Disturbances in mitochondrial bioenergetics and control quality and unbalanced redox homeostasis in the liver of a mouse model of mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II; Hunter syndrome) is a lysosomal storage disease caused by mutations in the gene encoding the enzyme iduronate 2-sulfatase (IDS) and biochemically characterized by the accumulation of glycosaminoglycans (GAGs) in different tissues. It is a multisystemic disorder that presents liver abnormalities, the pathophysiology of which is not yet established. In the present study, we evaluated bioenergetics, redox homeostasis, and mitochondrial dynamics in the liver of 6-month-old MPS II mice (IDS-). Our findings show a decrease in the activity of α-ketoglutarate dehydrogenase and an increase in the activities of succinate dehydrogenase and malate dehydrogenase. The activity of mitochondrial complex I was also increased whereas the other complex activities were not affected. In contrast, mitochondrial respiration, membrane potential, ATP production, and calcium retention capacity were not altered. Furthermore, malondialdehyde levels and 2',7'-dichlorofluorescein oxidation were increased in the liver of MPS II mice, indicating lipid peroxidation and increased ROS levels, respectively. Sulfhydryl and reduced glutathione levels, as well as glutathione S-transferase, glutathione peroxidase (GPx), superoxide dismutase, and catalase activities were also increased. Finally, the levels of proteins involved in mitochondrial mass and dynamics were decreased in knockout mice liver. Taken together, these data suggest that alterations in energy metabolism, redox homeostasis, and mitochondrial dynamics can be involved in the pathophysiology of liver abnormalities observed in MPS II.

In Vivo Intracerebral Administration of α-Ketoisocaproic Acid to Neonate Rats Disrupts Brain Redox Homeostasis and Promotes Neuronal Death, Glial Reactivity, and Myelination Injury.

Maple syrup urine disease (MSUD) is caused by severe deficiency of branched-chain α-keto acid dehydrogenase complex activity, resulting in tissue accumulation of branched-chain α-keto acids and amino acids, particularly α-ketoisocaproic acid (KIC) and leucine. Affected patients regularly manifest with acute episodes of encephalopathy including seizures, coma, and potentially fatal brain edema during the newborn period. The present work investigated the ex vivo effects of a single intracerebroventricular injection of KIC to neonate rats on redox homeostasis and neurochemical markers of neuronal viability (neuronal nuclear protein (NeuN)), astrogliosis (glial fibrillary acidic protein (GFAP)), and myelination (myelin basic protein (MBP) and 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase)) in the cerebral cortex and striatum. KIC significantly disturbed redox homeostasis in these brain structures 6 h after injection, as observed by increased 2',7'-dichlorofluorescein oxidation (reactive oxygen species generation), malondialdehyde levels (lipid oxidative damage), and carbonyl formation (protein oxidative damage), besides impairing the antioxidant defenses (diminished levels of reduced glutathione and altered glutathione peroxidase, glutathione reductase, and superoxide dismutase activities) in both cerebral structures. Noteworthy, the antioxidants N-acetylcysteine and melatonin attenuated or normalized most of the KIC-induced effects on redox homeostasis. Furthermore, a reduction of NeuN, MBP, and CNPase, and an increase of GFAP levels were observed at postnatal day 15, suggesting neuronal loss, myelination injury, and astrocyte reactivity, respectively. Our data indicate that disruption of redox homeostasis, associated with neural damage caused by acute intracerebral accumulation of KIC in the neonatal period may contribute to the neuropathology characteristic of MSUD patients.

Disturbance of mitochondrial functions caused by N-acetylglutamate and N-acetylmethionine in brain of adolescent rats: Potential relevance in aminoacylase 1 deficiency.

Aminoacylase 1 (ACY1) deficiency is a rare genetic disorder that affects the breakdown of short-chain aliphatic N-acetylated amino acids, leading to the accumulation of these amino acid derivatives in the urine of patients. Some of the affected individuals have presented with heterogeneous neurological symptoms such as psychomotor delay, seizures, and intellectual disability. Considering that the pathological mechanisms of brain damage in this disorder remain mostly unknown, here we investigated whether major metabolites accumulating in ACY1 deficiency, namely N-acetylglutamate (NAG) and N-acetylmethionine (NAM), could be toxic to the brain by examining their in vitro effects on important mitochondrial properties. We assessed the effects of NAG and NAM on membrane potential, swelling, reducing equivalents, and Ca2+ retention capacity in purified mitochondrial preparations obtained from the brain of adolescent rats. NAG and NAM decreased mitochondrial membrane potential, reducing equivalents, and calcium retention capacity, and induced swelling in Ca2+-loaded brain mitochondria supported by glutamate plus malate. Notably, these changes were completely prevented by the classical inhibitors of mitochondrial permeability transition (MPT) pore cyclosporin A plus ADP and by ruthenium red, implying the participation of MPT and Ca2+ in these effects. Our findings suggest that NAG- and NAM-induced disruption of mitochondrial functions involving MPT may represent relevant mechanisms of neuropathology in ACY1 deficiency.

