The protective role of sesame oil against Parkinson's-like disease induced by manganese in rats.

Chronic exposure to manganese (Mn) results in motor dysfunction, biochemical and pathological alterations in the brain. Oxidative stress, inflammation, and dysfunction of dopaminergic and GABAergic systems stimulate activating transcription factor-6 (ATF-6) and protein kinase RNA-like ER kinase (PERK) leading to apoptosis. This study aimed to investigate the protective effect of sesame oil (SO) against Mn-induced neurotoxicity. Rats received 25 mg/kg MnCl2 and were concomitantly treated with 2.5, 5, or 8 ml/kg of SO for 5 weeks. Mn-induced motor dysfunction was indicated by significant decreases in the time taken by rats to fall during the rotarod test and in the number of movements observed during the open field test. Also, Mn resulted in neuronal degeneration as observed by histological staining. The striatal levels of lipid peroxides and reduced glutathione (oxidative stress markers), interleukin-6 and tumor necrosis factor-α (inflammatory markers) were significantly elevated. Mn significantly reduced the levels of dopamine and Bcl-2, while GABA, PERK, ATF-6, Bax, and caspase-3 were increased. Interestingly, all SO doses, especially at 8 ml/kg, significantly improved locomotor activity, biochemical deviations and reduced neuronal degeneration. In conclusion, SO may provide potential therapeutic benefits in enhancing motor performance and promoting neuronal survival in individuals highly exposed to Mn.

Preventive treatment with sodium para-aminosalicylic acid inhibits manganese-induced apoptosis and inflammation via the MAPK pathway in rat thalamus.

Excessive exposure to manganese (Mn) may lead to neurotoxicity, referred to as manganism. In several studies, sodium para-aminosalicylic acid (PAS-Na) has shown efficacy against Mn-induced neurodegeneration by attenuating the neuroinflammatory response. The present study investigated the effect of Mn on inflammation and apoptosis in the rat thalamus, as well as the underlying mechanism of the PAS-Na protective effect. The study consisted of sub-acute (Mn treatment for 4 weeks) and sub-chronic (Mn and PAS-Na treatment for 8 weeks) experiments. In the sub-chronic experiments, pro-inflammatory cytokines, namely tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and cyclooxygenase 2 (COX-2) were significantly increased in the Mn-exposed group compared to the control II. PAS-Na treatment led to a significant reduction in the Mn-induced neuroinflammation by inhibiting IL-1ß and COX-2 mRNA expression and reducing IL-1ß secretion and JNK/p38 MAPK pathway activity. Furthermore, immunohistochemical analysis showed that the expression of caspase-3 was significantly increased in both the sub-acute and sub-chronic experimental paradigms concomitant with a significant decrease in B-cell lymphoma 2 (Bcl-2) in the thalamus of Mn-treated rats. PAS-Na also decreased the expression levels of several apoptotic markers downstream of the MAPK pathway, including Bcl-2/Bax and caspase-3, while up-regulating anti-apoptotic Bcl-2 proteins. In conclusion, Mn exposure led to inflammation in the rat thalamus concomitant with apoptosis, which was mediated via the MAPK signaling pathway. PAS-Na treatment antagonized effectively Mn-induced neurotoxicity by inhibiting the MAPK activity in the same brain region.

N-Acetylcysteine Ameliorates Neurotoxic Effects of Manganese Intoxication in Rats: A Biochemical and Behavioral Study.

Clinical and experimental evidences reveal that excess exposure to manganese is neurotoxic and leads to cellular damage. However, the mechanism underlying manganese neurotoxicity remains poorly understood but oxidative stress has been implicated to be one of the key pathophysiological features related to it. The present study investigates the effects associated with manganese induced toxicity in rats and further to combat these alterations with a well-known antioxidant N-acetylcysteine which is being used in mitigating the damage by its radical scavenging activity. The study was designed to note the sequential changes along with the motor and memory dysfunction associated with biochemical and histo-pathological alterations following exposure and treatment for 2 weeks. The results so obtained showed decrease in the body weights, behavioral deficits with increased stress markers and also neuronal degeneration in histo-pathological examination after manganese intoxication in rats. To overcome the neurotoxic effects of manganese, N-acetylcysteine was used in the current study due to its pleiotropic potential in several pathological ailments. Taken together, N-acetylcysteine helped in ameliorating manganese induced neurotoxic effects by diminishing the behavioral deficits, normalizing acetylcholinesterase activity, and augmentation of redox status.

