Michael Woontner

Mechanism of age-dependent susceptibility and novel treatment strategy in glutaric acidemia type I

Glutaric acidemia type I (GA-I) is an inherited disorder of lysine and tryptophan metabolism presenting with striatal lesions anatomically and symptomatically similar to Huntington disease. Affected children commonly suffer acute brain injury in the context of a catabolic state associated with nonspecific illness. The mechanisms underlying injury and age-dependent susceptibility have been unknown, and lack of a diagnostic marker heralding brain injury has impeded intervention efforts. Using a mouse model of GA-I, we show that pathologic events began in the neuronal compartment while enhanced lysine accumulation in the immature brain allowed increased glutaric acid production resulting in age-dependent injury. Glutamate and GABA depletion correlated with brain glutaric acid accumulation and could be monitored in vivo by proton nuclear magnetic resonance (1H NMR) spectroscopy as a diagnostic marker. Blocking brain lysine uptake reduced glutaric acid levels and brain injury. These findings provide what we believe are new monitoring and treatment strategies that may translate for use in human GA-I.

Induction of oxidative stress in brain of glutaryl-CoA dehydrogenase deficient mice by acute lysine administration

In the present work we evaluated a variety of indicators of oxidative stress in distinct brain regions (striatum, cerebral cortex and hippocampus), the liver, and heart of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh(-/-)) mice. The parameters evaluated included thiobarbituric acid-reactive substances (TBA-RS), 2-7-dihydrodichlorofluorescein (DCFH) oxidation, sulfhydryl content, and reduced glutathione (GSH) concentrations. We also measured the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and glucose-6-phosphate dehydrogenase (G6PD). Under basal conditions glutaric (GA) and 3-OH-glutaric (3OHGA) acids were elevated in all tissues of the Gcdh(-/-) mice, but were essentially absent in WT animals. In contrast there were no differences between WT and Gcdh(-/-) mice in any of the indicators or oxidative stress under basal conditions. Following a single intra-peritoneal (IP) injection of lysine (Lys) there was a moderate increase of brain GA concentration in Gcdh(-/-) mice, but no change in WT. Lys injection had no effect on brain 3OHGA in either WT or Gcdh(-/-) mice. The levels of GA and 3OHGA were approximately 40% higher in striatum compared to cerebral cortex in Lys-treated mice. In the striatum, Lys administration provoked a marked increase of lipid peroxidation, DCFH oxidation, SOD and GR activities, as well as significant reductions of GSH levels and GPx activity, with no alteration of sulfhydryl content, CAT and G6PD activities. There was also evidence of increased lipid peroxidation and SOD activity in the cerebral cortex, along with a decrease of GSH levels, but to a lesser extent than in the striatum. In the hippocampus only mild increases of SOD activity and DCFH oxidation were observed. In contrast, Lys injection had no effect on any of the parameters of oxidative stress in the liver or heart of Gcdh(-/-) or WT animals. These results indicate that in Gcdh(-/-) mice cerebral tissue, particularly the striatum, is at greater risk for oxidative stress than peripheral tissues following Lys administration.

Marked reduction of Na(+), K(+)-ATPase and creatine kinase activities induced by acute lysine administration in glutaryl-CoA dehydrogenase deficient mice.