N-Acetylglutamate and N-acetylmethionine compromise mitochondrial bioenergetics homeostasis and glutamate oxidation in brain of developing rats: Potential implications for the pathogenesis of ACY1 deficiency.

Aminoacylase 1 (ACY1) deficiency is an inherited metabolic disorder biochemically characterized by high urinary concentrations of aliphatic N-acetylated amino acids and associated with a broad clinical spectrum with predominant neurological signs. Considering that the pathogenesis of ACY1 is practically unknown and the brain is highly dependent on energy production, the in vitro effects of N-acetylglutamate (NAG) and N-acetylmethionine (NAM), major metabolites accumulating in ACY1 deficiency, on the enzyme activities of the citric acid cycle (CAC), of the respiratory chain complexes and glutamate dehydrogenase (GDH), as well as on ATP synthesis were evaluated in brain mitochondrial preparations of developing rats. NAG mildly inhibited mitochondrial isocitrate dehydrogenase 2 (IDH2) activity, moderately inhibited the activities of isocitrate dehydrogenase 3 (IDH3) and complex II-III of the respiratory chain and markedly suppressed the activities of complex IV and GDH. Of note, the NAG-induced inhibitory effect on IDH3 was competitive, whereas that on GDH was mixed. On the other hand, NAM moderately inhibited the activity of respiratory complexes II-III and GDH activities and strongly decreased complex IV activity. Furthermore, NAM was unable to modify any of the CAC enzyme activities, indicating a selective effect of NAG toward IDH mitochondrial isoforms. In contrast, the activities of citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and of the respiratory chain complexes I and II were not changed by these N-acetylated amino acids. Finally, NAG and NAM strongly decreased mitochondrial ATP synthesis. Taken together, the data indicate that NAG and NAM impair mitochondrial brain energy homeostasis.

Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.

Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.

L-2-Hydroxyglutaric Acid Administration to Neonatal Rats Elicits Marked Neurochemical Alterations and Long-Term Neurobehavioral Disabilities Mediated by Oxidative Stress.

L-2-Hydroxyglutaric aciduria (L-2-HGA) is an inherited neurometabolic disorder caused by deficient activity of L-2-hydroxyglutarate dehydrogenase. L-2-Hydroxyglutaric acid (L-2-HG) accumulation in the brain and biological fluids is the biochemical hallmark of this disease. Patients present exclusively neurological symptoms and brain abnormalities, particularly in the cerebral cortex, basal ganglia, and cerebellum. Since the pathogenesis of this disorder is still poorly established, we investigated the short-lived effects of an intracerebroventricular injection of L-2-HG to neonatal rats on redox homeostasis in the cerebellum, which is mostly affected in this disorder. We also determined immunohistochemical landmarks of neuronal viability (NeuN), astrogliosis (S100B and GFAP), microglia activation (Iba1), and myelination (MBP and CNPase) in the cerebral cortex and striatum following L-2-HG administration. Finally, the neuromotor development and cognitive abilities were examined. L-2-HG elicited oxidative stress in the cerebellum 6 h after its injection, which was verified by increased reactive oxygen species production, lipid oxidative damage, and altered antioxidant defenses (decreased concentrations of reduced glutathione and increased glutathione peroxidase and superoxide dismutase activities). L-2-HG also decreased the content of NeuN, MBP, and CNPase, and increased S100B, GFAP, and Iba1 in the cerebral cortex and striatum at postnatal days 15 and 75, implying long-standing neuronal loss, demyelination, astrocyte reactivity, and increased inflammatory response, respectively. Finally, L-2-HG administration caused a delay in neuromotor development and a deficit of cognition in adult animals. Importantly, the antioxidant melatonin prevented L-2-HG-induced deleterious neurochemical, immunohistochemical, and behavioral effects, indicating that oxidative stress may be central to the pathogenesis of brain damage in L-2-HGA.

