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Originally published In Press as doi:10.1074/jbc.M502845200 on April 19, 2005

J. Biol. Chem., Vol. 280, Issue 23, 21830-21836, June 10, 2005
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Bioenergetics in Glutaryl-Coenzyme A Dehydrogenase Deficiency

A ROLE FOR GLUTARYL-COENZYME A*

Sven W. Sauer{ddagger}, Jürgen G. Okun{ddagger}, Marina A. Schwab{ddagger}, Linda R. Crnic{dagger}§, Georg F. Hoffmann{ddagger}, Stephen I. Goodman§, David M. Koeller¶, and Stefan Kölker{ddagger}||

From the {ddagger}Department of General Pediatrics, Division of Inborn Metabolic Diseases, University Children's Hospital of Heidelberg, D-69120 Heidelberg, Germany, the §Department of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262, and the Departments of Pediatrics, and Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon 97201

Received for publication, March 15, 2005 , and in revised form, April 11, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inherited deficiency of glutaryl-CoA dehydrogenase results in an accumulation of glutaryl-CoA, glutaric, and 3-hydroxyglutaric acids. If untreated, most patients suffer an acute encephalopathic crisis and, subsequently, acute striatal damage being precipitated by febrile infectious diseases during a vulnerable period of brain development (age 3 and 36 months). It has been suggested before that some of these organic acids may induce excitotoxic cell damage, however, the relevance of bioenergetic impairment is not yet understood. The major aim of our study was to investigate respiratory chain, tricarboxylic acid cycle, and fatty acid oxidation in this disease using purified single enzymes and tissue homogenates from Gcdh-deficient and wild-type mice. In purified enzymes, glutaryl-CoA but not glutaric or 3-hydroxyglutaric induced an uncompetitive inhibition of {alpha}-ketoglutarate dehydrogenase complex activity. Notably, reduced activity of {alpha}-ketoglutarate dehydrogenase activity has recently been demonstrated in other neurodegenerative diseases, such as Alzheimer, Parkinson, and Huntington diseases. In contrast to {alpha}-ketoglutarate dehydrogenase complex, no direct inhibition of glutaryl-CoA, glutaric acid, and 3-hydroxyglutaric acid was found in other enzymes tested. In Gcdh-deficient mice, respiratory chain and tricarboxylic acid activities remained widely unaffected, virtually excluding regulatory changes in these enzymes. However, hepatic activity of very long-chain acyl-CoA dehydrogenase was decreased and concentrations of long-chain acylcarnitines increased in the bile of these mice, which suggested disturbed oxidation of long-chain fatty acids. In conclusion, our results demonstrate that bioenergetic impairment may play an important role in the pathomechanisms underlying neurodegenerative changes in glutaryl-CoA dehydrogenase deficiency.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cerebral organic aciduria glutaryl-CoA dehydrogenase deficiency (synonym: glutaric aciduria/acidemia type I; MIM number 231670 [OMIM] ) is an autosomal recessive disease with an estimated median prevalence of 1:100,000 newborns (1). This disease is overrepresented in some cohorts, such as the Amish (2) and Canadian Oji-Cree (3). The glutaryl-CoA dehydrogenase (GCDH)1 gene on chromosome 19p13.2 contains 11 exons and spans 7 kb (4). GCDH (EC 1.3.99.7 [EC] ) is a key mitochondrial enzyme in the catabolic pathways of the amino acids tryptophan, lysine, and hydroxylysine, catalyzing the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA and CO2 (5). More than 150 disease-causing mutations have been identified (6–8). The biochemical hallmark of this disease is the accumulation of the organic acids glutaric (GA) and 3-hydroxyglutaric acids (3-OH-GA) in body fluids and tissues.

Clinically, GCDH deficiency is characterized by acute striatal degeneration that occurs during encephalopathic crises precipitated by intercurrent febrile illnesses or even routine vaccinations. The majority of untreated children develop severe clinical symptoms before age 36 months, suggesting that vulnerability is limited to a finite period of brain development (2, 9, 10). Frequent neuroradiologic findings include temporal hypoplasia, striatal degeneration, white matter abnormalities, subependymal pseudocysts, chronic subdural effusions, and hematomas. If diagnosed and treated early by a lysine-restricted diet, carnitine supplementation, and an intensified emergency treatment during infectious diseases, the development of a clinically significant acute encephalopathic crisis and subsequent striatal damage can be prevented in the majority of affected children (2, 9, 11).

