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Zika virus (ZIKV) is a neurotropic flavivirus that induces congenital Zika syndrome and neurodevelopmental disorders. Given that ZIKV can infect and replicate in neural cells, neurological complications in adult brain are also observed. Glial cells may emerge to delay and/or prevent the development of ZIKV-induced neurodegeneration. These cells actively participate in metabolic, inflammatory and redox processes, and consequently, in the pathophysiology of neurodegenerative diseases, including diabetic encephalopathy. In this sense, changes in glucose metabolism can support the inflammatory activity of astroglial cells; however, the effects of increased glucose concentration during ZIKV infection have not yet been explored in astroglial cells. Here, we evaluated functional parameters of astroglial cells exposed to ZIKV upon normal and high glucose concentrations, focusing on inflammatory profile, oxidative stress, and expression of critical genes for astroglial functions. High glucose potentiated the pro-inflammatory and oxidative effects of ZIKV, as well as potentiated the downregulation of signaling pathways, such as Nrf-2 (nuclear factor erythroid derived 2 like 2), sirtuin 1 (SIRT1), peroxisome proliferator activated receptor gamma coactivator 1-alpha (PGC-1α), and poly (ADP-ribose) polymerase (PARP). In summary, our results suggest that high glucose can favor the activation of inflammatory signaling while impairing cytoprotective pathways in astroglial cells exposed to ZIKV and reinforce the hypothesis that this virus is highly neurotrophic, with significant impact in glial cells., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to The Journal of NeuroVirology, Inc.)

Carnosine is composed of β-alanine and L-histidine and is considered to be an important neuroprotective agent with antioxidant, metal chelating, and antisenescence properties. However, children with serum carnosinase deficiency present increased circulating carnosine and severe neurological symptoms. We here investigated the in vitro effects of carnosine on redox and mitochondrial parameters in cultured cortical astrocytes from neonatal rats. Carnosine did not alter mitochondrial content or mitochondrial membrane potential. On the other hand, carnosine increased mitochondrial superoxide anion formation, levels of thiobarbituric acid reactive substances and oxidation of 2',7'-dichlorofluorescin diacetate (DCF-DA), indicating that carnosine per se acts as a pro-oxidant agent. Nonetheless, carnosine prevented DCF-DA oxidation induced by H 2 O 2 in cultured cortical astrocytes. Since alterations on mitochondrial membrane potential are not likely to be involved in these effects of carnosine, the involvement of N-Methyl-D-aspartate (NMDA) receptors in the pro-oxidant actions of carnosine was investigated. MK-801, an antagonist of NMDA receptors, prevented DCF-DA oxidation induced by carnosine in cultured cortical astrocytes. Astrocyte reactivity induced by carnosine was also prevented by the coincubation with MK-801. The present study shows for the very first time the pro-oxidant effects of carnosine per se in astrocytes. The data raise awareness on the importance of a better understanding of the biological actions of carnosine, a nutraceutical otherwise widely reported as devoid of side effects., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Non ketotic hyperglycinemia (NKH) is an inborn error of glycine metabolism caused by mutations in the genes encoding glycine cleavage system proteins. Classic NKH has a neonatal onset, and patients present with severe neurodegeneration. Although glycine accumulation has been implicated in NKH pathophysiology, the exact mechanisms underlying the neurological damage and white matter alterations remain unclear. We investigated the effects of glycine in the brain of neonatal rats and MO3.13 oligodendroglial cells. Glycine decreased myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) in the corpus callosum and striatum of rats on post-natal day (PND) 15. Glycine also reduced neuroglycan 2 (NG2) and N-methyl-d-aspartate receptor subunit 1 (NR1) in the cerebral cortex and striatum on PND15. Moreover, glycine reduced striatal glutamate aspartate transporter 1 (GLAST) content and neuronal nucleus (NeuN), and increased glial fibrillary acidic protein (GFAP) on PND15. Glycine also increased DCFH oxidation and malondialdehyde levels and decreased GSH concentrations in the cerebral cortex and striatum on PND6, but not on PND15. Glycine further reduced viability but did not alter DCFH oxidation and GSH levels in MO3.13 cells after 48- and 72-h incubation. These data indicate that impairment of myelin structure and glutamatergic system and induction of oxidative stress are involved in the neuropathophysiology of NKH., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Ethylmalonic encephalopathy (EE) is a severe inherited metabolic disorder that causes tissue accumulation of hydrogen sulfide (sulfide) and thiosulfate in patients. Although symptoms are predominantly neurological, chronic hemorrhagic diarrhea associated with intestinal mucosa abnormalities is also commonly observed. Considering that the pathophysiology of intestinal alterations in EE is virtually unknown and that sulfide and thiosulfate are highly reactive molecules, the effects of these metabolites were investigated on bioenergetic production and transfer in the intestine of rats. We observed that sulfide reduced NADH- and FADH 2 -linked mitochondrial respiration in the intestine, which was avoided by reduced glutathione (GSH) but not by melatonin. Thiosulfate did not change respiration. Moreover, both metabolites markedly reduced the activity of total, cytosolic and mitochondrial isoforms of creatine kinase (CK) in rat intestine. Noteworthy, the addition of GSH but not melatonin, apocynin, and Trolox (hydrosoluble vitamin E) prevented the change in the activities of total CK and its isoforms caused by sulfide and thiosulfate, suggesting a direct protein modification on CK structure by these metabolites. Sulfide further increased thiol content in the intestine, suggesting a modulation in the redox state of these groups. Finally, sulfide and thiosulfate decreased the viability of Caco-2 intestinal cells. Our data suggest that bioenergetic impairment caused by sulfide and thiosulfate is a mechanism involved in the gastrointestinal abnormalities found in EE., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 Fluoromyelin TM 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-1β (IL-1β), 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-1β, 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.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [ 3 H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [ 3 H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

