A Mouse Model of L-2-Hydroxyglutaric Aciduria, a Disorder of Metabolite Repair

Abstract

The purpose of the present work was to progress in our understanding of the pathophysiology of L-2-hydroxyglutaric aciduria, due to a defect in L-2-hydroxyglutarate dehydrogenase, by creating and studying a mouse model of this disease. L-2-hydroxyglutarate dehydrogenase-deficient mice (l2hgdh -/-) accumulated L-2-hydroxyglutarate in tissues, most particularly in brain and testis, where the concentration reached ≈ 3.5 μmol/g. Male mice showed a 30% higher excretion of L-2-hydroxyglutarate compared to female mice, supporting that this dicarboxylic acid is partially made in males by lactate dehydrogenase C, a poorly specific form of this enzyme exclusively expressed in testes. Involvement of mitochondrial malate dehydrogenase in the formation of L-2-hydroxyglutarate was supported by the commensurate decrease in the formation of this dicarboxylic acid when down-regulating this enzyme in mouse l2hgdh -/- embryonic fibroblasts. The concentration of lysine and arginine was markedly increased in the brain of l2hgdh -/- adult mice. Saccharopine was depleted and glutamine was decreased by ≈ 40%. Lysine-α-ketoglutarate reductase, which converts lysine to saccharopine, was inhibited by L-2-hydroxyglutarate with a Ki of ≈ 0.8 mM. As low but significant activities of the bifunctional enzyme lysine-α-ketoglutarate reductase/saccharopine dehydrogenase were found in brain, these findings suggest that the classical lysine degradation pathway also operates in brain and is inhibited by the high concentrations of L-2-hydroxyglutarate found in l2hgdh -/- mice. Pathological analysis of the brain showed significant spongiosis. The vacuolar lesions mostly affected oligodendrocytes and myelin sheats, as in other dicarboxylic acidurias, suggesting that the pathophysiology of this model of leukodystrophy may involve irreversible pumping of a dicarboxylate in oligodendrocytes. Neurobehavioral testing indicated that the mice mostly suffered from a deficit in learning capacity. In conclusion, the findings support the concept that L-2-hydroxyglutaric aciduria is a disorder of metabolite repair. The accumulation of L-2-hydroxyglutarate exerts toxic effects through various means including enzyme inhibition and glial cell swelling.

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

“…The findings presented here substantiate prior genetic evidence implicating LDH and MDH as cellular sources of L-2HG 16–18,21 . In hypoxia, cells selectively produce L-2HG in a manner primarily dependent on LDHA and MDH2 17,18 .…”

“…3b–f). Collectively, these findings substantiate genetic evidence suggesting that LDH and MDH enzymes contribute to cellular production of L-2HG 16–18,21 .…”

“…Importantly, biochemical assays demonstrated that L-2HG functions as a more potent inhibitor of αKG-dependent enzymes compared to D-2HG; thus cells may be quantitatively more sensitive to changes in L-2HG than D-2HG 11–13 . Indeed, deregulated L-2HG disposal causes significant developmental pathology and is associated with brain and kidney cancer 14–16 .…”

“…Genetic evidence suggests that lactate dehydrogenase and malate dehydrogenase enzymes are major sources of hypoxia-induced L-2HG 16–18,21 , but there has been no demonstration that these enzymes directly catalyze L-2HG production. Here we demonstrate that purified LDH and MDH enzymes can catalyze stereospecific reduction of αKG to L-2HG and that acidic reaction conditions dramatically enhance LDH- and MDH-mediated production of L-2HG in vitro and in cells.…”

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

“…Finally, there is evidence that wildtype IDH1 and IDH2 also slowly form D-2HG from ␣-ketoglutarate (15). A known source of L-2HG in mammalian cells is a low side activity of malate dehydrogenase, converting ␣-ketoglutarate to L-2HG at the expense of NADH (16,17). Such an L-2HG-producing side activity has also been demonstrated for the mitochondrial malate dehydrogenases of Arabidopsis thaliana (18).…”