Disruption of mitochondrial bioenergetics, calcium retention capacity and cell viability caused by D-2-hydroxyglutaric acid in the heart.

Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria type II (D2HGA2). D2HGA2 is a metabolic inherited disease caused by gain-of-function mutations in the gene isocitrate dehydrogenase 2. It is clinically characterized by neurological abnormalities and a severe cardiomyopathy whose pathogenesis is still poorly established. The present work investigated the potential cardiotoxicity D-2-HG, by studying its in vitro effects on a large spectrum of bioenergetics parameters in heart of young rats and in cultivated H9c2 cardiac myoblasts. D-2-HG impaired cellular respiration in purified mitochondrial preparations and crude homogenates from heart of young rats, as well as in digitonin-permeabilized H9c2 cells. ATP production and the activities of cytochrome c oxidase (complex IV), alpha-ketoglutarate dehydrogenase, citrate synthase and creatine kinase were also inhibited by D-2-HG, whereas the activities of complexes I, II and II-III of the respiratory chain, glutamate, succinate and malate dehydrogenases were not altered. We also found that this organic acid compromised mitochondrial Ca2+ retention capacity in heart mitochondrial preparations and H9c2 myoblasts. Finally, D-2-HG reduced the viability of H9c2 cardiac myoblasts, as determined by the MTT test and by propidium iodide incorporation. Noteworthy, L-2-hydroxyglutaric acid did not change some of these measurements (complex IV and creatine kinase activities) in heart preparations, indicating a selective inhibitory effect of the enantiomer D. In conclusion, it is presumed that D-2-HG-disrupts mitochondrial bioenergetics and Ca2+ retention capacity, which may be involved in the cardiomyopathy commonly observed in D2HGA2.

Disruption of mitochondrial functions involving mitochondrial permeability transition pore opening caused by maleic acid in rat kidney.

Propionic acid (PA) predominantly accumulates in tissues and biological fluids of patients affected by propionic acidemia that may manifest chronic renal failure along development. High urinary excretion of maleic acid (MA) has also been described. Considering that the underlying mechanisms of renal dysfunction in this disorder are poorly known, the present work investigated the effects of PA and MA (1-5 mM) on mitochondrial functions and cellular viability in rat kidney and cultured human embryonic kidney (HEK-293) cells. Mitochondrial membrane potential (∆ψm), NAD(P)H content, swelling and ATP production were measured in rat kidney mitochondrial preparations supported by glutamate or glutamate plus malate, in the presence or absence of Ca2+. MTT reduction and propidium iodide (PI) incorporation were also determined in intact renal cells pre-incubated with MA or PA for 24 h. MA decreased Δψm and NAD(P)H content and induced swelling in Ca2+-loaded mitochondria either respiring with glutamate or glutamate plus malate. Noteworthy, these alterations were fully prevented by cyclosporin A plus ADP, suggesting the involvement of mitochondrial permeability transition (mPT). MA also markedly inhibited ATP synthesis in kidney mitochondria using the same substrates, implying a strong bioenergetics impairment. In contrast, PA only caused milder changes in these parameters. Finally, MA decreased MTT reduction and increased PI incorporation in intact HEK-293 cells, indicating a possible association between mitochondrial dysfunction and cell death in an intact cell system. It is therefore presumed that the MA-induced disruption of mitochondrial functions involving mPT pore opening may be involved in the chronic renal failure occurring in propionic acidemia.

Pathophysiology of maple syrup urine disease: Focus on the neurotoxic role of the accumulated branched-chain amino acids and branched-chain α-keto acids.

Maple syrup urine disease (MSUD) is an autosomal recessive neurometabolic disorder caused by severe deficiency of branched-chain α-keto acid dehydrogenase complex activity, which catalyzes the oxidative decarboxylation of the branched-chain α-keto acids (BCKA). The metabolic blockage results in tissue accumulation and high urinary excretion of the branched-chain amino acids (BCAA) leucine, isoleucine and valine, as well as alloisoleucine, and their respective BCKA α-ketoisocaproic (α-KIC), α-ketoisovaleric and α-keto-ß-methylvaleric acids. Affected patients usually manifest acute episodes of encephalopathy associated with seizures, coma and life-threatening cerebral edema in the first weeks of life, which is followed by progressive neurological deterioration with motor delay, ataxia, intellectual disability and psychiatric symptoms. The pathophysiology of the brain damage in MSUD has been mainly focused on brain amino acid imbalance leading to deficient cerebral protein and neurotransmitter synthesis. However, the acute episodes of severe neurological symptoms accompanied by large increases of BCKA/BCAA levels suggest neurotoxic actions of these compounds. In this particular, mounting evidence from humans and animal models support an important role of particularly leucine and α-KIC on the pathogenesis of the brain injury in MSUD. In this review we will present the current knowledge of the major mechanisms presumably involved in MSUD neuropathology and highlight the neurotoxic properties of the BCAA and BCKA, disturbing brain bioenergetics and redox homeostasis, besides inducing neuroinflammation. We suggest that these pathomechanisms may contribute to the neurological sequelae of MSUD patients and hopefully allow the design of novel therapeutic strategies, including antioxidant and bioenergetics stimulating drugs targeting the mitochondria.

