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Journal of Trace Elements in Medicine and Biology 26 (2012) 183– 187

Contents lists available at SciVerse ScienceDirect

Journal of Trace Elements in Medicine and Biology

j ourna l homepage: www.elsev ier .de / j temb

ltered manganese homeostasis: Implications for BLI-3-dependent dopaminergiceurodegeneration and SKN-1 protection in C. elegans

udipta Chakrabortya,b,c,∗, Michael Aschnerb,c,d

Neuroscience Graduate Program, Vanderbilt University Medical Center, Nashville, TN, United StatesCenter in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, TN, United StatesDepartment of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, United StatesDepartment of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, United States

r t i c l e i n f o

rticle history:eceived 1 March 2012ccepted 24 March 2012

eywords:anganese

a b s t r a c t

The role of environmental factors in the etiology of neurodegenerative disorders, such as in Parkinson’sdisease (PD), has become increasingly imperative for examination, as genetics can only partially accountfor most cases. The heavy metal manganese (Mn) falls into this category of environmental contributors, asit is both essential but also neurotoxic upon overexposure and produces Parkinsonian symptomatology.In order to understand its toxicity, this review focuses on the various aspects of improper Mn homeo-

arkinson’s diseaseLI-3MT1/SMFKN-1/Nrf2

stasis and its consequences using the genetically amenable Caenorhabditis elegans model. Namely, theroles of Mn transporter homologs for the divalent metal transporter 1 (DMT1) will be discussed, as Mnhomeostasis is initially governed by proper cellular transport. Mn dyshomeostasis can result in enhancedoxidative stress through synergistic actions of dopamine oxidation that is dependent on the C. elegansdual oxidase BLI-3. Finally, neuroprotection conferred by the antioxidant transcription factor Nrf2 (C.elegans SKN-1) may signify a potential therapeutic approach against Mn toxicity.

© 2012 Elsevier GmbH. All rights reserved.

ntroduction

Manganese (Mn) is an essential heavy metal that is readily foundn the environment as the 5th most abundant metal and 12th mostbundant element, comprising approximately 0.1% of the earth’srust. Natural erosion results in the ubiquitous presence of Mnn the soil, water sources and in ambient air. Mn is physiologi-ally necessary for proper metabolic functioning and antioxidantesponses, and is found in several food sources as a vital componentf daily diet. It serves as an important cofactor for several criticaletalloenzymes, including: arginase for urea formation; pyruvate

arboylase for gluconeogenesis; superoxide dismutase (Mn-SOD)nd other peroxidases for detoxification of reactive oxygen speciesROS); and glutamine synthetase for the detoxification of ammo-ia and essential production of glutamine. Inorganic Mn is emitted

nto the environment via industrial sources, as Mn is a key compo-ent of steel, dry cell batteries, ceramics, glass, leather and other

extile manufacturing. Organic forms of Mn can be found in pesti-ides, fungicides, smoke inhibitors, as well as in fuel oil and gasolinenti-knock additives [1].

∗ Corresponding author at: Department of Pediatrics, 2215-B Garland Ave, 11415RB IV, Nashville, TN 37232, United States.

E-mail address: sudipta.chakraborty@vanderbilt.edu (S. Chakraborty).

946-672X/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jtemb.2012.03.011

Dietary intake is the primary source of human Mn exposure,with tight homeostatic control regulating the absorption of onlyabout 3% of ingested Mn. Once absorbed, Mn can be found bound toblood proteins like �2-macroglobulin and transferrin for regionaldistribution within the body, particularly in the brain. Mn pref-erentially accumulates in the caudate-putamen, globus pallidus,substantia nigra, and subthalamic nuclei [2]. Mn toxicity leads toirreversible damage that is referred to as manganism. Initially, itpresents with psychiatric disturbances that are later followed byataxia and an extrapyramidal syndrome. These symptoms highlyresemble that of the progressive, neurodegenerative brain disorderknown as Parkinson’s disease (PD). The development of man-ganism typically arises from environmental overexposure in anoccupational setting, such as that of welders, miners, smelters, andother industrial workers that are exposed to high levels of Mn infumes. Several studies suggest that industrial workers exposed toMn-containing fumes develop the cognitive, emotional and motordeficits that are characteristic of manganism and mirror PD symp-tomatology [1].