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity leading to accumulation of predominantly glutaric (GA) and 3-hydroxyglutaric (3HGA) acids in the brain and other tissues. Affected patients usually present with hypotonia and brain damage and acute encephalopathic episodes whose pathophysiology is not yet fully established. In this study we investigated important parameters of cellular bioenergetics in brain, heart and skeletal muscle from 15-day-old glutaryl-CoA dehydrogenase deficient mice (Gcdh(-/-)) submitted to a single intra-peritoneal injection of saline (Sal) or lysine (Lys - 8 μmol/g) as compared to wild type (WT) mice. We evaluated the activities of the respiratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and synaptic Na(+), K(+)-ATPase. No differences of all evaluated parameters were detected in the Gcdh(-/-) relatively to the WT mice injected at baseline (Sal). Furthermore, mild increases of the activities of some respiratory chain complexes (II-III and IV) were observed in heart and skeletal muscle of Gcdh(-/-) and WT mice after Lys administration. However, the most marked effects provoked by Lys administration were marked decreases of the activities of Na(+), K(+)-ATPase in brain and CK in brain and skeletal muscle of Gcdh(-/-) mice. In contrast, brain α-KGDH activity was not altered in WT and Gcdh(-/-) injected with Sal or Lys. Our results demonstrate that reduction of Na(+), K(+)-ATPase and CK activities may play an important role in the pathogenesis of the neurodegenerative changes in GA I.

Disruption of brain redox homeostasis in glutaryl-CoA dehydrogenase deficient mice treated with high dietary lysine supplementation.

Deficiency of glutaryl-CoA dehydrogenase (GCDH) activity or glutaric aciduria type I (GA I) is an inherited neurometabolic disorder biochemically characterized by predominant accumulation of glutaric acid and 3-hydroxyglutaric acid in the brain and other tissues. Affected patients usually present acute striatum necrosis during encephalopathic crises triggered by metabolic stress situations, as well as chronic leukodystrophy and delayed myelination. Considering that the mechanisms underlying the brain injury in this disease are not yet fully established, in the present study we investigated important parameters of oxidative stress in the brain (cerebral cortex, striatum and hippocampus), liver and heart of 30-day-old GCDH deficient knockout (Gcdh(-/-)) and wild type (WT) mice submitted to a normal lysine (Lys) (0.9% Lys), or high Lys diets (2.8% or 4.7% Lys) for 60 h. It was observed that the dietary supplementation of 2.8% and 4.7% Lys elicited noticeable oxidative stress, as verified by an increase of malondialdehyde concentrations (lipid oxidative damage) and 2-7-dihydrodichlorofluorescein (DCFH) oxidation (free radical production), as well as a decrease of reduced glutathione levels and alteration of various antioxidant enzyme activities (antioxidant defenses) in the cerebral cortex and the striatum, but not in the hippocampus, the liver and the heart of Gcdh(-/-) mice, as compared to WT mice receiving the same diets. Furthermore, alterations of oxidative stress parameters in the cerebral cortex and striatum were more accentuated in symptomatic, as compared to asymptomatic Gcdh(-/-) mice exposed to 4.7% Lys overload. Histopathological studies performed in the cerebral cortex and striatum of these animals exposed to high dietary Lys revealed increased expression of oxidative stress markers despite the absence of significant structural damage. The results indicate that a disruption of redox homeostasis in the cerebral cortex and striatum of young Gcdh(-/-) mice exposed to increased Lys diet may possibly represent an important pathomechanism of brain injury in GA I patients under metabolic stress.

Reduction of Na + ,K + -ATPase activity and expression in cerebral cortex of glutaryl-CoA dehydrogenase deficient mice: A possible mechanism for brain injury in glutaric aciduria type I

Mitochondrial dysfunction has been proposed to play an important role in the neuropathology of glutaric acidemia type I (GA I). However, the relevance of bioenergetics disruption and the exact mechanisms responsible for the cortical leukodystrophy and the striatum degeneration presented by GA I patients are not yet fully understood. Therefore, in the present work we measured the respiratory chain complexes activities I-IV, mitochondrial respiratory parameters state 3, state 4, the respiratory control ratio and dinitrophenol (DNP)-stimulated respiration (uncoupled state), as well as the activities of α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and Na+, K+-ATPase in cerebral cortex, striatum and hippocampus from 30-day-old Gcdh-/- and wild type (WT) mice fed with a normal or a high Lys (4.7%) diet. When a baseline (0.9% Lys) diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice as compared to WT animals. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na(+), K(+)-ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Furthermore, a high Lys (4.7%) diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed with a normal diet. Since Na(+), K(+)-ATPase activity is required for cell volume regulation and to maintain the membrane potential necessary for a normal neurotransmission, it is presumed that reduction of this enzyme activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by GA I.