Methylmalonic acidemia is a neurometabolic disorder biochemically characterized by the accumulation of methylmalonic acid (MMA) in different tissues, including the central nervous system (CNS). In this sense, it has been shown that high levels of this organic acid have a key role in the progressive neurological deterioration in patients. Astroglial cells actively participate in a wide range of CNS functions, such as antioxidant defenses and inflammatory response. Considering the role of these cells to maintain brain homeostasis, in the present study, we investigated the effects of MMA on glial parameters, focusing on redox homeostasis and inflammatory process, as well as putative mediators of these events in C6 astroglial cells. MMA decreased cell viability, glutathione levels, and antioxidant enzyme activities, increased inflammatory response, and changed the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear factor kappa B (NFκB), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and adenosine receptors, suggesting that these transcriptional factors and proteins may underlie the glial responses induced by MMA. Moreover, we also demonstrated the protective roles of melatonin and resveratrol against MMA-induced inflammation and decrease in glutathione levels. In summary, our findings support the hypothesis that astroglial changes are associated with pathogenesis of methylmalonic acidemia. In addition, we showed that these cells might be potential targets for preventive/therapeutic strategies by using molecules, such as melatonin and resveratrol, which mediated glioprotection in this inborn error of metabolism., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pulmonary arterial hypertension (PAH) is characterized by increased resistance of the pulmonary vasculature and afterload imposed on the right ventricle (RV). Two major contributors to the worsening of this disease are oxidative stress and mitochondrial impairment. This study aimed to explore the effects of monocrotaline (MCT)-induced PAH on redox and mitochondrial homeostasis in the RV and brain and how circulating extracellular vesicle (EV) signaling is related to these phenomena. Wistar rats were divided into control and MCT groups (60 mg/kg, intraperitoneal), and EVs were isolated from blood on the day of euthanasia (21 days after MCT injections). There was an oxidative imbalance in the RV, brain, and EVs of MCT rats. PAH impaired mitochondrial function in the RV, as seen by a decrease in the activities of mitochondrial complex II and citrate synthase and manganese superoxide dismutase (MnSOD) protein expression, but this function was preserved in the brain. The key regulators of mitochondrial biogenesis, namely, proliferator-activated receptor gamma coactivator 1-alpha and sirtuin 1, were poorly expressed in the EVs of MCT rats, and this result was positively correlated with MnSOD expression in the RV and negatively correlated with MnSOD expression in the brain. Based on these findings, we can conclude that the RV is severely impacted by the development of PAH, but this pathological injury may signal the release of circulating EVs that communicate with different organs, such as the brain, helping to prevent further damage through the upregulation of proteins involved in redox and mitochondrial function., (© 2021. The Author(s), under exclusive licence to The Japanese Society of Hypertension.)