It has been suggested that accumulating organic acids are involved in the pathogenesis of this disease, activating or facilitating an excitotoxic mechanism (12). A direct or indirect activation of N-methyl-D-aspartate receptors was demonstrated in both in vitro and in vivo studies (13–16). In support of this mechanism, a potential pathogenic role has been suggested for reduced glutamate uptake (17) and GABA production (18), Na+/K+-ATPase inhibition (19), and increased generation of reactive oxygen species (15, 20) and nitric oxide (21) induced by these organic acids. However, some studies of cultured primary neurons were unable to confirm a role for glutamate receptor activation (22–24), suggesting that non-excitotoxic mechanisms, such as secondary inhibition of mitochondrial energy metabolism may be important. It has been shown that 3-OH-GA induced a decrease in creatine phosphate (25) and mild inhibition of respiratory chain complexes II and V (26), although direct inhibition of respiratory chain complexes could not be confirmed in another study (15). The major aim of the present study was to comprehensively investigate whether the metabolites that accumulate in GCDH deficiency affect mitochondrial energy metabolism, including the respiratory chain, tricarboxylic acid (TCA) cycle, and {beta}-oxidation of fatty acids.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Tissue Extracts—The Gcdh–/– mice used for these experiments were generated via gene targeting in mouse embryonic stem cells, and have been previously described (27). Gcdh–/– and wt mice were sacrificed at age 56–200 days. Tissues (brain, liver, heart, and skeletal muscle) were immediately removed and chilled on ice in a buffer (0.1 ml/0.1 mg of tissue) containing 250 mmol/liter sucrose, 50 mmol/liter KCl, 5 mmol/liter MgCl2, 20 mmol/liter Tris-HCl (adjusted at pH 7.4)). Tissues were homogenized with a Potter-Elvehjem system and subsequently homogenates were snap-frozen in liquid nitrogen and stored at –80 °C. Animal care and experiments followed the official governmental guidelines and were approved by the governmental review board. Submitochondrial particles (SMPs) from bovine heart were prepared as previously described (28). Protein was determined according to Ref. 29 with modifications (30) using bovine serum albumin as a standard.

Spectrophotometric Analysis of Single Respiratory Chain Complexes I–V—Steady state activities of enzyme complexes were recorded using a computer tunable spectrophotometer (Spectramax Plus Microplate Reader, Molecular Devices, Sunnyvale, CA) operating in the dual wave-length mode. Samples were analyzed in temperature-controlled 96-well plates in a final volume of 300 µl. The catalytic activities of respiratory chain complexes I–V in SMPs were investigated as previously described (31, 32). The addition of standard respiratory chain inhibitors (complex I, 2-n-decylquinazolin-4-yl-amine (1 µmol/liter); complex II, thenoyltrifluoroacetone (8 mmol/liter); complex III, antimycin A (1 µmol/liter); complex IV, NaCN (2 mmol/liter); complex V, oligomycin (80 µmol/liter)) revealed good inhibitory responses (93–100% of control activity, p < 0.001 versus controls), confirming a high specific enzyme activity in our assay system. GA, 3-OH-GA, or glutaryl-CoA (all adjusted to pH 7.4) were added and effects on single enzyme activity were determined at concentrations up to 1 mmol/liter each. The same methods were used to measure single respiratory complex enzyme activities in tissue homogenates from Gcdh–/– and wt mice (complex I, 3 mg of protein/ml; complex II, 1 mg/ml; complex III, 0.5 mg/ml; complex IV, 0.2 mg/ml; complex V, 1 mg/ml).

Spectrophotometric Analysis of TCA Enzymes—Purified citrate synthase, malate dehydrogenase, fumarase, isocitric dehydrogenase, aconitase, {alpha}-ketoglutarate dehydrogenase complex, and pyruvate dehydrogenase complex from porcine heart were purchased from Sigma.

Citrate Synthase (CS)—CS (23 milliunits/ml; EC 2.3.3.1 [EC] ) was assayed in a buffer containing 10 mmol/liter potassium phosphate, 30 µmol/liter oxaloacetate, 80 µmol/liter acetyl-CoA, and 100 µmol/liter 5,5-dithiobis-(2-nitrobenzoate) (DTNB), which was adjusted to pH 7.4 (25 °C). CS activity was determined as DTNB reduction at {lambda} = 412 nm.

