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• W2083304827 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 [14C]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 [14C]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. 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 [14C]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 [14C]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. Glutaric aciduria type 1 (GA1) 3The abbreviations used are: GA1glutaric aciduria type 1DCdicarboxylic acidGAglutaric acidGCDHglutaryl-CoA dehydrogenaseGFAPglial fibrillary acidic protein3OHGA3-hydroxyglutaric acidL2OHGAl- 2-hydroxyglutaric acidNaCsodium-dependent dicarboxylate co-transporterNeuNneuron-specific nuclear proteinOATorganic anion transporterqRT-PCRquantitative RT-PCRTCAtricarboxylic acid. is caused by deficiency of the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH). This enzyme catalyzes the oxidative decarboxylation of glutaryl-CoA in the degradative pathway of the amino acids lysine, hydroxylysine and tryptophan. GCDH deficiency leads to the accumulation of the dicarboxylic acids (DC) glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in tissues and body fluids. Affected patients during a time window from birth to 36 months of age are at risk for the development of encephalopathic crises triggered by catabolic situations such as infectious diseases, fever, vomiting or diarrhea, accompanied with a further increase of GA and 3OHGA concentrations (1Goodman S.I. Frerman F.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Childs B. Kinzler K.W. Vogelstein B. 8th Ed. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York2001: 2195-2204Google Scholar, 2Strauss K.A. Puffenberger E.G. Robinson D.L. Morton D.H. Am. J. Med. Genet. 2003; 121C: 38-52Crossref PubMed Scopus (281) Google Scholar). These encephalopathic crises lead to the selective destruction of striatal neurons with a subsequent irreversible disabling movement disorder. glutaric aciduria type 1 dicarboxylic acid glutaric acid glutaryl-CoA dehydrogenase glial fibrillary acidic protein 3-hydroxyglutaric acid l- 2-hydroxyglutaric acid sodium-dependent dicarboxylate co-transporter neuron-specific nuclear protein organic anion transporter quantitative RT-PCR tricarboxylic acid. The pathophysiologic mechanisms underlying the cytotoxic effects of GA and 3OHGA are not fully understood. In cultured primary neuronal cells prepared from rat and chicken brains the activation of N-methyl-d-aspartate (NMDA) receptors has been reported upon incubation with GA and 3OHGA in vitro (3Ullrich K. Flott-Rahmel B. Schluff P. Musshoff U. Das A. Lücke T. Steinfeld R. Christensen E. Jakobs C. Ludolph A. Neu A. Röper R. J. Inherit. Metab. Dis. 1999; 22: 392-403Crossref PubMed Scopus (86) Google Scholar, 4Kölker S. Koeller D.M. Okun J.G. Hoffmann G.F. Ann. Neurol. 2004; 55: 7-12Crossref PubMed Scopus (104) Google Scholar), which could not be confirmed in other studies (5Freudenberg F. Lukacs Z. Ullrich K. Neurobiol. Dis. 2004; 16: 581-584Crossref PubMed Scopus (24) Google Scholar, 6Lund T.M. Christensen E. Kristensen A.S. Schousboe A. Lund A.M. J. Neurosci. Res. 2004; 77: 143-147Crossref PubMed Scopus (26) Google Scholar). Furthermore, inhibition of γ-aminobutyric acid (GABA) synthesis and the impairment of mitochondrial energy production due to inhibition of the α-ketoglutarate dehydrogenase complex and depletion of creatine phosphate are suggested to be relevant for neuronal death (3Ullrich K. Flott-Rahmel B. Schluff P. Musshoff U. Das A. Lücke T. Steinfeld R. Christensen E. Jakobs C. Ludolph A. Neu A. Röper R. J. Inherit. Metab. Dis. 1999; 22: 392-403Crossref PubMed Scopus (86) Google Scholar, 4Kölker S. Koeller D.M. Okun J.G. Hoffmann G.F. Ann. Neurol. 