Friedreich's Ataxia Causes Redistribution of Iron, Copper, and Zinc in the Dentate Nucleus

Friedreich's ataxia (FRDA) causes selective atrophy of the large neurons of the dentate nucleus (DN). High iron (Fe) concentration and failure to clear the metal from the affected brain tissue are potential risk factors in the progression of the lesion. The DN also contains relatively high amounts of copper (Cu) and zinc (Zn), but the importance of these metals in FRDA has not been established. This report describes nondestructive quantitative X-ray fluorescence (XRF) and "mapping" of Fe, Cu, and Zn in polyethylene glycol?Cdimethylsulfoxide (PEG/DMSO)-embedded DN of 10 FRDA patients and 13 controls. Fe fluorescence arose predominantly from the hilar white matter, whereas Cu and Zn were present at peak levels in DN gray matter. Despite collapse of the DN in FRDA, the location of the peak Fe signal did not change. In contrast, the Cu and Zn regions broadened and overlapped extensively with the Fe-rich region. Maximal metal concentrations did not differ from normal (in micrograms per milliliter of solid PEG/DMSO as means ± S.D.): Fe normal, 364?±?117, FRDA, 344?±?159; Cu normal, 33?±?13, FRDA, 33?±?18; and Zn normal, 32?±?16, FRDA, 33?±?19. Tissues were recovered from PEG/DMSO and transferred into paraffin for matching with immunohistochemistry of neuron-specific enolase (NSE), glutamic acid decarboxylase (GAD), and ferritin. NSE and GAD reaction products confirmed neuronal atrophy and grumose degeneration that coincided with abnormally diffuse Cu and Zn zones. Ferritin immunohistochemistry matched Fe XRF maps, revealing the most abundant reaction product in oligodendroglia of the DN hilus. In FRDA, these cells were smaller and more numerous than normal. In the atrophic DN gray matter of FRDA, anti-ferritin labeled mostly hypertrophic microglia. Immunohistochemistry and immunofluorescence of the Cu-responsive proteins Cu,Zn-superoxide dismutase and Cu⁺⁺-transporting ATPase ??-peptide did not detect specific responses to Cu redistribution in FRDA. In contrast, metallothionein (MT)-positive processes were more abundant than normal and contributed to the gliosis of the DN. The isoforms of MT, MT-1/2, and brain-specific MT-3 displayed only limited co-localization with glial fibrillary acidic protein. The results suggest that MT can provide effective protection against endogenous Cu and Zn toxicity in FRDA, similar to the neuroprotective sequestration of Fe in holoferritin.     

Roles of the metallothionein family of proteins in the central nervous system

Metallothioneins (MTs) constitute a family of proteins characterized by a high heavy metal [Zn(II), Cu(I)] content and also by an unusual cysteine abundance. Mammalian MTs are comprised of four major isoforms designated MT-1 trough MT-4. MT-1 and MT-2 are expressed in most tissues including the brain, whereas MT-3 (also called growth inhibitory factor) and MT-4 are expressed predominantly in the central nervous system and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions, such as zinc and copper metabolism, protection against reactive oxygen species, or adaptation to stress. In the case of MT-3, an additional involvement of this isoform in neuromodulatory events and in the pathogenesis of Alzheimer's disease has also been suggested. It is essential to gain insight into how MTs are regulated in the brain in order to characterize MT functions, both in normal brain physiology, as well as in pathophysiological states. The focus of this review concerns the biology of the MT family in the context of their expression and functional roles in the central nervous system.     

Metal changes in CSF and peripheral compartments of parkinsonian patients

BACKGROUND: Involvement of metals in the risk of developing Parkinson's disease (PD) has been suggested. In the present study, concentration of metals in cerebrospinal fluid (CSF), blood, serum, urine and hair of 91 PD patients and 18 controls were compared. METHODS: Blood and hair were microwave digested, while CSF, serum and urine were water-diluted. Elements quantification was achieved by Inductively Coupled Plasma Atomic Emission Spectrometry and Sector Field Inductively Coupled Plasma Mass Spectrometry. RESULTS: Some metal imbalances in PD were observed: i), in CSF, lower Fe and Si; ii), in blood, higher Ca, Cu, Fe, Mg and Zn; iii), in serum, lower Al and Cu; iv), in urine, lower Al and Mn, higher Ca and Fe; and v), in hair, lower Fe. The ROC analysis suggested that blood Ca, Fe, Mg and Zn were the best discriminators between PD and controls. In addition, hair Ca and Mg were at least 1.5 times higher in females than in males of patients and controls. A decrement with age of patients in hair and urine Ca and, with less extent, in urine Si was observed. Magnesium concentration in CSF decreased with the duration and severity of the disease. Elements were not influenced by the type of antiparkinsonian therapy. CONCLUSIONS: Variation in elements with the disease do not exclude their involvement in the neurodegeneration of PD.     

