Myelin Disruption, Neuroinflammation, and Oxidative Stress Induced by Sulfite in the Striatum of Rats Are Mitigated by the pan-PPAR agonist Bezafibrate.

Sulfite predominantly accumulates in the brain of patients with isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies. Patients present with severe neurological symptoms and basal ganglia alterations, the pathophysiology of which is not fully established. Therapies are ineffective. To elucidate the pathomechanisms of ISOD and MoCD, we investigated the effects of intrastriatal administration of sulfite on myelin structure, neuroinflammation, and oxidative stress in rat striatum. Sulfite administration decreased FluoromyelinTM and myelin basic protein staining, suggesting myelin abnormalities. Sulfite also increased the staining of NG2, a protein marker of oligodendrocyte progenitor cells. In line with this, sulfite also reduced the viability of MO3.13 cells, which express oligodendroglial markers. Furthermore, sulfite altered the expression of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1), indicating neuroinflammation and redox homeostasis disturbances. Iba1 staining, another marker of neuroinflammation, was also increased by sulfite. These data suggest that myelin changes and neuroinflammation induced by sulfite contribute to the pathophysiology of ISOD and MoCD. Notably, post-treatment with bezafibrate (BEZ), a pan-PPAR agonist, mitigated alterations in myelin markers and Iba1 staining, and IL-1ß, IL-6, iNOS and HO-1 expression in the striatum. MO3.13 cell viability decrease was further prevented. Moreover, pre-treatment with BEZ also attenuated some effects. These findings show the modulation of PPAR as a potential opportunity for therapeutic intervention in these disorders.

Sulfite Impairs Bioenergetics and Redox Status in Neonatal Rat Brain: Insights into the Early Neuropathophysiology of Isolated Sulfite Oxidase and Molybdenum Cofactor Deficiencies.

Isolated sulfite oxidase (ISOD) and molybdenum cofactor (MoCD) deficiencies are genetic diseases biochemically characterized by the toxic accumulation of sulfite in the tissues of patients, including the brain. Neurological dysfunction and brain abnormalities are commonly observed soon after birth, and some patients also have neuropathological alterations in the prenatal period (in utero). Thus, we investigated the effects of sulfite on redox and mitochondrial homeostasis, as well as signaling proteins in the cerebral cortex of rat pups. One-day-old Wistar rats received an intracerebroventricular administration of sulfite (0.5 µmol/g) or vehicle and were euthanized 30 min after injection. Sulfite administration decreased glutathione levels and glutathione S-transferase activity, and increased heme oxygenase-1 content in vivo in the cerebral cortex. Sulfite also reduced the activities of succinate dehydrogenase, creatine kinase, and respiratory chain complexes II and II-III. Furthermore, sulfite increased the cortical content of ERK1/2 and p38. These findings suggest that redox imbalance and bioenergetic impairment induced by sulfite in the brain are pathomechanisms that may contribute to the neuropathology of newborns with ISOD and MoCD. Sulfite disturbs antioxidant defenses, bioenergetics, and signaling pathways in the cerebral cortex of neonatal rats. CII: complex II; CII-III: complex II-III; CK: creatine kinase; GST: glutathione S-transferase; HO-1: heme oxygenase-1; SDH: succinate dehydrogenase; SO32-: sulfite.

Peroxisome proliferator-activated receptor (PPAR) agonists as a potential therapy for inherited metabolic disorders.

Inherited metabolic disorders (IMDs) are genetic disorders that cause a disruption of a specific metabolic pathway leading to biochemical, clinical and pathophysiological sequelae. While the metabolite abnormalities in body fluids and tissues can usually be defined by directed or broad-spectrum metabolomic analysis, the pathophysiology of these changes is often not obvious. Mounting evidence has revealed that secondary mitochondrial dysfunction, mainly oxidative phosphorylation impairment and elevated reactive oxygen species, plays a pivotal role in many disorders. Peroxisomal proliferator-activated receptors (PPARs) consist of a group of nuclear hormone receptors (PPARα, PPARß/δ, and PPARγ) that regulate multiple cellular functions and processes, including response to oxidative stress, inflammation, lipid metabolism, and mitochondrial bioenergetics and biogenesis. In this context, the activation of PPARs has been shown to stimulate oxidative phosphorylation and reduce reactive species levels. Thus, pharmacological treatment with PPAR activators, such as fibrates, has gained much attention in the last 15 years. This review summarizes preclinical (animal models and patient-derived cells) and clinical data on the effect of PPARs in IMDs.

Antioxidant system disturbances and mitochondrial dysfunction induced by 3-methyglutaric acid in rat heart are prevented by bezafibrate.

