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- 10.1093/biohorizons/hzy012
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Abstract
Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 BioscienceHorizons Volume 11 2018 10.1093/biohorizons/hzy012 ............................................................................................ ..................................................................... Research article Manganese toxicity and effects on polarized hepatocytes Luke Tillman Genetics and Complex Diseases, Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA *Corresponding author: University of Exeter Medical School, St Luke’s Campus, 79 Heavitree Rd, Exeter, Devon, EX1 1TX, UK. Email: Luketillman12@gmail.com Supervisors: Khristy Thompson, PhD and Marianne Wessling-Resnick, Professor of Nutritional Biochemistry, Harvard T.H. Chan School of Public Health ............................................................................................ ..................................................................... Manganese (Mn) is an essential metal involved in several cellular metabolic pathways including DNA synthesis, sugar meta- bolism and protein modification. The majority of Mn is obtained through the diet in food products such as nuts, whole grains and leafy greens. Abundant in most diets, Mn deficiency is rare while excess exposure in the occupational environment leads to cytotoxic levels. Labour workers commonly inhale Mn in settings like welding and mining. Metal inhalation bypasses many of the body’s homeostatic pathways leading to accumulation in the brain. Physiologically, the presence of Mn in Mn- sensitive brain regions, such as the globus pallidus, has been linked to neurodegeneration and induction of a Parkinsonian-like syndrome known as manganism. Mn homeostasis is therefore critical for brain health. The liver controls the redistribution of excess Mn to specific tissues/organs and hepatobiliary clearance. Mutations in Mn transporters, however, compromises homeosta- sis causing hepatic damage and surplus body Mn. Understanding Mn toxicity in hepatocytes is crucial for developing new medi- cines that prevent blood Mn build-up. To understand the molecular changes attributed to excess hepatic Mn, we sought to determine changes in hepatocyte viability under Mn hepatotoxicity. For these experiments, polarized hepatocytoma WIF-B cells were grown for 12–14 days to achieve maximal polarity. Immunocytochemistry, Western blot and MTT viability assays helped characterize Mn’s effect on the Golgi. We found that WIF-B cell viability was maintained during 4 h exposures of up to 100 μMMn. Under these conditions, we identified no change at the cis-Golgi but levels of the trans-Golgi marker TGN38 fell in a dose- dependent manner. Immunofluorescence (IF) images confirmed that Mn-induced TGN38 loss, while the cis-Golgi marker GM130 remained unaffected. Treatment with the lysosomal inhibitor Bafilomycin A for 16 h prevented degradation of TGN38 when cells were exposed to Mn for 4 h and increased its co-localization with the late endosomal marker mannose-6-phosphate receptor (M6PR). Our results suggest disrupting Mn homeostasis negatively affects the integrity of the Golgi apparatus, altering normal Mn trafficking in WIF-B cells. Understanding how Mn-induced changes in the Golgi architecture affect toxicity is key to developing therapeutic treatments for Mn toxicity. Key words: manganese, manganism, TGN38, GM130, WIF-B, toxicity Submitted on 25 July 2018; editorial decision on 2 November 2018 ............................................................................................ ..................................................................... Introduction The European Union’s Scientific Committee for Food has −1 −1 defined 1–10 mg of Mn person day to be a ‘safe and Mn is essential to the function of the immune system, bone adequate intake’ while the US National Research Council spe- development and lipid metabolism (Erikson et al.,2005; −1 cifies safe daily intake to be 0.3–1, 1–3 and 2–5mg day for Aschner et al.,2007; O’neal et al.,2014; Zizza et al., 2018). ............................................................................................... .................................................................. © The Author(s) 2019. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Research article Bioscience Horizons � Volume 11 2018 ............................................................................................... .................................................................. infants, children and adults, respectively. The majority of Mn in loss of function, display hypermanganesemia (Mn accumula- is obtained from the diet with daily intake commonly ranging tion in the liver and brain) (Tuschl et al., 2016a)while ZnT10 between 2 mg and 6 mg of which ~1.5% is absorbed (Davis, deficient mice display ∼20–60-fold increase in brain, liver and Zech and Greger, 1993). Due to its high abundance in the diet blood Mncomparedtocontrol(Hutchens et al.,2017). Thus, and regular clearance by the liver, blood Mn homeostasis is these findings elucidate the role of ZnT10 in Mn homeostasis. rarely compromised. Excess Mn, however, is commonly owed SLC39A8 encodes a protein known as ZIP8, a divalent to exposure in occupational settings. Workers employed in metal ion transporter that has been shown to transport Mn welding, battery manufacture and mining inhale toxic levels in vitro (He et al., 2006; Boycott et al., 2015). Patients with daily (Wang, DU and Zheng, 2008; Aschner et al.,2009). This SLC39A8 mutations suffer from severe Mn deficiency leading leads to motor dysfunction, neuropsychiatric defects and cog- to neurological and skeletal defects (Boycott et al., 2015; Park nitive disabilities by accumulating in the basal ganglia et al., 2015). According to a recent study, hepatic ZIP8 (Bakthavatsalam et al.,2014). Mn acts as a catalytic cofactor reclaims Mn from the bile in order to contribute to whole for many antioxidant enzymes in the mitochondria (Zhou body Mn homeostasis (Lin et al., 2017). In mutant ZIP8- et al.,2018). High levels, however, impair homeostasis and deficient mice, the same study noted a substantial decrease in mitochondrial function; for example, inhibiting respiration liver, kidney, brain and heart Mn concentrations (Lin et al., complexes I and II (Sriram et al.,2010) which is detrimental to 2017) suggesting a crucial role of ZIP8 in Mn cellular import. the survival of neurons (Zorov, Juhaszova and Sollott, 2014). Continued neuronal cell death then leads to the development Recent findings surrounding the Mn transporter ZIP14, a of a Parkinsonian-like syndrome named manganism. Patients homologue of ZIP8, revealed it as a key factor for preventing suffering from this disease display behavioural changes, tre- Mn accumulation in mouse brains (Jenkitkasemwong et al., mors and difficulty walking (Peres et al.,2016). 2018). Similar to ZIP8, ZIP14 is capable of transporting iron (Km = 6 μM), Mn (Km = 4.4 and 18.2 μM) and cadmium Mn enters the blood by intestinal absorption and is trans- (Km = 0.14 and 1.1 μM) (Girijashanker et al., 2008; Ji and ported by the portal vein to the liver where excess may be Kosman, 2015; Tuschl et al., 2016b), however, its association secreted into the bile (Barceloux, 1999). More than a dozen with Mn homeostasis was majorly advanced by the study of putative Mn transporters have been identified (Horning et al., humans carrying SLC39A14 mutations. In a recent study, 2015) but few have been well characterized. Several studies patients carrying mutated SLC39A14 displayed learning dis- have noted that perturbations in Mn uptake and efflux result abilities and Parkinsonian features. Magnetic resonance from disruption to the genes involved in maintaining homeosta- imaging displayed brain features characteristic of Mn accu- sis (Rentschler et al., 2012; Riley et al., 2017; Mukhopadhyay, mulation. Companion studies found zebra fish knockout 2018). Genes such as SLC30A10 (ZnT10), SLC39A14 models accumulated Mn in the brain but not in other com- (ZIP14), SLC39A8 (ZIP8) and ATP2C1 (SPCA1) are all monly affected organs such as the liver, spleen and kidneys involved in maintaining appropriate blood Mn levels. Recent (Tuschl et al., 2016b). A study using SLc39a14 knockout genetic studies have revealed the significant role of SLC30A10 mice observed similar characteristics coupled with impaired in Mn homeostasis. Patients with mutations in ZnT10, resulting 2+ 2+ Figure 1. Potential cellular model for Mn efflux in the WIF-B cell. At the sinusoidal or basolateral membrane Mn is thought to be pumped 2+ into the WIF-B cell by the metal transporter Zip14. Mn is stored at the trans-Golgi, however excess stores are sent to the plasma membrane of the bile canaliculus by the process of exocytosis. ............................................................................................... .................................................................. 2 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Bioscience Horizons � Volume 11 2018 Research article ............................................................................................... .................................................................. Mn elimination and increased tissue Mn accumulation 100 μM) prepared by serial dilution from an original 10mM (Aydemir et al., 2017). Thus, ZIP14 is considered a vital con- stock. Cells were incubated under stated conditions for either tributor to Mn homeostasis in mammals. 