“Manganese-induced neurotoxicity: a review of its behavioral consequences and neuroprotective strategies”

Membrane localized Mn importers

Membrane importers are the primary route of Mn transport into the CNS. These transporters include the divalent metal transporter 1 (DMT1), Zrt-like, Irt-like proteins ZIP8 (SLC39A8) and ZIP14 (SLC39A14), dopamine transporter (DAT), voltage-regulated, store-operated and ionotropic glutamate receptor Ca channels, choline transporters and the citrate transporter [49, 50]. These proteins are localized on cell membranes and are able to form a membrane pore to take up divalent Mn from the extracellular matrix. Moreover, Mn may block transient receptor potential channel (TRPC3), a receptor-operated plasma membrane channel of astrocytes that responds to ATP-induced Ca signaling, thus decreasing purinergic signaling [51].

DMT1 is the most representative and best studied one. It is also known as divalent cation transporter 1 (DCT1), natural resistance-associated macrophage protein 2 (NRAMP 2) or solute carrier family 11 member 2 (SLC11A2). Gunshin et al. (1997), first cloned and characterized DMT1 with a wide range of substrates, including Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Co²⁺, Cd²⁺, Ni²⁺ and Pb²⁺ [52]. Garrick et al. (2006), showed that Mn is DMT1 preferred substrate, with the following transport affinity (reflecting transport efficacy): Mn > Cd > Fe > Pb ~ Co ~ Ni > Zn [53]. Thus, although Fe has also been linked to PD pathology, Mn might play a more prominent role in this disease given its higher affinity for DMT1. In the brain, DMT1 is highly expressed in the basal ganglia, including SN, GP, hypothalamic nucleus and striatum [54–56], rendering these regions more susceptible to Mn accumulation and toxicity. DMT1 regulates Mn influx into neurons by two ways. One is via a direct transport mechanism whereby the membrane-localized DMT1 opens up a pore and allows the extracellular divalent Mn to enter neurons. The other way is via a transferrin (Tf)-dependent process, which will be discussed next.

Transferrin (Tf) and transferrin receptor (TfR)

While the majority of Mn in the body is in the divalent oxidation state, there is a small amount of trivalent Mn, which is not a substrate for the above referenced importers. Tf/TfR facilitates Mn³⁺ influx into the CNS from the blood stream [57]. Tf is synthesized in the liver and then released into the blood [58]. Mn exposure increases the expression of TfR by enhancing the binding of iron regulatory proteins (IRPs) to iron responsive element-containing RNA in vitro [59]. TfR is a membrane protein with high affinity for Mn, which is expressed in neurons, microglia, astrocytes and the endothelial cells of the BBB [60]. When TfR recognizes and binds to Tf, the cell membrane expands inwardly and forms an endocytic vesicle, which brings in the Mn [67, 74]. Mn³⁺ is a stronger oxidizing agent than Mn²⁺ and it may cause severe oxidative stress. Ferrireductase reduces Mn³⁺ into Mn²⁺, which is released into the cytosol by DMT1 localized on the endosomal membrane [50].

Mn export in the CNS

Efflux plays a fundamental role in regulating intracellular concentrations of Mn in the CNS. Compared with Mn import, efflux of Mn is less studied, partially due to limited proteins identified in Mn export. However, with the recent discovery of four proteins facilitating Mn export, the role of Mn export has begun to be elucidated. These four proteins include ferroportin (Fpn), SLC30A10 (solute carrier family 30 member 10), secretory pathway Ca²⁺-ATPase 1 (SPCA1) and ATPase 13A2 (ATP13A2 or PARK9). Among them, Fpn and SLC30A10 are able to directly export cytosolic Mn out of neurons, while SPCA1 and ATP13A2 indirectly regulate Mn efflux through the Golgi apparatus and lysosome, respectively. Together, these proteins maintain Mn homeostasis in the CNS and mutations in them have been associated with certain diseases.

Membrane localized Mn exporters

Currently, these exporters include Fpn and SCL30A10. Fpn was the first known Mn exporter, however, it was first identified as a Fe exporter. And that is why it is also known as iron-regulated transporter 1 or solute carrier family 40 member 1 (SLC40A1). In the brain, Fpn has been found in neurons, astrocytes, the endothelial cells of the BBB, oligodendrocytes, the choroid plexus and ependymal cells [61]. Fpn expression levels are increased in mice and human embryonic kidney cells in the presence of Mn [62]. Xenopus laevis oocytes expressing human Fpn showed lower intracellular Mn and higher extracellular Mn [63]. Although these results indicate Fpn may play an important role on Mn homeostasis in the CNS, a direct study to investigate brain Mn levels in human or animal models carrying Fpn mutations has not been reported yet.

