtsu, John H. Krystal, Linda Mayes, Ivan de Araujo, 7q inversion disrupting CNTNAP2 in a child with cognitive and Yu-Shin Ding, Matthew W. State, Christopher Pittenger* social delay. Our goals in developing this model are: 1) to leverage any distinctive reproducible and quantifiable phenotype for Yale University, New Haven, USA forward genetic studies that will help to elaborate conserved molecular mechanisms and pathways involving these susceptibility Background: Tourette syndrome (TS) is a neurodevelopmental genes; and 2) to test the relative ability of the wild type human gene disorder characterized by tics and sensory gating abnormalities. compared to constructs containing rare mutations identified in Available treatments are limited, and its neurobiological under- affected and unaffected individuals to rescue the identified pinnings are not well understood. Pathophysiological analysis of phenotype. such conditions in animal models can provide key insight; Methods: We identified the zebrafish orthologs of the human however, modeling complex neuropsychiatric conditions presents CNTNAP2 gene by conducting a search of the zebrafish genome daunting challenges. TS has a heritability of 30-60%, but causative (Zv7) in the National Center for Biotechnology Information mutations and risk alleles have proven elusive, attesting to a (NCBI) database. We analyzed the expression patterns of these complex, heterogeneous genetic architecture. In this setting, the paralogs in zebrafish embryos at 30 and 48 hours post fertilization investigation of rare, highly penetrant mutations is of particular by in situ hybridization. We utilized ZFN directed against exons 2 value. A recent study of a dense 2-generation TS pedigree and 3 of CNTNAP2a and CNTNAP2b, respectively, to generate identified a rare segregating nonsense mutation, Hdc W317X, in targeted germline deletions in each gene. Founders were generated the l-histidine decarboxylase (Hdc) gene (Ercan –Sencicek et al., by injecting mRNA encoding ZFN (Sigma-Aldrich) targeting either NEJM 2010). The causal connection between a reduction in HDC CNTNAP2a or 2b into embryos at the one-cell stage. Founders were activity and the symptoms of TS remains unclear. Here we analyze identified by screening the progeny of incrosses of ZFN-injected mice lacking one or both copies of the Hdc gene and TS patients adult fish by PCR followed by high-resolution fragment analysis. carrying the Hdc W317X mutation to establish that reduction in Founders were outcrossed to wild-type fish, and the heterozygous histamine (HA) biosynthesis produces key phenomenological and CNTNAP2a and 2b knockouts were incrossed to generate viable neurochemical features of TS. homozygous knockouts. Methods: 9 TS patients carrying the Hdc W317X mutation were Results: We identified two zebrafish orthologs of CNTNAP2, characterized clinically and in prepulse inhibition (PPI). Adult CNTNAP2a and 2b. In situ analysis of these transcripts between male HDC knockout mice, backcrossed multiple generations onto 30 and 48 hours after fertilization revealed expression of both the C57Bl/6 genetic background, were tested in parallel behavioral paralogs in the CNS, predominantly in the midbrain and assays (stereotypy; PPI). Striatal dopamine in knockout mice was hindbrain. These paralogs demonstrate distinct yet partially assessed by in vivo microdialysis. Dopamine D2/D3 receptors were overlapping expression patterns. Utilizing ZFN targeting each quantified in vivo in patients carrying the Hdc W317X mutation by gene, we generated multiple zebrafish founders with deleterious positron emission tomography (PET) with the D3-preferring germline mutations in both CNTNAP2a and 2b and outcrossed ligand PHNO, and in vitro in mice using the D2/D3 ligand these founders to wild-type fish, producing viable heterozygous raclopride. ACNP 50th Annual Meeting Abstracts S199 Results: All patients carrying the Hdc W317X mutation had current we hypothesized that they may also have poor attentional control. and/or past tics; patients also had a deficit in auditory PPI, as has Indeed, impulsivity may be conceptualized as a consequence of such been shown in TS more generally. Heterozygous and homozygous poor top-down control of attention. We therefore characterized the knockout mice had reduced brain histamine and exhibited parallel attentional capacities of STs and GTs using our translational behavioral symptomatology, with enhanced stereotypy after Sustained Attention Task (SAT; e.g., Demeter et al., 2008, 2011). amphetamine administration and a deficit in PPI. Mice showed Methods: Sprague-Dawley rats were screened using a Pavlovian increased dopamine in the dorsal striatum during the active (dark) Conditioned Approach (PCA) procedure and designated STs or phase of the circadian cycle, when histamine was high in wild-type GTs, as described previously (e.g., Lovic et al., 2011). Animals were controls. Both patients and mice showed an elevation in D2/D3 then trained on the SAT (see Demeter et al., 2008). This task receptor binding in the substantia nigra; this is consistent with the consists of random sequences of unpredictably occurring visual upregulation of D3 receptors in this structure seen in other signals of variable duration as well as trials during which no signal is contexts after chronic dopamine elevation. presented (blanks). Following a signal or non-signal event two levers Discussion: Parallel behavioral abnormalities in patients and mice are extended into the chambers for 4 sec, and subjects report the establish a casual link between disruption of the Hdc gene and presence or absence of a signal by pressing the appropriate lever. TS-like behavioral abnormalities, validating the unexpected connec- Hits and correct rejections are rewarded while misses and false tion first revealed by genetic linkage in a single family. Dysregulation alarms trigger a variable intertrial interval. Daily sessions lasted of striatal dopamine represents a plausible pathophysiological link 64 min and consisted of approximately 318 trials total. Animals were between histamine deficiency and functional abnormalities in the extensively trained until their performance
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