Cigarette Smoke Condensate Causes a Decrease of the Gene Expression of Cu�CZn Superoxide Dismutase, Mn Superoxide Dismutase, Glutathione Peroxidase, Catalase, and Free Radical-Induced Cell Injury in SH-SY5Y Human Neuroblastoma Cells

Cigarette smoking condensate (CSC) contains oxidant compounds able to generate superoxide. The aim of the present study was to investigate the effect of the exposure to CSC on: (1) free radical production, (2) the gene expression of the antioxidant enzymes Cu?CZn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), Glutathione Peroxidase (GPx), and catalase (CAT), and (3) cell survival in human neuroblastoma SH-SY5Y cells. The results showed that exposure (24?h) to different concentrations (10?C150???g/ml) of CSC caused a dose dependent cell injury that was coupled to the maximal increase of free radical production. These events were prevented by the addition to the incubation medium of the scavenger Vitamin E (50???M). Furthermore, CSC exposure caused a reduction of the gene expression of the antioxidant enzymes SOD1, SOD2, GPx, and CAT that was counteracted by Vitamin E (50???M). These results suggest that CSC exposure can induce a free radical overcharge that may be responsible for the inhibition of antioxidant enzymes expression and cell injury in SH-SY5Y human neuroblastoma cells. In fact the scavenger vitamin E can block both cell injury and inhibition of SOD1, SOD2, GPx, and CAT induced by CSC exposure.     

Antioxidant intervention attenuates oxidative stress in children and teenagers with Down syndrome

We previously demonstrated that systemic oxidative stress is present in Down syndrome (DS) patients. In the present study we investigated the antioxidant status in the peripheral blood of DS children and teenagers comparing such status before and after an antioxidant supplementation. Oxidative stress biomarkers were evaluated in the blood of DS patients (n = 21) before and after a daily antioxidant intervention (vitamin E 400 mg, C 500 mg) during 6 months. Healthy children (n = 18) without DS were recruited as control group. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), gamma-glutamyltransferase (GGT), glucose-6-phosphate dehydrogenase (G6PD) and myeloperoxidase (MPO), as well as the contents of reduced glutathione (GSH), uric acid, vitamin E, thiobarbituric acid reactive substances (TBARS), and protein carbonyls (PC) were measured. Before the antioxidant therapy, DS patients presented decreased GST activity and GSH depletion; elevated SOD, CAT, GR, GGT and MPO activities; increased uric acid levels; while GPx and G6PD activities as well as vitamin E and TBARS levels were unaltered. After the antioxidant supplementation, SOD, CAT, GPx, GR, GGT and MPO activities were downregulated, while TBARS contents were strongly decreased in DS. Also, the antioxidant therapy did not change G6PD and GST activities as well as uric acid and PC levels, while it significantly increased GSH and vitamin E levels in DS patients. Our results clearly demonstrate that the antioxidant intervention with vitamins E and C attenuated the systemic oxidative damage present in DS patients.     

Peripheral markers for oxidative stress in Parkinson’s disease patients of Eastern India

Oxidative stress is thought to play a major role in the pathogenesis of Parkinson’s disease (PD). Neurons are highly susceptible to a defective antioxidant scavenging system, thus inducing oxidative changes in human red blood cells (RBCs), in vivo and in vitro. Previous studies on oxidative stress in RBCs in patients with PD have yielded controversial results claiming unaltered activity to reduced activity. We have thus undertaken this study to investigate the possibility of oxidative damage to the RBCs in PD by measuring the cytosolic antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (G-Px). The biochemical parameters were measured in erythrocytes of 80 PD patients and 80 normal age-matched healthy controls. The enzymes activities were correlated with age of patients, age of onset of disease, duration of disease, United Parkinson’s Disease Rating Scale (UPDRS) and Hoehn and Yahr stage. Patients with PD had higher red blood corpuscle (RBC) activity of SOD. The CAT, and G-Px activities were significantly lower in patients with PD compared to the controls. Erythrocyte SOD, CAT and G-Px were markedly lower in those PD patients who were suffering for a greater duration of the disease and in advanced cases of PD. A significant (P < 0.05) negative correlation of enzyme activities with disease duration, UPDRS score and Hoehn and Yahr stage of the disease was found. Results of our present study concludes the implication of oxidative stress as one of the risk factors, which can initiate or promote neurodegeneration in PD by playing a role in dopaminergic neuronal loss and was correlated to the severity of the disease.     