In addition to those exposed to high Mn levels due to their occu-pations, there are other populations at risk for manganism. Mn is

found in the gasoline anti-knock additive methylcyclopentadienylmanganese tricarbonyl (MMT), thereby making urban areas sus-ceptible to Mn toxicity from heightened gasoline combustion. Onthe contrary, high levels of Mn may also affect rural populations,

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s several pesticides and fungicides containing Mn can often beound in agricultural run-off. There are also studies that have foundognitive deficits in populations exposed to high Mn levels in drink-ng wells and groundwater [1]. Interestingly, Mn toxicity can alsoe a health concern for patients dealing with iron (Fe) deficiency,s Mn can accumulate to a higher degree in areas of low Fe con-entration due to competition with the same transporter. Hepaticncephalopathy can also induce Mn toxicity, due to the necessity ofhe biliary system for proper Mn excretion. Similarly, infants given

n-supplemented total parenteral nutrition given to ill neonatesan also be at risk for Mn poisoning due to underdeveloped biliaryystems [2].

ltered Mn homeostasis

Due to the neurological potency of Mn toxicity, tight regula-ion must be enforced in order to obtain sufficient Mn for properunctioning without imposing a toxic condition. An interconnectedystem of transport mechanisms allows for this regulation fol-owing the absorption of Mn into the blood. Trivalent Mn is

ainly found in a complex with the transferrin protein, whichs then imported by the transferrin receptor. However, Mn is

ostly found in the divalent form, implicating the divalent metalransporter 1 (DMT1) as the primary mode of Mn import [3].MT1-mediated Mn import is dependent on a proton gradi-nt generated by a vaculoar-ATPase (v-ATPase). This gradientlso allows for the reacidification of endocytic vesicles, induc-ng the conversion of Mn3+ to Mn2+ for transport into the cellia DMT1 [4]. There are other Mn transporters, such as thelutamate ionotropic receptor, voltage-gated and store-operatedalcium channels, the solute carrier 39 family member zinc trans-orters, the citrate transporter and a choline transporter [5–7].ore recent evidence also suggests novel Mn transport roles for

oth the magnesium transporter HIP14 (Huntingtin-interactingrotein 14) [8], as well as the P-type transmembrane ATPaserotein ATP13A2 [9]. The wide variety of proteins that can trans-ort Mn further illustrates the need for proper regulation of Mnomeostasis.

Despite the presence of multiple Mn transporters, DMT1emains the primary Mn transporter. DMT1 was first identi-ed as a homolog for NRAMP1, or natural resistance-associatedacrophage protein, a protein that plays a role in host defense

gainst infection. It was then referred to as DCT1, or divalent cationransporter 1, due to its ability to transport a variety of cations10], before being renamed to DMT1 in 1999 [11]. Interestingly,MT1 in the brain shows highest expression in the basal ganglia

12], the same brain region affected by both PD and manganism.ecent evidence has found direct links between DMT1 and PD,s the postmortem brains of PD patients show up-regulation ofMT1 protein in the substantia nigra pars compacta (SNpc), as wells in the SNpc of brains from MPTP (1-methyl-4-phenyl-1,2,3,6-etrahydropyridine)-exposed mouse models. This MPTP-inducedncrease in DMT1 expression corresponds to increased DAergiceurodegeneration, with mice carrying mutated DMT1 showingrotection from MPTP toxicity in these cells [13]. Furthermore, inelation to occupational overexposure as a source of manganism,ecent evidence has found an increase in DMT1 mRNA expres-ion in combination with DAergic neurotoxicity in rats exposedo Mn-containing fumes at doses that mimic about 1–5 years oforker pulmonary exposure [14]. Recent evidence has also iden-

ified specific DMT1 polymorphisms that may be risk factors for

D [15]. Such connections of Mn exposure to a dopaminergic-pecific toxicity that involves changes in DMT1 expression warranturther investigation into the possible mechanisms behind suchhenomena.

