THE METAL TRANSPORTER SMF-3/DMT-1 MEDIATES ALUMINUM-INDUCED DOPAMINE NEURON DEGENERATION

Abstract

Aluminum (Al³⁺) is the most prevalent metal in the earth's crust, and is a known human neurotoxicant. Al³⁺ has been shown to accumulate in the substantia nigra of Parkinson's disease (PD) patients, and epidemiological studies suggest correlations between Al³⁺ exposure and the propensity to develop both PD and the amyloid plaque-associated disorder Alzheimer's disease (AD). Although Al³⁺ exposures have been associated with the development of the most common neurodegenerative disorders, the molecular mechanism involved in Al³⁺ transport in neurons and subsequent cellular death has remained elusive. In this study we show that a brief exposure to Al³⁺ decreases mitochondrial membrane potential and cellular ATP levels, and confers dopamine (DA) neuron degeneration in the genetically tractable nematode Caenorhabditis elegans (C. elegans). Al³⁺ exposure also exacerbates DA neuronal death conferred by the human PD-associated protein α-synuclein. DA neurodegeneration is dependent on SMF-3, a homologue to the human divalent metal transporter (DMT-1), as a functional null mutation partially inhibits the cell death. We also show that SMF-3 is expressed in DA neurons, Al³⁺ exposure results in a significant decrease in protein levels, and the neurodegeneration is partially dependent on the PD-associated transcription factor Nrf2/SKN-1 and caspase Apaf1/CED-4. Furthermore we provide evidence that the deletion of SMF-3 confers Al³⁺-resistance due to sequestration of Al³⁺ into an intracellular compartment. This study describes a novel model for Al³⁺-induced DA neurodegeneration and provides the first molecular evidence of an animal Al³⁺ transporter.

Introduction

Al³⁺ is a highly abundant and ubiquitously distributed environmental and industrial toxicant (Vasudevaraju et al. 2008). Human exposure to Al³⁺ occurs through a number of mechanisms including soil and fertilizers, cookware, water from purification systems, as well as pharmaceutical and cosmetic preparations (Verstraeten et al. 2008). Al³⁺ is readily absorbed in the gastrointestinal tract, skin, and olfactory system, and can interact with a number of biological systems including the liver, kidney, bone, and brain. Bone can contain approximately 70% of the total Al³⁺ in the body (Crisponi et al. 2011). A physiological requirement for Al³⁺ has not been identified (Verstraeten et al. 2008).

High exposure to Al³⁺ is toxic to the central nervous system, and epidemiological and molecular evidence suggests a link between exposures, cell Al³⁺ concentrations, and the propensity to develop several neurological disorders (Vasudevaraju et al. 2008, Yokel 2006). Al³⁺ exposure has been reported to be a risk factor for the development of the most common neurodegenerative disorder Alzheimer's disease (AD). Al³⁺ has been found in senile plaques and neurofibrillary tangles in degenerating neurons in AD brains, and exposures can induce neurofibrillary tangles in vertebrate AD animal models (Vasudevaraju et al. 2008, Kawahara & Kato-Negishi 2011). Al³⁺ exposures have been associated with the development of Parkinson's disease (PD), which is characterized by motor deficits and the loss of dopamine (DA) neurons in the substantia nigra (SN) (Zayed et al. 1990a). Combinatorial long-term exposure of Al³⁺, Fe²⁺, and Mn²⁺ has also been associated with increasing the probability of developing PD (Zayed et al. 1990a). Al³⁺ has been shown to accumulate in the SN in PD and parkinsonism patients, affect tyrosine hydroxylase (TH) activity and inhibit DA synthesis (Erazi et al. 2011). Furthermore, the formation of the toxic moiety of the PD-associated protein α-synuclein has shown to be accelerated in the presence of Al³⁺, and PD Lewy bodies have been shown to contain Al³⁺ (Uversky et al. 2001).

Although the molecular basis of Al³⁺-induced toxicity is not well defined, oxidative stress appears to play a significant role in the cellular pathology (Verstraeten et al. 2008, Sanchez-Iglesias et al. 2009). Exposure of cells to Al³⁺ can increase reactive oxygen species (ROS) formation by interfering directly with mitochondrial function, or by increasing the redox activity of Fe²⁺ through the Fenton reaction (Vasudevaraju et al. 2008). Al³⁺ exposure can also increase oxidative stress by causing protein misfolding, reduced microtubule transport of neuronal vesicles, DNA damage, and lipid peroxidation resulting in apoptosis (Kumar et al. 2009, Yokel 2006).