Effect of Valsartan on Cerebellar Adrenomedullin System Dysregulation During Hypertension

Adrenomedullin (AM) and its receptors components, calcitonin-receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3) are expressed in cerebellum. Cerebellar AM, AM binding sites and receptor components are altered during hypertension, suggesting a role for cerebellar AM in blood pressure regulation. Thus, we assessed the effect of valsartan, on AM and its receptor components expression in the cerebellar vermis of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Additionally, we evaluated AM action on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS) production in cerebellar vermis. Animals were treated with valsartan or vehicle for 11 days. Rats were sacrificed by decapitation; cerebellar vermis was dissected; and AM, CRLR, RAMP1, RAMP2, and RAMP3 expression was quantified by Western blot analysis. CAT, SOD, and GPx activity was determined spectrophotometrically and blood pressure by non-invasive plethysmography. We demonstrate that AM and RAMP2 expression was lower in cerebellum of SHR rats, while CRLR, RAMP1, and RAMP3 expression was higher than those of WKY rats. AM reduced cerebellar CAT, SOD, GPx activities, and TBARS production in WKY rats, but not in SHR rats. Valsartan reduced blood pressure and reversed the altered expression of AM and its receptors components, as well the loss of AM capacity to reduce antioxidant enzyme activity and TBARS production in SHR rats. These findings demonstrate that valsartan is able to reverse the dysregulation of cerebellar adrenomedullinergic system; and they suggest that altered AM system in the cerebellum could represent the primary abnormality leading to hypertension.     

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.     

Divalent metals differentially block cloned T-type calcium channels

We tested divalent metals including Cu2+, Pb2+, and Zn2+ to determine their pharmacological profiles for blockade of cloned T-type Ca2+ channels (alpha1G, alpha1 H, and alpha1I). Effects of the metals were also evaluated for native low and high voltage-activated Ca2+ channels in rat sympathetic pelvic neurons. Cu2+ and Zn2+ blocked three T-type channel isoforms in a concentration-dependent manner with a higher affinity for alpha1H currents (IC50 = 0.9 microM and 2.3 microM). In pelvic neurons, only Zn2+ showed strong selectivity for T-type Ca2+ currents over high voltage-activated Ca2+ currents. Conversely, Pb2+ block on Ca2+ channels did not show distinctive selectivity. Taken together, these results suggest that besides Ni2+, Cu2+ and Zn2+ can be used as selective blockers of alpha1 H at low concentrations.     

Persistence of the benefit of an antioxidant therapy in children and teenagers with Down syndrome

This study examined the effect of an antioxidant intervention in biomarkers of inflammation and oxidative stress (OS) in the blood of Down syndrome (DS) children and teenagers during four different stages. A control group was composed by healthy children (n = 18), assessed once, and a Down group composed by DS patients (n = 21) assessed at the basal period (t₀), as well as after 6 months of antioxidant supplementation (t₁), after 12 months (after interruption of the antioxidant intervention for 6 months) (t₂), and again after further 6 months of antioxidant supplementation (t₃). Biomarkers of inflammation (myeloperoxidase activity – MPO and levels of IL-1β and TNF-α) and OS (thiobarbituric acid reactive substances – TBARS, protein carbonyls – PC), reduced glutathione (GSH), uric acid (UA) and vitamin E levels, as well as antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and gamma-glutamyltransferase (GGT) activities, were measured after each period. After the antioxidant supplementation, the activities of SOD, CAT, GPx, GR, GGT and MPO were downregulated, while TBARS contents were strongly decreased, the contents of GSH and vitamin E were significantly increased, and no changes in G6PD and GST activity as well as in UA and PC levels were detected. After the interruption of the antioxidant therapy for 6 months, DS patients showed elevated GPx and GGT activities and also elevated UA and TBARS levels. No changes in SOD, CAT, GR, GST, G6PD and MPO activities as well as in GSH, vitamin E, PC, TNF-α and IL-1β levels were detected. The results showed that the antioxidant intervention persistently attenuated the systemic oxidative damage in DS patients even after a relatively long period of cessation of the antioxidant intervention.     