This study evaluated the effect of lacosamide (LCM) on biochemical and mitochondrial parameters after PTZ kindling in mice. Male mice were treated on alternative days for a period of 11 days with LCM (20, 30, or 40 mg/kg), saline, or diazepam (2 mg/kg), before PTZ administration (50 mg/kg). The hippocampi were collected to evaluate free radicals, the activities of superoxide dismutase (SOD), catalase (CAT), and the mitochondrial complexes I-III, II, and II-III, as well as Bcl-2 and cyclo-oxygenase-2 (COX-2) expressions. Hippocampi, blood, and bone marrow were collected for genotoxic and mutagenic evaluations. LCM 40 mg/kg increased latency and decreased percentage of seizures, only on the 3rd day of observation. The dose of 30 mg/kg only showed positive effects on the percentage of seizures on the 2nd day of observation. LCM decreased free radicals and SOD activity and the dose of 40 mg/kg were able to increase CAT activity. LCM 30 and 40 mg/kg improved the enzymatic mitochondrial activity of the complex I-III and LCM 30 mg/kg improved the activity of the complex II. In the comet assay, the damage induced by PTZ administration was reduced by LCM 20 and 30 mg/kg. The dose of 20 mg/kg increased COX-2 expression while the highest dose used, 40 mg/kg, was able to reduce this expression when compared to the group treated with LCM 20 mg/kg. Although LCM did not produce the antiepileptogenic effect in vivo, it showed the neuroprotective effect against oxidative stress, bioenergetic dysfunction, and DNA damage induced by the repeated PTZ administration., (© 2020 Société Française de Pharmacologie et de Thérapeutique.)

Trichlorfon is an organophosphate insecticide that is widely used on fish farms to control parasitic infections. It has been detected in freshwater ecosystems as well as in fishery products. There is a growing body of evidence to suggest that certain feed additives may reduce or prevent pesticide-induced toxicity in fish. The aim of the present study was to determine whether acute exposure to trichlorfon would alter bioenergetic homeostasis and alter fatty acid profiles in muscles of silver catfish (Rhamdia quelen). We also sought to determine whether rutin prevents or reduces these effects. Cytosolic and mitochondrial creatine kinase (CK) and activities of complexes II-III and IV in muscle were significantly inhibited by exposure to 11 mg/L trichlorfon for 48 h compared to effects in the unexposed group. Total content of polyunsaturated fatty acids (omega-3 and omega-6) were significantly lower in muscle of silver catfish exposed to 11 mg/L trichlorfon for 48 h than in the unexposed group. Addition of 3 mg rutin/kg feed increased CK activity and prevented inhibition of complex IV activity, as well as preventing all alterations of muscle fatty acid profiles elicited by exposure to trichlorfon. No significant differences were observed between groups with respect to muscle adenylate kinase or pyruvate kinase activities, as well as total content of saturated and monounsaturated fatty acids. Our findings suggest that exposure (48 h) to 11 mg trichlorfon/L water inhibits cytosolic and mitochondrial CK activity in muscle. Trichlorfon also affects activities of complexes II-III and IV in respiratory chain, with important consequences for adenosine triphosphate production. The pesticide alters fatty acid profiles in the fish and endangers human consumers of the product. The most important finding of the present study is that inclusion of rutin improves bioenergetic homeostasis and muscle fatty acid profiles, suggesting that it reduces trichlorfon-induced muscle damage., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest We declare no conflict of interest., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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.

Phenylketonuria (PKU) is a metabolic disorder accumulating phenylalanine (Phe) and its metabolites in plasma and tissues of the patients. Regardless of the mechanisms, which Phe causes brain impairment, are poorly understood, energy deficit may have linked to the neurotoxicity in PKU. It is widely recognized that creatine is involved in maintaining of cerebral energy homeostasis. Because of this, in a previous work, we incorporated it into liposomes and this increased the concentration of creatine in the cerebral cortex. Here, we examined the effect of creatine nanoliposomes on parameters of oxidative stress, enzymes of phosphoryl transfer network, and activities of the mitochondrial respiratory chain complexes (RCC) in the cerebral cortex of young rats chemically induced hyperphenylalaninemia (HPA). HPA was induced with L-phenylalanine (5.2 µmol/g body weight; twice a day; s.c.), and phenylalanine hydroxylase inhibitor, α-methylphenylalanine (2.4 µmol/g body weight; once a day; i.p.), from the 7th to the 19th day of life. HPA reduced the activities of pyruvate kinase, creatine kinase, and complex II + III of RCC in the cerebral cortex. Creatine nanoliposomes prevented the inhibition of the activities of the complexes II + III, caused by HPA, and changes oxidative profile in the cerebral cortex. Considering the importance of the mitochondrial respiratory chain for brain energy production, our results suggesting that these nanoparticles protect against neurotoxicity caused by HPA, and can be viable candidates for treating patients HPA.