4 or 16 h in a tissue culture incubator. SPCA1 is a Mn transporter involved in cellular detoxifica- Cell viability was determined using the TOX-1 assay kit tion. Sepulveda et al. (2012) observed Mn accumulation in (Invitrogen). After treatment with MnCl , cells were treated the brain, pituitary and thyroid glands after a 3-week treat- with 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium −1 ment of dietary Mn in mice (Sepulveda et al., 2012). Leitch Bromide (MTT; 0.5 mg ml ) for 4 h. The crystal products et al. reported high Mn sensitivity in ATP2C1 negative WIF-B formed were dissolved in isopropyl alcohol containing 0.1 N −/− hepatocytes. SPCA1 cells displayed a 30% reduction in hydrochloric acid causing the solution to change colour. +/+ growth rate compared to control (SPCA1 )(Leitch et al., Absorbance values were measured at a wavelength of 570 nm 2011). These findings suggest functional expression of SPCA1 using a Gen 1.05 biotech plate reader. Background absorb- is important for maintaining Mn homeostasis in the brain and ance, measured at 690 nm, was subtracted from the 570 nm plays a role in effective WIF-B cell growth and polarization. measurement. Cell viability was calculated as a percentage of control. A need to better understand the intracellular homeostasis and effects of Mn was raised by these studies. To do this, we Indirect immunofluorescence microscopy elected to use WIF-B cells, a derivative of the WIFI2-1 cell line (hybrid mix of rat hepatoma and human fibroblast, Fao) that Cells were grown on coverslips for 12–14 days. MnCl was display a highly polarized structure (Ihrke et al., 1993). Up to added to the media at a final concentration of 0–100 μM for 90% of confluent WIF-B cells form apical domains that 4 h. WIF-B cells were then fixed using methanol with 4% enclose spherical bile canalicular-like spaces (BCs, Fig. 1) paraformaldehyde on ice for 1 min, before mixing with cold while also growing in a monolayer. (−20°C) methanol for an additional minute. The solution was aspirated, and cells were exposed to 1 ml of cold (−20°C) Whilst examining the effects of time-dependent Mn contact methanol for a further 10 min on ice. Three, five-min intervals on WIF-B cells, we noted that cell viability was maintained dur- of exposure to phosphate-buffered saline (PBS, Sigma Life ing 4 h exposure of up to 500 μM Mn but diminished after Sciences) were conducted to rehydrate cells 16 h. Within 4 h, the trans-Golgi marker TGN38 was degraded in a dose-dependent manner while the cis-Golgi marker GM130 For immunostaining, cells were blocked in 1 ml of 1% was maintained. These changes in Golgi morphology led us to Bovine serum albumin (BSA, Sigma Life Sciences)/PBS mix for study the role of TGN38 in WIF-B cells. Further research of Mn 30 min at room temperature. Primary antibodies were diluted dependent protein degradation may develop understanding of to the desired concentration (see Antibody section) in 1% Mn homeostasis. The goals of this study were to define Mn tox- BSA/PBS of which ~100 μl was placed on the coverslip in a icity in WIF-B cells, to characterize the level and distribution of moistened chamber for 1 h. Cells were washed three times in TGN38 in response to Mn and to identify the contribution of a 0.1% BSA/PBS mix before treatment with the secondary TGN38toMnmetabolisminWIF-B cells. antibody (diluted in 1% BSA/PBS solution) for 30 min. Cells received three washes before being mounted onto slides. Prior to fixation, images were counter stained using a NucBlue live Materials and Methods cell stain (Invitrogen) to visualize nuclei. Coverslips display- ing the dose-dependent response of TGN38 and GM130 were Cell culture viewed using a Zeiss apotome Axioscope at 63x oil magnifi- WIF-B cells were supplied by Dr Pamela Tuma of The Catholic cation. Images obtained for our co-localization study were University of America (Washington DC, USA) and grown obtained using a Yokogawa CSU-X1 spinning disk confocal according to the methods of Ihrke et al. (1998). All cells were system with a Nikon Ti-E inverted microscope. Focus was grown at 37°C in an F12 Coons media mix containing 1% peni- obtained using a 60× Plan Apo objective lens with Zyla cillin streptomycin, 1.1 μM amphotericin B, 10 μM hypoxan- cMOS camera using 561 and 488 lasers. Nikon Instruments thine–aminopterin–thymidine and 5% foetal bovine serum in a Software elements were used for acquisition parameters, shut- humidified 7% CO incubator at pH 7.0. Cells used for experi- 2 ters, filter positions and focus control. mental procedure were plated on glass coverslips (22 mm × 4 −1 22 mm, 2.4 × 10 cells well ) while cells required for further Western blot 2 5 −1 passage were grown on 10 cm dishes (3 × 10 cells coverslip ) Thirteen days after seeding, cells were exposed to MnCl (0, and were grown for 7–8daystoprevent BC formation. Cells 5, 10, 50,100 μM) for 4 h and subsequently lysed using Radio intended for experimental procedure were not used prior to this immunoprecipitation assay (RIPA) buffer (25 mM Tris, time to allow formation of functional BCs. 150 mM sodium chloride, 1% sodium deoxychlorate, 1% Nonident P40, 1 mM PMSF, pH 7.6). BSA standards were Cell viability assay −1 prepared (4, 8,16, 32 and 64 μgml ) using a 1% BSA stock On Day 13, WIF-B cells grown on coverslips were moved to in millipure water. Samples were then diluted (1:200) before new wells with media containing MnCl (0, 5, 10, 25, 50 and mixing with acidified Coomassie Brilliant Blue G-250 dye ............................................................................................... .................................................................. 