Methionine-Mediated Protein Phosphatase 2A Catalytic Subunit (PP2Ac) Methylation Ameliorates the Tauopathy Induced by Manganese in Cell and Animal Models.

The molecular mechanism of Alzheimer-like cognitive impairment induced by manganese (Mn) exposure has not yet been fully clarified, and there are currently no effective interventions to treat neurodegenerative lesions related to manganism. Protein phosphatase 2 A (PP2A) is a major tau phosphatase and was recently identified as a potential therapeutic target molecule for neurodegenerative diseases; its activity is directed by the methylation status of the catalytic C subunit. Methionine is an essential amino acid, and its downstream metabolite S-adenosylmethionine (SAM) participates in transmethylation pathways as a methyl donor. In this study, the neurotoxic mechanism of Mn and the protective effect of methionine were evaluated in Mn-exposed cell and rat models. We show that Mn-induced neurotoxicity is characterized by PP2Ac demethylation accompanied by abnormally decreased LCMT-1 and increased PME-1, which are associated with tau hyperphosphorylation and spatial learning and memory deficits, and that the poor availability of SAM in the hippocampus is likely to determine the loss of PP2Ac methylation. Importantly, maintenance of local SAM levels through continuous supplementation with exogenous methionine, or through specific inhibition of PP2Ac demethylation by ABL127 administration in vitro, can effectively prevent tau hyperphosphorylation to reduce cellular oxidative stress, apoptosis, damage to cell viability, and rat memory deficits in cell or animal Mn exposure models. In conclusion, our data suggest that SAM and PP2Ac methylation may be novel targets for the treatment of Mn poisoning and neurotoxic mechanism-related tauopathies.

Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity.

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week-old YY1 flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 µg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.

Activation of protein kinase R in the manganese-induced apoptosis of PC12 cells.

Manganese neurotoxicity leads to Parkinson-like symptoms associated with the apoptotic cell death of dopaminergic neurons. Protein kinase R (PKR) is a serine/threonine-specific protein kinase that has been implicated in several cellular signal transduction pathways, including the induction of apoptosis. Here, we investigated the role of PKR in the manganese-induced apoptosis of dopamine-producing pheochromocytoma PC12 cells. Manganese (0.5 mM) induced the proteolytic cleavage of PKR and caspase-3, DNA fragmentation, and cell death, which were prevented by the co-treatment of PC12 cells with a PKR specific inhibitor, C16 in a concentration-dependent manner. C16 did not affect the manganese-induced activation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) pathway, indicating that PKR functions downstream of JNK and p38 MAPK. In contrast, C16 triggered the activation of the p44/42 MAPK (ERK1/2) pathway and induced hemoxygenase-1, both in the absence and presence of manganese. PKR is reportedly involved in endoplasmic reticulum (ER) stress-induced apoptosis. Manganese activated all three branches of the unfolded protein response in PC12 cells; however, this effect was very weak compared with the ER stress induced by the well-known ER stress inducers thapsigargin and tunicamycin. Moreover, C16 did not affect manganese-induced ER stress at concentrations that almost prevented caspase-3 activation and DNA fragmentation. These results suggest that PKR is involved in manganese-induced apoptotic cell death and stress response, such as the activation of the p44/42 MAPK pathway and the induction of hemoxygenase-1. Although manganese induced a faint, but typical, ER stress, these events contributed little to manganese-induced apoptosis.

The role of zinc, copper, manganese and iron in neurodegenerative diseases.