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., (Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

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.

Alzheimer's disease (AD) is the main aging-associated neurodegenerative disorder and is characterized by mitochondrial dysfunction, oxidative stress, synaptic failure, and cognitive decline. It has been a challenge to find disease course-modifying treatments. However, several studies demonstrated that regular physical activity and exercise are capable of promoting brain health by improving the cognitive function. Maternal lifestyle, including regular exercise during pregnancy, has also been shown to influence fetal development and disease susceptibility in adulthood through fetal metabolism programming. Here, we investigated the potential neuroprotective role of regular maternal swimming, before and during pregnancy, against amyloid-β neurotoxicity in the adult offspring. Behavioral and neurochemical analyses were performed 14 days after male offspring received a single, bilateral, intracerebroventricular (icv) injection of amyloid-β oligomers (AβOs). AβOs-injected rats of the sedentary maternal group exhibited learning and memory deficits, along with reduced synaptophysin, brain-derived neurotrophic factor (BDNF) levels, and alterations of mitochondrial function. Strikingly, the offspring of the sedentary maternal group had AβOs-induced behavioral alterations that were prevented by maternal exercise. This effect was accompanied by preventing the alteration of synaptophysin levels in the offspring of exercised dams. Additionally, offspring of the maternal exercise group exhibited an augmentation of functional mitochondria, as indicated by increases in mitochondrial mass and membrane potential, α-ketoglutarate dehydrogenase, and cytochrome c oxidase enzymes activities. Moreover, maternal exercise during pregnancy induced long-lasting modulation of fusion and fission proteins, Mfn1 and Drp1, respectively. Overall, our data demonstrates a potential protective effect of exercise during pregnancy against AβOs-induced neurotoxicity in the adult offspring brain, by mitigating the neurodegenerative process triggered by Alzheimer-associated AβOs through programming the brain metabolism.

Non-ketotic hyperglycinemia (NKH) is a severe neurological disorder caused by defects in glycine (GLY) catabolism and characterized by a high cerebrospinal fluid/plasma GLY ratio. Treatment is often ineffective and limited to the control of symptoms and detoxification of GLY. In the present work, we investigated the in vivo effects of GLY intracerebroventricular administration on oxidative stress parameters in rat striatum, cerebral cortex, and hippocampus. In vitro effects of GLY were also evaluated in striatum. The effects of bezafibrate (BEZ), a potential neuroprotective agent, on the possible alterations caused by GLY administration were further evaluated. Our in vivo results showed that GLY increased the activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) in striatum. Furthermore, GLY decreased the concentrations of total glutathione and reduced glutathione (GSH), as well as GSH/oxidized glutathione ratio in vivo in hippocampus. In vitro data also showed that GLY induced lipid peroxidation and decreased GSH in striatum. Regarding the effects of BEZ, we found that GLY-induced increase of GPx, SOD, and GR activities was attenuated or prevented by this compound. However, BEZ did not alter GLY-induced decrease of GSH in hippocampus. We hypothesize that GLY-induced increase of the activities of antioxidant enzymes in striatum occurs as a mechanism to avoid accumulation of reactive oxygen species and consequent oxidative damage. Furthermore, since BEZ prevented GLY-induced alterations, it might be considered as an adjuvant therapy for NKH.

Sulfite oxidase, molybdenum cofactor, and ETHE1 deficiencies are autosomal recessive disorders that affect the metabolism of sulfur-containing amino acids. Patients with these disorders present severe neurological dysfunction and basal ganglia abnormalities, accompanied by high levels of thiosulfate in biological fluids and tissues. Aiming to better elucidate the pathophysiology of basal ganglia damage in these disorders, we evaluated the in vivo effects of thiosulfate administration on bioenergetics, oxidative stress, and neural damage in rat striatum. The in vitro effect of thiosulfate on creatine kinase (CK) activity was also studied. In vivo findings showed that thiosulfate administration decreased the activities of CK and citrate synthase, and increased the activity of catalase 30 min after administration. Activities of other antioxidant enzymes, citric acid cycle, and respiratory chain complex enzymes, as well as glutathione concentrations and markers of neural damage, were not altered by thiosulfate 30 min or 7 days after its administration. Thiosulfate also decreased the activity of CK in vitro in striatum of rats, which was prevented by the thiol reducing agents dithiothreitol (DTT), the antioxidants glutathione (GSH), melatonin, trolox (hydrosoluble analogue of vitamin E), and lipoic acid. DTT and GSH further prevented thiosulfate-induced decrease of the activity of a purified CK in a medium devoid of biological samples. These data suggest that thiosulfate inhibits CK activity by altering critical sulfhydryl groups of this enzyme. It may be also presumed that bioenergetics impairment and ROS generation induced by thiosulfate are mechanisms underlying the neuropathophysiology of disorders in which this metabolite accumulates.