The molecular basis of Al³⁺ transport into neurons and between intracellular compartments is largely unknown (Yokel 2002, Yokel 2006). Al³⁺ may bind to proteins and the plasma membrane and potentially enter the cell through diffusion, although in vitro transport kinetics do not support this mechanism. The physiochemical similarities (ionic radius and hydration shells) between Fe²⁺ and Al³⁺ suggest that Al³⁺ may bind to Fe²⁺-containing proteins and Fe⁺² transporters (Martin 1986, Vasudevaraju et al. 2008). Al³⁺ has also been proposed to enter cells through transferrin receptor mediated endocytosis (TfR-ME) yet this does not appear to be the primary route of Al³⁺ influx (Yokel 2002, Yokel 2006). In vertebrates, the natural resistance-associated macrophage protein (NRAMP) family consists of Nramp1 and Nramp2 (divalent metal transporter 1, DMT-1) that are involved in the transport of divalent metals across plasma and intracellular membranes (Cellier et al. 1995). DMT-1 has been implicated in metal-associated DA neuronal death in PD (Salazar et al. 2008). In yeast, the DMT-1 homologues Smf2p and Smf3p transport Mn²⁺ across intracellular membranes (Portnoy et al. 2000). Recently a DMT-1 homologue in rice, Nrat1, has been shown to transport Al³⁺, and is involved in Al³⁺-associated cellular toxicity (Xia et al. 2010). Caenorhabditis elegans (C. elegans) contains 3 homologues to the vertebrate DMT-1, SMF-1–3 (Settivari et al. 2009). SMF-1 and SMF-2 are expressed in DA neurons and play a role in PD-associated 6-hydroxydopamine (6-OHDA) and Mn²⁺-induced DA neuron degeneration (Settivari et al. 2009). SMF-3 GFP fusions indicate expression in a variety of tissues in the nematode, including neurons, although localization to specific neuronal types has not been previously described (Bandyopadhyay et al. 2009). SMF-3 does not play a role in Mn²⁺-induced DA neuron degeneration (Settivari et al. 2009).

The nematode Caenorhabditis elegans (C. elegans) is a useful model system to explore the molecular basis of PD and DA neuron vulnerability to metals (Nass et al. 2008, Nass & Settivari 2008). All of the genes responsible for DA neurotransmission as well as apoptosis-associated genes are present in the nematode, and DA neuron specific expression of the green fluorescent protein (GFP) allows the eight DA neurons to be clearly visible in vivo under a fluorescent dissecting microscope (Nass & Blakely 2003). The DA neurons are also sensitive to PD-associated toxicants, and the expression of human α-synuclein confers DA neuronal death (Nass et al. 2001, Nass et al. 2002, Lakso et al. 2003, Cooper et al. 2006, Vartiainen et al. 2006). The DA neurons also express the PD-associated transcription factor Nrf2/SKN-1 that inhibits MeHg-induced neuronal death (Vanduyn et al. 2010).

Considering the strong conservation of genes and proteins between C. elegans and humans, and the high similarities on the molecular level in how both organisms respond to neurotoxicants, we asked whether the nematode C. elegans may be a useful genetic model to explore the molecular basis of Al³⁺-induced DA neurodegeneration in vivo. In this study we show that C. elegans recapitulates key molecular attributes of Al³⁺-induced vertebrate DA neuron toxicity, identify Al³⁺-associated DA neuron death effectors, and identify and characterize a novel Al³⁺ transporter that modulates intracellular Al³⁺ and inhibits DA neurodegeneration.

Methods

C. elegans strains and maintenance

The following strains have been described previously: BY250 (P_dat-1∷GFP); RJ907 (Pdat-1∷GFP; smf-1(eh5)); RJ938 (Pdat-1∷GFP; smf-2(gk133)); RJ934 (Pdat-1∷GFP; smf-3(ok1035)); BY273 (Pdat-1∷GFP; Pdat-1∷WT_α-synuclein); and RJ928 (Pdat-1∷GFP; rrf-3(pk1426)) (Settivari et al. 2009, Nass et al. 2002, Lakso et al. 2003). C. elegans strains were cultured on OP50 or NA22 bacterial lawns on NGM or 8P plates, respectively, at 20°C according to standard methods (Brenner 1974, Hope 1999).

Toxicant exposures

To obtain synchronized L1 stage worms, gravid adults were treated with hypochlorite and the embryos were incubated in M9 buffer for 18 h and washed 3× in dH₂0 using standard protocols (Nass et al. 2002, Nass & Hamza 2007). For acute exposures, L1 stage worms (10 worms/μl) were incubated with dH₂0 +/− aluminum chloride (AlCl₃; Fisher Scientific, Fair Lawn, NJ), as previously described (Nass & Hamza 2007) for 30 min at room temperature (~ 22°C) with gentle mixing every 10 min. After exposure, the worms were placed onto NGM/OP50 plates and allowed to recover for 72 h at 20°C. After recovery, 50–60 worms were immobilized on 2% agarose pads with 2% sodium azide and were scored for DA neurodegeneration under a fluorescent microscope (Leica MZ 16FA, Switzerland). Worms were scored positive for DA neuron degeneration when GFP in any part of the four cephalic dendrites (CEP; which run from nerve ring to tip of the nose) was absent (Nass et al. 2002, Settivari et al. 2009). Each of the experiments was performed at least in triplicate.

Mitochondrial membrane potential analysis

Mitochondrial membrane potential was measured using the lipophilic cationic dye tetramethyl rhodamine ethyl ester (TMRE, Sigma, St. Louis, MO) as previously described (Yoneda et al. 2004, Ehrenberg et al. 1988, Settivari et al. 2009). L1 stage worms were exposed to 100 μM AlCl₃ for 30 min and then were allowed to recover on NGM plates containing 0.1 μM TMRE for 48 h. A Leica MZ 16FA fluorescent microscope was used to obtain fluorescent images of the head region of each animal (from tip of nose to posterior end of terminal bulb) and the amount of dye accumulated was quantified using Image Pro Plus v6.2 software (Media Cybernetics, MD). The membrane potential of at least 20 live animals was evaluated in triplicate for each experimental condition.