ts in Medicine and Biology 26 (2012) 183– 187

C. elegans model system

The use of vertebrate models has greatly improved our under-standing of the pathophysiology behind brain diseases like PDand manganism. However, the inherent intricacies of the verte-brate brain have hampered faster advances in understanding themolecular mechanisms behind these disorders. Recent interestshave turned to alternative models that allow for a more high-throughput, mechanistic approach. Consequently, we turned tothe advantageous Caenorhabditis elegans (C. elegans) model sys-tem in order to understand the mechanisms behind Mn toxicityand transport regulation. This model offers several advantages,such as a quick, 50-hour life cycle from egg to egg. C. elegans arealso inexpensive and easy to maintain, as the nematodes typicallygrow on Escherichia coli bacterial lawns at 20–25 ◦C and can alsobe frozen for storage [16]. Another benefit of this model is easein genetic manipulation, as the population is primarily composedof hermaphrodites whose self-fertilization allows for rapid geneticbreeding and screens [17]. Moreover, their complete cell lineage iswell-mapped, as worms are composed of 959 cells (302 of whichare neurons). Notably, the whole C. elegans genome is characterizedand shows high homology with the human genome, with about70% of human genes having a clear C. elegans homolog. In regardsto using C. elegans to study Mn-induced DAergic neurotoxicity, themodel also usefully contains all homologs for genes necessary fordopamine synthesis, packaging, reuptake and signaling [18].

C. elegans Mn transport

Although DMT1 may be known as the primary Mn transporter,mechanisms behind both Mn transport and DMT1 protein functionremain unclear. This is primarily due to the existence of differentisoforms of DMT1 that are both transcriptionally and translationallyregulated in a tissue-specific manner that is also dependent on var-ious conditions (e.g., iron levels) [19]. Accordingly, our laboratorydecided to use the C. elegans model system to further understandthe complex regulation of Mn transport. We were able to iden-tify, clone and characterize three worm homologs for DMT1: SMF1,SMF2 and SMF3. Protein sequence analysis was conducted to findthat all three homologs show high homology to human DMT1 con-taining both the characteristic 12 transmembrane domains anda consensus transport sequence (CTS). Deletion mutants of thethree homologs from the Caenorhabditis Genetic Center (CGC) werethen used to assess whether loss-of-function or down-regulationof these transporters confers sensitivity to Mn exposure. Both smf-1(eh5) and smf-3(ok1035) deletion mutants exhibited heightenedresistance to an acute (30 minutes) MnCl2 exposure when com-pared to wild-type Bristol N2 worms. This was evident with asignificantly higher LD50, or the lethal dose at which 50% of thetreated worms are dead, for these mutants 24 hours after treat-ment. smf-1(eh5) mutants were almost twice as resistant to Mnas wild-type worms, with an LD50 of 94 mM MnCl2 compared tothe N2 LD50 of 47 mM. smf-3(ok1035) deletion mutants showedan even higher level of resistance, with an LD50 of 126 mM [20].These data suggest that these two homologs somehow mediate Mnuptake, as the lack of functional SMF1 and SMF3 does not increasesensitivity to Mn compared to wildtype worms that contain func-tional SMF1 and SMF3. On the other hand, smf-2(gk133) deletionmutants exhibited hypersensitivity to Mn exposure compared towild-type worms (LD50 of 26 mM), suggesting that functional SMF2is somehow protective against Mn toxicity in worms.

To further corroborate the role of the SMF proteins in regulat-ing Mn homeostasis, metal content analysis was conducted usinggraphite furnace atomic absorption spectrometry (GFAAS). Allstrains showed increases in Mn accumulation in a dose-dependent

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anner. smf-1(eh5) deletion mutants accumulated less Mn thanildtype worms, though not significant at any of the concentra-

ions assessed. However, smf-3(ok1035) mutants accumulated theeast amount of Mn compared to all other strains, reaching signif-cantly lower levels at 100 mM and 150 mM MnCl2. Interestingly,he smf-2 deletion mutants exhibited significantly higher Mn con-ent than the other strains upon exposure to 35 mM, 100 mM and50 mM MnCl2 [20]. This Mn content data paralleled the Mn toxi-ity evidence: without SMF1 and SMF3, the treated worms cannotccumulate Mn upon exposure, indicating a prominent role forptake.