2004; 55: 7-12Crossref PubMed Scopus (104) Google Scholar, 7Stokke O. Goodman S.I. Moe P.G. Clin. Chim. Acta. 1976; 66: 411-415Crossref PubMed Scopus (82) Google Scholar, 8Das A.M. Lücke T. Ullrich K. Mol. Genet. Metab. 2003; 78: 108-111Crossref PubMed Scopus (40) Google Scholar, 9Sauer S.W. Okun J.G. Schwab M.A. Crnic L.R. Hoffmann G.F. Goodman S.I. Koeller D.M. Kölker S. J. Biol. Chem. 2005; 280: 21830-21836Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In addition, it has been shown that GA and 3OHGA impair the integrity of endothelial barriers in vitro and in vivo (10Mühlhausen C. Ott N. Chalajour F. Tilki D. Freudenberg F. Shahhossini M. Thiem J. Ullrich K. Braulke T. Ergün S. Pediatr. Res. 2006; 59: 196-202Crossref PubMed Scopus (28) Google Scholar). The physiologic significance of these studies, however, is unclear, because they have been performed with cells exhibiting enzymatically active endogenous GCDH. Gcdh−/− mice display a biochemical phenotype similar to human GA1 patients with an accumulation of GA and 3OHGA in tissues and body fluids (11Koeller D.M. Woontner M. Crnic L.S. Kleinschmidt-DeMasters B. Stephens J. Hunt E.L. Goodman S.I. Hum. Mol. Genet. 2002; 11: 347-357Crossref PubMed Google Scholar). The administration of a high protein diet to young Gcdh−/− mice leads to the induction of an encephalopathic crisis accompanied by vacuolation in the brain and death within 3–5 days. Under these conditions, a further increase of GA and 3OHGA concentrations in urine, serum, and tissues is observed (12Zinnanti W.J. Lazovic J. Wolpert E.B. Antonetti D.A. Smith M.B. Connor J.R. Woontner M. Goodman S.I. Cheng K.C. Brain. 2006; 129: 899-910Crossref PubMed Scopus (96) Google Scholar, 13Keyser B. Glatzel M. Stellmer F. Kortmann B. Lukacs Z. Kölker S. Sauer S.W. Muschol N. Herdering W. Thiem J. Goodman S.I. Koeller D.M. Ullrich K. Braulke T. Mühlhausen C. Biochim. Biophys. Acta. 2008; 1782: 385-390Crossref PubMed Scopus (27) Google Scholar). Recently, we identified the sodium-dependent dicarboxylate co-transporter 3 (NaC3) and organic anion transporters (OAT) 1 and 4 as low and high affinity transporters, respectively, for the translocation of GA and 3OHGA through membranes (14Stellmer F. Keyser B. Burckhardt B.C. Koepsell H. Streichert T. Glatzel M. Jabs S. Thiem J. Herdering W. Koeller D.M. Goodman S.I. Lukacs Z. Ullrich K. Burckhardt G. Braulke T. Mühlhausen C. J. Mol. Med. 2007; 85: 763-770Crossref PubMed Scopus (36) Google Scholar, 15Hagos Y. Krick W. Braulke T. Mühlhausen C. Burckhardt G. Burckhardt B.C. Pflugers Arch. 2008; 457: 223-231Crossref PubMed Scopus (33) Google Scholar). The concerted action of NaC3, OAT1, and OAT4 in renal proximal tubule cells may be important for the urinary excretion of GA and 3OHGA (16Mühlhausen C. Burckhardt B.C. Hagos Y. Burckhardt G. Keyser B. Lukacs Z. Ullrich K. Braulke T. J. Inherit. Metab. Dis. 2008; 31: 188-193Crossref PubMed Scopus (24) Google Scholar). How these metabolites are transported in the brain, however, and which transporters are involved are still unclear. In addition to renal proximal tubule cells, NaC3 and OAT1 have been reported to be expressed in murine and human choroid plexus, respectively (17Pajor A.M. Gangula R. Yao X. Am. J. Physiol. Cell Physiol. 2001; 280: C1215-C1223Crossref PubMed Google Scholar, 18Alebouyeh M. Takeda M. Onozato M.L. Tojo A. Noshiro R. Hasannejad H. Inatomi J. Narikawa S. Huang X.L. Khamdang S. Anzai N. Endou H. J. Pharmacol. Sci. 2003; 93: 430-436Crossref PubMed Scopus (90) Google Scholar). More recently, it has been concluded from mRNA and immunocytochemical expression studies in cultured primary cells from rat brain that NaC3 is expressed in astrocytes, whereas in neurons NaC2 is present (19Yodoya E. Wada M. Shimada A. Katsukawa H. Okada N. Yamamoto A. Ganapathy V. Fujita T. J. Neurochem. 