Behavioral changes in G72/G30 transgenic mice

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Abstract

Genetic studies have implicated the evolutionary novel, primates-specific gene locus G72/G30 in schizophrenia, bipolar and panic disorders. It encodes for a protein LG72 whose function has been controversially discussed as putative regulator of the peroxisomal enzyme D-amino-acid-oxidase (DAO), or as a mitochondrial protein, which promotes robust mitochondrial fragmentation in mammalian cell lines including human and rat primary neurons. Because of this conserved function we here have generated “humanized” BAC transgenic mice (G72Tg) expressing alternatively spliced G72 and G30 transcripts, and the LG72 protein. G72 expression is prominent in granular cells of the cerebellum, the hippocampus, the cortex and the olfactory bulb. Most strikingly, G72Tg mice displayed deficits in sensorimotor gating which could be reversed with haloperidol, increased sensitivity to PCP, motor-coordination deficits, increased compulsive behaviors and deficits in smell identification. These results demonstrate that expression of the human G72/G30 gene locus in mice produces behavioral phenotypes that are relevant to psychiatric disorders.

Introduction

Schizophrenia (MIM 181500) is a debilitating mental disorder with a complex symptomatology that comprises positive (e.g. hallucinations, delusions), as well as enduring negative (e.g. emotional blunting) symptoms, cognitive and motor dysfunctions and catatonic behaviors. The disease affects approximately 1% of the adult population with similar prevalence among different ethnic groups (Bromet and Fennig, 1999). Family , twin , and adoption studies have clearly demonstrated that genetic risk factors contribute to the disease etiology (Tsuang, 2000). Among all schizophrenia linkage findings listed by the Human Gene Nomenclature Committee, SCZD7 on chromosome 13q32-q33 (MIM 603176) is considered one of the promising candidate regions. A total of 9 genome-wide studies in schizophrenia have independently reported linkage on SCZD7 (Abou Jamra et al., 2006). A systematic linkage disequilibrium (LD) mapping further supported and highlighted SCZD7. In two independent schizophrenia samples, Chumakov et al. (Chumakov et al., 2002) found significant associations at a locus on SCZD7 containing two overlapping genes, G72 and G30. This finding was followed by significant replications in 12 independent schizophrenia samples (Abou Jamra et al., 2006). Consistent with the replication findings, a meta-analysis combining 10 association samples found p-values between p < 0.0001 and p = 0.0413 for several genetic markers on G72/G30 (Detera-Wadleigh and Mcmahon, 2006). Based on the G72/G30 results, this locus is considered one of the few true-positive and most robust association findings in schizophrenia so far. Additionally, several studies report evidence for linkage at 13q32-q33 with BPAD (Detera-Wadleigh et al., 1999, Kelsoe et al., 2001, Liu et al., 2001, Stine et al., 1997) and panic disorder (Hamilton et al., 2003).

G72 and G30 are transcribed from overlapping opposite DNA strands (Fig. 1A-B; reviewed in (Abou Jamra et al., 2006)). These genes appeared late during primate evolution and G72 is only found in anthropoid primates (Chumakov et al., 2002). It seems to represent an exceptional case of a primate-specific gene with a rapidly changing protein structure, most probably connected with a rapid evolution of underlying brain function (Kvajo et al., 2008). The transcripts are very weakly expressed and encoded proteins have no known functional motifs. LG72, the longest splice variant, has been demonstrated to be located in mitochondria and to alter mitochondrial morphology without affecting mitochondrial integrity. In this study the mitochondrial localization and function were not only found in Wt Hela cells and LG72 transfected Cos7 cells but also in LG72 transfected rat primary neurons, thus demonstrating that LG72 function is preserved in rodents (Kvajo et al., 2008). Yeast two-hybrid and biochemical studies possibly contradict a mitochondrial function as they demonstrated that LG72 affects DAO activity, the main degrading enzyme of D-serine, an allosteric co-activator of NMDA receptors. (Chumakov et al., 2002, Sacchi et al., 2008). To address behavioral consequences of G72, we have generated transgenic mice carrying the entire human G72/G30 genomic region. Intriguingly, these mice display behavioral symptoms related to psychiatric diseases.

Section snippets

Generation of the G72Tg mice and analysis of transgenic mice

The BAC-plasmid BAC RP11-111A8, containing the whole G73/G30 genomic region was obtained from the Children's Hospital Oakland Research Institute. BAC DNA was prepared using the Large Construct Kit from Qiagen (Hilden Germany), diluted in oocyte injection buffer (5 mM Tris, pH 7.4, 0.2 mM EDTA, 100 mM NaCl) to 1 ng/µl and injected into pronuclei of fertilized oocytes from CD1 mice. The transgenic lines were crossed to CD1 WT mice and were maintained and analyzed as heterozygote lines. Transgenic

G72/G30-derived transcripts in BAC transgenic mice

We established two transgenic mouse lines (G72Tg1 and G72Tg2) carrying the human BAC clone RP11-111-A8, respectively. Animals of both lines appeared healthy, were fertile, cared for their offspring, and transmitted the transgene with the expected Mendelian frequency.

BAC transgenes often accurately recapitulate the expression pattern of the cognate endogenous gene. To date, several differentially spliced G72 transcripts have been described from human brain and testes tissues (UCSC database).

Discussion

We have generated G72/G30 BAC-transgenic mice in order to study the function of this primate-specific gene locus on the modulation of animal behaviors. This transgenic approach is currently the only possible method to address the in vivo G72/G30 functions, because pharmacological tools are not yet available and genetic manipulations of anthropoid primates, the only animal species that has this gene locus, are not feasible. Mice are an appropriate model organism for the expression of G72 since

Role of the funding sources

This work was supported by grants from the Federal Ministry of Education and Research (NGFN2, and 01GW0511). These granting source had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors

David-M. Otte, Andras Bilkei-Gorzo, and Öznur Yilmaz planned and performed most of the experiments and analyzed the data. Martin Holst and Karl Schilling conducted in situ hybridization analysis. Isabel Benzel provided the G72 antibody. Rami Abou Jamra and Johannes Schumacher contributed to the initial concept of the study. Heinz Beck and Wolfram S. Kunz assisted in carrying out the study. Christoph Turck and Michaela Filiou performed the 2D Western blot analysis. Andreas Zimmer planned and

Conflict of interest

Author Isabel Benzel was by GlaxoSmithKline in the Psychiatry Discovery Technology Group, Harlow, United Kingdom. All authors declare that they have no conflicts of interest.

Acknowledgements

We thank Peter Propping for encouraging this project, continuous support and suggestions, Jürgen Schmidt for pronucleus injections, Kerstin Michel for help with behavioral experiments, and Daniela Mauer for help with isolation and purification of BAC-plasmids.

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