In order to investigate differential expression patterns, trans-enic worms expressing GFP under the control of the smf1, smf2,r smf3 promoters were created, in addition to strains express-ng GFP-tagged SMF1, SMF2 and SMF3. Using both transcriptionalnd translational fusion strains, both SMF1 and SMF3 show local-zation to major epithelial tissues (intestine, rectal gland cells,terus, vulva, epidermis and sensory organs), whereas SMF2emains in minor epithelial (pharynx, pharyngeal-intestinal valve)issues. At the cellular level, SMF1 and SMF3 also both local-ze to the apical membrane, while SMF2 is mostly cytoplasmic.pon Mn exposure, SMF1::GFP and SMF2::GFP reporter strains

how no change in localization or expression. SMF3::GFP, how-ver, shows a remarkable change in both localization and GFPntensity upon acute exposure to Mn. One hour post-treatment,MF3::GFP worms show a dramatic translocation of SMF3 into api-al vesicular compartments in the intestine. However, five hoursfter treatment, this intestinal GFP signal significantly decreasesnd is no longer localized to apical vesicles. This process is com-letely reversible, as SMF3::GFP worms return their expression

evel and intracellular localization to normal by 30 hours post-reatment [20]. Thus, these studies indicate an interconnectedetwork of Mn homeostatic control through three homologs: SMF1nd SMF3 are the DMT1 isoforms responsible for Mn uptake in. elegans, with SMF3 being the most DMT1-like homolog. Thesendings also suggest that SMF2 is involved with Mn sensingnd regulation via protective inhibition of pharyngeal pump-ng.

electivity in Mn-induced neurodegeneration

Upon establishment of how Mn is imported in C. elegans,echanisms behind Mn toxicity in this model were still unclear.sing transgenic strains expressing GFP under the promoters ofifferent neurotransmitter systems, it was determined that neu-odegeneration upon Mn exposure is selective toward DAergiceurons. In particular, using worms expressing GFP under the con-rol of the dopamine reuptake transporter 1 promoter (pdat-1::GFP),euronal processes of the DAergic CEP mechanosensory neuronsxhibited distinctive puncta and discontinuous GFP signal in aose-dependent manner. However, these effects were not seen

n strains expressing GFP as driven by GABAergic, cholinergic andlutamatergic-specific promoters. Further confirming the selectiv-ty in toxicity toward the DAergic system, wildtype worms treated

ith a sub-lethal treatment of 10 mM dopamine showed sensi-ization to Mn treatment, with an LD50 of 25 mM compared to7 mM for wildtype worms. However, DA pre-treatment alone didot affect viability. Strains lacking functional DAT-1 also exhibitedignificant hypersensitivity to Mn exposure, with a dramaticallyowered LD50 of 9 mM [21]. This correlates with previous workhat has found that C. elegans dat-1 is necessary for DAergic-specific

euronal ablation using the toxin 6-OHDA [22]. This indicates theecessity of dopamine for Mn neurotoxicity to occur, and thatomehow, Mn and dopamine work synergistically to produce selec-ive DAergic neurodegeneration in vivo.

ts in Medicine and Biology 26 (2012) 183– 187 185

In order to determine which part of the dopamine pathwayis affecting Mn sensitivity, strains lacking functional dopaminereceptors (DOP1-3) and DAT-1 were used to represent regula-tors of extracellular dopamine, whereas strains lacking vesicularmonoamine transporter 2 (VMAT2, or CAT-1 in worms), and tyro-sine hydroxylase (TH, or CAT-2 in worms) where employed asregulators of intracellular dopamine levels. High performance liq-uid chromatography (HPLC) analysis found that loss of both DAT-1and the dopamine receptors increased the levels of dopamine inthe worms, whereas loss of the ability to package dopamine in vesi-cles (cat-1 mutants), and more significantly, the ability to producedopamine (cat-2 mutants) resulted in decreased dopamine levels.Upon Mn treatment, both cat-1 and cat-2 mutants showed signif-icant resistance to Mn toxicity, with LD50s of 108 mM and 95 mM,respectively, compared to wildtype worms. Meanwhile, in addi-tion to the dat-1 mutants showing a significant leftward shift in thedose–response curve as previously mentioned (LD50 of 9 mM), thedopamine receptor homolog mutants also showed a significantlydecreased LD50 of 27 mM. There was also a rescue in the resistancetoward Mn toxicity in worms lacking tyrosine hydroxylase (cat-2 mutants) pre-treated with 10 mM dopamine [21]. Collectively,these data suggested that increased extracellular, and not intracel-lular, dopamine is responsible for hypersensitivity to Mn exposure,whereas decreased dopamine levels are protective against Mn toxi-city. This suggests that Mn neurotoxicity must occur in the presenceof dopamine in vivo, and that increased extracellular dopamineenhances this selective toxicity.