Metals are involved in different pathophysiological mechanisms associated with neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), Parkinson's disease (PD) and multiple sclerosis (MS). The aim of this study was to review the effects of the essential metals zinc (Zn), copper (Cu), manganese (Mn) and iron (Fe) on the central nervous system (CNS), as well as the mechanisms involved in their neurotoxicity. Low levels of Zn as well as high levels of Cu, Mn, and Fe participate in the activation of signaling pathways of the inflammatory, oxidative and nitrosative stress (IO&NS) response, including nuclear factor kappa B and activator protein-1. The imbalance of these metals impairs the structural, regulatory, and catalytic functions of different enzymes, proteins, receptors, and transporters. Neurodegeneration occurs via association of metals with proteins and subsequent induction of aggregate formation creating a vicious cycle by disrupting mitochondrial function, which depletes adenosine triphosphate and induces IO&NS, cell death by apoptotic and/or necrotic mechanisms. In AD, at low levels, Zn suppresses ß-amyloid-induced neurotoxicity by selectively precipitating aggregation intermediates; however, at high levels, the binding of Zn to ß-amyloid may enhance formation of fibrillar ß-amyloid aggregation, leading to neurodegeneration. High levels of Cu, Mn and Fe participate in the formation α-synuclein aggregates in intracellular inclusions, called Lewy Body, that result in synaptic dysfunction and interruption of axonal transport. In PD, there is focal accumulation of Fe in the substantia nigra, while in AD a diffuse accumulation of Fe occurs in various regions, such as cortex and hippocampus, with Fe marginally increased in the senile plaques. Zn deficiency induces an imbalance between T helper (Th)1 and Th2 cell functions and a failure of Th17 down-regulation, contributing to the pathogenesis of MS. In MS, elevated levels of Fe occur in certain brain regions, such as thalamus and striatum, which may be due to inflammatory processes disrupting the blood-brain barrier and attracting Fe-rich macrophages. Delineating the specific mechanisms by which metals alter redox homeostasis is essential to understand the pathophysiology of AD, PD, and MS and may provide possible new targets for their prevention and treatment of the patients affected by these NDDs.

Metabolomic Responses to Manganese Dose in SH-SY5Y Human Neuroblastoma Cells.

Manganese (Mn)-associated neurotoxicity has been well recognized. However, Mn is also an essential nutrient to maintain physiological function. Our previous study of human neuroblastoma SH-SY5Y cells showed that Mn treatment comparable to physiological and toxicological concentrations in human brain resulted in different mitochondrial responses, yet cellular metabolic responses associated with such different outcomes remain uncharacterized. Herein, SH-SY5Y cells were examined for metabolic responses discriminated by physiological and toxicological levels of Mn using high-resolution metabolomics (HRM). Before performing HRM, we examined Mn dose (from 0 to100 µM) and time effects on cell death. Although we did not observe any immediate cell death after 5 h exposure to any of the Mn concentrations assessed (0-100 µM), cell loss was present after a 24-h recovery period in cultures treated with Mn ≥ 50 µM. Exposure to Mn for 5 h resulted in a wide range of changes in cellular metabolism including amino acids (AA), neurotransmitters, energy, and fatty acids metabolism. Adaptive responses at 10 µM showed increases in neuroprotective AA metabolites (creatine, phosphocreatine, phosphoserine). A 5-h exposure to 100 µM Mn, a time before any cell death occurred, resulted in decreases in energy and fatty acid metabolites (hexose-1,6 bisphosphate, acyl carnitines). The results show that adjustments in AA metabolism occur in response to Mn that does not cause cell death while disruption in energy and fatty acid metabolism occur in response to Mn that results in subsequent cell death. The present study establishes utility for metabolomics analyses to discriminate adaptive and toxic molecular responses in a human in vitro cellular model that could be exploited in evaluation of Mn toxicity.

Heme Oxygenase-1 protects astroglia against manganese-induced oxidative injury by regulating mitochondrial quality control.

Heme Oxygenase-1 (HO-1), a stress- responsive enzyme which catalyzes heme degradation into iron, carbon monoxide, and biliverdin, exerts a neuroprotective role involving many different signaling pathways. In Parkinson disease patients, elevated HO-1 expression levels in astrocytes are involved in antioxidant defense. In the present work, employing an in vitro model of Mn2+-induced Parkinsonism in astroglial C6 cells, we investigated the role of HO-1 in both apoptosis and mitochondrial quality control (MQC). HO-1 exerted a protective effect against Mn2+ injury. In fact, HO-1 decreased both intracellular and mitochondrial reactive oxygen species as well as the appearance of apoptotic features. Considering that Mn2+ induces mitochondrial damage and a defective MQC has been implicated in neurodegenerative diseases, we hypothesized that HO-1 could mediate cytoprotection by regulating the MQC processes. Results obtained provide the first evidence that the beneficial effects of HO-1 in astroglial cells are mediated by the maintenance of both mitochondrial fusion/fission and biogenesis/mitophagy balances. Altogether, our data demonstrate a pro-survival function for HO-1 in Mn2+-induced apoptosis that involves the preservation of a proper MQC. These findings point to HO-1 as a new therapeutic target linked to mitochondrial pathophysiology in Manganism and probably Parkinson´s disease.

