Mitochondrial dysfunction, oxidative stress, ER stress and mitochondria-ER crosstalk alterations in a chemical rat model of Huntington's disease: Potential benefits of bezafibrate.

Redox homeostasis, mitochondrial functions, and mitochondria-endoplasmic reticulum (ER) communication were evaluated in the striatum of rats after 3-nitropropionic acid (3-NP) administration, a recognized chemical model of Huntington's disease (HD). 3-NP impaired redox homeostasis by increasing malondialdehyde levels at 28 days, decreasing glutathione (GSH) concentrations at 21 and 28 days, and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione S-transferase at 7, 21, and 28 days, catalase at 21 days, and glutathione reductase at 21 and 28 days. Impairment of mitochondrial respiration at 7 and 28 days after 3-NP administration was also observed, as well as reduced activities of succinate dehydrogenase (SDH) and respiratory chain complexes. 3-NP also impaired mitochondrial dynamics and the interactions between ER and mitochondria and induced ER-stress by increasing the levels of mitofusin-1, and of DRP1, VDAC1, Grp75 and Grp78. Synaptophysin levels were augmented at 7 days but reduced at 28 days after 3-NP injection. Finally, bezafibrate prevented 3-NP-induced alterations of the activities of SOD, GPx, SDH and respiratory chain complexes, DCFH oxidation and on the levels of GSH, VDAC1 and synaptophysin. Mitochondrial dysfunction and synaptic disruption may contribute to the pathophysiology of HD and bezafibrate may be considered as an adjuvant therapy for this disorder.

Neuronal Death, Glial Reactivity, Microglia Activation, Oxidative Stress and Bioenergetics Impairment Caused by Intracerebroventricular Administration of D-2-hydroxyglutaric Acid to Neonatal Rats.

D-2-hydroxyglutaric acid (D-2-HG) accumulates and is the biochemical hallmark of D-2-hydroxyglutaric acidurias (D-2-HGA) types I and II, which comprehend two inherited neurometabolic diseases with severe cerebral abnormalities. Since the pathogenesis of these diseases is poorly established, we tested whether D-2-HG could be neurotoxic to neonatal rats. D-2-HG intracerebroventricular administration caused marked vacuolation in cerebral cortex and striatum. In addition, glial fibrillary acidic protein (GFAP), S-100 calcium binding protein B (S100B) and ionized calcium-binding adapter molecule 1 (Iba-1) staining was increased in both brain structures, suggesting glial reactivity and microglial activation. D-2-HG also provoked a reduction of NeuN-positive cells in cerebral cortex, signaling neuronal death. Considering that disturbances in redox homeostasis and energy metabolism may be involved in neuronal damage and glial reactivity, we assessed whether D-2-HG could induce oxidative stress and bioenergetics impairment. D-2-HG treatment significantly augmented reactive oxygen and nitrogen species generation, provoked lipid peroxidation and protein oxidative damage, diminished glutathione concentrations and augmented superoxide dismutase and catalase activities in cerebral cortex. Increased reactive oxygen species generation, lipoperoxidation and protein oxidation were also found in striatum. Furthermore, the antagonist of NMDA glutamate receptor MK-801 and the antioxidant melatonin were able to prevent most of D-2-HG-induced pro-oxidant effects, implying the participation of these receptors in D-2-HG-elicited oxidative damage. Our results also demonstrated that D-2-HG markedly reduced the respiratory chain complex IV and creatine kinase activities. It is presumed that these deleterious pathomechanisms caused by D-2-HGA may be involved in the brain abnormalities characteristic of early-infantile onset D-2-HGA.

Ethylmalonic acid impairs bioenergetics by disturbing succinate and glutamate oxidation and induces mitochondrial permeability transition pore opening in rat cerebellum.

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.

Free Radical Scavengers Prevent Argininosuccinic Acid-Induced Oxidative Stress in the Brain of Developing Rats: a New Adjuvant Therapy for Argininosuccinate Lyase Deficiency?