Malate Dehydrogenase (MDH)—MDH (16 milliunits/ml; EC 1.1.1.37 [EC] ) was assayed in a buffer containing 400 milliunits of CS, 2 mmol/liter of malate, 10 mmol/liter of potassium phosphate, 330 µmol/liter of NAD, and 80 µmol/liter of acetyl-CoA, which was adjusted to pH 7.4 (25 °C). MDH activity was determined as NAD reduction at {lambda} = 340–400 nm.

Fumarase—Fumarase (650 milliunits/ml; EC 4.2.1.2 [EC] ) was assayed in a buffer containing 660 milliunits/ml of MDH, 400 milliunits of CS, 2 mmol/liter of fumarate, 10 mmol/liter of potassium phosphate, 330 µmol/liter of NAD, and 80 µmol/liter of acetyl-CoA, which was adjusted to pH 7.4 (25 °C). Fumarase activity was determined as NAD reduction at {lambda} = 340–400 nm.

Isocitrate Dehydrogenase (IDH)—IDH (1 unit/ml; EC 1.1.1.41 [EC] ) was assayed in a buffer containing 2 mmol/liter of isocitrate, 10 mmol/liter of potassium phosphate, and 0.2 mmol/liter of NADP, which was adjusted to pH 7.4 (25 °C). IDH activity was determined as NADP reduction at {lambda} = 340–400 nm.

Aconitase—Aconitase (EC 4.2.1.3 [EC] ) activity was measured according to Ref. 33 with modifications. Aconitase (0.70 mg) was assayed in a buffer containing 0.7 unit of IDH, 36 mmol/liter of Tris-HCl, 0.07 mmol/liter of citric acid, 0.18 mmol/liter of NADP, 1.3 mmol/liter of manganese sulfate, 0.8 µmol/liter of ferrous ammonium sulfate, and 0.08 mmol/liter of L-cysteine, which was adjusted to pH 7.4 (25 °C). Aconitase activity was determined as NADP reduction at {lambda} = 340–400 nm.

{alpha}-Ketoglutarate Dehydrogenase Complex (KGDHc)—KGDHc activity was measured according to Ref. 34 with modifications. KGDHc (650 milliunits/ml) was assayed in a buffer containing 35 mmol/liter of potassium phosphate, 5 mmol/liter of MgCl2, 0.5 mmol/liter of EDTA, 0.5 mmol/liter of NAD, 0.2 mmol/liter of thiamine pyrophosphate, 0.04 mmol/liter of CoA-SH, and 2 mmol/liter of {alpha}-ketoglutarate, which was adjusted to pH 7.4 (30 °C). KGDHc activity was determined as NAD reduction at {lambda} = 340–400 nm.

{alpha}-Ketoglutarate Dehydrogenase (E1k, EC 1.2.4.2 [EC] )—KGDHc (650 milliunits/ml) was assayed in a buffer containing 35 mmol/liter of potassium phosphate, 0.5 mmol/liter of MgSO4, 0.5 mmol/liter of EDTA, 1 mmol/liter of thiamine pyrophosphate, 0.5 mmol/liter of 2,6-dichlorophenolindophenol, and 2 mmol/liter of {alpha}-ketoglutarate, which was adjusted to pH 7.4 (30 °C). E1k activity was determined as 2,6-dichlorophenolindophenol reduction at {lambda} = 610–750 nm.

Dihydrolipoyl Succinyltransferase (E2k, EC 2.3.1.61 [EC] )—KGDHc (650 milliunits/ml) was assayed in a buffer containing 35 mmol/liter of potassium phosphate, 0.5 mmol/liter of EDTA, 1 mmol/liter of glutaryl-CoA/succinyl-CoA, and 0.1 mmol/liter of DTNB, which was adjusted to pH 7.4 (30 °C). E2k activity was determined as the rate of production of free CoA, as indicated by DTNB reduction at {lambda} = 412 nm.

Dihydrolipoyl Dehydrogenase (E3, EC 1.8.1.4 [EC] )—KGDHc (650 milliunits/ml) was assayed in a buffer containing 35 mmol/liter of potassium phosphate, 0.5 mmol/liter of EDTA, 0.1 mmol/liter of NADH, and 0.4 mmol/liter of lipoamide, which was adjusted to pH 7.4 (30 °C). E3 activity was determined as NADH oxidation at {lambda} = 340–400 nm.