ATP analysis

ATP levels were determined using the ATP determination kit (Molecular Probes, Eugene, OR) following the manufacturer's instructions with minor modifications. Briefly, synchronized L1 stage nematodes were exposed to water or 100 μM AlCl₃ for 30 min and allowed to recover on NGM plates for 24 h at 20°C. The nematodes were collected from the plates and washed three times. The worm pellet was immediately frozen in liquid nitrogen, thawed, and sonicated in TE buffer (100 mM Tris-Cl, pH 7.6, 4 mM EDTA). ATPase activity was inhibited by incubating the lysate at 85°C for 7 min. The lysate was spun at 14000 g for 30 min, the supernatant was collected and protein concentration was determined using the Bradford assay with bovine gamma globulin as the standard. 10 μl of the supernatant was added to 100 μl of the standard reaction solution (provided with the kit) and light intensities were measured using a luminometer (Spectra Fluor Plus, Tecan). ATP levels within each sample were background subtracted and normalized to the protein content. All assays were performed in triplicate.

Antibodies and Western analysis

An antibody to amino acids 100–154 from the putative C. elegans SMF-3 protein (WP:CE36370) was generated using Genomic Antibody Technology at Strategic Diagnostics Inc. (SDI, Newark, DE). Rabbit polyclonal antibodies were further purified at SDI. GAPDH (ab36840 Abcam, Cambridge, MA) was used as a loading control for Western analysis. To prepare protein for Western blot analysis, synchronized L1 stage worms were exposed to 100 μM AlCl₃ for 30 min and allowed to recover for 24 h on NGM plates. Following recovery, worms were pelleted from media plates as described above. 150 μl of mito buffer (20 mM HEPES, pH 7.5, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1.5 mM MgCl₂, 1 mM DTT, 0.1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 2 μg/ml aprotinin) was added to 300–400 μl of pelleted worms and the tubes were frozen at −20°C until protein purification. Worm samples stored at −20°C were thawed and homogenized on ice with 50–60 strokes with in a 2 ml glass homogenizer. The lysate was spun at 400 g at 4°C for 4 min, the supernatant was collected in a sterile tube, and protein concentration was determined using the Bradford assay with bovine gamma globulin as the standard. The samples were diluted in NuPAGE LDS buffer (Invitrogen, Carlsbad, CA), heated at 85°C for 15 min, and total cell lysates (50 μg protein) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk dissolved in TBST (tris-buffered saline, 0.1% Tween-20) for 2 h at room temperature, followed by incubation with the appropriate primary antibody dilution (anti-SMF-3 at 1:80,000; anti-GAPDH at 1:20,000) at 4°C overnight. The membranes were washed 3 times at room temperature for 15 min, and incubated with HRP-conjugated secondary anti-rabbit IgG (611–1302 Rockland, Gilbertsville, PA). The membrane was developed using enhanced chemiluminescence (ECL) (Amersham Biosciences, Pittsburgh, PA), captured using Bio-Rad ChemiDoc XRS, and total protein intensities were measured using QuantityOne software (Bio-Rad, Hercules, CA).

Immunohistochemistry and intracellular localization analysis

Primary C. elegans cultures were prepared as previously described with slight modifications (Bianchi & Driscoll 2006, Settivari et al. 2009). Briefly, BY250 gravid adults were lysed with the synchronization solution, and the egg pellet was washed using egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 25 mM HEPES). The eggs were then separated from the debris using a 60% sucrose solution, digested using 4 mg/ml chitinase (Sigma, St. Louis, MO), and the embryonic cells were dissociated using a syringe. The embryonic cells were then resuspended in L-15 medium (containing 10% FBS and 1% pen/strep) and grown on polylysine-coated cover slips at 20°C. Following growth for 72 h, cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and incubated in blocking buffer containing 2% BSA and 20% normal goat serum for 1 h at room temperature. The cells were then incubated with SMF-3 primary antibody (1:100) at 4°C overnight (14 h), followed by incubation with an Alexa Fluor 594 conjugated goat anti-rabbit secondary antibody (Invitrogen; 1:200) at room temperature for 1 h. Images were captured using confocal microscopy (Olympus FV1000-MPE Confocal/Multiphoton Microscope). To confirm intracellular localization of SMF-3 staining we performed spatial overlap analysis using the Measure Colocalization Application in MetaMorph imaging software (Molecular Devices, Downingtown, PA). A projection image was created from Z-stack slices and the region of interest was chosen to include the GFP expressing dopamine neuron. The area of overlap between GFP (green) and SMF-3 (red) channels was calculated as a percentage of red pixels above threshold colocalizing with green pixels above threshold and the percentage of green pixels above threshold that colocalize with red pixels above threshold. SMF-3 immunofluorescence was analyzed in three primary GFP expressing DA neurons.

RNA interference

RNA-mediated interference using the RNAi sensitive strain RJ928 was carried out on NGM plates containing 1 mM isopropyl β-d-thiogalactoside (IPTG) and 100 μg/ml ampicillin and seeded with HT115 (DE3), an RNase III-deficient E. coli strain carrying L4440 vector with the gene fragment (skn-1 or ced-4) (GeneService, Source BioScience, PLC, Nottingham, UK) or empty vector (Addgene, Cambridge, MA) (Timmons & Fire 1998). Synchronized L1 stage RJ928 worms were transferred onto RNAi plates and grown to adulthood following the feeding protocol with slight modifications (Kamath & Ahringer 2003). Gravid adults grown on RNAi bacteria were treated with hypochlorite to obtain synchronized L1 stage worms. The L1 staged worms were exposed to 100 μM AlCl₃ as described above and allowed to recover on fresh RNAi plates for 72 h and dopamine neuronal death was evaluated as previously described (Nass et al., 2002). As skn-1 knockdown results in embryonic lethality, first-generation synchronized L1 larvae were exposed to AlCl₃ and DA neuron vulnerability was evaluated following 72 h recovery.