Role of Caenorhabditis elegans AKT-1/2 and SGK-1 in Manganese Toxicity.

Excessive levels of the essential metal manganese (Mn) may cause a syndrome similar to Parkinson's disease. The model organism Caenorhabditis elegans mimics some of Mn effects in mammals, including dopaminergic neurodegeneration, oxidative stress, and increased levels of AKT. The evolutionarily conserved insulin/insulin-like growth factor-1 signaling pathway (IIS) modulates worm longevity, metabolism, and antioxidant responses by antagonizing the transcription factors DAF-16/FOXO and SKN-1/Nrf-2. AKT-1, AKT-2, and SGK-1 act upstream of these transcription factors. To study the role of these proteins in C. elegans response to Mn intoxication, wild-type N2 and loss-of-function mutants were exposed to Mn (2.5 to 100 mM) for 1 h at the L1 larval stage. Strains with loss-of-function in akt-1, akt-2, and sgk-1 had higher resistance to Mn compared to N2 in the survival test. All strains tested accumulated Mn similarly, as shown by ICP-MS. DAF-16 nuclear translocation was observed by fluorescence microscopy in WT and loss-of-function strains exposed to Mn. qRT-PCR data indicate increased expression of γ-glutamyl cysteine synthetase (GCS-1) antioxidant enzyme in akt-1 mutants. The expression of sod-3 (superoxide dismutase homologue) was increased in the akt-1 mutant worms, independent of Mn treatment. However, dopaminergic neurons degenerated even in the more resistant strains. Dopaminergic function was evaluated with the basal slowing response behavioral test and dopaminergic neuron integrity was evaluated using worms expressing green fluorescent protein (GFP) under the dopamine transporter (DAT-1) promoter. These results suggest that AKT-1/2 and SGK-1 play a role in C. elegans response to Mn intoxication. However, tissue-specific responses may occur in dopaminergic neurons, contributing to degeneration.

Association of exposure to manganese and iron with relaxation rates R1 and R2*- magnetic resonance imaging results from the WELDOX II study.

OBJECTIVE: Magnetic resonance imaging is a non-invasive method that allows the indirect quantification of manganese (Mn) and iron (Fe) accumulation in the brain due to their paramagnetic features. The WELDOX II study aimed to explore the influence of airborne and systemic exposure to Mn and Fe on the brain deposition using the relaxation rates R1 and R2* as biomarkers of metal accumulation in regions of interest in 161 men, including active and former welders. MATERIAL AND METHODS: We obtained data on the relaxation rates R1 and R2* in regions that included structures within the globus pallidus (GP), substantia nigra (SN), and white matter of the frontal lobe (FL) of both hemispheres, as well as Mn in whole blood (MnB), and serum ferritin (SF). The study subjects, all male, included 48 active and 20 former welders, 41 patients with Parkinson's disease (PD), 13 patients with hemochromatosis (HC), and 39 controls. Respirable Mn and Fe were measured during a working shift for welders. Mixed regression models were applied to estimate the effects of MnB and SF on R1 and R2*. Furthermore, we estimated the influence of airborne Mn and Fe on the relaxation rates in active welders. RESULTS: MnB and SF were significant predictors of R1 but not of R2* in the GP, and were marginally associated with R1 in the SN (SF) and FL (MnB). Being a welder or suffering from PD or HC elicited no additional group effect on R1 or R2* beyond the effects of MnB and SF. In active welders, shift concentrations of respirable Mn>100µg/m3 were associated with stronger R1 signals in the GP. In addition to the effects of MnB and SF, the welding technique had no further influence on R1. CONCLUSIONS: MnB and SF were significant predictors of R1 but not of R2*, indicative of metal accumulation, especially in the GP. Also, high airborne Mn concentration was associated with higher R1 signals in this brain region. The negative results obtained for being a welder or for the techniques with higher exposure to ultrafine particles when the blood-borne concentration was included into the models indicate that airborne exposure to Mn may act mainly through MnB.

Excess Manganese-Induced Apoptosis in Chicken Cerebrums and Embryonic Neurocytes.