Pyruvate Dehydrogenase Complex (PDHc)—PDHc activity (EC 1.2.4.1 [EC] ) was investigated according to a recently described method (35). In brief, PDHc (160 milliunits/ml) was assayed in a buffer containing 0.05 M potassium phosphate, 2.5 mmol/liter of NAD, 0.2 mmol/liter of thiamine pyrophosphate, 0.1 mmol/liter of CoA, 0.1% Triton X-100, 1 mmol/liter of MgCl2, 1 mg/ml of bovine serum albumin, 0.6 mmol/liter of p-iodonitrotetrazolium violet, and 6.5 µmol/liter of phenazine methosulfate, which was adjusted to pH 7.4 (25 °C). PDHc activity was determined as p-iodonitrotetrazolium violet reduction at {lambda} = 500–750 nm.

The effects of GA, 3-OH-GA, and glutaryl-CoA (up to 2 mmol/liter, all adjusted to pH 7.4) on the above listed enzymes were determined using purified enzymes and the assay conditions described above. These assay conditions were also used for the analysis of bovine heart SMPs.

Glutathione Content of Tissue Homogenates—Glutathione concentrations were determined according to a modification of Ref. 36. Glutathione reductase (EC 1.8.1.7 [EC] ) activity in liver and brain homogenates was assayed in a buffer (adjusted to pH 7.5; 25 °C) containing 180 mmol/liter of disodium hydrogen phosphate, 6 mmol/liter of Na-EDTA, 0.3 mmol/liter of NADPH, 6 mmol/liter of DTNB, and 0.3 units/ml of glutathione reductase (Sigma). Enzyme activity was determined as DTNB reduction at {lambda} = 412 nm. Glutathione content was determined by correlating the detected enzyme activities with a calibration curve generated with samples of defined glutathione concentration.

Spectrophotometric Analysis of FAD-dependent Acyl-CoA Dehydrogenases—FAD-dependent acyl-CoA dehydrogenases, such as short-chain (SCAD, EC 1.3.99.2 [EC] ), medium-chain (MCAD, EC 1.3.99.3 [EC] ), very long-chain (VLCAD, EC 1.3.99.13 [EC] ), and GCDH (EC 1.3.99.7 [EC] ), were assayed at a concentration of 1 mg of protein/ml in a buffer (adjusted to pH 7.5; 37 °C) containing 250 mmol/liter of sucrose, 120 mmol/liter of potassium phosphate, 5 mmol/liter of MgCl2, 0.05% (w/v) laurylmaltoside, 0.2 mmol/liter of ferricenium hexafluorophosphate, 0.01 mmol/liter of FAD, and 0.5 mmol/liter of N-ethylmaleimide. Activity was determined by following the rate of reduction of ferricenium hexafluorophosphate at {lambda} = 300–617 nm. Butyryl-CoA (50 µmol/liter; for SCAD activity), octanoyl-CoA (50 µmol/liter; for MCAD activity), and palmitoyl-CoA (50 µmol/liter; for VLCAD activity) were used as physiological substrates. Changes in the activity of these enzymes were determined by comparison of liver homogenates from Gcdh–/– and wt mice. In further experiments, the effects of GA, 3-OH-GA, and glutaryl-CoA (up to 1 mmol/liter; adjusted to pH 7.5) on SCAD, MCAD, and VLCAD activities were investigated.

Statistical Analysis—Data were expressed as mean ± S.D. All experiments were performed at least in triplicates. Enzyme activity was normalized to the protein concentration of the same sample. A Mann-Whitney U-test was calculated using SPSS for Windows 11.0 software. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activities of Respiratory Chain Complexes I–V Remain Unchanged—To evaluate whether abnormalities in the mitochondrial respiratory chain contributes to the pathology of GCDH deficiency, we investigated the activity of single respiratory chain complexes I–V in homogenates (brain, liver, heart muscle, and skeletal muscle) from Gcdh–/– (n = 7–14) and wt mice (wt; n = 6–14) by spectrophotometric analysis. No significant differences in the activity of the five multiprotein complexes were found between Gcdh–/– and wt mice, except for a slight increase of complex II activity in liver homogenates of Gcdh–/– mice (p = 0.004 versus wt; Table I).


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  		TABLE I
Spectrophotometric analysis of single respiratory chain complexes I–V in tissue homogenates from Gcdh–/– and wild type (wt) mice

Activities are given as milliunits/mg of protein. Data are mean ± S.D. (n = 14).