ICP-MS analysis

Wild type (BY250) and smf-3 mutant (RJ934) synchronized L1 stage worms were obtained by hypochlorite treatment of gravid adults followed by incubation of the embryos in M9 buffer for 18 h. L1s were washed 3 times with H₂O, placed on NGM plates and grown at 20°C for 48 h. After 48 h, L4 stage worms were washed off of the plates with W5 H₂O (Fisher Scientific, submicron filtered HPLC grade H₂O to minimize contamination of aluminum from diH₂O) and washed three times to remove bacteria. Worms of each strain were divided among 6 tubes and exposed to W5 H₂O or 100 μM AlCl₃ for 30 min at room temperature. Following the exposure, ice-cold W5 H₂O was added to each tube to stop the reaction, worms were moved to pre-weighed tubes, and additionally washed 4 more times with ice-cold W5 H₂O. After the last wash, all water was removed from the top of the worm pellet and the worms were frozen at −80°C. To prepare the samples for analysis, worms were digested using the MARS Xpress system (CEM, Matthews, NC). Worm samples were dried overnight (12–14 h) at 60°C and the dry weight of each sample was determined and then added to the digestion vessel. Samples were digested in 45% HNO₃, 5% HCl at 200°C for 15min. Digested samples were diluted with W5 H₂O to achieve a solution containing 2% acid. Total aluminum content of each sample was determined by analysis with the X Series ICP-MS (Thermo Fisher Scientific), using beryllium and gallium as internal standards, and data was normalized to the dry weight.

Statistics

All data are expressed as the mean ± SEM. The difference between control and treated groups was evaluated with a student's t-test, or one-way anova for multiple treatment groups. Two-way anova was used for experiments involving treatment of wild type (WT) and mutant animals. Statistical analysis was carried out with GraphPad Prism software. Differences were considered statistically significant when p < 0.05, unless otherwise indicated.

Results

Al³⁺ exposure decreases mitochondrial membrane potential and ATP levels in C. elegans

In order to determine whether a brief 30 min exposure to Al³⁺ may be toxic to C.elegans mitochondria, we exposed WT first larval stage animals (L1) to 100 μM AlCl₃, and evaluated mitochondrial membrane potential changes in the head region following growth on bacteria and tetramethylrhodamine ethyl ester (TMRE) for 48 h (Settivari et al. 2009). TMRE is a mitochondrial-specific fluorescent dye whose rate of uptake is dependent on the mitochondria membrane potential (Yoneda et al. 2004). Under these exposure conditions the animals appeared to develop normally and there was no decrease in animal viability. A single exposure to 100 μM AlCl₃ resulted in a significant decrease in mitochondria membrane potential within the head relative to non-exposed nematodes (Fig. 1a). These results suggest that as in vertebrate systems, Al³⁺ may impair mitochondria function. To further explore whether Al³⁺ may be toxic to mitochondria in C. elegans, we determined total ATP levels in control and Al³⁺ exposed animals. Exposure to 100 μM AlCl₃ resulted in a 40% decrease in cellular ATP levels relative to controls (Fig. 1b). These results indicate that exposure of C. elegans to Al³⁺ is deleterious to mitochondria, and suggests that Al³⁺ may also be toxic to cells that are sensitive to mitochondria-associated neurotoxicants.

Figure 1. Al³⁺ exposure decreases mitochondrial membrane potential and ATP production, and confers DA neuron degeneration.

Synchronized L1 nematodes were exposed to 100 μM AlCl₃ for 30 min and allowed to recover on plates containing TMRE for 48 h. The mitochondrial membrane potential was evaluated by measuring the fluorescence intensity per area for the head region of at least 20 animals per replicate and the experiment was performed three times. Shown are mean values ± SEM of three individual replicates. p values were calculated using t test analysis. Asterisk indicates p ≤ 0.05 (a). ATP levels were evaluated following a 30 min exposure of L1 stage worms to 100 μM AlCl₃ and a 24h recovery on NGM plates. Asterisk indicates p ≤ 0.05 (b). ATP was quantified by a luminescent ATP determination kit and normalized to protein content for three independent replicates. To determine the effect of Al³⁺ exposure on DA neurons, L1 stage worms were exposed to 100 μM AlCl₃ for 30 min and allowed to recover for 72 h before examining the integrity of GFP-expressing dopamine neurons (CEPs). Asterisk indicates p ≤ 0.01 (c). Transgenic worms expressing human α-synuclein are more sensitive to Al³⁺ exposure as indicated by a significant increase in DA neuron degeneration with 100 μM AlCl₃. Asterisk indicates a difference between WT and α-synuclein, # indicates a difference between control and AlCl₃ exposure, p ≤ 0.001 (d).