S-adenosylmethionine induces mitochondrial dysfunction, permeability transition pore opening and redox imbalance in subcellular preparations of rat liver.

S-adenosylmethionine (AdoMet) predominantly accumulates in tissues and biological fluids of patients affected by liver dysmethylating diseases, particularly glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies, as well as in some hepatic mtDNA depletion syndromes, whose pathogenesis of liver dysfunction is still poorly established. Therefore, in the present work, we investigated the effects of S-adenosylmethionine (AdoMet) on mitochondrial functions and redox homeostasis in rat liver. AdoMet decreased mitochondrial membrane potential and Ca2+ retention capacity, and these effects were fully prevented by cyclosporin A and ADP, indicating mitochondrial permeability transition (mPT) induction. It was also verified that the thiol-alkylating agent NEM prevented AdoMet-induced ΔΨm dissipation, implying a role for thiol oxidation in the mPT pore opening. AdoMet also increased ROS production and provoked protein and lipid oxidation. Furthermore, AdoMet reduced GSH levels and the activities of aconitase and α-ketoglutarate dehydrogenase. Free radical scavengers attenuated AdoMet effects on lipid peroxidation and GSH levels, supporting a role of ROS in these effects. It is therefore presumed that disturbance of mitochondrial functions associated with mPT and redox unbalance may represent relevant pathomechanisms of liver damage provoked by AdoMet in disorders in which this metabolite accumulates.

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.

Recent Advances in the Pathophysiology of Fatty Acid Oxidation Defects: Secondary Alterations of Bioenergetics and Mitochondrial Calcium Homeostasis Caused by the Accumulating Fatty Acids.

Deficiencies of medium-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein, isolated long-chain 3-hydroxyacyl-CoA dehydrogenase, and very long-chain acyl-CoA dehydrogenase activities are considered the most frequent fatty acid oxidation defects (FAOD). They are biochemically characterized by the accumulation of medium-chain, long-chain hydroxyl, and long-chain fatty acids and derivatives, respectively, in tissues and biological fluids of the affected patients. Clinical manifestations commonly include hypoglycemia, cardiomyopathy, and recurrent rhabdomyolysis. Although the pathogenesis of these diseases is still poorly understood, energy deprivation secondary to blockage of fatty acid degradation seems to play an important role. However, recent evidence indicates that the predominant fatty acids accumulating in these disorders disrupt mitochondrial functions and are involved in their pathophysiology, possibly explaining the lactic acidosis, mitochondrial morphological alterations, and altered mitochondrial biochemical parameters found in tissues and cultured fibroblasts from some affected patients and also in animal models of these diseases. In this review, we will update the present knowledge on disturbances of mitochondrial bioenergetics, calcium homeostasis, uncoupling of oxidative phosphorylation, and mitochondrial permeability transition induction provoked by the major fatty acids accumulating in prevalent FAOD. It is emphasized that further in vivo studies carried out in tissues from affected patients and from animal genetic models of these disorders are necessary to confirm the present evidence mostly achieved from in vitro experiments.

Disruption of mitochondrial functions and oxidative stress contribute to neurologic dysfunction in organic acidurias.

Organic acidurias (OADs) are inherited disorders of amino acid metabolism biochemically characterized by accumulation of short-chain carboxylic acids in tissues and biological fluids of the affected patients and clinically by predominant neurological manifestations. Some of these disorders are amenable to treatment, which significantly decreases mortality and morbidity, but it is still ineffective to prevent long-term neurologic and systemic complications. Although pathogenesis of OADs is still poorly established, recent human and animal data, such as lactic acidosis, mitochondrial morphological alterations, decreased activities of respiratory chain complexes and altered parameters of oxidative stress, found in tissues from patients and from genetic mice models with these diseases indicate that disruption of critical mitochondrial functions and oxidative stress play an important role in their pathophysiology. Furthermore, organic acids that accumulate in the most prevalent OADs were shown to compromise bioenergetics, by decreasing ATP synthesis, mitochondrial membrane potential, reducing equivalent content and calcium retention capacity, besides inducing mitochondrial swelling, reactive oxygen and nitrogen species generation and apoptosis. It is therefore presumed that secondary mitochondrial dysfunction and oxidative stress caused by major metabolites accumulating in OADs contribute to tissue damage in these pathologies.