2006; 97: 162-173Crossref PubMed Scopus (65) Google Scholar). Both transporters have a broad substrate specificity and translocate dicarboxylates such as succinate, α-ketoglutarate, malate, and fumarate with high affinity (17Pajor A.M. Gangula R. Yao X. Am. J. Physiol. Cell Physiol. 2001; 280: C1215-C1223Crossref PubMed Google Scholar, 20Inoue K. Fei Y.J. Zhuang L. Gopal E. Miyauchi S. Ganapathy V. Biochem. J. 2004; 378: 949-957Crossref PubMed Scopus (68) Google Scholar). Neurons depend on the anaplerotic supply of these dicarboxylates from astrocytes required as substrates for the tricarboxylic acid (TCA) cycle to maintain energy metabolism and the synthesis of the neurotransmitters glutamate and GABA (21Hertz L. Peng L. Dienel G.A. J. Cereb. Blood Flow Metab. 2007; 27: 219-249Crossref PubMed Scopus (444) Google Scholar, 22Jitrapakdee S. St. Maurice M. Rayment I. Cleland W.W. Wallace J.C. Attwood P.V. Biochem. J. 2008; 413: 369-387Crossref PubMed Scopus (300) Google Scholar), which might involve sodium-dependent dicarboxylate co-transporters (19Yodoya E. Wada M. Shimada A. Katsukawa H. Okada N. Yamamoto A. Ganapathy V. Fujita T. J. Neurochem. 2006; 97: 162-173Crossref PubMed Scopus (65) Google Scholar, 23Schousboe A. Westergaard N. Waagepetersen H.S. Larsson O.M. Bakken I.J. Sonnewald U. Glia. 1997; 21: 99-105Crossref PubMed Scopus (175) Google Scholar). Based on the ability of NaC3 to transport GA and 3OHGA (14Stellmer F. Keyser B. Burckhardt B.C. Koepsell H. Streichert T. Glatzel M. Jabs S. Thiem J. Herdering W. Koeller D.M. Goodman S.I. Lukacs Z. Ullrich K. Burckhardt G. Braulke T. Mühlhausen C. J. Mol. Med. 2007; 85: 763-770Crossref PubMed Scopus (36) Google Scholar), we hypothesized that the increased concentrations of GA and 3OHGA in the brain of GA1 patients during an encephalopathic crisis, and induced in Gcdh−/− mice, impair the anaplerotic supply of TCA cycle intermediates from astrocytes to neurons in a competitive manner. In the present study, we have demonstrated for the first time that GA and 3OHGA inhibit the uptake of [14C]succinate into primary neuronal cells of wild-type and Gcdh−/− mice. Most important, the efflux of [14C]succinate from Gcdh-deficient astrocytic cells has been found to be strongly retarded compared with wild-type cells. GA and succinate were purchased from Fluka (Taufkirchen, Germany). 3OHGA was synthesized as described previously (14Stellmer F. Keyser B. Burckhardt B.C. Koepsell H. Streichert T. Glatzel M. Jabs S. Thiem J. Herdering W. Koeller D.M. Goodman S.I. Lukacs Z. Ullrich K. Burckhardt G. Braulke T. Mühlhausen C. J. Mol. Med. 2007; 85: 763-770Crossref PubMed Scopus (36) Google Scholar). 1,4-[14C]-Labeled succinate was obtained from Moravek Biochemicals (Brea, CA). DNase I, papain, and AraC were purchased from Sigma. Minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), neurobasal A medium, and horse serum were from Invitrogen. Fetal calf serum (FCS) was from PAA Laboratories (Pasching, Austria), and B27 supplement was obtained from Invitrogen. All other chemicals were of analytical grade or higher. Rabbit anti-glial fibrillary acidic protein (GFAP) antibody was purchased from DAKO Cytomation (Glostrup, Denmark), and mouse anti-neuron-specific nuclear protein (NeuN) antibody was from Millipore. Peroxidase-conjugated goat anti-rabbit IgG was from Dianova (Hamburg, Germany). Sheep anti-mouse IgG coupled to fluorescein isothiocyanate (FITC) and anti-rabbit IgG-Cy3 were from Sigma. Gcdh−/− or wild-type mice were bred from homozygous (Gcdh−/− or Gcdh+/+, respectively) parents and sacrificed at P0–P2. The genetic background in all mice groups used in this study was C57BL6/SJ129 hybrid. Genotypes were confirmed by PCR and measurement of glutarylcarnitine concentration in dried blood spots as described