Hypothyroidism alters antioxidant defence system in rat brainstem during postnatal development and adulthood

The present investigation was carried out to evaluate alterations in oxidative stress parameter [lipid peroxidation (LPx)] and antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] in rat brainstem in response to neonatal hypothyroidism during development (from birth to 7, 15 and 30 days old) and adulthood (90 days old). Hypothyroidism in rats was induced by feeding the lactating mothers (from the day of parturition till weaning, 25 days old) or directly to the pups with 0.05 % [6-n-propyl 2-thiouracil (PTU)] in drinking water. Increased level of LPx was observed in brainstem of 7 days old hypothyroid rats, accompanied by augmented activities of SOD and GPx. In 15 and 30 days old hypothyroid rat brainstem, a significant decline in LPx was observed. Significantly increased activities of CAT and GPx were observed in 15 and 30 days PTU-treated rats. Decreased level of LPx was observed in brainstem of rats treated with PTU from birth to 30 days followed by withdrawal up to 90 days of age (transient hypothyroidism) as compared to control and persistent treatment of PTU up to 90 days of age. Activities of CAT and GPx were decreased in persistent hypothyroid rats of 90 days old with respect to control and transient hypothyroid rats. On the other hand, SOD activity was decreased in both persistent and transient hypothyroid rats with respect to control rats. These results suggest that the PTU-induced neonatal hypothyroidism modulates the antioxidant defence system during postnatal development and adulthood in brainstem of rats.     

Effect of ingested sulfite on hippocampus antioxidant enzyme activities in sulfite oxidase competent and deficient rats

Animal tissues are exposed to sulfite used as a preservative in food and drugs, and generated from the catabolism of sulfur-containing amino acids. Sulfite, which is a very reactive and potentially toxic molecule, is detoxified by the enzyme sulfite oxidase (SOX). Laboratory animals can be made deficient in SOX by the administration of a high-tungsten/low molybdenum regimen. It has been suggested that SOX deficient rats might be used as a model for the prediction of sulfite toxicity in humans. The aim of this study was to investigate the effects of ingested sulfite on hippocampus superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in SOX competent and deficient rats. Hippocampus SOD, CAT and GPx activities were found to be significantly increased by sulfite treatment in SOX competent groups. On the other hand, exposure to sulfite had no effect on antioxidant status in hippocampus of SOX deficient rats. In conclusion, these results suggest that hippocampus antioxidant capacity where defense mechanism against the oxidative challenge is up regulated by sulfite in SOX competent rats. This up regulation mechanism in antioxidant enzymes against to sulfite related oxidative stress is not observed in SOX deficient rats and remains to be explained.     

Altered Antioxidant Defenses in Drug-Naive First Episode Patients with Schizophrenia Are Associated with Poor Treatment Response to Risperidone: 12-Week Results from a Prospective Longitudinal Study

Abnormal redox regulation is thought to contribute to schizophrenia (SCZ). Accumulating studies have shown that the plasma antioxidant enzyme activity is closely associated with the course and outcome in antipsychotics-naïve first-episode (ANFE) patients with SCZ. The main purpose of this study was to investigate the effect of risperidone on oxidative stress markers in ANFE patients and the relationship between risperidone response and changes in oxidative stress markers. Plasma activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) enzyme, total antioxidant status (TAS), and malondialdehyde (MDA) levels were measured in 354 ANFE patients and 152 healthy controls. The clinical symptoms were evaluated by the Positive and Negative Syndrome Scale (PANSS). Patients received risperidone monotherapy for 12 weeks and oxidative stress markers and PANSS were measured at baseline and at follow-up. Compared with healthy controls, the patients exhibited higher activities of SOD, CAT, and TAS levels, but lower MDA levels and GPx activity. A comparison between 168 responders and 50 non-responders at baseline and 12-week follow-up showed that GPx activity decreased in both groups after treatment. Moreover, GPx activity decreased less in responders and was higher in responders than in non-responders at follow-up. These results demonstrate that the redox regulatory system and antioxidant defense enzymes may have predictive value for the response of ANFE patients to risperidone treatment.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01036-3.Key Words: Schizophrenia, Oxidative stress, Antioxidant defense system, Clinical response, Risperidone     