Aeromonas caviae is a Gram-negative bacterium rarely found in fish but it can be associated to high mortality of infected animals. The disease pathogenesis in fish associated to liver and kidney lesions directly linked to the initiation and progression of the disease remains poorly understood. Thus, the aim of this study was to evaluate whether A. caviae infection causes oxidative stress in liver and kidney of silver catfish Rhamdia quelen, and its involvement in disease pathogenesis. Reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) levels increased in liver and kidney of fish experimentally infected by A. caviae compared to the control uninfected group. On the other hand, non-protein sulfhydryl (NPSH) levels decreased in both tissues of infected animals, while the glutathione S-transferase (GST) activity decreased only in the hepatic tissue. No difference was observed between groups in both tissues regarding superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) activities and glutathione (GSH) levels. In summary, the disturbance of hepatic and renal antioxidant/oxidant equilibrium contributes to the pathophysiology of the disease in fish experimentally infected by A. caviae., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Inborn errors of metabolism (IEM) comprise a group of over 600 disorders, each with a specific metabolic impairment due to a genetic defect. Urea cycle disorders (UCD) are IEM that affect the nitrogen disposal system, leading to hyperammonemia and the accumulation of other toxic metabolites in tissues of affected patients. UCD arise from mutations in the genes coding any of the enzymes participating in the urea cycle, either directly or as regulators of this pathway, causing severe respiratory alkalosis. Considering that the exact mechanisms underlying the damage found in UCD, the purpose of this minireview is to obtain data and search for links between UCD and oxidative stress, a phenomenon common to several IEM. In vitro studies and animal models of UCD suggest that, not only the accumulation of ammonia, but also of the other metabolites involved in each UCD may impair redox status. Nitric oxide metabolism also seems to play an interesting role in the maintenance of redox balance in these conditions. Clinical research provides little information on the subject, but, studies appear to support the role of oxidative stress in pathologic mechanisms of UCD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

High glycine (GLY) levels have been suggested to induce neurotoxic effects in the central nervous system of patients with nonketotic hyperglycinemia (NKH). Since the mechanisms involved in the neuropathophysiology of NKH are not totally established, we evaluated the effect of a single intracerebroventricular administration of GLY on the content of proteins involved in neuronal damage and inflammatory response, as well as on the phosphorylation of the MAPK p38, ERK1/2, and JNK in rat striatum and cerebral cortex. We also examined glial fibrillary acidic protein (GFAP) staining, a marker of glial reactivity. The parameters were analyzed 30 min or 24 h after GLY administration. GLY decreased Tau phosphorylation in striatum and cerebral cortex 30 min and 24 h after its administration. On the other hand, synaptophysin levels were decreased in striatum at 30 min and in cerebral cortex at 24 h after GLY injection. GLY also decreased the phosphorylation of p38, ERK1/2, and JNK 30 min after its administration in both brain structures. Moreover, GLY-induced decrease of p38 phosphorylation in striatum was attenuated by N-methyl-D-aspartate receptor antagonist MK-801. In contrast, synuclein, NF-κB, iκB, inducible nitric oxide synthase and nitrotyrosine content, and GFAP immunostaining were not altered by GLY infusion. It may be presumed that the decreased phosphorylation of MAPK associated with alterations of markers of neuronal injury induced by GLY may contribute to the neurological dysfunction observed in NKH.