Consequences of extracellular dopamine-dependent Mntoxicity

A shared hallmark behind cell death in most neurodegen-erative diseases is increased oxidative stress, although specificproteins and pathways involved may not be clearly understood yet.Using the ROS-sensitive 2′7′-dichlorodihydrofluorescein diacetate(H2DCF-DA) fluorescent dye, wild-type worms showed a signifi-cant increase in DCF fluorescence at the sub-lethal concentrationof 10 mM MnCl2, while dat-1 mutants exhibited significance at thelower dose of 1 mM. However, cat-2;dat-1 double mutants did notshow a similar increase in fluorescence up to 30 mM. These resultswere confirmed by also assaying for lipid peroxidation by measur-ing isoprostane levels, which are oxidation products of arachidonicacid (AA) that are released from membranes upon an oxidativeinsult. Isoprostane analysis in C. elegans corroborated that dat-1mutants exhibited significantly higher oxidant levels upon expo-sure to MnCl2. Once again, cat-2;dat-1 double mutants rescuedthe increased oxidative stress effect [21]. These data collectivelysuggest that Mn exposure induces increased oxidative stress in C.elegans that is dependent on levels of extracellular dopamine.

Dopamine itself is known to be a naturally strong reactivemolecule, with dopamine auto-oxidation resulting in the cre-ation of dopamine-quinones that damage lipids, proteins andDNA. The Fenton reaction is also well known in employing metalcations to oxidize biogenic amines [23,24]. Our data support thisphenomenon, with Mn working with extracellular dopamine toincrease peroxidation of lipids, as indicated by increased iso-prostane levels in dat-1 mutants. Therefore, the next goal was toidentify why Mn and dopamine may work together to increaseoxidative stress by identifying any genetic modifiers that may beinvolved in this process. Outside of the electron transport chain inmitochondria, we know that NADPH oxidases are another major

source of ROS production in cells. In C. elegans, the bli-3 geneencodes a dual oxidase homolog for vertebrate DUOX1 and DUOX2that is involved in di-tyrosine bond formation in the cuticle, theworm’s tough, outer barrier, to maintain its integrity. Consequently,

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li-3(e767) mutants possess a blistered cuticle that renders the ani-al highly susceptible to toxicants that would otherwise not be

ble to penetrate the cuticle [25]. Interestingly, bli-3(e767) mutantshowed a hyper-resistant phenotype to Mn toxicity, with a signifi-antly higher LD50 of 83 mM. When these worms were pre-treatedith sub-lethal 10 mM DA, there was no significant change in

ethality upon Mn exposure in bli-3 mutants, in addition to no sig-ificant change in ROS production from 1 to 30 mM MnCl2 (similaro cat-2 mutants) [21]. Therefore, due to the structural similarity inopamine and tyrosine, we propose that BLI-3 is a mediator in theelectivity of Mn toxicity toward DAergic cells, with Mn-inducedxidation of DA and its reactive metabolites producing oxidizedpecies that may serve as substrates for BLI-3. This warrants fur-her investigation as a major overlap between manganism and PD,s previous data indicate BLI-3 is up-regulated in the SNpc of PDatients and MPTP-exposed mice [26].

ntioxidant responses against DA-dependent Mn toxicity

Next, potential antioxidant pathways that could protect againstxidative insults in C. elegans were explored. Previous data hashown that the oxidative stressor sodium arsenite produces aarked translocation of the antioxidant gene skn-1, the C. elegans

omolog of the vertebrate transcription factor nrf2. SKN-1 in wormss required for the typical 2–3 week lifespan of wildtype worms,nd is a necessary force of resistance against stress [27]. Conse-uently, our lab investigated whether skn-1 plays a role in theA-dependent, Mn-induced oxidative stress seen in our model sys-

em. Worms over-expressing SKN-1 show a significant resistanceo Mn toxicity, with an LD50 of 114 mM, while worms contain-ng mutated skn-1 exhibit a hypersensitive phenotype, with anD50 of 34 mM. Although GFP expression of SKN-1::GFP worms didot increase in intensity, there was a remarkable translocation ofKN-1 into the nucleus, as evident from a distinctive, nuclear punc-ate pattern in ASI neurons compared to diffuse GFP expressionn untreated worms [21]. These data indicate that SKN-1 medi-tes Mn toxicity in vivo by re-localizing to the nucleus, where itp-regulates antioxidant genes to combat increased Mn-inducedxidative stress.