Barth syndrome (BTHS) and dilated cardiomyopathy with ataxia syndrome (DCMA) are biochemically characterized by high levels of 3-methylglutaric acid (MGA) in the urine and plasma of affected patients. Although cardiolipin abnormalities have been observed in these disorders, their pathophysiology is not fully established. We evaluated the effects of MGA administration on redox homeostasis and mitochondrial function in heart, as well as on vascular reactivity in aorta of Wistar rats without cardiolipin genetic deficiency. Potential cardioprotective effects of a pretreatment with bezafibrate (BEZ), a pan-PPAR agonist that induces mitochondrial biogenesis, were also determined. Our findings showed that MGA induced lipid peroxidation, altered enzymatic and non-enzymatic antioxidant defenses and reduced respiratory chain function in rat heart. MGA also increased Drp1 and reduced MFN1 levels, suggesting mitochondrial fission induction. Moreover, MGA altered MAPK and Akt signaling pathways, and had a strong tendency to reduce Sirt1 and PGC-1α, indicative of mitochondrial biogenesis impairment. Aorta vascular reactivity was further altered by MGA. Additionally, BEZ mitigated most alterations on antioxidant defenses and mitochondrial quality control proteins provoked by MGA. However, vascular reactivity disturbances were not prevented. It may be presumed that oxidative stress, mitochondrial bioenergetics and control quality disturbances, and vascular reactivity impairment caused by MGA may be involved in the cardiac failure observed in BTHS and DCMA, and that BEZ should be considered as a pharmacological candidate for the treatment of these disorders.

The mitochondrial-targeted reactive species scavenger JP4-039 prevents sulfite-induced alterations in antioxidant defenses, energy transfer, and cell death signaling in striatum of rats.

Sulfite oxidase (SO) deficiency is a disorder caused either by isolated deficiency of SO or by defects in the synthesis of its molybdenum cofactor. It is characterized biochemically by tissue sulfite accumulation. Patients present with seizures, progressive neurological damage, and basal ganglia abnormalities, the pathogenesis of which is not fully established. Treatment is supportive and largely ineffective. To address the pathophysiology of sulfite toxicity, we examined the effects of intrastriatal administration of sulfite in rats on antioxidant defenses, energy transfer, and mitogen-activated protein kinases (MAPK) and apoptosis pathways in rat striatum. Sulfite administration decreased glutathione (GSH) concentration and glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione S-transferase, and glutathione reductase activities in striatal tissue. Creatine kinase (CK) activity, a crucial enzyme for cell energy transfer, was also decreased by sulfite. Superoxide dismutase-1 (SOD1) and catalase (CAT) proteins were increased, while heme oxygenase-1 (HO-1) was decreased. Additionally, sulfite altered phosphorylation of MAPK by decreasing of p38 and increasing of ERK. Sulfite further augmented the content of GSK-3ß, Bok, and cleaved caspase-3, indicating increased apoptosis. JP4-039 is a mitochondrial-targeted antioxidant that reaches higher intramitochondrial levels than other traditional antioxidants. Intraperitoneal injection of JP4-039 before sulfite administration preserved activity of antioxidant enzymes and CK. It also prevented or attenuated alterations in SOD1, CAT, and HO-1 protein content, as well as changes in p38, ERK, and apoptosis markers. In sum, oxidative stress and apoptosis induced by sulfite injection are prevented by JP4-039, identifying this molecule as a promising candidate for pharmacological treatment of SO-deficient patients.

In vivo evidence that bezafibrate prevents oxidative stress and mitochondrial dysfunction caused by 3-methylglutaric acid in rat liver.

High urinary excretion and tissue accumulation of 3-methylglutaric acid (MGA) are observed in patients affected by 3-hydroxy-3-methylglutaric (HMGA) and 3-methylglutaconic (MGTA) acidurias. The pathomechanisms underlying the hepatic dysfunction commonly observed in these disorders are not fully elucidated so that we investigated here the effects of intraperitoneal administration of MGA on redox homeostasis, mitochondrial bioenergetics, biogenesis and dynamics in rat liver. The effects of a pre-treatment with the protective compound bezafibrate (BEZ) were also determined. Our data showed that MGA induced lipid peroxidation and altered enzymatic and non-enzymatic antioxidant defenses in liver, indicating redox homeostasis disruption. BEZ prevented most of these alterations induced by MGA. MGA also decreased the activities of the respiratory chain complexes II and IV and increased of II-III, whereas BEZ prevented the alteration in complex II activity. Furthermore, MGA decreased levels of nuclear PGC-1α and Sirt1, and increased levels of AMPKα1 and cytosolic PPARγ, which were blocked by BEZ. MGA augmented the levels of mitofusin-1 and dynamin-related protein 1, suggesting that both fusion and fission mitochondrial processes are enhanced by MGA. BEZ was able to prevent only the changes in mitofusin-1 levels. Collectively, these findings indicate that oxidative stress and mitochondrial dysfunction are mechanisms involved in the hepatic dysfunction found in HMGA and MGTA. It is also presumed that mitochondrial biogenesis stimulation may constitute an attractive approach to reduce MGA toxicity in liver of individuals affected by HMGA and MGTA.

