βOHB protective pathways in Aralar-KO neurons and brain: an alternative to ketogenic diet.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest and epilepsy. We have investigated whether β-hydroxybutyrate (βOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-KO neurons and mice. We report that βOHB efficiently recovers aralar-KO neurons from deficits in basal- and glutamate-stimulated respiration, effects requiring βOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development; but this approach was unfeasible. Therefore, aralar-KO pups, were treated without distinction of gender with daily intraperitoneal injections of βOHB during 5 days. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), DOPAC/DA ratio and VMAT2 protein. Regarding postnatal myelination, MBP and MAG myelin proteins were markedly increased in the cortices of βOHB-treated aralar-KO mice. Although brain aspartate and NAA levels did not change by βOHB administration, a 4-day βOHB treatment to aralar-KO, but not to control neurons led to a substantial increase in aspartate (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain aspartate and NAA is possibly due to its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The efectiveness of βOHB as a therapeutic treatment in AGC1-deficiency deserves further investigation. SIGNIFICANCE STATEMENT:Aralar deficiency induces a fatal phenotype in humans and mice, and is associated with impaired neurodevelopment, epilepsy and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Herein, we find that βOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. βOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-KO mice, βOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The βOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.