Exposure to sub-lethal concentrations of Al³⁺ confers DA neurodegeneration in WT and human α-synuclein expressing animals

The DA neurons in vertebrates are particularly sensitive to oxidative stress and heavy metals, and exposures have been associated with the development of Parkinson's disease and parkinsonism. We previously generated transgenic worms that express the green fluorescent protein (GFP) in the eight DA neurons in the hermaphrodite that allows clear visualization of the neurons under a fluorescent dissecting microscope (Nass et al. 2002). Our studies and others have shown that brief exposures to PD-associated neurotoxicants including 6-OHDA, MPP⁺, rotenone, and Mn²⁺ can result in DA neuron cell death (Settivari et al. 2009, Nass et al. 2002, Nass & Settivari 2008). To determine whether a brief exposure to Al³⁺ can induce DA neuron degeneration in C. elegans, we exposed the animals to various concentrations of toxicant ranging from 0 to 500 μM for 30 min and transferred the animals to agar plates. We scored these animals similar to our earlier studies in which the CEP processes that cannot be visually followed from the cell body to the tip of the nose are considered to have degeneration (Nass et al. 2002, Settivari et al. 2009, Vanduyn et al. 2010). We find that a brief 30 min exposure to Al³⁺ caused a loss of DA neurons within 72 h at all concentrations tested, and approximately 15% of the animals displayed DA neuron degeneration following exposure to 100 μM AlCl₃ (Fig. 1c). This exposure does not result in any apparent long-term changes in whole animal morphology or behavior, which suggests there is not large-scale cell death, and the DA neurodegeneration appears similar to our earlier studies in which we characterized 6-OHDA-induced DA cell death by loss of dendritic GFP and loss of neuronal integrity by electron microscopy (Nass et al. 2002). Al³⁺ exposure has been associated with the development of PD, and has been shown to increase PD-associated α-synuclein aggregation and fibrillation in vitro which suggests that the toxicant may interact with α-synuclein to increase DA neuron dysfunction and pathology (Uversky et al. 2001, Zayed et al. 1990b). In order to determine whether Al³⁺ may amplify the toxicity of α-synuclein-induced DA neuronal death in vivo, we exposed nematodes expressing human WT α-synuclein to 100 μM AlCl₃ for 30 min, and found a significant increase in DA neuronal death (Fig. 1d). These results indicate that short-term Al³⁺ exposure can confer DA neuron degeneration in C. elegans, and α-synuclein expression amplifies the cell death.

SMF-3 is highly conserved with the rice Al³⁺ transporter Nrat1 and the yeast intracellular metal transporter Smf2p

A number of divalent metals enter eukaryotic cells and are transported between cellular compartments through the DMTs (Papp-Wallace & Maguire 2006, Portnoy et al. 2000). Recently a DMT-1 homologue, Nramp aluminum transporter 1 (Nrat1) in rice was shown to function as a trivalent Al³⁺ transporter (Xia et al. 2010). A BLAST search with the rice Nrat1, and a sequence alignment of the results using ClustalW2 indicated that the C. elegans SMF-3 is highly conserved with Nrat1 (53% similar, 34% identical) (Fig. 2) (Altschul 1991, Larkin et al. 2007). There is also high sequence homology between SMF-3 and the yeast intracellular DMT Smf2p (49% similar, 31% identical) (Portnoy et al. 2000, Portnoy et al. 2002). We have previously shown that SMF-3 is highly homologous with the human DMT-1 (Settivari et al. 2009), and the strong conservation of SMF-3 with Nrat1 and Smfp2 suggests that SMF-3 may also transport intracellular Al³⁺ to facilitate Al³⁺-induced DA neurodegeneration.

Figure 2. Sequence alignment of the rice Al³⁺ transporter Nrat1, yeast and mammalian DMTs, and C. elegans SMF-3.

ClustalW was used to align the protein sequences of the rice aluminum transporter Nrat1 (accession BAJ22943.1), human DMT-1 (accession NP_000608), yeast Smf2p (accession NP_011917) and C. elegans SMF-3 (accession NP_500235). Putative transmembrane domains are highlighted in gray with the arrow indicating the orientation relative to the membrane (right-directed arrowhead, N-terminal to C-terminal, extracellular to intracellular). The consensus transport sequence is highlighted in black. An asterisk indicates that the amino acids in that column are identical, a colon indicates that the amino acids are highly conserved and a period indicates that the amino acids are similar.

SMF-3 is expressed within DA neurons

To determine the location of SMF-3 expression in C. elegans, we generated antibodies to the nematode SMF-3. The antigenic sequence is unique to SMF-3 and spans between transmembrane domains two and four. To determine whether the DA neurons express SMF-3, we generated primary cultures from BY250, since GFP is strongly expressed in DA neurons both in vivo and in vitro (Carvelli et al. 2004, Nass et al. 2002). We incorporated affinity-purified anti-SMF-3 to evaluate cellular SMF-3 expression levels. As can be seen in Figure 3c, SMF-3 immunoreactivity is observed in all DA neurons, as well as other cell types (Fig. 3a–d, data not shown). To determine whether SMF-3 may be expressed intracellularly, we utilized the Measure Colocalization Application in MetaMorph imaging software. The degree of overlap between GFP and SMF-3 signal was calculated. On average 96% of SMF-3 immunofluorescence overlapped with GFP fluorescence, while only 74% of GFP overlapped with SMF-3 fluorescence (data not shown). Based on protein sequence, SMF-3 is likely a membrane protein. Furthermore, GFP is a soluble protein and would likely be found only in the cytoplasm and intracellular compartments (ie, not the plasma membrane). Considering that nearly all of the SMF-3 signal overlaps with the GFP signal suggests that the majority of SMF-3 does not reside on the plasma membrane. Taken together, these results indicate that SMF-3 is expressed in DA neurons and likely in an intracellular compartment.

