The Use of Drosophila to Study ASD Candidate Gene Function

Background: The number of ASD candidate genes has increased greatly in recent years with the completion of high resolution CNV analyses on simplex and multiplex families. That number is likely to increase further as genome sequencing efforts on patients with ASDs reach completion. However, there has been a lag between the identification of these candidate genes and our understanding of the pathophysiology of ASDs that is due to our ignorance of the biological functions of many of these genes. For a few candidate genes, the use of animal models has been informative, but it is impractical to make mouse knockouts of all of the ASD candidates. The fruit fly, Drosophila melanogaster, is a well characterized genetic model organism that has previously been used to gain insight about human diseases, particularly neurodegenerative disorders and cancer. The low cost, short generation time, and ease of genetic manipulation make Drosophila an ideal system for examining the biological functions of many ASD candidate genes as well as assessing the biological impact of human disease variants.

Objectives: We aim to show that Drosophila can be used to effectively study the functions of ASD candidate genes from the standpoint of neural development rather than behavior. To accomplish this, we have chosen to study Neurexin IV (the Drosophila homolog of a highly penetrant ASD candidate gene, CNTNAP2) as a proof of principle. We will examine the effects of loss of Neurexin IV on CNS neurons, identify biochemical interaction partners, and assess the impact of evolutionarily conserved rare variants (missense mutations) in Neurexin IV that are linked to cases of Autism.

Methods: We have generated a molecularly defined loss of function allele of Neurexin IV that can allow us to selectively remove Neurexin IV in select populations of neurons. We have also generated transgenic flies that have tagged versions of Neurexin IV that will allow us to identify binding parters through mass spectroscopy. We have also generated transgenic flies that express the human CNTNAP2 gene under the control of the Drosophila Neurexin IV locus that will allow us to assess to what degree the human gene can rescue loss of Neurexin IV.  We are currently generating transgenic flies that have ASD related variants of Neurexin IV and will assess their function in a Neurexin IV mutant background.

Results: Loss of Neurexin IV in the Drosophila eye results in defects in eye development as well as the loss of known Neurexin IV binding partners, Contactin and Coracle. We are currently investigating if loss of Neurexin IV leads to defects in synapse formation or function. Biochemical experiments to identify further binding partners are underway, as are experiments to determine the biological significance of ASD related rare variants.

Conclusions:  We estimate that about 60% of current ASD candidate genes have a high degree of evolutionary conservation between humans and Drosophila. Based on our experiences studying Neurexin IV, Drosophila can be used to effectively probe the biological function of many ASD candidate genes and thereby increase our understanding of ASD pathophysiology.