Interestingly, the recently identified SLC30A10 has been well known to play a critical role in regulating CNS Mn homeostasis. Currently, it is the only known protein associated with the first hereditary or familial form of Mn-induced parkinsonism. People carrying mutations in SLC30A10 suffer from hypermanganesemia with dystonia, polycythemia and hepatic cirrhosis [22, 64, 65]. The patients have ~10-fold increase in blood Mn levels and magnetic resonance imaging (MRI) studies show high levels of Mn accumulated in the basal ganglia without a history of exposure to elevated Mn from environmental or occupational sources [66]. The mechanisms by which mutations in SLC30A10 mediate Mn accumulation were recently characterized in rat-derived differentiated γ-aminobutyric acid (GABA) ergic AF5 cells, primary mice midbrain neurons and C. elegans. Leyva-Illades, Chen et al. (2014), found that wild type (WT) SLC30A10 is localized on the cell membrane, while 5 mutant transporters are all trapped in the endoplasmic reticulum (ER) or in the cytoplasm [67]. While the WT protein is able to protect from Mn induced DAergic neurodegeneration and cell toxicity, the mislocalization deprives these mutants of this essential efflux with ensuing retention of high Mn concentrations in the plasma.

Mn efflux mediated by SPCA1 and ATP13A2

SPCA1 is a Golgi-localized Ca/Mn ion pump, which belongs to the P-type ATPase family, with highest expression in keratinocytes but also in other tissues including liver and brain [68]. In HeLa cells, SPCA1 is required for transport of Mn into the Golgi, followed by secretion via exocytosis as a bona-fide Mn efflux pathway [69].

ATP13A2 (PARK9) is a transmembrane cation transporting ATPase localized on the membrane of vacuoles and lysosomes [67]. ATP13A2 has been associated with early-onset parkinsonism and Kufor-Rakeb syndrome [70–72]. In primary rat neurons, ATP13A2 levels were increased in the presence of excess Mn, while expression of wild-type ATP13A2 lowered intracellular Mn levels and prevented Mn-induced neuronal death [73].

Recently, a high throughput screening approach was carried out to identify small molecules responsible for intracellular regulation of Mn homeostasis at physiologically relevant levels. It’s suggested that intracellular Mn levels are actively controlled by the cell and not exclusively by the BBB or blood-cerebrospinal fluid barrier. Furthermore, mechanisms regulating Mn content might be developmentally regulated in DAergic neurons reflecting the changing physiological demand [74].

Mn exposure by inhalation in occupational settings

It is estimated that over one million workers in the USA perform welding as part of their job. The pipes used in heating and ventilation systems as well as industrial process piping often require welding, which is also essential for ductwork, laboratory hoods, tanks, boilers, and process vessels. Welding produces respirable fumes that may contain Mn as well as other chemicals, such as chromium, arsenic, iron and nickel. The level of Mn exposure varies dependent upon the type of welding activity performed, ranging from 0.01 to 2.0 mg/m³ [116]. In contrast, the world health organization (WHO) recommends that levels of Mn do not exceed 30 μg/m³. It has been demonstrated that the use of ventilation systems reduces these values and could be an effective approach to minimize Mn exposure [116].

Using rats to model Mn exposure via inhalation, it has been demonstrated that the inhalation route is more efficient than ingestion at delivering Mn to the brain [117]. Mn is taken up via the olfactory tract and transferred along olfactory neurons processes through the cribriform plate to synaptic junctions with olfactory bulb neurons, thus bypassing the BBB. Once in the brain, Mn can continue to traverse synapses and be transported along neuronal tracts to other sites of the brain [118, 119]. Furthermore, the accumulation of Mn in the blood after intranasal instillation is much greater than via the oral route because Mn bypasses the biliary excretion [120]. DMT-1 is important for Mn transport across the olfactory epithelium into the brain of rats and can be influenced by Fe status [121]. Other transporters may regulate Mn uptake from the olfactory epithelium. Candidates are SLC30A10 or Mn binding proteins [120]. DMT-1 also plays a role in lung uptake of inhaled Mn [122].