previously (13Keyser B. Glatzel M. Stellmer F. Kortmann B. Lukacs Z. Kölker S. Sauer S.W. Muschol N. Herdering W. Thiem J. Goodman S.I. Koeller D.M. Ullrich K. Braulke T. Mühlhausen C. Biochim. Biophys. Acta. 2008; 1782: 385-390Crossref PubMed Scopus (27) Google Scholar). Mice were housed in the animal facility of the University Medical Center with a 12-h light-dark cycle and allowed water and food ad libitum. Animal care and experiments were carried out in accordance with institutional guidelines as approved by local authorities. The immortalized murine astrocytoma cell line 11+/+ was cultured as described previously (24Matzner U. Habetha M. Gieselmann V. Gene Ther. 2000; 7: 805-812Crossref PubMed Scopus (30) Google Scholar). For the preparation of primary neuronal cells from mouse cortex, Gcdh−/− or wild-type pups were sacrificed at P0–P1. Preparation and maintenance of cells were performed according to Ref. 25Quitsch A. Berhörster K. Liew C.W. Richter D. Kreienkamp H.J. J. Neurosci. 2005; 25: 479-487Crossref PubMed Scopus (80) Google Scholar. After decapitation and removal of vessels and meninges, cortices were removed and washed in Hanks' buffered salt solution (1 mm MgCl2, 5.5 mm glucose, 137 mm NaCl, 5.4 mm KCl, 0.4 mm KH2PO4, 2.7 mm Na2HPO4·2H2O, 4.2 mm NaHCO3, 0.8 mm MgSO4, 1.7 mm CaCl) and subsequently incubated in papain solution (10 mm phosphate-buffered saline (PBS), 10 mm glucose, 50 μl of DNase I, 2.5 mg of papain) for 30 min at 37 °C on a shaker. Afterward, cortices were washed three times in plating medium (0.6% glucose, 10% horse serum in MEM) followed by homogenization of tissue by repeated resuspension through yellow pipette tips. Subsequently, cells were counted and plated at a density of ∼500 cells/mm2 on glass coverslips coated with poly-l-lysine, 12-well plates, or 35-mm dishes. After 4 h, medium was changed to medium 1 (neurobasal A medium supplemented with 2% B27, 0.5 mm glutamine, 25 μm glutamate, 20 units/ml penicillin, and 20 μg/ml streptomycin). Cells were grown in medium 1 for 3 days, maintained another 4 days in medium 2 (medium 1 without glutamate) supplemented with 10 μm AraC, and another 3 days in medium 2 without AraC. Primary neuronal cells were used for experiments after a total of 10 days in culture. For preparation of astrocytic cells, whole brains of Gcdh−/− or wild-type mice at P0–P2 were excised, vessels and meninges removed, and brains washed three times in Hanks' buffered salt solution. Subsequently, brains were dissected in small pieces and homogenized in 5 ml of medium (DMEM supplemented with 10% FCS, 0.6% glucose, 25 mm NaHCO3, 200 nm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin) at 37 °C by passing the tissue repeatedly through a Pasteur pipette. Cells were separated by sequentially passing the cell suspension through 180-, 140-, and 30-μm nylon net filters (Millipore) with a 10-ml syringe. Finally, cells were plated at a density of ∼1,000 cells/mm2. Medium was changed every 2–3 days, and cells were used for experiments 7 days after preparation. For uptake experiments, either primary neuronal cells cultured for 10 days or astrocytic cells cultured for 7 days on 12-well plates were used. After washing with prewarmed (37 °C) transport buffer (25 mm HEPES, pH 7.4, containing 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgSO4, 5 mm glucose), cells were incubated for 10 or 20 min at 37 °C/5% CO2 with 0.3 ml of uptake buffer (transport buffer supplemented with 0.1 μCi of [14C]-labeled succinate in the absence or presence of 2 mm effectors GA, 3OHGA, or nonlabeled succinate). After removal of uptake buffer, cells were immediately washed three times with ice-cold transport buffer and lysed in 0.2 m NaOH. The amount of cell-associated radioactivity was determined by scintillation counting (Tri-Carb 