Increased Glutamate Receptor and Transporter Expression in the Cerebral Cortex and Striatum of Gcdh-/- Mice: Possible Implications for the Neuropathology of Glutaric Acidemia Type I

We determined mRNA expression of the ionotropic glutamate receptors NMDA (NR1, NR2A and NR2B subunits), AMPA (GluR2 subunit) and kainate (GluR6 subunit), as well as of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum of wild type (WT) and glutaryl-CoA dehydrogenase deficient (Gchh -/-) mice aged 7, 30 and 60 days. The protein expression levels of some of these membrane proteins were also measured. Overexpression of NR2A and NR2B in striatum and of GluR2 and GluR6 in cerebral cortex was observed in 7-day-old Gcdh -/-. There was also an increase of mRNA expression of all NMDA subunits in cerebral cortex and of NR2A and NR2B in striatum of 30-day-old Gcdh -/- mice. At 60 days of life, all ionotropic receptors were overexpressed in cerebral cortex and striatum of Gcdh -/- mice. Higher expression of GLAST and GLT1 transporters was also verified in cerebral cortex and striatum of Gcdh -/- mice aged 30 and 60 days, whereas at 7 days of life GLAST was overexpressed only in striatum from this mutant mice. Furthermore, high lysine intake induced mRNA overexpression of NR2A, NR2B and GLAST transcripts in striatum, as well as of GluR2 and GluR6 in both striatum and cerebral cortex of Gcdh -/- mice. Finally, we found that the protein expression of NR2A, NR2B, GLT1 and GLAST were significantly greater in cerebral cortex of Gcdh -/- mice, whereas NR2B and GLT1 was similarly enhanced in striatum, implying that these transcripts were translated into their products. These results provide evidence that glutamate receptor and transporter expression is higher in Gcdh -/- mice and that these alterations may be involved in the pathophysiology of GA I and possibly explain, at least in part, the vulnerability of striatum and cerebral cortex to injury in patients affected by GA I.

Experimental evidence that overexpression of NR2B glutamate receptor subunit is associated with brain vacuolation in adult glutaryl-CoA dehydrogenase deficient mice: A potential role for glutamatergic-induced excitotoxicity in GA I neuropathology

Glutaric aciduria type I (GA I) is biochemically characterized by accumulation of glutaric and 3-hydroxyglutaric acids in body fluids and tissues, particularly in the brain. Affected patients show progressive cortical leukoencephalopathy and chronic degeneration of the basal ganglia whose pathogenesis is still unclear. In the present work we investigated parameters of bioenergetics and redox homeostasis in various cerebral structures (cerebral cortex, striatum and hippocampus) and heart of adult wild type (Gcdh(+/+)) and glutaryl-CoA dehydrogenase deficient knockout (Gcdh(-/-)) mice fed a baseline chow. Oxidative stress parameters were also measured after acute lysine overload. Finally, mRNA expression of NMDA subunits and GLT1 transporter was determined in cerebral cortex and striatum of these animals fed a baseline or high lysine (4.7%) chow. No significant alterations of bioenergetics or redox status were observed in these mice. In contrast, mRNA expression of the NR2B glutamate receptor subunit and of the GLT1 glutamate transporter was higher in cerebral cortex of Gcdh(-/-) mice. Furthermore, NR2B expression was markedly elevated in striatum of Gcdh(-/-) animals receiving chronic Lys overload. These data indicate higher susceptibility of Gcdh(-/-) mice to excitotoxic damage, implying that this pathomechanism may contribute to the cortical and striatum alterations observed in GA I patients.

New Cases of DHTKD1 Mutations in Patients with 2-Ketoadipic Aciduria.