Several studies point to a strong correlation between occupational Mn exposure and an increased risk of PD [123]. Parkinsonian symptoms in welders attributed to Mn exposure have been reported in numerous studies. A statistically significant difference in the age of PD onset between welders (46 years of age) and a control group (63 years) [124] has been noted. Alpha-synuclein (α-Syn), the major component of Lewy bodies and the hallmark of PD, contains metal binding sites, and its activity is not yet fully understood. It has been proposed that α-Syn attenuates Mn-induced DAergic degeneration in the early stage, but after prolonged exposure, Mn promotes α-Syn aggregation [125]. In C. elegans, α-Syn attenuates Mn-induced toxicity in the background of PD-associated genes [126]. Recently, α-Syn has been proposed to act as a intracellular Mn store [127].

Because of its paramagnetic properties, Mn accumulation can be visualized using T1-weighted magnetic resonance imaging (MRI) [128]. In a study of 193 subjects exposed to welding activities from the Midwestern USA it was shown that Mn accumulates throughout the basal ganglia, with a diffused T1 signal as well as elevated blood Mn levels when compared to age and gender matched controls. However, it was found that the MRI data not always correlated with clinical symptomatology [129, 130]. This may occur because modern occupational exposure to Mn occurs at much lower levels than reported in the past, resulting in a less distinguishable clinical phonotype. Even asymptomatic welder apprentices display increased T1 signal in the basal ganglia, but when evaluated in the Grooved Pegboard (for dexterity and fine motor control) or the unified PD rating scale motor subsection 3 (UPDRS3-for parkinsonian signs such as rest and postural tremor, bradykinesia and gait disturbance), the subjects performed within the reference range [131]. Nevertheless important neuropathological alterations have been observed even in the absence of motor symptoms [129, 132, 133]. It is not clear from the clinical studies, however, whether Mn facilitates the development of PD or induces a distinct parkinsonian syndrome. Future studies should address this issue by clearly diagnosing either PD or manganism based on the known distinctions between the two diseases.

To better understand the significance of MRI findings, an ex vivo study correlated imaging with neuropathology in 19 mine workers and 10 race- and sex-matched controls from South Africa (where 80 % of the world’s Mn reserves are located). It was found an inverse relationship between the T1 intensity indices and neuronal density in the caudate and putamen, suggesting neuronal loss. The authors also noted increased microglial cell density in the basal ganglia. Based on this and their previous study [133] they propose that the pre-clinical stage of Mn-induced neurotoxicity is marked by an initial inflammatory response that may progress to astrocyte isruption and neuronal injury [132]. This would be in agreement with in vitro findings that report a 50 fold higher accumulation of Mn in astrocytes, which may alter their neurotrophic actions and contribute no neuronal injury [134–137]. Astrocytes are initially affected in manganism showing changes in the expression of glial fibrilary acidic protein (GFAP) preceding neuronal death [138]. Increased GFAP expression is observed in the striatum of rats, which indicates glial activation in response to Mn [139, 140]. Microglial cells are also affected by Mn with increased release of proinflammatory cytokines [134] and may activate astrocytes to release inflammatory mediators such as prostaglandin E2 and nitric oxide [141].

Environmental Mn exposure

Contaminated air or water pose a risk of Mn intoxication to the general population. Mn exposure from environmental sources has also been associated with a higher prevalence of Parkinsonian disturbances [142]. For instance, near foundries, Mn concentrations may reach 200–300 ng/m³, contrasting to normal levels of Mn in the air that are around 10–30 ng/m³ according to the WHO. Recently, a study by Bowler et al. (2015) was performed to assess cognitive function in adults environmentally exposed to Mn in Ohio, USA, in two towns identified as having high levels of air-Mn from industrial sources. The authors report that non-occupational environmental Mn exposure appears to be associated with lower performance on neuropsychological tests measuring a variety of cognitive functions [143].