Sulfite accumulates in tissues of patients affected by sulfite oxidase (SO) deficiency, a neurometabolic disease characterized by seizures and progressive encephalopathy, often resulting in early death. We investigated the effects of sulfite on mitochondrial function, antioxidant system, glial reactivity and neuronal damage in rat striatum, as well as the potential protective effects of bezafibrate on sulfite-induced toxicity. Thirty-day-old rats were intrastriatally administered with sulfite (2μmol) or NaCl (2μmol; control) and euthanized 30min after injection for evaluation of biochemical parameters and western blotting, or 7days after injection for analysis of glial reactivity and neuronal damage. Treatment with bezafibrate (30 or 100mg/kg/day) was performed by gavage during 7days before (pre-treatment) or after sulfite administration. Sulfite decreased creatine kinase and citrate synthase activities, mitochondrial mass, and PGC-1α nuclear content whereas bezafibrate pre-treatment prevented these alterations. Sulfite also diminished cytochrome c oxidase (COX) IV-1 content, glutathione levels and the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH). On the other hand, catalase activity was increased by sulfite. Bezafibrate pre-treatment prevented the reduction of GPx, GR, GST and G6PDH activities. Finally, sulfite induced glial reactivity and neuronal damage, which were prevented by bezafibrate when administered before or after sulfite administration. Our findings provide strong evidence that sulfite induces neurotoxicity that leads to glial reactivity and neuronal damage. Since bezafibrate exerts neuroprotective effects against sulfite toxicity, it may be an attractive agent for the development of novel therapeutic strategies for SO-deficient patients., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Patients affected by sulfite oxidase (SO) deficiency present severe seizures early in infancy and progressive neurological damage, as well as tissue accumulation of sulfite, thiosulfate and S-sulfocysteine. Since the pathomechanisms involved in the neuropathology of SO deficiency are still poorly established, we evaluated the effects of sulfite on redox homeostasis and bioenergetics in cerebral cortex, striatum, cerebellum and hippocampus of rats with chemically induced SO deficiency. The deficiency was induced in 21-day-old rats by adding 200ppm of tungsten, a molybdenum competitor, in their drinking water for 9weeks. Sulfite (70mg/kg/day) was also administered through the drinking water from the third week of tungsten supplementation until the end of the treatment. Sulfite decreased reduced glutathione concentrations and the activities of glutathione reductase and glutathione S-transferase (GST) in cerebral cortex and of GST in cerebellum of SO-deficient rats. Moreover, sulfite increased the activities of complexes II and II-III in striatum and of complex II in hippocampus, but reduced the activity of complex IV in striatum of SO-deficient rats. Sulfite also decreased the mitochondrial membrane potential in cerebral cortex and striatum, whereas it had no effect on mitochondrial mass in any encephalic tissue evaluated. Finally, sulfite inhibited the activities of malate and glutamate dehydrogenase in cerebral cortex of SO-deficient rats. Taken together, our findings indicate that cerebral cortex and striatum are more vulnerable to sulfite-induced toxicity than cerebellum and hippocampus. It is presumed that these pathomechanisms may contribute to the pathophysiology of neurological damage found in patients affected by SO deficiency., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Accumulation of glycine (GLY) is the biochemical hallmark of glycine encephalopathy (GE), an aminoacidopathy characterized by severe neurological dysfunction that may lead to early death. In the present study, we evaluated the effect of a single intracerebroventricular administration of GLY on bioenergetics, redox homeostasis, and histopathology in brain of neonatal rats. Our results demonstrated that GLY decreased the activities of the respiratory chain complex IV and creatine kinase, induced reactive species generation, and diminished glutathione (GSH) levels 1, 5, and 10 days after GLY injection in cerebral cortex of 1-day-old rats. GLY also increased malondialdehyde (MDA) levels 5 days after GLY infusion in this brain region. Furthermore, GLY differentially modulated the activities of superoxide dismutase, catalase, and glutathione peroxidase depending on the period tested after GLY administration. In contrast, bioenergetics and redox parameters were not altered in brain of 5-day-old rats. Regarding the histopathological analysis, GLY increased S100β staining in cerebral cortex and striatum, and GFAP in corpus callosum of 1-day-old rats 5 days after injection. Finally, we verified that melatonin prevented the decrease of complex IV and CK activities and GSH concentrations, and the increase of MDA levels and S100β staining caused by GLY. Based on our findings, it may be presumed that impairment of redox and energy homeostasis and glial reactivity induced by GLY may contribute to the neurological dysfunction observed in GE.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency is characterized by tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), and 3-methylglutaric (MGA) acids. Affected patients present cardiomyopathy, whose pathomechanisms are not yet established. We investigated the effects of HMG and MGA on energy and redox homeostasis in rat heart using in vivo and in vitro models. In vivo experiments showed that intraperitoneal administration of HMG and MGA decreased the activities of the respiratory chain complex II and creatine kinase (CK), whereas HMG also decreased the activity of complex II-III. Furthermore, HMG and MGA injection increased reactive species production and carbonyl formation, and decreased glutathione concentrations. Regarding the enzymatic antioxidant defenses, HMG and MGA increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities, while only MGA diminished the activities of superoxide dismutase (SOD) and catalase, as well as the protein content of SOD1. Pre-treatment with melatonin (MEL) prevented MGA-induced decrease of CK activity and SOD1 levels. In vitro results demonstrated that HMG and MGA increased reactive species formation, induced lipid peroxidation and decreased glutathione. We also verified that reactive species overproduction and glutathione decrease provoked by HMG and MGA were abrogated by MEL and lipoic acid (LA), while only MEL prevented HMG- and MGA-induced lipoperoxidation. Allopurinol (ALP) also prevented reactive species overproduction caused by both metabolites. Our data provide solid evidence that bioenergetics dysfunction and oxidative stress are induced by HMG and MGA in heart, which may explain the cardiac dysfunction observed in HL deficiency, and also suggest that antioxidant supplementation could be considered as adjuvant therapy for affected patients.