There were many studies about the effect of excess manganese (Mn) on nervous system apoptosis; however, Mn-induced apoptosis in chicken cerebrums and embryonic neurocytes was unclear. The purpose of this study was to investigate the effect of excess Mn on chicken cerebrum and embryonic neurocyte apoptosis. Seven-day-old Hyline male chickens were fed either a commercial diet or three levels of manganese chloride (MnCl2)-added commercial diets containing 600-, 900-, and 1800-mg/kg-Mn diet, respectively. On the 30th, 60th, and 90th days, cerebrums were collected. Fertilized Hyline chicken eggs were hatched for 6-8 days and were selected. Embryonic neurocytes with 0, 0.5, 1, 1.5, 2, 2.5, and 3 mM Mn were collected and were cultured for 12, 24, 36, and 48 h, respectively. The following research contents were performed: superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities; tumor protein p53 (p53), B cell lymphoma-2 (Bcl-2), B cell lymphoma extra large (Bcl-x), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), fas, and caspase-3 messenger RNA (mRNA) expression; and morphologic observation. The results indicated that excess Mn inhibited SOD and T-AOC activities; induced p53, Bax, Bak, fas, and caspase-3 mRNA expression; and inhibited Bcl-2 and Bcl-x mRNA expression in chicken cerebrums and embryonic neurocytes. There were dose-dependent manners on all the above factors at all the time points and time-dependent manners on SOD activity of 1800-mg/kg-Mn group, T-AOC activity, and apoptosis-related gene mRNA expression in all the treatment groups in chicken cerebrums. Excess Mn induced chicken cerebrum and embryonic neurocyte apoptosis.

Effects of chronic manganese exposure on the learning and memory of rats by observing the changes in the hippocampal cAMP signaling pathway.

Chronic manganese exposure can produce cognitive deficits; however, the underlying mechanism remains unclear; reliable peripheral biomarker of Mn neurotoxicity have not yet been fully developed. Hence, this study aimed to investigate the mechanism of Mn-induced cognitive deficits and the potential biomarker of Mn neurotoxicity in rats. Thirty-two male Sprague Dawley rats were divided into four groups; these groups received intraperitoneal injections of 0, 5, 10 and 20 mg Mn/kg once daily, five days/week for 18 weeks. Learning and memory were assessed via Morris water maze test. Hippocampal and plasma Mn concentrations were measured through graphite furnace atomic absorption spectrometry. The levels of plasma BDNF, hippocampal BDNF, cAMP, protein kinase A, and pCREB were assessed through ELISA or Western blot. Results showed that the Mn concentrations in the hippocampus and plasma of the Mn-treated rats were higher than those of the control rats. Mn exposure impaired the learning and memory of rats. Plasma BDNF levels and hippocampal BDNF, cAMP, protein kinase A, and pCREB levels were significantly lower in the Mn-treated rats than in the control rats. Plasma BDNF levels were negatively correlated with the escape latency and the hippocampal and plasma Mn concentrations. By contrast, plasma BDNF levels were positively correlated with the number of platform crossings and the hippocampal cAMP and BDNF levels. Therefore, Mn impaired learning and memory probably by inhibiting the hippocampal cAMP signaling pathway in rats. Plasma BDNF levels may also be a potential effect biomarker of Mn neurotoxicity.

Involvement of dysregulated Wip1 in manganese-induced p53 signaling and neuronal apoptosis.

Overexposure to manganese (Mn) has been known to induce neuronal death and neurodegenerative symptoms. However, the precise mechanisms underlying Mn neurotoxicity remain incompletely understood. In the present study, we established a Mn-exposed rat model and found that downregulation of wild type p53-induced phosphatase 1 (Wip1) might contribute to p53 activation and resultant neuronal apoptosis following Mn exposure. Western blot and immunohistochemical analyses revealed that the expression of Wip1 was markedly decreased following Mn exposure. In addition, immunofluorescence assay demonstrated that Mn exposure led to significant reduction in the number of Wip1-positive neurons. Accordingly, the expression of Mdm2 was progressively decreased, which was accompanied with markedly increased expression of p53, as well as the ratio of Bax/Bcl-xl. Furthermore, we showed that Mn exposure decreased the viability and induced apparent apoptosis in NFG-differentiated neuron-like PC12 cells. Importantly, the expression of Wip1 decreased progressively, whereas the level of cellular p53 and the ratio of Bax/Bcl-xl were elevated, which resembled the expression of the proteins in animal model studies. Depletion of p53 significantly ameliorated Mn-mediated cytotoxic effect in PC12 cells. In addition, ectopic expression of Wip1 attenuated Mn-induced p53 signaling as well as apoptosis in PC12 cells. Finally, we observed that depletion of Wip1 augmented Mn-induced apoptosis in PC12 cells. Collectively, these findings suggest that downregulated Wip1 expression plays an important role in Mn-induced neuronal death in the brain striatum via the modulation of p53 signaling.