Tissue accumulation and high urinary excretion of argininosuccinate (ASA) is the biochemical hallmark of argininosuccinate lyase deficiency (ASLD), a urea cycle disorder mainly characterized by neurologic abnormalities, whose pathogenesis is still unknown. Thus, in the present work, we evaluated the in vitro and in vivo effects of ASA on a large spectrum of oxidative stress parameters in brain of adolescent rats in order to test whether disruption of redox homeostasis could be involved in neurodegeneration of this disorder. ASA provoked in vitro lipid and protein oxidation, decreased reduced glutathione (GSH) concentrations, and increased reactive oxygen species generation in cerebral cortex and striatum. Furthermore, these effects were totally prevented or attenuated by the antioxidants melatonin and GSH. Similar results were obtained by intrastriatal administration of ASA, in addition to increased reactive nitrogen species generation and decreased activities of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase. It was also observed that melatonin and N-acetylcysteine prevented most of ASA-induced in vivo pro-oxidant effects in striatum. Taken together, these data indicate that disturbance of redox homeostasis induced at least in part by high brain ASA concentrations per se may potentially represent an important pathomechanism of neurodegeneration in patients with ASLD and that therapeutic trials with appropriate antioxidants may be an adjuvant treatment for these patients.

ETHE1 and MOCS1 deficiencies: Disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts.

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

In vivo intracerebral administration of L-2-hydroxyglutaric acid provokes oxidative stress and histopathological alterations in striatum and cerebellum of adolescent rats.

Patients affected by L-2-hydroxyglutaric aciduria (L-2-HGA) are biochemically characterized by elevated L-2-hydroxyglutaric acid (L-2-HG) concentrations in cerebrospinal fluid, plasma, and urine due to a blockage in the conversion of L-2-HG to α-ketoglutaric acid. Neurological symptoms associated with basal ganglia and cerebelar abnormalities whose pathophysiology is still unknown are typical of this neurometabolic disorder. In the present study we evaluated the early effects (30min after injection) of an acute in vivo intrastriatal and intracerebellar L-2-HG administration on redox homeostasis in rat striatum and cerebellum, respectively. Histological analyses of these brain structures were also carried out 7 days after L-2-HG treatment (long-term effects). L-2-HG significantly decreased the concentrations of reduced (GSH) and total glutathione (tGS), as well as of glutathione peroxidase (GPx) and reductase (GR) activities, but did not change the activities of superoxide dismutase and catalase in striatum. Furthermore, the concentrations of oxidized glutathione (GSSG) and malondialdehyde (MDA), as well as 2',7'-dichlorofluorescein (DCFH) oxidation and hydrogen peroxide (H2O2) production, were increased, whereas carbonyl formation and nitrate plus nitrite concentrations were not altered by L-2-HG injection. It was also found that the melatonin, ascorbic acid plus α-tocopherol, and creatine totally prevented most of these effects, whereas N-acetylcysteine, the noncompetitive glutamate NMDA antagonist MK-801, and the nitric oxide synthase inhibitor L-NAME were not able to normalize the redox alterations elicited by L-2-HG in striatum. L-2-HG intracerebellar injection similarly provoked a decrease of antioxidant defenses (GSH, tGS, GPx, and GR) and an increase of the concentrations of GSSG, MDA, and H2O2 in cerebellum. These results strongly indicate that the major accumulating metabolite in L-2-HGA induce oxidative stress by decreasing the antioxidant defenses and enhancing reactive oxygen species in striatum and cerebellum of adolescent rats. Regarding the histopathological findings, L-2-HG caused intense vacuolation, lymphocyte and macrophage infiltrates, eosinophilic granular bodies, and necrosis in striatum. Immunohistochemistry revealed that L-2-HG treatment provoked an increase of GFAP and a decrease of NeuN immunostaining, indicating reactive astroglyosis and reduction of neuronal population, respectively, in striatum. Similar macrophage infiltrates, associated with less intense vacuolation and lymphocytic infiltration, were observed in cerebellum. However, we did not observe necrosis, eosinophilic granular bodies, and alteration of GFAP and NeuN content in L-2-HG-teated cerebellum. From the biochemical and histological findings, it is presumed that L-2-HG provokes striatal and cerebellar damage in vivo possibly through oxidative stress induction. Therefore, we postulate that antioxidants may serve as adjuvant therapy allied to the current treatment based on a protein-restricted diet and riboflavin and L-carnitine supplementation in patients affected by L-2-HGA.

