Functional Dissection of the Autism Susceptibility Gene Slc25a12 in Model Systems

Background: SLC25A12 encodes a mitochondrial aspartate/glutamate carrier (AGC1), which is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and ATP production. The gene has been identified as a susceptibility gene for autism spectrum disorders (ASDs) (e.g., Ramoz et al, Am J Psychiatry, 2004) and is responsible for a neurodevelopmental syndrome when both alleles are mutated (Wibom et al, NEJM, 2009). Objectives: To understand the role of AGC1 in brain development as a means of relating alterations in this gene to neurodevelopmental disorders. Methods: We have developed mice with a targeted disruption of the Slc25a12 gene and we have developed BAC transgenic mice overexpressing this gene. Mice are characterized in biochemical, neuropathological and behavioral studies, followed by confirmatory in vitro studies. Results: Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In P13-14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetyl aspartate (NAA) and/or alterations in the NADH/NAD+ ratio, resulting in myelin defects. AGC1 heterozygotes showed reduction of Mag expression in brains, and showed mild spatial learning impairment in Morris water maze testing, and alterations in social behavior. We have also developed mice overexpressing AGC1 using BAC transgenic technology that ensures transgene expression in a pattern similar to the endogenous AGC1. We confirmed 2-3 fold over expression of AGC1 both at RNA and protein levels. We did not observe overexpression of oligodendrcyte/myelin genes in these mice. Currently, we plan to analyze these mice morphologically and behaviorally. Conclusions: Our data implicate AGC1 activity in myelination and in neuronal structure, and indicate that while complete loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development contributing to increased autism susceptibility. Studies relating genotype at the SLC25A12 locus with head circumference and cognitive measures in human subjects are underway. Supported by the Seaver Foundation.