3-Hydroxy-3-Methylglutaric Acid Impairs Redox and Energy Homeostasis, Mitochondrial Dynamics, and Endoplasmic Reticulum-Mitochondria Crosstalk in Rat Brain.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a neurometabolic disorder characterized by predominant accumulation of 3-hydroxy-3-methylglutaric acid (HMG) in tissues and biological fluids. Patients often present in the first year of life with metabolic acidosis, non-ketotic hypoglycemia, hypotonia, lethargy, and coma. Since neurological symptoms may be triggered or worsened during episodes of metabolic decompensation, which are characterized by high urinary excretion of organic acids, this study investigated the effects of HMG intracerebroventricular administration on redox homeostasis, citric acid cycle enzyme activities, dynamics (mitochondrial fusion and fission), and endoplasmic reticulum (ER)-mitochondria crosstalk in the brain of neonatal rats euthanized 1 (short term) or 20 days (long term) after injection. HMG induced lipid peroxidation and decreased the activities of glutathione peroxidase (GPx) and citric acid cycle enzymes, suggesting bioenergetic and redox disruption, 1 day after administration. Levels of VDAC1, Grp75, and mitofusin-1, proteins involved in ER-mitochondria crosstalk and mitochondrial fusion, were increased by HMG. Furthermore, HMG diminished synaptophysin levels and tau phosphorylation, and increased active caspase-3 content, indicative of cell damage. Finally, HMG decreased GPx activity and synaptophysin levels, and changed MAPK phosphorylation 20 days after injection, suggesting that long-term toxicity is further induced by this organic acid. Taken together, these data show that HMG induces oxidative stress and disrupts bioenergetics, dynamics, ER-mitochondria communication, and signaling pathways in the brain of rats soon after birth. It may be presumed that these mechanisms underlie the onset and progression of symptoms during decompensation occurring in HL-deficient patients during the neonatal period.

Bezafibrate In Vivo Administration Prevents 3-Methylglutaric Acid-Induced Impairment of Redox Status, Mitochondrial Biogenesis, and Neural Injury in Brain of Developing Rats.

3-Methylglutaric acid (MGA) is an organic acid that accumulates in 3-methylglutaconic (MGTA) and 3-hydroxy-3-methylglutaric (HMGA) acidurias. Patients affected by these disorders present with neurological dysfunction that usually appears in the first years of life. In order to elucidate the pathomechanisms underlying the brain injury in these disorders, we evaluated the effects of MGA administration on redox homeostasis, mitochondrial respiratory chain activity, and biogenesis in the cerebral cortex of developing rats. Neural damage markers and signaling pathways involved in cell survival, and death were also measured after MGA administration. Furthermore, since the treatment for MGTA and HMGA is still limited, we tested whether a pre-treatment with the pan-peroxisome proliferator-activated receptor (PPAR) agonist bezafibrate could prevent the alterations caused by MGA. MGA provoked lipid peroxidation, increased heme oxygenase-1 content, and altered the activities of antioxidant enzymes, strongly suggestive of oxidative stress. MGA also impaired mitochondrial function and biogenesis by decreasing the activities of succinate dehydrogenase and various respiratory chain complexes, as well as the nuclear levels of PGC-1α and NT-PGC-1α, and cell content of Sirt1. AMPKα1 was further increased by MGA. Neural cell damage was also observed following the MGA administration, as verified by decreased Akt and synaptophysin content and reduced ERK phosphorylation, and by the increase of active caspase-3 and p38 and Tau phosphorylation. Importantly, bezafibrate prevented MGA-elicited toxic effects towards mitochondrial function, redox homeostasis, and neural cell injury, implying that this compound may be potentially used as an adjunct therapy for MGTA and HMGA and other disorders with mitochondrial dysfunction.

Evidence that thiol group modification and reactive oxygen species are involved in hydrogen sulfide-induced mitochondrial permeability transition pore opening in rat cerebellum.

