Glutaric Aciduria Type 1 Metabolites Impair the Succinate Transport from Astrocytic to Neuronal Cells

Jessica Lamp,Britta Keyser,David M Koeller,Kurt Ullrich,Thomas Braulke,Chris Mühlhausen

doi:10.1074/jbc.m111.232744

Abstract

The inherited neurodegenerative disorder glutaric aciduria type 1 (GA1) results from mutations in the gene for the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to elevations of the dicarboxylates glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in brain and blood. The characteristic clinical presentation of GA1 is a sudden onset of dystonia during catabolic situations, resulting from acute striatal injury. The underlying mechanisms are poorly understood, but the high levels of GA and 3OHGA that accumulate during catabolic illnesses are believed to play a primary role. Both GA and 3OHGA are known to be substrates for Na(+)-coupled dicarboxylate transporters, which are required for the anaplerotic transfer of the tricarboxylic acid cycle (TCA) intermediate succinate between astrocytes and neurons. We hypothesized that GA and 3OHGA inhibit the transfer of succinate from astrocytes to neurons, leading to reduced TCA cycle activity and cellular injury. Here, we show that both GA and 3OHGA inhibit the uptake of [(14)C]succinate by Na(+)-coupled dicarboxylate transporters in cultured astrocytic and neuronal cells of wild-type and Gcdh(-/-) mice. In addition, we demonstrate that the efflux of [(14)C]succinate from Gcdh(-/-) astrocytic cells mediated by a not yet identified transporter is strongly reduced. This is the first experimental evidence that GA and 3OHGA interfere with two essential anaplerotic transport processes: astrocytic efflux and neuronal uptake of TCA cycle intermediates, which occur between neurons and astrocytes. These results suggest that elevated levels of GA and 3OHGA may lead to neuronal injury and cell death via disruption of TCA cycle activity.

Highlights

Glutaric aciduria type 1 (GA1)3 is caused by deficiency of the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH)
Production of neurotransmitters glutamate and GABA in neurons requires a continuous supply of tricarboxylic acid cycle (TCA) cycle intermediates such as ␣-ketoglutarate and succinate
Normal neuron function is dependent on the import of metabolites produced by astrocytes to replenish TCA cycle intermediates

Summary

EXPERIMENTAL PROCEDURES

Materials—GA and succinate were purchased from Fluka (Taufkirchen, Germany). 3OHGA was synthesized as described previously [14]. 1,4-[14C]-Labeled succinate was obtained from Moravek Biochemicals (Brea, CA). For preparation of astrocytic cells, whole brains of GcdhϪ/Ϫ or wild-type mice at P0 –P2 were excised, vessels and meninges removed, and brains washed three times in Hanks’ buffered salt solution. HPLC Analyses—The content of cell pellets and supernatants of [14C]succinate efflux assays was determined by anion exchange HPLC analyses using a Mono Q PC 1.6/5 column connected to a SMART system (GE Healthcare). Supernatants from efflux experiments were centrifuged at 20,000ϫ g for 10 min at 4 °C to remove cell debris and divided into four aliquots of 50 ␮l. Primary astrocytic or neuronal cells were grown on poly-L-lysine-coated glass coverslips, fixed, permeabilized, and stained using rabbit anti-GFAP (1:250) and mouse anti-NeuN (1:50) as primary antibodies as described previously [27]. Calculations were operated using SPSS௡ 15.0 (SPSS, Chicago, IL) and Microsoft௡ Office Excel 2003 software

RESULTS

Findings

DISCUSSION