2900TR liquid scintillation analyzer; Packard, Groningen, The Netherlands or Rotiszint eco plus liquid scintillation mixture; Roth, Karlsruhe, Germany) and related to the protein content (Roti-Quant protein assay; Roth). Cells were incubated with [14C]succinate for 20 min as described above. After removal of the radioactive buffer, cells were further incubated in transport buffer for the indicated times. The supernatant was collected, and cells were lysed in 0.2 m NaOH. Radioactivity was determined in supernatant and cell lysates and related to cellular protein content. 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). A mobile phase consisting of 10 mm Tris-HCl, pH 8.5, operated at a flow rate of 100 μl/min at ambient temperature was utilized with a gradient of 0–0.25 m NaCl within 20 min. Supernatants from efflux experiments were centrifuged at 20,000× g for 10 min at 4 °C to remove cell debris and divided into four aliquots of 50 μl. Cell pellets from efflux experiments were sonicated for 3 × 20 s, and proteins were denatured by incubation of samples for 5 min at 95 °C. Cell debris and denatured proteins were sedimented by centrifugation at 20,000× g for 10 min at 4 °C, and the resulting cell extract was divided into four aliquots of 50 μl each. Aliquots of efflux supernatants and cell extracts were diluted 1:20 in mobile phase buffer (10 mm Tris, pH 8.5) and loaded onto the HPLC column. Bound anions were eluted by an NaCl gradient, and fractions of 100 μl were collected. Radioactivity in pooled HPLC fractions of four sequential HPLC runs was determined by liquid scintillation counting. Elution profiles of unlabeled standard anions (1–100 nmol; aspartic acid, fumaric acid, glutamic acid, α-ketoglutaric acid, oxaloacetic acid, sodium citrate, sodium succinate; Sigma) were recorded at 210 nm. Total RNA was prepared from cell pellets with a TRI®-Reagent RNA preparation kit (Sigma). RNA (1 μg) was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). For qRT-PCR, 6-carboxy-fluorescein dye-labeled murine TaqMan MGB probes (Applied Biosystems) were used in 96-well optical reaction plates, and triplicates were quantified in an Mx3000P® qRT-PCR system (Stratagene). TaqMan assay ID numbers are as follows: NaC2, Mm01334459_m1; NaC3, Mm00475280_m1; Oat1, Mm00456258_m1; β-actin, Mm00607939_s1; GAPDH, Mm99999914_g1. The relative level of each mRNA was determined using the DART-PCR method and software (26Peirson S.N. Butler J.N. Foster R.G. Nucleic Acids Res. 2003; 31: e73Crossref PubMed Scopus (711) Google Scholar). Protein concentration was determined with the Roti-Quant protein assay (Roth). For double immunofluorescence microscopy, 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 (27Keyser B. Mühlhausen C. Dickmanns A. Christensen E. Muschol N. Ullrich K. Braulke T. Hum. Mol. Genet. 2008; 17: 3854-3863Crossref PubMed Scopus (28) Google Scholar). For staining of nuclei with 4,6-diamidino-2-phenylindol (DAPI; Sigma), cells were washed with PBS and subsequently incubated with 200 μl of 5 μg/ml DAPI in PBS for 5 min. Data were analyzed using either one-way analysis of variance followed by Scheffé's test or unpaired two-tailed Student's t tests as applicable. Significance was accepted at p < 0.05. Calculations were operated using SPSS® 15.0 (SPSS, Chicago, IL) and Microsoft® Office Excel 2003 software. Astrocytic cells were isolated from wild-type mouse brain and cultured for 7 days. Double immunofluorescence staining of cultures with astrocyte-specific GFAP and NeuN antibodies was performed. About 80–90% of cells were GFAP-positive, and no anti-NeuN immunoreactivity was observed (supplemental Fig. S1). Staining of primary astrocytic cells from Gcdh−/− mice showed similar results (data not shown). Incubation of primary astrocytic cells prepared from