Sulfite oxidase (SOX) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation and high urinary excretion of sulfite and thiosulfate. Affected patients present severe neurological dysfunction accompanied by seizures, whose pathophysiology is poorly known. In the present study we evaluated the in vitro effects of sulfite and thiosulfate on important parameters of glutamatergic neurotransmission and redox homeostasis in rat cerebral cortex slices. We verified that sulfite, but not thiosulfate, significantly decreased glutamate uptake when cerebral cortex slices were exposed during 1h to these metabolites. We also observed that thiosulfate inhibited glutamine synthetase (GS) activity. A pronounced trend toward GS inhibition induced by sulfite was also found. Regarding redox homeostasis, sulfite, at the concentration of 10 μM, increased thiobarbituric acid-reactive substances and decreased glutathione concentrations after 1h of exposure. In contrast, thiosulfate did not alter these parameters. We also found that 500 μM sulfite increased sulfhydryl group content in rat cerebral cortex slices and increased GSH levels in a medium containing oxidized GSH (GSSG) and devoid of cortical slices, suggesting that sulfite reacts with disulfide bonds to generate sulfhydryl groups. Moreover, sulfite and thiosulfate did not alter the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) after 1h of incubation. However, sulfite inhibited the activities of GPx, GST and G6PDH when cortical slices were exposed for 3h to sulfite. We finally verified that sulfite did not induce cell death after 1h of incubation. Our data show that sulfite impairs glutamatergic neurotransmission and redox homeostasis in cerebral cortex. Therefore, it may be presumed that these pathomechanisms contribute, at least in part, to the seizures observed in patients affected by SOX deficiency., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Patients with non-ketotic hyperglycinemia (NKH) present severe neurological symptoms and brain abnormalities involving cerebellum. Although the pathomechanisms underlying the cerebellum damage have not been studied, high tissue levels of glycine (GLY), the biochemical hallmark of this disorder have been suggested to contribute to the neuropathology of this disease. We investigated the in vitro effects of GLY on important parameters of oxidative stress and energy metabolism in cerebellum of 30-day-old rats. Our results show that GLY increased 2',7'-dichlorofluorescin oxidation, suggesting that reactive species production are augmented by GLY in the cerebellum. However, hydrogen peroxide generation was not altered by GLY. GLY also increased thiobarbituric acid-reactive substances (TBA-RS) levels and reduced the glutathione (GSH) content, indicating that this amino acid provokes lipid oxidative damage and compromises the non-enzymatic antioxidant defenses, respectively, in cerebellum. The antioxidants melatonin and trolox (the hydrosoluble analog of vitamin E) prevented the GLY-induced increase of TBA-RS and decrease of GSH in cerebellum, indicating the involvement of hydroxyl and peroxyl radicals in these effects. The NMDA receptor antagonist MK-801 also attenuated GLY-induced decrease of GSH, suggesting that this effect is mediated through NMDA receptor. In contrast, GLY did not alter the protein carbonyl formation and total and protein-bound sulfhydryl group content, as well as catalase and superoxide dismutase activities. Furthermore, GLY did not alter the activities of the respiratory chain complexes and creatine kinase. Our present data indicate that oxidative stress elicited by GLY in vitro may be a potential pathomechanism involved in the cerebellar dysfunction observed in NKH.