 
In SMPs, we investigated the effect of GA, 3-OH-GA, and glutaryl-CoA on single respiratory chain activities. In accordance with the results obtained in tissue homogenates, no direct inhibitory effect of GA, 3-OH-GA, or glutaryl-CoA (up to a final concentration of 1 mmol/liter each) on single respiratory chain complexes I–V was found in SMPs (Table II). In both homogenates and SMPs, addition of standard respiratory chain inhibitors revealed good inhibitory responses (93–100% of control activity, p < 0.001 versus controls), confirming the specificity of our results.


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  		TABLE II
Spectrophotometric analysis of single respiratory chain complexes I-V in SMPs from bovine heart after incubation with glutaric acid, 3-hydoxyglutaric acid, or glutaryl-CoA

Activities are given as milliunits/mg of protein. Data are mean ± S.D. (n = 4). Statistical analysis revealed no significant effect of GA, 3-OH-GA, and glutaryl-CoA on complexes I-V (the results of lower concentrations than 1 mmol/liter are not shown), whereas these complexes revealed a good inhibitory response to standard inhibitors.

 
Glutaryl-CoA Inhibits {alpha}-Ketoglutarate Dehydrogenase Complex—Next, we investigated the activity of purified single TCA cycle enzymes from porcine heart during incubation with GA, 3-OH-GA, or glutaryl-CoA. Notably, glutaryl-CoA inhibited KGDHc in a concentration-dependent way (Fig. 1A). Varying the concentrations of {alpha}-ketoglutarate and glutaryl-CoA demonstrated that inhibition of KGDHc by glutaryl-CoA was uncompetitive to {alpha}-ketoglutarate (Vmax, 54.7 mOD/min; Km, 0.15 mmol/liter; Kis, 0.35 mmol/liter; Fig. 1A). Glutaryl-CoA (1 mmol/liter) was degraded in the reverse reaction of the KGDHc subunit E2k (50% of activity with succinyl-CoA as substrate) indicating that glutaryl-CoA is a product of this subunit, whereas it showed no influence on the E1k (126% of control activity) and the E3 (106% of control activity) subunits. Inhibition of KGDHc by glutaryl-CoA was also found using 2-ketoadipate as an alternative substrate (data not shown). The specificity of our finding was confirmed by additional experiments in SMPs, demonstrating the same results (data not shown). In comparison with KGDHc, glutaryl-CoA induced a less pronounced inhibition of PDHc (Fig. 1B).

Next, we investigated additional enzymes of the TCA cycle. Glutaryl-CoA showed no significant effect on MDH, fumarase, aconitase, and IDH, and revealed only a weak inhibition of CS (Fig. 2). The apparent inhibition of MDH and fumarase was most likely an artifact because of the coupling of the malate dehydrogenase and fumarase assays with citrate synthase, which was weakly inhibited by glutaryl-CoA. Neither GA nor 3-OH-GA showed a significant effect on KGDHc (GA 1 mmol/liter, 96% of control; 3-OH-GA 1 mmol/liter, 98% of control), PDHc (GA 1 mmol/liter, 95% of control; 3-OH-GA 1 mmol/liter, 90% of control) aconitase, IDH, fumarase, or MDH, but both slightly activated CS (Fig. 2).

Mitochondrial {beta}-Oxidation of Long-chain Fatty Acids Is Reduced in Gcdh–/– Mice—A comparison of the activities of SCAD, MCAD, and VLCAD in liver homogenates from Gcdh–/– and wt mice demonstrated a mild but significant decrease in the activity of VLCAD, whereas MCAD and SCAD activities remained unchanged (Fig. 3A). In accordance with these results, quantitative acylcarnitine profiling demonstrated increased concentrations of some long-chain fatty acids (Table III) in the bile of the Gcdh–/– mice. These elevations were not observed in acylcarnitine profiles of either serum or dried blood spots from these animals (data not shown). To determine the basis for the reduction of VLCAD activity in the Gcdh–/– mice we investigated the effect of GA, 3-OH-GA, and glutaryl-CoA on VLCAD activity in liver homogenates from Gcdh–/– and control mice. We found no significant effect of any of these metabolites (up to a final concentration of 1 mmol/liter) on VLCAD activity (Fig. 3B). Furthermore, we could not find a significant effect of these compounds on SCAD or MCAD activity (Fig. 3B). Therefore, decreased VLCAD activity in Gcdh–/– mice is most likely because of regulatory changes, rather than a direct inhibition by GA, 3-OH-GA, or glutaryl-CoA.