Relationship between brain accumulation of manganese and aberration of hippocampal adult neurogenesis after oral exposure to manganese chloride in mice.

We previously found persistent aberration of hippocampal adult neurogenesis, along with brain manganese (Mn) accumulation, in mouse offspring after developmental exposure to 800-ppm dietary Mn. Reduction of parvalbumin (Pvalb)(+) γ-aminobutyric acid (GABA)-ergic interneurons in the hilus of the dentate gyrus along with promoter region hypermethylation are thought to be responsible for this aberrant neurogenesis. The present study was conducted to examine the relationship between the induction of aberrant neurogenesis and brain Mn accumulation after oral Mn exposure as well as the responsible mechanism in young adult animals. We used two groups of mice with 28- or 56-day exposure periods to oral MnCl2·xH2O at 800 ppm as Mn, a dose sufficient to lead to aberrant neurogenesis after developmental exposure. A third group of mice received intravenous injections of Mn at 5-mg/kg body weight once weekly for 28 days. The 28-day oral Mn exposure did not cause aberrations in neurogenesis. In contrast, 56-day oral exposure caused aberrations in neurogenesis suggestive of reductions in type 2b and type 3 progenitor cells and immature granule cells in the dentate subgranular zone. Brain Mn accumulation in 56-day exposed cases, as well as in directly Mn-injected cases occurred in parallel with reduction of Pvalb(+) GABAergic interneurons in the dentate hilus, suggesting that this may be responsible for aberrant neurogenesis. For reduction of Pvalb(+) interneurons, suppression of brain-derived neurotrophic factor-mediated signaling of mature granule cells may occur via suppression of c-Fos-mediated neuronal plasticity due to direct Mn-toxicity rather than promoter region hypermethylation of Pvalb.

Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis.

Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-µ (PFT-µ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-µ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H2O2 production. Finally, we observed that pretreatment with PFT-α and PFT-µ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death.

The outcome of the movement disorder in methcathinone abusers: clinical, MRI and manganesemia changes, and neuropathology.

BACKGROUND AND PURPOSE: There is limited knowledge regarding the long-term outcome of the methcathinone/manganese-induced movement disorder. Our purpose was to define prognosis in intravenous methcathinone abusers affected by this distinctive disorder attributed to manganese (Mn) toxicity. Also, neuropathology from a globus pallidus region biopsy from a former user is reported. METHODS: Eighteen methcathinone abusers were categorized as active (five), discontinued (four) or former (nine) users. They were reassessed after a median of 32.5 months (range 3.4-59.6) clinically, on rating scales, and with MRI and blood Mn levels. The biopsy was examined ultrastructurally. RESULTS: Overall the group showed a slight tendency to deterioration at follow-up on clinical assessment of motor functioning, especially the active users. No significant change occurred on parkinsonian rating scale reassessment. Significant reduction in Mn levels occurred in former users, and decreased T1-weighted hyperintensity on basal ganglia MRI occurred in 3 of 4 former and 2 of 3 discontinued users, despite lack of clinical improvement. The biopsy consisted of white matter showing decompacted myelin sheaths and frequent abnormalities of mitochondria. CONCLUSIONS: No improvement in this Mn-induced movement disorder occurs after cessation of methcathinone abuse despite improvement of Mn blood levels and/or MRI abnormalities. Ultrastructural abnormalities in a former user confirm structural damage to white matter is associated with the disorder. Methcathinone/Mn toxicity is an important, disabling and permanent medical sequel of intravenous drug abuse in the former Soviet Union.

Evaluation of neurobehavioral and neuroinflammatory end-points in the post-exposure period in rats sub-acutely exposed to manganese.