Increased oxidative stress and altered activities of erythrocyte free radical scavenging enzymes in autism

Abstract. There is great evidence in recent years that oxygen free radicals play an important role in the pathophysiology of many neuropsychiatric disorders. The present study was performed to assess the changes in red blood cells thiobarbituric acid-reactive substances (TBARS) levels, and superoxide dismutase (SOD), catalase (CAT), adenosine deaminase (ADA) and xanthine oxidase (XO) activities in patients with autism (n = 27) compared to age- and sex-matched normal controls (n = 26). In the autistic group, increased TBARS levels (p < 0.001) and XO (p < 0.001) and SOD (p < 0.001) activity, decreased CAT (p < 0.001) activity and unchanged ADA activity were detected. It is proposed that antioxidant status may be changed in autism and this new situation may induce lipid peroxidation. These findings indicated a possible role of increased oxidative stress and altered enzymatic antioxidants, both of which may be relevant to the pathophysiology of autism.     

Patterns of levels of biological metals in CSF differ among neurodegenerative diseases

We measured the levels of some biological metals: copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) in the cerebrospinal fluid (CSF) in patients with neurodegenerative diseases (52 patients with amyotrophic lateral sclerosis (ALS)), 21 patients with Alzheimer's disease (AD), and 20 patients with Parkinson's disease (PD) by inductively coupled plasma mass spectrometry (ICP-MS). The diagnoses were additionally supported by neuroimaging techniques for AD and PD. In ALS, the levels of Mg (p<0.01 significant difference), Fe, Cu (p<0.05), and Zn (p<0.10) in CSF were higher than those in controls. Some patients showed very high levels of Cu and Zn before the critical deterioration of the disease. In AD, the levels of Cu and Zn in CSF were significantly higher in patients with late-onset AD (p<0.01). In PD, we found significantly increased levels of especially Cu and Zn in particular (p<0.01) and Mn (p<0.05) in CSF. A multiple comparison test suggested that the increased level of Mg in ALS and that of Mn in PD were the pathognomonic features. These findings suggest that Cu and Zn in particular play important roles in the onset and/or progression of ALS, AD, and PD. Therefore, Cu-chelating agents and modulators of Cu and Zn such as metallothionein (MT) can be new candidates for the treatment of ALS, AD, and PD.     

Association Study Between Metallothionein-3 Protein Polymorphisms and Autism

Genetic susceptibility to high mercury body burden has been suggested as an autism risk factor in children. Metallothionein III (MT3) is the brain-specific form of the metallothionein family, which plays a key role in metal metabolism. We therefore looked for genetic variations in the MT3 gene that might increase the predisposition to autism. DNA was extracted from 132 autistic children and 132 age and gender-matched unrelated controls. All the samples were analyzed for nine single nucleotide polymorphisms (SNPs) with minor allele frequency >?10% in the MT3 gene. The mRNA levels of MT3 in white blood cells were evaluated by real-time PCR. We did not detect any association between these MT3 polymorphisms and the mRNA levels of MT3. We did not detect any association between MT3 polymorphisms and autism risk. However, we detected four novel MT3 SNPs that are not in the human SNP database. The clinical importance of these SNPs needs further investigation. Our data suggest that MT3 gene polymorphisms are not associated with autism.     

Regional metal concentrations in Parkinson's disease, other chronic neurological diseases, and control brains

Metal deficiency or toxicity states have been recognized as a cause of several neurological disorders and are suspected in others. We analyzed four brain regions (frontal cortex, caudate nucleus, substantia nigra, and cerebellum) in 36 human brains for concentrations of 24 metals (Ag, Al, As, B, Be, Ca, Cd, Co, Cr, Cu, Fe, K, Pb, Mg, Mn, Mo, Na, Ni, P, Se, Ti, V, W, Zn). Regional metal concentrations, measured using atomic absorption and atomic emission spectroscopy, were compared between 9 Parkinson's disease (PD) brains, 15 brains from patients with other chronic neurological diseases, and 12 control brains. No significant metal concentration differences were noted between brains from PD and other chronic neurologic disease. However, parkinsonian brains (PD and parkinsonism secondary to neurofibrillary tangle disease) showed lower concentrations of magnesium in the caudate nucleus and copper in the substantia nigra than control brains. These findings may represent an etiologically important clue to parkinsonism.