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  		TABLE III
Analysis of long-chain acylcarnitines in bile of Gcdh-deficient mice

Concentrations are given as micromole/liter. Data are mean ± S.D. (Gcdh–/– = 7, Gcdh+/– = 11).

 



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  		FIG. 1.
Inhibition of KGDHc by glutaryl-CoA. A, glutaryl-CoA inhibits KGDHc activity in an uncompetitive way. KGDHc activity was assayed with varying concentrations of {alpha}-ketoglutarate (2, 1, 0.2, 0.1, and 0.05 mmol/liter) and glutaryl-CoA (2, 1, 0.5, and 0.25 mmol/liter). A Hanes-Woolf plot demonstrated an uncompetitive inhibition of KGDHc by glutaryl-CoA with regard to the substrate {alpha}-ketoglutarate (Vmax, 54.7 mOD/min; Km, 0.15 mmol/liter; Kis, 0.35 mmol/liter). B, glutaryl-CoA-induced inhibition of PDHc activity is less pronounced than of KGDHc activity. KGDHc and PDHc were incubated with varying concentrations of glutaryl-CoA (0.025–2 mmol/liter). In both enzyme complexes, glutaryl-CoA induced a concentration-dependent decrease in activity, however, inhibition of KGDHc activity was much more pronounced than of PDHc activity. All data are expressed as mean ± S.D., experiments were performed in quadruplicates.

 
Glutathione Concentrations Are Decreased in Gcdh–/– Mice—To estimate changes in oxidative stress of Gcdh–/– mice compared with wt mice, glutathione contents in tissue homogenates were investigated. Glutathione concentrations in brain and liver homogenates were significantly decreased in Gcdh–/– mice, suggesting increased oxidative stress in these animals (Fig. 4, A and B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major aim of the present study was to investigate whether inhibition of mitochondrial energy metabolism by GA, 3-OH-GA, or glutaryl-CoA is involved in the pathogenesis of GCDH deficiency. The chronic effects of these metabolites on endogenous enzyme activity were determined by comparing tissues from wt and Gcdh–/– mice, whereas short-term effects were measured using a combination of purified enzymes, bovine heart SMPs, and tissue homogenates. These are the first investigations to evaluate the potential role of glutaryl-CoA, the physiological substrate of GCDH, in the pathophysiology of GCDH deficiency.

Reduction of Flux through TCA Cycle via Inhibition of KGDHc by Glutaryl-CoA—The most important result of the present study is the demonstration of an uncompetitive inhibition of the KGDHc by glutaryl-CoA. The KGDHc is a key and most likely rate-controlling enzyme of the TCA cycle and consists of three subunits: {alpha}-ketoglutarate dehydrogenase (E1k, EC 1.2.4.2 [EC] .), dihydrolipoyl succinyltransferase (E2k, EC 2.3.1.61 [EC] ), and dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4 [EC] ). E1k and E2k are unique to KGDHc, whereas E3 is also a component of PDHc and the branched-chain {alpha}-ketoacid dehydrogenase complex. Glutaryl-CoA inhibited the E2k subunit of KGDHc, but did not affect the E1k or E3 subunits. KGDHc inhibition in patients with GCDH deficiency is supported by the presence of increased urinary excretion of {alpha}-ketoglutarate, in particular during metabolic decompensation (37).2 In comparison to KGDHc, PDHc activity was less affected by glutaryl-CoA. Additional experiments were performed using {alpha}-ketoadipate as substrate for KGDHc, which revealed similar results, demonstrating {alpha}-ketoadipate dehydrogenase activity of KGDHc. Notably, the product of this reaction is glutaryl-CoA.

KGDHc Activity Is Reduced in Common Neurodegenerative Diseases—There is accumulating evidence that reduced KGDHc activity is involved in pathomechanisms underlying a variety of common neurodegenerative diseases, such as Parkinson (38, 39) and Alzheimer diseases (40). Recently, a strongly reduced KGDHc activity was also demonstrated in the putamina of patients with Huntington disease (41) sharing neuropathological similarities with GCDH deficiency (42). Genetic studies considered polymorphisms in the DLST (encoding E2k) or DLD (encoding E3) genes to be a risk factor for the manifestation of Parkinson or Alzheimer diseases (43, 44). Increased oxidative stress is one plausible link between KGDHc deficiency and neurodegeneration, because KGDHc contains a labile Fe-S center, and is sensitive to a number of oxidants including 4-hydroxy-2-nonenal, H2O2, NO, peroxynitrite, hypochlorous acid, and mono-N-chloramine (34, 45–47).