Lipopolysaccharide-Elicited Systemic Inflammation Induces Selective Vulnerability of Cerebral Cortex and Striatum of Developing Glutaryl-CoA Dehydrogenase Deficient (Gcdh-/-) Mice to Oxidative Stress.

We investigated redox homeostasis in cerebral and peripheral tissues of wild type (WT) and glutaryl-CoA dehydrogenase knockout mice (Gcdh-/-) submitted to inflammation induced by lipopolysaccharide (LPS) since patients with glutaric aciduria type I (GA I) manifest acute encephalopathy during catabolic events triggered by inflammation. WT and Gcdh-/- mice fed a low (0.9%) or high (4.7%) Lys chow were euthanized 4 h after LPS intraperitoneal injection. Cerebral cortex of Lys-restricted Gcdh-/- animals presented no alterations of redox homeostasis, whereas those fed a high Lys chow showed increased malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity, compared to WT mice. Furthermore, Gcdh-/- mice receiving low Lys and injected with LPS presented elevated MDA levels and decreased reduced glutathione (GSH) concentrations, glutathione peroxidase (GPx), and glutathione reductase (GR) activities in cerebral cortex. LPS administration also decreased GSH values, as well as GPx and GR activities in cerebral cortex of Gcdh-/- mice receiving Lys overload. Further experiments performed in WT and Gcdh-/- mice injected with LPS and receiving either a low or high Lys chow revealed increased MDA levels and decreased GSH concentrations in cerebral cortex and striatum, but not in hippocampus, liver and heart of Gcdh-/- mice, suggesting a selective vulnerability of these cerebral structures to oxidative stress during an inflammatory process. LPS administration also increased S100B and NF-κF protein levels in brain of Gcdh-/- mice receiving high Lys. These data support the hypothesis that low Lys diet is beneficial in GA I by preventing redox imbalance, whereas a high Lys diet or systemic inflammation per se or combined induce oxidative stress in striatum and cerebral cortex that are mainly damaged in this disorder.

The Effect of Periodontal Diseases and Cognitive Deficit on Behavioral State, Oxidative Stress Parameters and Alveolar Bone Loss in Rats.

AIMS: To evaluate the effect of periodontal disease (PD) and cognitive deficit (CD) on behavioral state, oxidative stress parameters, and alveolar bone loss (ABL). MATERIALS AND METHODS: Fifty male Wistar rats were randomly divided into five groups: control; scopolamine; periodontal disease (PD); PD+scopolamine evaluated by the Morris water maze behavioral test; PD+scopolamine assessed by the inhibitory avoidance behavioral test. PD and CD were, respectively, induced by ligature and scopolamine. Both the maxilla and mandible were morphometrically analyzed for ABL. For all animal groups, the study duration was one month. For the ligated animals, the final evaluation was performed 14 days after the ligature placement. Reduced glutathione (GSH), total sulfhydryl (TS), thiobarbituric acid, catalase, and superoxide essays were assessed in the brain tissues (cortex and hippocampus). RESULTS: Both groups with induced PD+CD (0.46±0.04mm and 0.47±0.04mm for maze and inhibitory avoidance behavioral tests, respectively) presented a significantly higher ABL in comparison to the group that received only scopolamine (0.22±0.01mm). PD and PD+scopolamine groups presented significantly lower GSH and TS in comparison to the control group. Regarding the memory latency tests, there were no statistically significant differences among groups. CONCLUSION: The induction of PD was capable to modulate the CD, decreasing the action of the antioxidant agents in the brain. However, PD was not capable of impairing the short and long-term spatial memory retention behavioral.

Guanosine Neuroprotection of Presynaptic Mitochondrial Calcium Homeostasis in a Mouse Study with Amyloid-ß Oligomers.