wild-type mice with [14C]succinate revealed a time-dependent internalization of radioactivity (Fig. 1). Logarithmic regression analysis generated a fitted curve, where the time point of 10 min was located in the linear slope region of the curve, whereas the rate of [14C]succinate internalized after 30 min reached the saturation level. Further uptake experiments were performed by incubating cells with [14C]succinate for 10 and/or 20 min as indicated. The uptake of [14C]succinate into wild-type astrocytic cells required the presence of Na+ ions (supplemental Fig. S2A). An excess of unlabeled succinate (2 mm) inhibited the uptake of [14C]succinate both into wild-type and Gcdh−/− astrocytic cells to 8–10% of control during an incubation time of 10 min (Fig. 2A), indicating the specificity of [14C]succinate transport into these cells. In the presence of GA (40 μm), succinate (25 μm), and the GA derivative l-2-hydroxyglutaric acid (L2OHGA, 267 μm), concentrations corresponding to the Km values of NaC3 for these effectors (15Hagos Y. Krick W. Braulke T. Mühlhausen C. Burckhardt G. Burckhardt B.C. Pflugers Arch. 2008; 457: 223-231Crossref PubMed Scopus (33) Google Scholar), the uptake of [14C]succinate was reduced to 55, 44, and 56%, respectively, of control as expected (Table 1 and supplemental Fig. S2B). In contrast, incubation of cells in the presence of 930 μm 3OHGA (corresponding to Km of 3OHGA for NaC3) showed an inhibition of [14C]succinate uptake to 77.3 ± 22.1% of control. Therefore, the concentration-dependent inhibition of [14C]succinate into primary astrocytic cells derived from wild-type mice in the presence of 3OHGA was tested. The strongest inhibition of [14C]succinate uptake (approximately 50%) was observed in the presence of 2 mm 3OHGA (supplemental Fig. S2A). In the presence of 2 mm GA, the uptake of [14C]succinate into wild-type and Gcdh−/− astrocytic cells was significantly reduced to 14–20% of controls (Fig. 2A). These data suggest that uptake of [14C]succinate into astrocytic cells is sodium-dependent and can be inhibited in a competitive manner by dicarboxylates such as succinate, GA, and 3OHGA.TABLE 1Dicarboxylate-dependent inhibition of [14C]succinate uptake into astrocytic cellsCompoundKmaAccording to Ref. 15.[14C]Succinate uptakeb[14C]succinate uptake was determined in the absence (control) or presence of dicarboxylates at concentrations corresponding to their Km values for NaC3.mm% of controlGA0.04 ± 0.0254.9 ± 12.93OHGA0.93 ± 0.2577.3 ± 22.1Succinate0.02544.1 ± 9.3L2OHGA0.267 ± 0.04855.9 ± 12.2a According to Ref. 15Hagos Y. Krick W. Braulke T. Mühlhausen C. Burckhardt G. Burckhardt B.C. Pflugers Arch. 2008; 457: 223-231Crossref PubMed Scopus (33) Google Scholar.b [14C]succinate uptake was determined in the absence (control) or presence of dicarboxylates at concentrations corresponding to their Km values for NaC3. Open table in a new tab Next, the uptake of [14C]succinate was determined in neuronal cells. Primary neuronal cells prepared from wild-type and Gcdh−/− mice cultured for 10 days were 80–90% NeuN-positive (supplemental Fig. S1). The uptake of [14C]succinate into neuronal cells during an incubation time of 10 min was strongly inhibited by an excess of unlabeled succinate and GA to 2–6% of controls (Fig. 2B), whereas 2 mm 3OHGA reduced the uptake only to 42–44%. There were no significant differences between astrocytic or neuronal cells from wild-type and Gcdh−/− mice in the relative (percent of respective control, Fig. 2) or absolute amount of radioactivity (cpm/mg of protein, data not shown) accumulated. To investigate the efflux of [14C]succinate from the intracellular compartment, primary wild-type or Gcdh−/− astrocytic or neuronal cells were preincubated with [14C]succinate for 20 min. After washing, cells were incubated