North America’s longest operating ferromanganese refinery is located in Marietta, Ohio, USA. To address the population leading environmental public health concern, a study was conducted to evaluate children’s cognitive function. It was found that both high and low blood and hair levels of Mn could negatively impact children’s IQ, consistent with the notion that Mn is both a nutrient and a neurotoxicant. Of note, lead (Pb) and cotinine (a nicotine metabolite) were also measured in children’s blood, serum or hair since environmental exposures to toxic chemicals rarely occur isolated. Pb levels in blood of that study population were similar to mean blood Pb of children in the USA and did not influence IQ scores. Cotinine levels were significantly associated with IQ scores, demonstrating that secondhand tobacco smoke can negatively impact child cognitive function [144]. Airborne Mn also detrimentally influenced children’s postural stability in this population [145]. Mn has been identified as a developmental neurotoxicant associated with hyperactivity, lower intellectual function, impaired motor skills and reduced olfactory function in children [146, 147]. In animal models, the immature CNS is more susceptible to Mn neurotoxicity compared to the adult [148] and experimental evidence suggests that exposure to this metal during development can affect neurological function in adulthood [139, 140, 149, 150].

The presence of excessive Mn levels in drinking water has been associated with poorer memory and attention [14], and hyperactive behavior [151] in school-aged children. Consumption of water containing elevated Mn levels had adverse effects on 10-year-old children’s cognitive function [152]. Children exposed to elevated airborne Mn in an area close to a ferromanganese alloy plant in Brazil presented lower I.Q., impairment of verbal skills [153] and lower neuropsychological performance in tests of executive function of inhibition responses, strategic visual formation and verbal working memory [154].

Mn and parenteral nutrition

Mn is present in parenteral nutrition formulations both as an essential element but also as a contaminant, thus posing as an important source of excessive exposure to Mn. The content of Mn in TPN varies from 0.18 μmol/d (0.01 mg/d) to 40 μmol/d (2.2 mg/d) [21]. Toxicity to Mn has been observed in adults receiving >500 μg/d and in pediatric patients receiving >40 μg/kg/d. Furthermore, duration of TPN treatment is associated with increased blood and brain concentrations of Mn [155–157]. Thus, current guidelines recommend monitoring patients for Mn toxicity if they receive TPN longer than 30 days [158].

Parenteral administration bypasses the regulatory mechanisms of the gastrointestinal tract. The bioavailability of Mn in parenteral fluid is 100 %, compared to only 5 % for enteral dietary Mn. For newborns, the Mn burden derived from parenteral nutrition can be 100 times greater than human milk. Of particular importance, the hepatic mechanisms responsible for Mn excretion are not completely developed in newborns. This factor combined with the high bioavailability of the metal in TPN increases the risk of Mn overload. That is also true for patients with hepatic dysfunction [17, 18, 21, 157].

Behavioral studies of Mn intoxication

In C. elegans, Mn exposure has been shown to result specifically in DAergic neurodegeneration [174]. In C. elegans DAergic neurons are considered mechanosensory and any condition impairing DA signaling will affect the ability to sense or respond to changes in its environment. DA signaling plays an important role in learning and regulation of locomotor behavior, including basal slowing response, ethanol preference, area-restricted searching, habituation task/tap withdrawal response, egg laying, dauer movement, pharyngeal pumping and thrashing behaviors [175, 176]. Among these behaviors, basal slowing response is DA-specific, and other behaviors are usually controlled by DA along with other neurotransmitters, such as serotonin, glutamate or GABA, etc. To date, basal slowing response and dauer movement have been studied with Mn exposure [175, 177, 178]. Levya-Illades, Chen et al. (2014), have shown that Mn exposure resulted in decreased basal slowing response, while expression of Mn exporter SLC30A10 exclusively in DAergic neurons rescued this behavioral defect together with decreased DAergic neurodegeneration [67]. In WT dauer worms, the locomotion was increased in the presence of Mn, indicating DA signaling is damaged by Mn exposure [176]. Similarly, the movement in djr-1.2 (homolog of mammalian DJ-1) worms was increased, indicating that loss of DJ-1 function resulted in abnormal DAergic neurons.

Vitamin E and GSH

Vitamin E and trolox (a hydrophilic analog of vitamin E) have been reported to protect the CNS of rodents and cultured cells from the toxic effects of Mn [183–185]. I.p. exposure of lactating rats to Mn caused striatal and hippocampal oxidative stress and motor impairments, which were prevented by trolox co-administration [183]. GSH and N-Acetylcysteine (NAC), a precursor of GSH, can also decrease the toxicity of Mn in vitro [186]; however, the protective mechanism involved in NAC and GSH has yet to be fully studied. It is likely that these compounds serve as indirect antioxidants since GSH is a substrate of glutathione peroxidase (GPx) enzymes.