Amyloid-ß oligomers (AßOs) toxicity causes mitochondrial dysfunction, leading to synaptic failure in Alzheimer's disease (AD). Considering presynaptic high energy demand and tight Ca2+ regulation, impairment of mitochondrial function can lead to deteriorated neural activity and cell death. In this study, an AD mouse model induced by ICV (intracerebroventricular) injection of AßOs was used to investigate the toxicity of AßOs on presynaptic function. As a therapeutic approach, GUO (guanosine) was given by oral route to evaluate the neuroprotective effects on this AD model. Following 24 h and 48 h from the model induction, behavioral tasks and biochemical analyses were performed, respectively. AßOs impaired object recognition (OR) short-term memory and reduced glutamate uptake and oxidation in the hippocampus. Moreover, AßOs decreased spare respiratory capacity, reduced ATP levels, impaired Ca2+ handling, and caused mitochondrial swelling in hippocampal synaptosomes. Guanosine crossed the BBB, recovered OR short-term memory, reestablished glutamate uptake, recovered mitochondrial Ca2+ homeostasis, and partially prevented mitochondrial swelling. Therefore, this endogenous purine presented a neuroprotective effect on presynaptic mitochondria and should be considered for further studies in AD models.

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats.

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05-5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients.

Impairment of mitochondrial bioenergetics and permeability transition induction caused by major long-chain fatty acids accumulating in VLCAD deficiency in skeletal muscle as potential pathomechanisms of myopathy.

cis-5-Tetradecenoic (cis-5) and myristic (Myr) acids predominantly accumulate in patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. They commonly manifest myopathy with muscular pain and rhabdomyolysis, whose underlying mechanisms are poorly known. Thus, in the present study we investigated the effects of cis-5 and Myr on mitochondrial bioenergetics and Ca2+ homeostasis in rat skeletal muscle. cis-5 and Myr decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration, especially when mitochondria were supported by NADH-linked as compared to FADH2-linked substrates. In contrast, these fatty acids increased resting respiration (state 4). Similar effects were observed in skeletal muscle fibers therefore validating the data obtained with isolated mitochondria. Furthermore, cis-5 and Myr markedly decreased mitochondrial membrane potential and Ca2+ retention capacity that were avoided by cyclosporin A plus ADP and ruthenium red, indicating that cis-5 and Myr induce mitochondrial permeability transition (MPT). Finally, docosanoic acid did not disturb mitochondrial homeostasis, indicating selective effects for Myr and cis-5. Taken together, our findings indicate that major long-chain fatty acids accumulating in VLCAD deficiency behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and MPT inducers. It is presumed that these pathomechanisms contribute to the muscular symptoms and rhabdomyolysis observed in patients affected by VLCAD deficiency.

Disturbance of mitochondrial functions associated with permeability transition pore opening induced by cis-5-tetradecenoic and myristic acids in liver of adolescent rats.

Patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency commonly present liver dysfunction whose pathogenesis is poorly known. We demonstrate here that major metabolites accumulating in this disorder, namely cis-5-tetradecenoic acid (Cis-5) and myristic acid (Myr), markedly impair mitochondrial respiration, decreasing ATP production in liver mitochondrial preparations from adolescent rats. Other parameters of mitochondrial homeostasis such as membrane potential (ΔΨm) and Ca2+retention capacity were strongly compromised by these fatty acids, involving induction of mitochondrial permeability transition. The present data indicate that disruption of mitochondrial bioenergetics and Ca2+homeostasis may contribute to the liver dysfunction of VLCAD deficient patients.

Pathogenesis of brain damage in glutaric acidemia type I: Lessons from the genetic mice model.

Glutaric acidemia type I (GA I) is an inherited neurometabolic disease caused by deficient activity of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH), resulting in predominant accumulation of glutaric and 3-hydroxyglutaric acids derived from lysine (Lys), hydroxylysine, and tryptophan catabolism. GA I patients usually present progressive cortical leukodystrophy and frequently develop acute striatal degeneration during encephalopathic crises during the first three years of life. The pathophysiology of the neurodegeneration observed in GA I is still partly known, although the development of the genetic mice model of GA I (Gcdh-/-) has contributed to clarify potential underlying mechanisms involved in brain damage in this disease. In this review we will summarize the knowledge acquired from studies using this animal model indicating that disruption of redox homeostasis, glutamatergic neurotransmission and bioenergetics, as well as vascular alterations, blood-brain barrier breakage and altered myelination underlie the cortical and striatum abnormalities and white matter changes observed in GA I patients. Elucidation of these pathomechanisms potentially offers new standpoints for the development of novel therapeutic strategies for this disease.