Figure 3. SMF-3 is expressed in C. elegans DA neurons.

Primary cultures were generated from WT nematodes expressing GFP in the DA neurons. Differential interference contrast (DIC) image of a DA neuron (a) which was identified by the expression of GFP (b). Immunofluorescence was performed with a primary antibody to SMF-3 and an Alexa Fluor 594 secondary antibody to observe the expression of SMF-3 (c). The merge image indicates that SMF-3 is expressed in DA neurons (d). Final images were created in Image J from a Z-stack of images obtained with an Olympus2 confocal microscope. Scale bar represents 5 μm.

SMF-3 contributes to Al³⁺-induced DA neuron degeneration

Considering that Al³⁺ confers DA neurodegeneration and SMF-3 is expressed in DAergic neurons, we asked if SMF-3 plays a role in Al³⁺-associated neuropathology. To determine if SMF-3 contributes to Al³⁺-associated DA neuron degeneration, we crossed our P_dat-1∷GFP transgenic animals (BY250) into the C. elegans strain that contains a mutation in SMF-3, RB1074 smf-3(ok1035) resulting in strain RJ934 (P_dat-1∷GFP; smf-3(ok1035)). Sequencing of the smf-3(ok1035) mutant revealed a 2,067 bp deletion that spans from amino acid 154 to amino acid 378 and results in a truncation of the protein beginning in transmembrane domain 4 (data not shown). The mutation likely results in a null mutant since at least 80% of the consensus transport sequence as well as the C-terminal of the protein has been deleted. The morphology of the DA neurons in animals containing the deletion mutation appear identical to those of BY250 ((Settivari et al. 2009); data not shown), suggesting that SMF-3 does not play a significant role in maintaining DA neuron integrity. To determine if SMF-3 contributes to Al³⁺-induced DA neuron degeneration, we exposed the smf-3 mutants to various concentrations of AlCl₃ for 30 min and evaluated DA neuronal integrity 72 h later. As can be seen in Figure 4a, the smf-3 mutants were significantly more resistant to 100, 250 and 500 μM AlCl₃ relative to WT. We also asked whether SMF-1 or SMF-2 may contribute to Al³⁺-induced degeneration and we did not find any significant change in Al³⁺-induced cell death (Fig. 4b). These results indicate that the expression of SMF-3 increases DA neuron vulnerability to Al³⁺, and suggest that animals containing the deletion likely do not express a functional protein.

Figure 4. SMF-3 contributes to Al³⁺-induced DA neurodegeneration.

Synchronized L1 stage WT or smf-3 mutant (Δsmf-3) nematodes were exposed to increasing concentrations of AlCl₃ for 30 min and allowed to recover for 72 h on NGM plates. DA neuron degeneration was evaluated in WT and mutant animals at each concentration as described in Methods. Asterisk indicates p ≤ 0.01 (a). There is no significant difference in the DA neuron sensitivity to Al³⁺ in smf-1 or smf-2 mutant worms (b).

SMF-3 protein expression is downregulated following exposure to Al³⁺

We have previously shown that acute exposure to Mn²⁺ reduces gene expression of the C. elegans DMTs, likely to protect against oxidative stress (Settivari et al. 2009). In order to determine if Al³⁺ exposure results in a decrease in SMF-3 protein levels, we examined transporter expression levels in control and Al³⁺-exposed nematodes. Young nematodes exposed to 100 μM AlCl₃ for 30 min showed a dramatic decrease in SMF-3 protein levels within 24 h (Fig. 5a). These results indicate that SMF-3 protein levels are exquisitely sensitive to Al³⁺. We also do not observe immunoreactive bands in the unexposed or Al³⁺-exposed mutant at the WT SMF-3 molecular weight size, or lower molecular weights that could represent a truncated protein (Fig. 5a, data not shown). Taken together, these results suggest that the decrease in SMF-3 expression may be an attempt to limit intracellular exposure to Al³⁺.

Figure 5. SMF-3 protein expression is reduced following Al³⁺ exposure and contributes to cellular Al levels.

Western blotting with a SMF-3 antibody was used to evaluate protein levels following a 30 min exposure to 100 μM AlCl₃ and 24 h recovery. No SMF-3 immunoreactivity is observed in smf-3 mutant worms. GAPDH is used as a loading control (a). Whole animal aluminum levels in L4 stage animals were determined following a 30 min exposure to 100 μM AlCl₃ and analyzed on an ICP-MS. smf-3 mutant worms accumulate higher levels of Al than wild type worms. Asterisk indicates p ≤ 0.001 (b).

SMF-3 modulates cellular Al³⁺ levels

The high homology between the C. elegans SMF-3 protein and the rice Nrat1 and yeast Smf2p/Smf3p transporters suggests that SMF-3 may also be involved in Al³⁺ homeostasis. In order to determine if SMF-3 expression can modulate whole animal Al³⁺ levels, we treated L4 WT or smf-3 mutant animals with either water or 100 μM AlCl₃ (pH 4.6) for 30 min and determined whole animal Al³⁺ concentrations by ICP-MS. As can be seen in Figure 5b, an acute Al³⁺ exposure resulted in a 40% increase in Al in the smf-3 mutant relative to WT. We also found a slightly lower level of Al³⁺ in the non-Al³⁺ exposed mutant animals relative to WT. Although this low amount is statistically insignificant from WT animals, this difference may be due changes in general cellular metal homeostasis (eg, Fe²⁺ or Cu²⁺ levels) that could compete for the transporter or TfR-Me in a low Al³⁺ environment (Wu et al. 2012, Yokel 2006, Becaria et al. 2002). Taken together these results indicate the SMF-3 transporter modulates cellular Al³⁺ levels and is involved in cellular retention of Al³⁺.