in fresh nonradioactive medium for various time periods. Subsequently, radioactivity was measured in medium and cell lysates (Fig. 3). [14C]Succinate disappeared from primary astrocytic cells of wild-type mice with a half-efflux time (t½ efflux) of 5.6 ± 2.6 min upon incubation at 37 °C (Fig. 3A). Between 20 and 25% of the initial radioactivity remained intracellular, and 75 to 80% was released into the medium. In contrast, the t½ efflux rate of [14C]succinate in astrocytic cells of Gcdh−/− mice was significantly retarded to 20.2 ± 8.6 min (p < 0.05; Fig. 3B). In addition, after 30 min of incubation, only 60% of the initial cell-associated radioactivity was released into the medium. The t½ efflux rates of [14C]succinate from neuronal cells prepared from wild-type and Gcdh−/− mice were not significantly different (13.5 ± 9 min versus 12.6 ± 2.7 min, respectively; Fig. 3, C and D). Of note, the percentage of radioactivity remaining in neuronal cells after 30 min comprised approximately 40% in both wild-type and Gcdh−/− cells, respectively. The direct comparison of t½ efflux rates of [14C]succinate from astrocytic and neuronal cells of wild-type and Gcdh−/− mice is summarized in supplemental Fig. S3. To elucidate whether the measured radioactivity effused from astrocytic and neuronal cells represents [14C]succinate or metabolized [14C]-labeled compounds, HPLC analyses of efflux media and cell extracts were performed. First, anionic exchange HPLC conditions were established allowing the separation of the TCA cycle intermediates succinate (fractions 9 and 10), α-ketoglutarate (fractions 11 and 12), fumarate (fraction 13), and citrate (fractions 15 and 16) as well as aspartate/glutamate (both co-eluting in overlapping fractions 4 and 5; supplemental Fig. S4). [14C]Succinate co-eluted with unlabeled succinate in fractions 9 and 10. HPLC analyses of cell extracts of efflux experiments from wild-type astrocytic cells revealed that after loading (efflux t = 0 min) 69% of radioactivity bound to the column represented intracellular [14C]succinate, whereas 17, 2, and 5% of total column-bound radioactivity co-eluted with aspartate/glutamate, fumarate, and citrate, respectively. These data indicate that intracellular [14C]succinate was partially metabolized to these compounds (Fig. 4A). After 10 and 20 min (data not shown) and 30 min of efflux (Fig. 4B), the amount of intracellular radioactivity co-eluting with succinate was reduced to 33–39% of total column-bound radioactivity, whereas the proportion of bound radioactivity co-eluting with aspartate/glutamate, fumarate, and citrate was 35–43, 3, and 7%, respectively. HPLC analyses of efflux media from wild-type astrocytic cells after 10- and 20-min (data not shown) and 30-min efflux (Fig. 4C) showed that the majority (86–91%) of radioactivity co-eluted with unlabeled succinate in fractions 9 and 10. These data provide evidence that virtually all of the effused radioactivity represents [14C]succinate. HPLC analyses of media and cell pellets from Gcdh−/− astrocytic cells revealed similar results (data not shown). HPLC analyses of efflux media and cell extracts from wild-type neuronal cells revealed that the majority of effused radioactivity in the media after 10 and 20 min (data not shown), and 30 min (Fig. 4F) corresponds to [14C]succinate, representing 67–82% of total column-bound radioactivity. Compared with wild-type astrocytic cells, a higher proportion of effused radioactivity co-eluted with aspartate/glutamate (neuronal cells, Fig. 4F: 9–15%, versus astrocyt" @default.
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• W2083304827 title "Glutaric Aciduria Type 1 Metabolites Impair the Succinate Transport from Astrocytic to Neuronal Cells" @default.
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