Manganese (Mn) can cause manganism, a neurological disorder similar to Parkinson' Disease (PD). The neurobehavioral and neuroinflammatory end-points in the Mn post exposure period have not been studied yet. Rats were injected on alternate days with 8 doses of MnCl2 (25mg/kg) or saline, then euthanized 1, 10, 30 or 70 days following the last dose. Whole-blood (WB) (p<0.05), urine (p<0.05) and brain cortical (p<0.0001) Mn levels were significantly increased 24h after the last dose. Decreases in the rats' ambulation were noted 1, 10 and 30 days after the last Mn dose (p<0.001; p<0.05; p<0.001, respectively) and also in the rearing activity at the four time-points (p<0.05). Cortical glial fibrillary acid protein immunoreactivity (GFAP-ir) was significantly increased at 1, 10, 30 (p<0.0001) and 70 (p<0.001) days after the last Mn dose, as well as tumor necrosis α (TNF-α) levels (p<0.05) but just on day 1. Taken together, the results show that, during the 70-day clearance phase of Mn, the recovery is not immediate as behavioral alterations and neuroinflammation persist long after Mn is cleared from the cortical brain compartment.

Oxidative stress involvement in manganese-induced alpha-synuclein oligomerization in organotypic brain slice cultures.

Overexposure to manganese (Mn) has been known to induce neuronal damage. However, little is known of the role that reactive oxygen species (ROS) play in protein aggregation resulting from Mn exposure. The current study investigated whether oxidative stress is involved in manganese-induced alpha-synuclein oligomerization in organotypic brain slices. After application of Mn (0-400µM) for 24h, there was a dose-dependent increase in average percentage of propidium iodide positive (PI(+)) nuclei in slices and levels of lactate dehydrogenase (LDH) in the culture medium. Moreover, the treatment with Mn resulted in a dose-dependent increase in neurocyte apoptosis, ROS level, and decrease in superoxide dismutase (SOD) activity. Mn also caused oxidative damage in cell lipid and protein. At the same time, the exposure of Mn leaded to significantly increase in the expression of alpha-synuclein mRNA and protein. Alpha-synuclein oligomerization occurred in Mn-treated slices, especially on membrane-bound form. It indicated that alpha-synuclein oligomers were more likely to combination cell membranes and resulting in membrane damage. Mn-induced neurocyte damage and alpha-synuclein oligomerization were also partially alleviated by the pretreatment with GSH and aggravated by H2O2 pretreatment. The findings revealed Mn might exert its neurotoxic effects by oxidative stress-mediated alpha-synuclein oligomerization in organotypic brain slices.

Manganese-induced oxidative DNA damage in neuronal SH-SY5Y cells: attenuation of thymine base lesions by glutathione and N-acetylcysteine.

Manganese (Mn) is an essential trace element required for normal function and development. However, exposure to this metal at elevated levels may cause manganism, a progressive neurodegenerative disorder with neurological symptoms similar to idiopathic Parkinson's disease (IPD). Elevated body burdens of Mn from exposure to parental nutrition, vapors in mines and smelters and welding fumes have been associated with neurological health concerns. The underlying mechanism of Mn neurotoxicity remains unclear. Accordingly, the present study was designed to investigate the toxic effects of Mn(2+) in human neuroblastoma SH-SY5Y cells. Mn(2+) caused a concentration dependent decrease in SH-SY5Y cellular viability compared to controls. The LD50 value was 12.98 µM Mn(2+) (p<0.001 for control vs. 24h Mn treatment). Both TUNEL and annexin V/propidium iodide (PI) apoptosis assays confirmed the induction of apoptosis in the cells following exposure to Mn(2+) (2 µM, 62 µM or 125 µM). In addition, Mn(2+) induced both the formation and accumulation of DNA single strand breaks (via alkaline comet assay analysis) and oxidatively modified thymine bases (via gas chromatography/mass spectrometry analysis). Pre-incubation of the cells with characteristic antioxidants, either 1mM N-acetylcysteine (NAC) or 1mM glutathione (GSH) reduced the level of DNA strand breaks and the formation of thymine base lesions, suggesting protection against oxidative cellular damage. Our findings indicate that (1) exposure of SH-SY5Y cells to Mn promotes both the formation and accumulation of oxidative DNA damage, (2) SH-SY5Y cells with accumulated DNA damage are more likely to die via an apoptotic pathway and (3) the accumulated levels of DNA damage can be abrogated by the addition of exogenous chemical antioxidants. This is the first known report of Mn(2+)-induction and antioxidant protection of thymine lesions in this SH-SY5Y cell line and contributes new information to the potential use of antioxidants as a therapeutic strategy for protection against Mn(2+)-induced oxidative DNA damage.