SKN-1 and CED-4 modulate Al³⁺-induced DA neurodegeneration

SKN-1, the homologue to the PD-associated vertebrate Nrf2, has recently been shown to be expressed in C. elegans DA neurons and to inhibit methylmercury-induced DA neuronal death (Vanduyn et al. 2010). In order to determine if SKN-1 may also inhibit Al³⁺-induced DA neuron death in C. elegans, we knocked down skn-1 gene expression using RNAi. As genetic knockdown of skn-1 is embryonic lethal, first generation L1 larvae were exposed to 100 μM Al³⁺ for 30 min, and then allowed to recover on the RNAi bacteria for 72 h before evaluating DA neuron morphology. Animals in which skn-1 gene expression was reduced showed a small yet significant increase in DA neuron death (Fig. 6a), indicating that SKN-1 plays a neuroprotective role in Al³⁺-induced DA neurodegeneration. In vertebrates, Al³⁺ has been demonstrated to induce cell death via apoptosis (Banasik et al. 2005, Lukiw et al. 2005). In order to determine if apoptosis may also play a role in Al³⁺-induced cell death in C. elegans, we knocked down gene expression of the apoptotic gene Apaf1/ced-4 using RNAi as described above except the second generation L1s were exposed to AlCl₃. Genetic knockdown resulted in a decrease in DA neuronal death (Fig. 6b), indicating that apoptosis is likely contributing to the Al³⁺-induced cell death.

Figure 6. skn-1 and ced-4 modulate Al³⁺-induced DA neuron degeneration.

RNAi was used to knockdown gene expression of skn-1 (a) or ced-4 (b) and worms were exposed to 100 μM AlCl₃ for 30 min, and the RNAi feeding was continued during the 72 h recovery period. DA neuron degeneration was evaluated as described in Methods. Asterisk indicates p ≤ 0.05.

DISCUSSION

Overexposure to Al³⁺ has been associated epidemiologically with the development of a number of human neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Gulf War syndrome, Parkinsonism dementia, AD and PD (Kawahara & Kato-Negishi 2011, Petrik et al. 2007). In PD, post-mortem metal analysis of brains demonstrates increased Al³⁺ levels in the substantia nigra relative to healthy controls (Yasui et al. 1992, Hirsch et al. 1991). High exposure to Al³⁺ is reported to cause oxidative stress, mitochondrial dysfunction, ATP depletion, and apoptosis, although the genetic and molecular basis for the toxicity is poorly defined (Kumar et al. 2008, Lemire et al. 2009). A difficulty in identifying the molecular components involved in Al³⁺-induced neuropathology has been the cellular complexity of vertebrate models and the lack of in vivo genetic models to dissect the molecular pathways involved in the cellular dysfunction. In this study we describe a novel model for Al³⁺-induced DA neurodegeneration. We show that a brief 30 min exposure of C. elegans to 100 μM Al³⁺ reduces mitochondrial membrane potential, ATP levels, and confers DA neuron degeneration partially through apoptosis (Fig. 1, 6b). The concentrations of Al³⁺ that C. elegans were exposed in this study are environmentally relevant and similar to vertebrate studies, as well as the estimated dietary Al levels consumed by Americans (Becaria et al. 2002, Greger 1993, Walton 2012). Taken together, this model recapitulates key characteristics of Al³⁺-associated toxicity in vertebrates, and suggests the nematode will be a valuable genetic tool to explore the relationship between Al³⁺ exposures and toxicity and the subsequent neuropathology (Kumar et al. 2009, Sanchez-Iglesias et al. 2009).

Our studies show that Al³⁺-induced DA neurodegeneration is partially dependent on a homologue of human DMT-1, SMF-3 (Fig 4). Until recently DMTs were believed to exclusively transport divalent cations. The biophysical characteristics of Al³⁺ suggest though that the metal can interact with divalent metal binding sites. Al³⁺'s small ionic radius (54 pm) relative to other DMT substrates (74 pm – 175 pm), as well as an ionic radius-to-charge ratio similar to Fe²⁺ (0.16 vs. 0.17) indicates that Al³⁺ should be able to compete with Fe²⁺ for binding on macromolecules (Kawahara & Kato-Negishi 2011, Bharathi & Rao 2008). Consistent with these properties, Al³⁺ has been shown to compete with divalent metals including Fe²⁺, Ca²⁺, Mg²⁺, and Zn²⁺ in binding with biomolecules (Martin 1986). The identification of the rice plasma membrane Al³⁺ transporter Nrat1, which has high amino acid sequence conservation with the C. elegans SMF-3 (Fig. 2), suggests that SMF-3 may also transport Al³⁺ (Xia et al. 2010). Indeed our studies show a significant increase in whole animal Al-levels in the functional null mutant, consistent with a role of SMF-3 in cellular Al³⁺ regulation.

