Generation and characterization of the adult neuron-specific Aralar/AGC1 knock-out mice.

Abstract

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease (OMIM #612949, also named early infantile epileptic encephalopathy 39) characterized by hypomyelination, hypotonia, developmental arrest and epilepsy. In mice, the global aralar knockout shows hyperreactivity, growth retardation, motor discoordination, seizures and hypomyelination; thus, recapitulating the pathology of the human disease. Aralar brain expression is mainly restricted to neurons, while glial expression of Aralar has been demonstrated to be scarce. However, Aralar deficiency manifests alteration in fundamental glial functions such as glutamine and myelination synthesis by astrocytes and oligodendrocytes respectively. In order to dissect the role of neuronal Aralar in the disease mechanism of AGC1 deficiency, we have ablated the expression of Aralar in mature (from PND30 onward) excitatory cortical and hippocampal neurons using the CaMKIIα-driven Cre recombinase expression in Aralarlox/lox mice. Here, we present the characterization of the mature neuron-specific aralar knock out mice. We demonstrated that the excision of exon 3 aralar starts from PND30 and continues to occur during mouse adulthood. Consequently, Aralar protein levels gradually decay across the 2nd to 6th month up to a 70- 75% decrease in the cortex and a 50% decrease in the hippocampus. The drop in Aralar levels drive an alteration in cortical metabolites causing a 25% and 50% decrease in aspartate and serine levels, the metabolites most affected in the brain of the global aralar-KO mice. This partial decreases in ARALAR and metabolites caused by cortical CaMKIIα-driven aralar ablation are not accompanied by growth or behavioral alterations in a battery of motor, memory and cognitive tests. The results suggest that the absence of Aralar in CaMKIIα expressing neurons (cortical and hippocampal mature excitatory neurons), does not mimic the alterations caused by global deficiency of Aralar/AGC1. However, since the levels of cortical ARALAR in this model resemble those of healthy aralar+/- mice, further experiments are being carried out to explore the origin (or location) of the ARALAR protein remnants and the possible metabolic compensation by other neurons and/or cell types in these mice. Without ruling out either that the ARALAR-MAS pathway could play a more critical role during neurodevelopment than adulthood in brain physiology and metabolism.