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.

Alpha-ketoisocaproic acid and leucine provoke mitochondrial bioenergetic dysfunction in rat brain.

Patients affected by maple syrup urine disease (MSUD) present severe neurological symptoms and brain abnormalities, whose pathophysiology is poorly known. In the present study we investigated the in vitro effects of leucine (Leu), alpha-ketoisocaproic acid (KIC) and alpha-hydroxyisovaleric acid (HIV), respectively, the branched-chain amino, keto and hydroxy acids that most accumulate in MSUD, on brain bioenergetic homeostasis, evaluating respiratory parameters obtained by oxygen consumption, membrane potential (Psim), NAD(P)H content, swelling and citric acid cycle enzyme activities in mitochondrial preparations from rat forebrain using glutamate plus malate, succinate or alpha-ketoglutarate as respiratory substrates. KIC increased state 4 and decreased the respiratory control ratio with all substrates, in contrast with Leu and HIV. Furthermore, KIC and Leu, but not HIV, decreased state 3 using alpha-ketoglutarate. A KIC-induced selective inhibition of alpha-ketoglutarate dehydrogenase activity was also verified, with no alteration of the other citric acid cycle activities. The ADP/O ratio and the mitochondrial NAD(P)H levels were also reduced by KIC using glutamate/malate and alpha-ketoglutarate. In addition, KIC caused a reduction in the Psim when alpha-ketoglutarate was the substrate. Finally, KIC was not able to induce mitochondrial swelling. The present data indicate that KIC acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor possibly through its inhibitory effect on alpha-ketoglutarate dehydrogenase activity, while Leu acts as a metabolic inhibitor. It is suggested that impairment of mitochondrial homeostasis caused by the major metabolites accumulating in MSUD may be involved in the neuropathology of this disease.

Creatine administration prevents Na+,K+-ATPase inhibition induced by intracerebroventricular administration of isovaleric acid in cerebral cortex of young rats.

Isovaleric acidemia (IVAcidemia) is an inborn error of metabolism due to deficiency of isovaleryl-CoA dehydrogenase activity, leading to predominant accumulation of isovaleric acid (IVA). Patients affected by IVAcidemia suffer from acute episodes of encephalopathy, whose underlying mechanisms are poorly known. In the present study we investigated whether an intracerebroventricular injection of IVA could compromise energy metabolism in cerebral cortex of young rats. IVA administration significantly inhibited (14)CO(2) production from acetate (22%) and citrate synthase activity (20%) in cerebral cortex homogenates prepared 24 h after injection. However, no alterations of these parameters were observed 2 h after injection. In contrast, no significant differences were found in the activities of succinate dehydrogenase, isocitrate dehydrogenase, electron transfer chain complexes or creatine kinase in rats sacrificed 2 or 24 h after IVA administration. Moreover, IVA injection significantly inhibited Na(+),K(+)-ATPase activity (25%) in cerebral cortex of rats 2 or 24 h after IVA administration, while pre-treatment of rats with creatine completely prevented the inhibitory effects of IVA on Na(+),K(+)-ATPase. In conclusion, in vivo administration of IVA inhibits the citric acid cycle probably through the enzyme citrate synthase, as well as Na(+),K(+)-ATPase, a crucial enzyme responsible for maintaining the basal potential membrane necessary for a normal neurotransmission. It is presumed that inhibition of these activities may be involved in the pathophysiology of the neurological dysfunction of isovaleric academic patients. The present findings are of particular interest because treatment with creatine supplementation may represent a potential novel adjuvant therapeutic strategy in IVAcidemia.