Recently, in vitro and in vivo studies demonstrated increased formation of reactive oxygen and nitrogen species following exposure to GA or 3-OH-GA, respectively (20, 21, 48, 49). Reduced glutathione concentrations in Gcdh–/– mice support a role for increased oxidative stress in GCDH deficiency. Inhibition of KG-DHc by glutaryl-CoA might be amplified by increased oxidative stress, resulting in a synergistic reduction of flux through the TCA cycle. If reduced KGDHc activity contributes to the neuropathogenesis of GCDH deficiency, treatment with thiamine and {alpha}-lipoic acid in concert with other antioxidants, such as {alpha}-tocopherol and N-acetylcysteine, might be helpful to (partially) restore KGDHc function and glutathione levels.

Suggested Mechanisms of KGDHc-mediated Neuronal Damage—The exact mechanism of neuronal damage induced by inhibition of KGDHc is not yet completely understood. A recent in vitro study showed that partial inhibition of KGDHc by {alpha}-keto-{beta}-methyl-n-valeric acid induced cytochrome c release, caspase-3 activation, and necrotic cell death but, notably, did not alter mitochondrial membrane potential (50). The same authors speculated that prominent thiol groups in KGDHc subunits support cellular antioxidant capacity through thioredoxin and suggested a functional coupling between KGDHc inhibition and opening of the mitochondrial permeability transition pore, which is also regulated by the disulfite redox state (51, 52). Such a mechanism is supported by the thioredoxin-deficient mice producing similar effects of KGDHc inhibition (53). Furthermore, thioredoxin can be anchored in the E3 subunit and can directly interact with the lipoyl domain of the E2k subunit (54), providing an additional mechanism to handle cellular oxidative stress (55). An analogous mechanism was demonstrated in Mycobacterium tuberculosis (56).



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  		FIG. 2.
Effect of glutaric acid, 3-hydroxyglutaric acid, and glutaryl-CoA on TCA cycle enzymes. Activities of CS, malate dehydrogenase (MD), fumarase, aconitase, and isocitrate dehydrogenase (ISD) were assayed in the presence or absence of GA, 3-OH-GA, and glutaryl-CoA (each 1 mmol/liter). CS was slightly activated by GA (129% of control) and 3-OH-GA (123% of control), whereas MDH, fumarase, aconitase, and ISD activities remained unaffected. Glutaryl-CoA slightly decreased activities of CS, MDH, and fumarase. The apparent inhibition of MDH and fumarase is most likely an artifact because of the coupling of the MDH and fumarase assays with CS. Data are expressed as mean ± S.D., experiments were performed in quadruplicates. Dotted line indicates control activity.

 



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  		FIG. 3.
Mitochondrial {beta}-oxidation of long-chain fatty acids is reduced in liver of Gcdh–/– mice. A, FAD-dependent acyl-CoA dehydrogenases of mitochondrial {beta}-oxidation of fatty acids, SCAD, MCAD, and VLCAD, were assayed in liver homogenates of Gcdh–/– (n = 13), GCDH+/– (n = 18), and wt (n = 16) mice. VLCAD activity was mildly decreased in Gcdh–/– mice compared with wt (p < 0.05), whereas MCAD and SCAD activities remained unchanged. B, GA, 3-OH-GA, and glutaryl-CoA (each 1 mmol/liter) did not directly inhibit activities of SCAD, MCAD, or VLCAD in liver homogenates of wt mice (n = 4) and Gcdh–/– mice (n = 4). All data are expressed as mean ± S.D. A Mann-Whitney-U test was used for statistical analysis.

 



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  		FIG. 4.
Glutathione concentrations are decreased in brain and liver of Gcdh–/– mice. We investigated glutathione concentrations in Gcdh–/– mice to estimate the intracellular oxidative capacity. Glutathione concentrations were detected in brain (A), respectively, and liver (B) homogenates of Gcdh–/– mice (n = 6), Gcdh+/– mice (n = 5) and wt mice (n = 9). In both, brain (A) and liver (B) homogenates of Gcdh–/– mice, glutathione concentrations were decreased compared with wt mice (p < 0.05). Glutathione decrease was more pronounced in brain than liver. Data are expressed as mean ± S.D. A Mann-Whitney-U test was used for statistical analysis.