Plant extracts

Plant extracts have been demonstrated to confer protection against Mn neurotoxicity after in vitro [81] and in vivo exposure in mice [187]. Acai (Euterpe oleracea) methanolic extract protected astrocytes from Mn-induced oxidative stress. The protective effects may be associated with the antioxidant and anti-inflammatory effects of its anthocyanin components [81]. Similarly, crude aqueous extracts of Melissa officinalis blunted the Mn-induced striatal and hippocampal lipid peroxidation [187]. Purified flavonoids, such as, silymarin (obtained from Silybum marianum, a plant with hepatoprotecive properties) protected neuroblastoma cells [188] and prevented Mn-induced oxidative stress in brain, liver, and kidney of rats [189–191]. Lycopene has also been reported to decrease the neurotoxicity of Mn in rats [192].

Chelating agents

Because of the chemical similarities between Mn and Fe, it is possible that the neurotoxic effects of Mn might be associated with competition with Fe for “non-redox” domains in proteins [193]. Consequently, compounds with Fe chelating properties or those interfering with the Fenton’s reaction, such as polyphenol compounds, can be of potential pharmacological importance in the treatment of Mn toxicity [194–196]. Indeed, the treatment with a calcium disodium salt of the chelator EDTA (CaNa₂EDTA) reduced Mn-induced DA autooxidation in vitro [197], enhanced urinary excretion of Mn in humans [198] and reduced Mn levels in the brain and liver of Mn-exposed rats [199]. However, there is still controversy regarding the amelioration provided by this chelating therapy [200, 201].

Synthetic compounds

Synthetic molecules have also been reported to reduce Mn toxicity. For instance, several organochalcogens (i.e. organocompounds containing selenium or tellurium atoms bound to carbon) have been reported to possess antioxidant and anti-inflammatory properties [202]. The protective effects of organoselenide and telluride compounds against Mn-induced neurotoxicity, including ebselen, have been reported [184]. One proposed mechanism might be related to a direct scavenger activity against ROS produced by Mn as most of these compounds have thiol-peroxidase activity catalyzed by glutathione-peroxidase isoforms [202]. Using the complementary animal model C. elegans, it was shown that these compounds could modulate the transcription factor DAF-16 (FOXO in mammals), increasing its translocation to the nucleus. In turn, the expression of antioxidant enzymes such as superoxide dismutase increased, thus protecting the worms from Mn-induced toxicity [203, 204]. An additional proposed mechanism is the anti-inflammatory action of some of these compounds, e.g. ebselen. Consequently, in addition to counteracting free radicals and modulating gene expression, ebselen and related compounds could decrease Mn toxicity via anti-inflammatory properties. Of note, anti-inflammatory agents have been reported to decrease Mn neurotoxicity in vitro and after in vivo exposure. For instance, Santos et al. (2013) demonstrated in vitro that 5- aminosalicylic acid (5-ASA) and para-aminosalicylic acid (4-PAS) increased mitochondrial and cell viability following Mn exposure [205]. Ibuprofen, a nonsteroidal anti-inflammatory drug, protected striatal neurons from dendritic atrophy and spine loss in rats treated for 2 weeks with the drug prior to Mn exposure [184].

The indirect pro-oxidative effects of Mn have been linked to disruption of synaptic glutamate homeostasis by interfering with glutamate uptake in astrocytes [206]. The increase in extracellular glutamate can cause excitotoxicity, which is associated with oxidative stress in neurons [206]. Furthermore, Mn decreases astrocytic glutamate uptake and expression of the astrocytic glutamate/aspartate transporter (GLAST) via disruption of intracellular signaling [207]. Of potential clinical significance, estrogen and tamoxifen have been reported to increase the expression of glutamate transporters (both GLAST and GLT-1) in astrocytes, potentially decreasing Mn toxicity [77, 207–210]. Raloxifene, which is a selective estrogen receptor modulator, also attenuates the reduction of GLT-1 and GLAST expression and the glutamate uptake induced by Mn in astrocytes [211], thus confirming how promising this class of molecules might be.

Finally, preventing or reducing Mn exposure is essential. For instance, methodologies by which welding fumes generation rate and/or welding practices can be modified to reduce toxic workplace exposures should be sought. In this context, a recent study of Sriram et al. (2015) demonstrated that rats exposed by whole body inhalation to an altered welding process (parameters: voltage, current and shielding gas) showed absence of neurotoxicity when compared to the rats exposed to regular welding process [11]. Reducing Mn levels in infant milk formulas and on parenteral nutrition should also be a strategy as safety policy.