2-Ketoadipic aciduria (OMIM 204750), a defect in the catabolic pathway of tryptophan, lysine, and hydroxylysine, is characterized by elevations in 2-ketoadipic, 2-aminoadipic, and 2-hydroxyadipic acids. Patients with the aforementioned biochemical profile have been described with a wide range of clinical presentations, from early-onset developmental delay, epilepsy, ataxia, and microcephaly to completely normal. This broad range of phenotypes has led some to question whether 2-ketoadipic aciduria represents a true disease state or if the biochemical abnormalities found in these patients merely reflect an ascertainment bias. We present four additional individuals from two families, with 2-ketoadipic aciduria with compound heterozygous or homozygous mutations in DHTKD1, three of which remain asymptomatic.

Experimental evidence that bioenergetics disruption is not mainly involved in the brain injury of glutaryl-CoA dehydrogenase deficient mice submitted to lysine overload

Bioenergetics dysfunction has been postulated as an important pathomechanism of brain damage in glutaric aciduria type I, but this is still under debate. We investigated activities of citric acid cycle (CAC) enzymes, lactate release, respiration and membrane potential (ΔΨm) in mitochondrial preparations from cerebral cortex and striatum of 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh-/-) and wild type mice fed a baseline or a high lysine (Lys, 4.7%) chow for 60 or 96h. Brain histological analyses were performed in these animals, as well as in 90-day-old animals fed a baseline or a high Lys chow during 30 days starting at 60-day-old. A moderate reduction of citrate synthase and isocitrate dehydrogenase activities was observed only in the striatum from 30-day-old Gcdh-/- animals submitted to a high Lys chow. In contrast, the other CAC enzyme activities, lactate release, the respiratory parameters state 3, state 4, the respiratory control ratio and CCCP-stimulated (uncoupled) state, as well as ΔΨm were not altered in the striatum. Similarly, none of the evaluated parameters were changed in the cerebral cortex from these animals under baseline or Lys overload. On the other hand, histological analyses revealed the presence of intense vacuolation in the cerebral cortex of 60 and 90-day-old Gcdh-/- mice fed a baseline chow and in the striatum of 90-day-old Gcdh-/- mice submitted to Lys overload for 30 days. Taken together, the present data demonstrate mild impairment of bioenergetics homeostasis and marked histological alterations in striatum from Gcdh-/- mice under a high Lys chow, suggesting that disruption of energy metabolism is not mainly involved in the brain injury of these animals.

Neurodevelopmental and cognitive behavior of glutaryl-CoA dehydrogenase deficient knockout mice

AIMS The establishment of a genetic knockout murine model of glutaric acidemia type I (GAI) with complete loss of glutaryl-CoA dehydrogenase (GCDH) activity has been used to investigate the pathological mechanisms underlying neurological symptoms in this disorder. However, very little has been reported on the neurobehavior of GCDH deficient mice (Gcdh(-/-)). MAIN METHODS In the present study we evaluated physical (body and weight gain) and neuromotor development (appearance of coat, upper incisor eruption, eye-opening day, motor coordination, muscular strength and climbing), as well as cognitive behavior (inhibitory avoidance) in Gcdh(-/-), as compared to wild type (WT) mice. KEY FINDINGS We found that Gcdh(-/-) mice did not differ in body and weight gain, appearance of coat, upper incisor eruption, motor coordination and muscular strength, but had a significant delayed eye opening, implying a mild impairment of neurodevelopment in these animals. Furthermore, the climbing behavior was significantly higher in Gcdh(-/-) as compared to WT mice, suggesting an altered dopaminergic function. Finally, Gcdh(-/-) mice presented a deficit of short- and long-term memories in the inhibitory avoidance task. SIGNIFICANCE Although it is difficult to extrapolate the present findings to the human condition, our present data are particularly interesting in view of the psychomotor/mental delay that occurs in a significant number of GAI patients with no previous history of acute encephalopathy with striatum destruction. Strict and early treatment possibly associated with novel therapies seems therefore important to prevent learning/memory disabilities in GAI patients.