Autism is a neurodevelopmental disorder that affects approximately 0.6% of the U.S. population. The causes of autism spectrum disorders are under intense investigation, with strong evidence for genetic substrates. Lifetime costs of caring for autistic individuals are high, both in terms of a) quality of life for the affected individuals and their families;b) financial expenses to the families, educational systems, and health care agencies. Discovery of multiple gene mutations, copy number variants, and epigenetic factors in people with autism has spurred the development of mouse models with the homologous mutation. Genetic manipulations in mice offer an optimized experimental strategy to understand the consequences of candidate gene mutations. Effective treatments for the core symptoms of autism are currently limited to early behavioral interventions. Discovery of effective pharmacological treatments requires a greater understanding of the genes, biological mechanisms, and environmental factors that contribute to the causes of autism. Animal models with robust phenotypes relevant to the diagnostic symptoms of autism offer an optimized experimental strategy to test the efficacy and safety of proposed treatments. Our Laboratory of Behavioral Neuroscience (LBN) is an international leader in behavioral assays for transgenic and knockout mice with mutations in genes expressed in brain pathways involved in neuropsychiatric disorders. We collaborate with a large number of molecular genetics laboratories that contribute mutant lines of mice with mutations in risk genes for autism to our research program. In FY2010 we tested shank1 knockout mice generated by Morgan Sheng at the Massachusetts Institute of Technology, shank3 knockout mice gnerated by Joseph Buxbaum at Mt. Sinai School of Medicine in New York, neuroligin2 and neuroligin4 knockout mice generated by Nils Brose at the Max Planck Institute, and BDNF trangenic mice in our laboratory and collaboration with Postdoctoral fellow Francesco Papaleo in Danny Weinberger's CBDB group, IRP, NIMH. A new line of engrailed2 mice recently arrived from Manny DiCicco-Bloom and Jim Millonig at RW Johnson Medical School in New Jersey. Neuroligins and shanks are families of cell adhesion proteins that regulate synapse maturation. Single mutations in several different neuroligin and shank genes have been detected in a small number of autistic individuals. During FY2010, shank1, shank3, neuroligin2, and neuroligin4 mice were evaluated on social and repetitive behavioral assays, and control measures of general health, by Research Fellow Dr. Mu Yang, Senior Research Associate Dr. Jill Silverman, Postbaccalaureates Adam Katz and Sarah Turner, and HHMI student interns Leuk Woldeyohannes and Dieynaba Diagne. Results to date indicate normal adult sociability but reductions in specific juvenile social interactions in some of these shank and neuroligin mutant mice, relevant to the first diagnostic symptom of autism. Because shank3 mutations also appear in the chromosomal deletion that defines Phelan-McDermid syndrome, Postbaccalaureate Danielle Abrams and HHMI student intern Harry Simon are testing sensory and motor functions and cognitive abilities relevant to this co-morbid neurodevelopmental disorder in the shank3 mice. Postdoctoral fellow Florence Roullet and Postbaccalaureate Roheeni Saxena analyzed social olfactory cues and responses using a novel scent marking task. Results indicate generally normal social olfactory communication in most cases in these lines of mutant mice. Postbaccalaureate Mark Harris worked with former postdoctoral fellows Maria Luisa Scattoni and Markus Wohr to analyze ultrasonic vocalizations in social settings, including pup separation, male/female interactions, and exposure to social olfactory cues. Significantly fewer social vocalizations were seen in some of these lines of mice, indicating abnormalities in social communication, relevant to the second diagnostic symptom of autism. During 2006-2008, we discovered autism-relevant phenotypes of the inbred mouse strain BTBR T+tf/J (BTBR). Lack of sociability was confirmed in multiple independently bred cohorts, on multiple social tasks including juvenile play, adult social approach, adult reciprocal social interactions, and social transmission of food preference (Yang et al.,2007a,b, 2009;McFarlane et al., 2008), relevant to the first diagnostic symptom of autism, abnormal social interactions. During 2009-2010, postdoctoral fellows Maria Luisa Scattoni, Florence Roullet,and Markus Wohr discovered that BTBR emitted fewer ultrasonic vocalizations in many social settings as compared to B6, and emitted less olfactory scent marking in some social settings (Roullet et al., 2010;Scattoni et al., 2010;Wohr et al., 2010), relevant to the second diagnostic symptom of autism. High levels of repetitive self-grooming are routinely detected in cohorts of BTBR (Yang et al., 2007a,b, 2009;McFarlane et al., 2008), relevant to the third diagnostic symptom of autism, repetitive behaviors. The absence of a corpus callosum in BTBR is unlikely to explain their phenotypes, as corpus callosum lesions did not affect social behaviors in C57BL/6J (B6), a standard control strain of mice with high sociability (Yang et al., 2009). More global underlying disruptions of white matter connectivity may contribute to the BTBR phenotypes. Genetic mechanisms responsible for the autism-like phenotypes in BTBR were pursued in FY2010. In collaboration with Dr. Elliott Sherr, University of California San Francisco, Dr. Yang and Postbaccalaureate Adam Katz completed the behavioral phenotyping for a quantitative trait loci linkage analysis (QTL) of the BTBR x B6 cross, designed to discover genes in the BTBR background that correlate with their autism-like phenotypes. 400 F2 mice were scored for social and repetitive behaviors. Tailsnips of the 400 behaviorally tested F2 mice are currently being genotyped in Dr. Sherr's laboratory. Biological mechanisms underlying the high levels of repetitive self-grooming in BTBR were evaluated by Dr. Silverman, in collaboration with Dr. Jim Koenig at the University of Maryland Psychiatric Research Center. High circulating corticosterone suggested a stress-related mechanism. However, low responses by BTBR on stressful behavioral tasks, and similar levels of corticotropin releasing factor in BTBR and B6 brain, indicate that repetitive self-grooming in BTBR is not mediated centrally by a brain response to stress. A major translational component of our Project MH-002179 is the preclinical search for potential treatments. Robust and highly replicated social deficits and repetitive self-grooming in BTBR provide a good model system for testing the ability of drugs and behavioral treatments to reverse and prevent autism-like symptoms. During FY2009, Dr. Silverman and Postbaccalaureate Sarah Turner discovered that MPEP, an mGluR5 antagonist reported to reverse phenotypes in Fragile X mice, blocked repetitive self-grooming in BTBR at doses that were not sedative. During FY2010, Sarah Turner and summer student intern Seda Tolu replicated this effect of MPEP. Their preliminary data indicate the same amelioration of repetitive behavior with a more selective mGluR5 antagonist, MTEP. Postbaccalaureate Sarah Turner tested CX546, a prototypic ampakine modulator, and detected significant improvement in sociability in BTBR in the first study. Postbaccalaureate Mike Karras is now repeating the CX546 experiments with a different drug vehicle and a different route of administration. These pharmacology studies provide evidence for the translational value of our assays in optimized mouse models of autism, for preclinical discovery of novel therapeutics to treat the diagnostic symptoms of autism spectrum disorders.
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