3 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Research article Bioscience Horizons � Volume 11 2018 ............................................................................................... .................................................................. (Bradford reagent, 1:4). After a 5-min incubation period at of Variance (ANOVA) test. For co-localization analysis, data room temperature, absorbance was measured at 595 nm and were collected from individual slides and an ANOVA was protein concentration determined from a standard curve. performed to determine significance. ImageJ software and the JACoP plugin were used to determine Mander’s coefficient. WIF-B protein lysates (50 μg) were mixed with 2-β- Mercaptoethanol (Sigma Life Sciences) in 2× Laemmli sample buffer (BIO-RAD, 1:20). Samples were then heated to 72°C Results for 10 min. Electrophoresis was conducted using a 10% Mini-PROTEAN TGX Precast Gel (BIO-RAD). WIF-B cell viability decreased at low-Mn Blots were blocked in a 5% non-fat milk solution in tris- concentrations buffered saline (TBS) containing Polysorbate 20 (TBSTW20, After exposure to Mn for 4 h, we observed a non-significant Fisher Scientific) for 30 min. The membrane was then stained reduction in cell viability across the full range of Mn concen- for 1 h (see Antibody section). Membranes were washed in trations (Fig. 2a). After 16 h of exposure to low and high con- TBSTW20 before addition of secondary antibodies for 1 h at centrations of Mn, cell viability was significantly reduced (P < room temperature. Glyceraldehyde 3-phosphate dehydrogen- 0.05). At intermediate concentrations of 10 μM and 25 μM, ase (GAPDH) was probed as a loading control. Blots were however, cell viability was approximately equal to control quantified using ODYSSEY LI-COR Image Studio Software after 16 h. These results suggest that Mn toxicity is time- (version 5.2). dependent. The greater viability of cells exposed to intermedi- ate concentrations may potentially be explained by a time and Antibodies dose-dependent adaptive homeostatic mechanism, requiring a certain concentration of Mn to be induced. At excessive Antibodies used in the IF study were mouse anti-rat TGN38 (BD Biosciences, 1:100), purified mouse anti-GM130 (BD Biosciences, 1:100) and anti-M6PR (Abcam, 1:1000). Secondary antibodies included Alexa488 green or Alexa568 red anti-mouse. During western analysis, primary antibodies included mouse anti-rat TGN38 and mouse anti-GM130 antibodies (BD Biosciences, 1:1000). GAPDH was detected using mouse anti-GAPDH (Sigma 1:2000). IgG IRDye 800 CW donkey anti-mouse or anti-rabbit (1:3000) or IRDye 680 RD donkey anti-mouse or donkey anti-rabbit (1:3000) were used for detection. Statistical analysis All statistical analyses were performed using GraphPad PRISM software (version 7.00). Toxicology assay data was analysed by means of a paired t-test against control. Western blot protein assays were compared using One-Way Analysis Figure 2. Cell viability assay of WIF-B cells exposed to variable Mn concentrations. Cells were incubated for (a) 4 h (n = 3–5) and (b) 16 h Figure 3. TGN38 levels decreased with increased concentration of (n = 3), respectively, in media containing indicated Mn concentrations. MnCl . WIF-B hepatocytes were grown on coverslips and treated with Significance values were determined using the paired t-test; *P < 0.05. (a and e) 0 μM, (b and f) 10 μM, (c and g) 50 μM, (d and h) 100 μM, Data are means ± SEM. MnCl (in standard media) for 4 h (bar, 10 μm). ............................................................................................... .................................................................. 