Sulfite oxidase (SO) deficiency is biochemically characterized by the accumulation of sulfite, thiosulfate and S-sulfocysteine in tissues and biological fluids of the affected patients. The main clinical symptoms include severe neurological dysfunction and brain abnormalities, whose pathophysiology is still unknown. The present study investigated the in vitro effects of sulfite and thiosulfate on mitochondrial homeostasis in rat brain mitochondria. It was verified that sulfite per se, but not thiosulfate, decreased state 3, CCCP-stimulated state and respiratory control ratio in mitochondria respiring with glutamate plus malate. In line with this, we found that sulfite inhibited the activities of glutamate and malate (MDH) dehydrogenases. In addition, sulfite decreased the activity of a commercial solution of MDH, that was prevented by antioxidants and dithiothreitol. Sulfite also induced mitochondrial swelling and reduced mitochondrial membrane potential, Ca(2+) retention capacity, NAD(P)H pool and cytochrome c immunocontent when Ca(2+) was present in the medium. These alterations were prevented by ruthenium red, cyclosporine A (CsA) and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. We further observed that N-ethylmaleimide prevented the sulfite-elicited swelling and that sulfite decreased free thiol group content in brain mitochondria. These findings indicate that sulfite acts directly on MPT pore containing thiol groups. Finally, we verified that sulfite reduced cell viability in cerebral cortex slices and that this effect was prevented by CsA. Therefore, it may be presumed that disturbance of mitochondrial energy homeostasis and MPT induced by sulfite could be involved in the neuronal damage characteristic of SO deficiency., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sulfite oxidase (SO) deficiency is biochemically characterized by tissue accumulation and high urinary excretion of sulfite, thiosulfate and S-sulfocysteine. Affected patients present severe neurological symptoms and cortical atrophy, whose pathophysiology is still poorly established. Therefore, in the present work we investigated the in vitro effects of sulfite and thiosulfate on important parameters of energy metabolism in the brain of young rats. We verified that sulfite moderately inhibited the activity of complex IV, whereas thiosulfate did not alter any of the activities of the respiratory chain complexes. It was also found that sulfite and thiosulfate markedly reduced the activity of total creatine kinase (CK) and its mitochondrial and cytosolic isoforms, suggesting that these metabolites impair brain cellular energy buffering and transfer. In contrast, the activity of synaptic Na(+),K(+)-ATPase was not altered by sulfite or thiosulfate. We also observed that the inhibitory effect of sulfite and thiosulfate on CK activity was prevented by melatonin, reduced glutathione and the combination of both antioxidants, as well as by the nitric oxide synthase N(ω)-nitro-l-arginine methyl ester, indicating the involvement of reactive oxygen and nitrogen species in these effects. Sulfite and thiosulfate also increased 2',7'-dichlorofluorescin oxidation and hydrogen peroxide production and decreased the activity of the redox sensor aconitase enzyme, reinforcing a role for oxidative damage in the effects elicited by these metabolites. It may be presumed that the disturbance of cellular energy and redox homeostasis provoked by sulfite and thiosulfate contributes to the neurological symptoms and abnormalities found in patients affected by SO deficiency., (© 2013.)

High tissue levels of glycine (GLY) are the biochemical hallmark of nonketotic hyperglycinemia (NKH), an inherited metabolic disease clinically characterized by severe neurological symptoms and brain abnormalities. Considering that the mechanisms underlying the neuropathology of this disease are not fully established, the present work investigated the in vivo effects of intracerebroventricular administration of GLY on important parameters of energy metabolism in cerebral cortex and striatum from young rats. Our results show that GLY reduced CO₂ production using glucose as substrate and inhibited the activities of citrate synthase and isocitrate dehydrogenase in striatum, whereas no alterations of these parameters were verified in cerebral cortex 30 min after GLY injection. We also observed that GLY diminished the activities of complex IV in cerebral cortex and complex I-III in striatum at 30 min and inhibited complex I-III activity in striatum at 24 h after its injection. Furthermore, GLY reduced the activity of total and mitochondrial creatine kinase in both brain structures 30 min and 24 h after its administration. In contrast, the activity of Na⁺, K⁺-ATPase was not altered by GLY. Finally, the antioxidants N-acetylcysteine and creatine, and the NMDA receptor antagonist MK-801 attenuated or fully prevented the inhibitory effects of GLY on creatine kinase and respiratory complexes in cerebral cortex and striatum. Our data indicate that crucial pathways for energy production and intracellular energy transfer are severely compromised by GLY. It is proposed that bioenergetic impairment induced by GLY in vivo may contribute to the neurological dysfunction found in patients affected by NKH.