 
In dihydrolipoamide dehydrogenase-deficient mice, reduced activity of KGDHc contributed to increased vulnerability of heterozygous animals (Dld+/–) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, malonate, and 3-nitropropionate (41). In addition, decreased complex II activity was found in isolated mitochondria of these mice. We did not observe any decrease in complex II activity in brain tissue from the Gcdh–/– mice, but an increase in liver complex II activity. However, enzymatic analyses of the mouse tissues were performed under standard conditions (32, 59), thus it could not be determined whether the reduced KGHDc activity was affecting the flux through complex II under resting conditions.

Because KGDHc feeds NADH as substrate into complex I and succinyl-CoA/succinate as substrate into complex II, an inhibition of KGDHc activity could secondarily reduce the activity of complex I/II (46). Reduction of spare respiratory capacity via reduced KGDHc activity and secondarily decreased complex I and II activities could increase the risk for neuronal damage in susceptible regions. Notably, the striatum that is predominantly affected in GCDH deficiency is one of the cerebral structures with the lowest spare capacity for complexes I and II (57). Another bioenergetic risk factor is that KGDHc is considered as a late developer among the TCA enzymes postnatally. In rat striatum, KGDHc activity increases between P10 and P17 and attains adult levels at P30 (58). Late up-regulation of KGDHc during postnatal development increases the susceptibility to glutaryl-CoA-induced inhibition during this time period, which can be suggested as a relevant risk factor for the precipitation of acute encephalopathic crises, which peaks during early infancy.

A Role for Impaired Mitochondrial Oxidation of Long-chain Fatty Acids?—We demonstrated that mitochondrial {beta}-oxidation of long-chain fatty acids is slightly impaired in Gcdh–/– mice, however, excluded a direct inhibition of VLCAD activity by GA, 3-OH-GA, and glutaryl-CoA. The pathophysiological relevance of these findings remains to be elucidated. A possible link between decreased VLCAD activity in Gcdh–/– mice and GCDH deficiency is the inhibitory potency of long-chain acyl-CoA esters, in particular palmitoyl-CoA, on energy metabolism including KGDHc (60), PDHc (61), and complex I (62). Interestingly, rhabdomyolysis, which is a characteristic complication of long-chain fatty acid oxidation defects, was described during acute crises in two patients with GCDH deficiency (63, 64), and fatty infiltration of the liver has been found in post-mortem investigations after fatal crises (65, 66). In addition, secondary carnitine depletion following increased urinary loss of glutarylcarnitine could further impair oxidation of long-chain fatty acids.

In conclusion, the present study demonstrates that inhibition of the TCA cycle and mitochondrial fatty acid oxidation may contribute to the pathophysiology of GCDH deficiency. This study significantly adds to previously hypothesized mechanisms, such as excitotoxicity and oxidative stress, supporting the notion that cerebral damage in GCDH deficiency is most likely the result of synergistic mechanisms induced by accumulating organic acids and CoA esters during a vulnerable period of brain development, rather than the result of a single mechanism alone.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant KO 2010/2-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{dagger} This study is dedicated to Dr. Linda Crnic, Ph.D., who was instrumental in developing and characterizing the glutaryl-CoA dehydrogenase-deficient mouse. She was the director of the Mental Retardation and Developmental Disabilities Research Center at the University of Colorado Health Sciences Center, and an internationally known researcher in mouse models of human mental retardation syndromes. Her work and passion for science will be missed. Back

|| To whom correspondence should be addressed: Dept. of General Pediatrics, Division of Inborn Metabolic Diseases, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany. Tel.: 49-6221-561714; Fax: 49-6221-565565; E-mail: Stefan_Koelker{at}med.uni-heidelberg.de.

1 The abbreviations used are: GCDH, glutaryl-CoA dehydrogenase; GA, glutaric acid; 3-OH-GA, 3-hydroxyglutaric acid; TCA, tricarboxylic acid cycle; SMPs, submitochondrial particles; CS, citrate synthase; DTNB, 5,5'-dithiobis-(2-nitrobenzoate); MDH, malate dehydrogenase; IDH, isocitrate dehydrogenase; KGDHc, {alpha}-ketoglutarate dehydrogenase complex; PDHc, pyruvate dehydrogenase complex; SCAD, short-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; VLCAD, very long-chain acyl-CoA dehydrogenase; E1k, {alpha}-ketoglutarate dehydrogenase; E2k, dihydrolipoyl succinyltransferase; E3, dihydrolipoyl dehydrogenase. Back

2 S. Kölker, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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