Little is known about the growing number of gene mutations implicated in Autism Spectrum Disorder (ASD). Their mutations need to be studied in order to predict in vivo effects. We hypothesize that mutations found in ASD genes will cause dysregulation of GABAergic interneuron development. The aim of this project is to utilize a moderate throughput assay to identify in vivo functional changes that are caused by nonsynonymous mutations in candidate ASD genes. We will focus on CNTNAP2, an ASD susceptibility gene that has multiple mutant alleles that were identified in ASD populations. CNTNAP2 dysfunction may contribute to neuropsychiatric diseases such as ASD, schizophrenia, and epilepsy.

In order to develop an in vivo screening assay, GABAergic neural stem cells from the medial ganglionic eminence (MGE) are being utilized as a model system. The MGE has been found to produce GABAergic progenitors that migrate into the neocortex to differentiate into interneurons. These MGE cells can be easily transduced via lentiviruses and transplanted into wild type cortices to study the effect of expressing a gene in these cells as they develop. Cell autonomous functions of CNTNAP2 in developing cortical interneurons will be assessed by transducing lentiviruses that express human CNTNAP2 wild type (WT) or a CNTNAP2 mutant into MGE cells that lack the CNTNAP2 gene to assess the ability of CNTNAP2 mutants to functionally complement the CNTNAP2 loss of function phenotypes. The CNTNAP2 gene will be inserted into a lentiviral vector that includes a Dlxi12b enhancer specific to GABAergic cells of the forebrain and the minimal promoter beta globin. These elements drive expression of both GFP and CNTNAP2 within GABAergic neural precursors of the MGE. CNTNAP2 WT mice will be used as a control. Control and CNTNAP2-/- mice will be generated for MGE dissection, viral transduction, and cortical transplantation. The MGE cells that have been transduced with CNTNAP2 alleles will be studied at four developmental time points to assess migration, cell fate, cell morphology, connectivity and physiology. MGE cells obtained from embryonic day (E)12.5 will be transduced with Dlx12b-GFP-T2a-MCS lentivirus to express GFP and CNTNAP2 WT or mutant to asses the ability to rescue the lack of CNTNAP2.

This assay will be a novel way to bridge the increasing number of mutant ASD alleles being discovered in genetic screens with functional in vivo readout of allele function in mammalian neocortex. This will accelerate our ability to functionally assess if mutations enriched in ASD populations alter protein function in vivo. It will also nullify the need for time intensive and expensive knock in mouse models for each ASD mutant allele. This complementation assay can be modified to include different enhancers/promoters to study other cell types for moderate throughput region/cell-type specific in vivo screening.