We report here the effects of hydrogen sulfide (sulfide), that accumulates in ETHE1 deficiency, in rat cerebellum. Sulfide impaired electron transfer and oxidative phosphorylation. Sulfide also induced mitochondrial swelling, and decreased ΔΨm and calcium retention capacity in cerebellum mitochondria, which were prevented by cyclosporine A (CsA) plus ADP, and ruthenium red, suggesting mitochondrial permeability transition (mPT) induction. Melatonin (MEL) and N-ethylmaleimide also prevented sulfide-induced alterations. Prevention of sulfide-induced decrease of ΔΨm and viability by CsA and MEL was further verified in cerebellum neurons. The data suggest that sulfide induces mPT pore opening via thiol modification and ROS generation.

Structure and intracellular antioxidant activity of pectic polysaccharide from acerola (Malpighia emarginata).

Malpighia emarginata is a tropical fruit plant, found naturally in the Caribbean islands and South America that produces an edible fruit known as acerola or Barbados Cherry. Its polysaccharides were obtained by aqueous extraction, subjected to a freezing and thawing process and ultrafiltration. A homogeneous fraction (ACWS-01E) was analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analyses. The results showed an arabinan-rich pectic polysaccharide, with 6.1×104g/mol and formed mainly by a high methyl esterified (DM=86%) homogalacturonan and branched arabinan. This latter is anchored in type I rhamnogalacturonan regions. The main chain of arabinan consisted of (1→5)-linked α-Araf, branched only at O-3. The potential ACWS-01E intracellular antioxidant activity against H2O2-induced oxidative stress in murine fibroblast cell line (3T3) was determined by DCFH-DA assay. The treatment with ACWS-01E significantly reduced H2O2-induced cytotoxic effect and the levels of reactive oxygen species (ROS). These findings suggested that ACWS-01E protected and improved NIH 3T3 cell viability from H2O2-induced toxicity by decreasing intracellular levels of ROS.

Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant pathomechanisms underlying the neurological dysfunction characteristic of ethylmalonic encephalopathy.

Hydrogen sulfide (sulfide) accumulates at high levels in brain of patients with ethylmalonic encephalopathy (EE). In the present study, we evaluated whether sulfide could disturb energy and redox homeostasis, and induce mitochondrial permeability transition (mPT) pore opening in rat brain aiming to better clarify the neuropathophysiology of EE. Sulfide decreased the activities of citrate synthase and aconitase in rat cerebral cortex mitochondria, and of creatine kinase (CK) in rat cerebral cortex, striatum and hippocampus supernatants. Glutathione prevented sulfide-induced CK activity decrease in the cerebral cortex. Sulfide also diminished mitochondrial respiration in cerebral cortex homogenates, and dissipated mitochondrial membrane potential (ΔΨm) and induced swelling in the presence of calcium in brain mitochondria. Alterations in ΔΨm and swelling caused by sulfide were prevented by the combination of ADP and cyclosporine A, and by ruthenium red, indicating the involvement of mPT in these effects. Furthermore, sulfide increased the levels of malondialdehyde in cerebral cortex supernatants, which was prevented by resveratrol and attenuated by glutathione, and of thiol groups in a medium devoid of brain samples. Finally, we verified that sulfide did not alter cell viability and DCFH oxidation in cerebral cortex slices, primary cortical astrocyte cultures and SH-SY5Y cells. Our data provide evidence that bioenergetics disturbance and lipid peroxidation along with mPT pore opening are involved in the pathophysiology of brain damage observed in EE.

Disruption of Energy Transfer and Redox Status by Sulfite in Hippocampus, Striatum, and Cerebellum of Developing Rats.

Patients with sulfite oxidase (SO) deficiency present severe brain abnormalities, whose pathophysiology is not yet elucidated. We evaluated the effects of sulfite and thiosulfate, metabolites accumulated in SO deficiency, on creatine kinase (CK) activity, mitochondrial respiration and redox status in hippocampus, striatum and cerebellum of developing rats. Our in vitro results showed that sulfite and thiosulfate decreased CK activity, whereas sulfite also increased malondialdehyde (MDA) levels in all brain structures evaluated. Sulfite further diminished mitochondrial respiration and increased DCFH oxidation and hydrogen peroxide production in hippocampus. Sulfite-induced CK activity decrease was prevented by melatonin (MEL), resveratrol (RSV), and dithiothreitol while increase of MDA levels was prevented by MEL and RSV. Regarding the antioxidant system, sulfite increased glutathione concentrations in hippocampus and striatum. In addition, sulfite decreased the activities of glutathione peroxidase in all brain structures, of glutathione S-transferase in hippocampus and cerebellum, and of glutathione reductase in cerebellum. In vivo experiments performed with intrahippocampal administration of sulfite demonstrated that this metabolite increased superoxide dismutase activity without altering other biochemical parameters in rat hippocampus. Our data suggest that impairment of energy metabolism and redox status may be important pathomechanisms involved in brain damage observed in individuals with SO deficiency.