A homologue of SMF-3, Smf2p, is expressed in yeast intracellular vesicles and transports Fe²⁺ and Mn²⁺ out of vesicles into the cytosol where the metals are subsequently transported into the Golgi or mitochondria (Luk & Culotta 2001, Portnoy et al. 2000, Portnoy et al. 2002). Smf2p is tightly regulated post-translationally, as excess Mn²⁺ results in Smf2p vacuole-associated degradation (Portnoy et al. 2000, Luk & Culotta 2001). SMF-3 may also be regulated post-translationally as the addition of Al³⁺ resulted in a dramatic decrease in protein levels within 24 h following Al³⁺ exposure (Fig. 5a). Furthermore, similar to DMTs and Smf2p, SMF-3 may reside in an intracellular compartment to transport metals into the cytoplasm, as the immunofluorescence data suggests that the vast majority of SMF-3 is located within the cell (Portnoy et al. 2002, Burdo et al. 2001). Consistent with SMF-3 residing in an intracellular compartment, a SMF-3 functional null mutant inhibits Al³⁺-induced DA neuron degeneration and retains 40% more Al³⁺ relative to controls. It is unlikely that SMF-1 and SMF-2 also transport Al³⁺, since a genetic deletion of either transporter does not affect Al³⁺-induced DA neuron vulnerability (Fig 4), but this cannot be ruled out until a more detailed Al³⁺ transport study of the cloned gene is performed. Taken together, these results suggest that SMF-3 contributes to Al³⁺-induced DA neurodegeneration through Al³⁺ efflux from an intracellular compartment within the DA neuron (Fig 7).

Figure 7. A model for SMF-3-associated Al³⁺ trafficking and toxicity in C. elegans DA neurons.

SMF-3 resides in an intracellular compartment and is predicted to transport Al³⁺ into the cytosol. Cytoplasmic Al³⁺ can increase α-synuclein aggregation, activate stress response pathways through the transcription factor SKN-1, and activate mitochondria associated-cell death pathways. The mechanism by which Al³⁺ is transported into the DA neurons and the intracellular vesicles is not known, but may involve receptor-mediated endocytosis, simple diffusion, or facilitated diffusion through ion channels (Becaria et al. 2002).

The PD-associated proteins DMT-1, α-synuclein and Nrf2 can interact functionally with each other and can contribute to DA neuron degeneration. DMT-1 expression has been shown to increase in PD patients and with age, and has been shown to increase in PD cell models (Salazar et al. 2008, Zhang et al. 2009). α-synuclein, the presynaptic protein whose aggregation and fibrillation contributes to familial and idiopathic PD, has been shown to facilitate DMT-1-associated DA neuron cell death (Chew et al. 2011). Nrf2 haplotypes have been associated with the development of PD, and Nrf2 deficiency has recently been shown to exacerbate α-synuclein-induced aggregation and cell death (Lastres-Becker et al. 2012). Furthermore, the aggregation and fibrillation of α-synuclein increases dramatically in the presence of Al³⁺ (Uversky et al. 2001). Our studies are consistent with the role these proteins likely play in Al³⁺-associated DA neuron vulnerability as overexpression of human α-synuclein or genetic knockdown of Nrf2/SKN-1 results in an increase in Al³⁺-induced DA neuropathology.

Al³⁺ induces cell death in vertebrates through the initiation of the apoptotic pathway (Vasudevaraju et al. 2008). Mitochondria are likely an intracellular target of Al³⁺ as the metal inhibits Na⁺/Ca²⁺ exchange resulting in an increase in Ca²⁺ in the mitochondria and ROS levels, and leads to the release of cytochrome c and subsequent apoptosis (Vasudevaraju et al. 2008, Kawahara & Kato-Negishi 2011). Al³⁺ has been shown to induce the opening of the mitochondrial transition pore resulting in apoptosis (Toninello et al. 2000). Al³⁺ also can cause oxidative stress and apoptosis by increasing the free radical damage associated with Fe²⁺ (Vasudevaraju et al. 2008, Zatta et al. 2003). Our studies are consistent with the role of apoptosis in Al³⁺-induced cell death in the nematode as a reduction in gene expression of the vertebrate apoptotic caspase homologue Apaf1, ced-4, results in a significant increase in viable DA neurons in the nematode.

In summary, we describe a novel model for Al³⁺ toxicity and show that the C. elegans transporter SMF-3 plays a significant role in modulating Al³⁺-induced DA neuron degeneration through the intracellular sequestration of Al³⁺. We also show that SMF-3 expression is sensitive to Al³⁺, and the PD-associated proteins α-synuclein, Nrf2/SKN-1, and Apaf1/CED-4 modulate Al³⁺-associated DA neuron cell death. This novel genetic model should facilitate identification of molecular pathways and potential therapeutic targets involved in Al³⁺-associated DA neuron pathology.

Abbreviations used

AD

Alzheimer's disease

DA

dopamine

DMT

divalent metal transporter

ECL

enhanced chemiluminescence

GFP

green fluorescent protein

ICP-MS

inductively coupled plasma mass spectrometry

NRAMP

natural resistance-associated macrophage protein

PAGE

polyacrylamide gel electrophoresis

PD

Parkinson's disease

ROS

reactive oxygen species

SN

substantia nigra

TfR-ME

transferrin receptor mediated endocytosis

TH

tyrosine hydroxylase

TMRE

tetramethyl rhodamine ethyl ester

WT

wild type