uture implications

In summary, we have identified a complex network of trans-orters regulating Mn import in C. elegans, with SMF-3 as therimary, most DMT1-like Mn transporter. Upon import, we pro-ose that Mn, in combination with BLI-3, induces oxidation ofxtracellular dopamine, resulting in an increase in DA-oxidizedpecies. DAT-1 also seems to be necessary in mediating Mn-inducedAergic neurodegeneration. Thus, we also propose a novel role for

he DAT-1 in taking up DA-oxidized species into DAergic neuronso induce Mn-induced neurodegeneration. Connections between

n and DAT demand further examination, in addition to BLI-3.ecent evidence has found a role for NADPH oxidase in zinc- and-hyroxydopamine-induced DAergic neurodegeneration [28,29],hich mirrors previous findings that an NADPH oxidase 1 inhibitor,

pocynin, can block against paraquat-induced DAergic cell loss [30].It would be interesting to determine whether the involvement

f dual-oxidases in DA oxidation is similar in vertebrates. In termsf PD treatment, the most common form of treatment involvessing the dopamine precursor L-3,4-dihydroxyphenylalanine, or-DOPA. However, in addition to debilitating side effects such as

yskinesia, previous in vitro findings have found that l-DOPA canlso increase oxidized reactive species, and that Mn can actuallynhance l-DOPA-induced apoptosis [31]. This increase in oxidizedpecies could be counter-productive and result in exacerbation

ts in Medicine and Biology 26 (2012) 183– 187

or acceleration of degeneration of the remaining DAergic neuronsover a longer term. Although these results have not been consistentin animal studies and human trials, the ineffectiveness of l-DOPAas a cure-all for PD still remains a concern. Interestingly, recentevidence has found that l-DOPA is incorporated into brain proteinsof PD patients treated with l-DOPA due to its structural analogyto l-tyrosine, resulting in misfolded proteins and aggregation thatis resistant to antioxidants [32]. If l-DOPA is oxidized by dualoxidases like BLI-3, it would be important to design alternative DAanalogs that could maintain high affinity for DA receptors and DAT,but would act as poor substrates for the pro-oxidant activity ofdual oxidases. Following our evidence on the role of dual-oxidasesas oxidative stress instigators in Mn-induced DAergic neurode-generation, the findings of protection against Mn toxicity throughSKN-1 over-expression is promising and warrants further inves-tigation. Recent findings in Drosophila corroborate our findings,with genetic up-regulation of Nrf2 being sufficient to mitigateneurodegeneration-associated phenotypes in a PD model, includ-ing amelioration of locomotor deficits and DAergic neuron loss [33].

An interesting connection between Mn transport and oxidativestress in C. elegans centers on immunity. Our findings reveal thatBLI-3 and DA-dependent ROS production are either precipitatedor intensified by Mn exposure, which is regulated by the DMT1homologs SMF1-3. Recent evidence has found that BLI-3-inducedROS production is involved in C. elegans defense against bacterialand fungal infections. Moreover, the DMT family of transportershas a well-characterized role in vertebrate innate immunity, asevidenced by its former family name of NRAMP. In fact, wormslacking functional DMT homologs show a hypersensitive pheno-type toward Staphylococcus aureus infection, which are rescued bytreatment with Mn [34]. Interestingly, following our studies on pro-tection from SKN-1 overexpression, recent evidence in C. elegansexposed to bacterial pathogens demonstrate an increase in BLI-3-mediated ROS production that results in the activation of SKN-1via p38 MAPK signaling as a protective response against infection[35]. The role of Mn toxicity mediated by SMF transporters and BLI-3-induced oxidation in DAergic neurodegeneration implicates thepossibility of DA playing a direct role in the nematode’s immunesystem, with the potential of neuroprotection from the infection-resistant forces of SKN-1. Further investigation into the role ofMn-induced BLI-3 oxidation of DA in vertebrates poses an inter-esting avenue for vertebrate research, as inflammation and innateimmunity have been considered as potential contributors in themysterious etiology of PD [36].

Acknowledgments

This review was supported in part by Grants from the NationalInstitute of Environmental Health Sciences ESR01-10563, R01-07331, ES T32-007028 and the Molecular Toxicology Center ES P30000267.

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