4 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Bioscience Horizons � Volume 11 2018 Research article ............................................................................................... .................................................................. concentrations, this mechanism may be unable preserve cell Mn compared to Mn alone. IF showed clustering of TGN38 viability. post BAF treatment (Fig. 6d), while cells treated with Mn alone displayed no such change (Fig. 6a). We hypothesize that Levels of TGN38 decreased with elevated this difference is due to TGN38 accumulation in the lysosome post BAF + Mn treatment. Mn Excess Mn uptake into the Golgi apparatus reduces function Inhibition of the lysosome did not affect of the secretory pathway thereby interrupting homeostasis levels of GM130 (Witzleben et al., 1968; Barceloux, 1999). As cellular viability remained relatively stable over the 4 h period, we elected to TGN38 is a Type I integral membrane protein that constitu- test for intracellular changes at these concentrations. TGN38 tively cycles between the TGN and plasma membrane but at expression was measured at Mn ranges where cells were steady state is localized predominantly to the TGN (Stephens known to maintain ≥80% viability for 4 h. Preliminary and Banting, 1999). As levels of the protein notably decreased experiments showed expression of the trans-Golgi marker with Mn treatment, we elected to test the same conditions on TGN38 decreased with increased Mn exposure, indicating the cis-Golgi marker GM130. possible interference with the Golgi membrane structure. IF GM130 is a cis-Golgi matrix protein that plays an integral microscopy confirmed Mn exposure reduced levels of TGN38 role in the stacking of the cisternae and maintenance of Golgi (Fig. 3a–d). To test whether this Mn-induced sensitivity was architecture (Nakamura et al., 1995). Following lysosomal isolated to the trans-Golgi, we stained for the cis-Golgi inhibition using BAF and subsequent Mn exposure, GM130 marker GM130. IF showed no change in GM130 levels under showed no visible clustering (as was seen with TGN38, any of the Mn treated conditions (Fig. 3e–h). Fig. 6h). Mn did not decrease levels of GM130 (Fig. 6f) nor Western blot analysis identified a significant decrease in was it degraded in the lysosome of the WIF-B cells. We there- TGN38 (P < 0.05, Fig. 4a) while levels of GM130 were fore elected to use it as a control for Golgi degradation. Given unchanged (Fig. 4b). All data were compared to the loading these results and the integral role GM130 plays in conserva- control (GAPDH). Our data confirmed that Mn decreased tion of Golgi structure, we concluded that Mn did not disturb levels of TGN38 in WIF-B cells. cis-Golgi morphology in the WIF-B cells. Inhibition of the lysosome reduced TGN38 TGN38 is degraded in the lysosome at 100 degradation μM Mn exposure To determine whether the decrease in TGN38 was due to To determine if TGN38 was being trafficked toward the lyso- lysosomal degradation, cells were treated with the lysosomal some we performed a co-stain of the late endosomal marker inhibitor Bafilomycin A (BAF) at a working concentration of M6PR with TGN38. IF staining identified increased co- 50 nM for 16 h before incubation with 100 μM Mn for an localization of TGN38 and M6PR after treatment with BAF andMn(Fig. additional 4 h. Western blot analysis identified 74% (Fig. 5) 7). Pearson’s correlation coefficient (PCC) was −1 higher levels of TGN38 in WIF-B cells exposed to BAF and performed randomly (n = 3–5 images slide ). Each condition Figure 4. TGN38 decreases with increased MnCl . WIF-B cells were treated with indicated MnCl concentrations in standard media for 4 h. IF 2 2 detected (a) TGN38 or (b) GM130. GAPDH is loading control. TGN38 levels appeared significantly reduced compared with control between the concentrations of 10–100 μM. Graphs displays ratio of desired protein/GAPDH from three separate experiments ± SEM. Significance was determined using the ANOVA test. ............................................................................................... .................................................................. 5 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Research article Bioscience Horizons � Volume 11 2018 ............................................................................................... .................................................................. Figure 5. Treatment with BAF decreased Mn-induced TGN38 degradation. WIF-B cells grown on coverslips were exposed to 50 nM Bafilomycin for 16 h before treatment with 100 μM MnCl for 4 h. Shown are means ± SEM (n = 3): a, different from control *P < 0.05; b, different from a **P < 0.005. Significance was determined using the ANOVA test. was performed in triplicate. PCC identified no significant differ- ence between any of the tested conditions. We then investigated co-localization using Mander’s overlap coefficient (MOC). Although both measures are mathematically similar PCC accounts for variation surrounding the mean whilst MOC ana- lyses absolute intensity of the selected signal (Adler and Parmryd, 2010). MOC identified a significant increase in co- localization of M6PR and TGN38 between control (0.296) and BAF + Mn (0.675) treated slides (P < 0.0005). Our data eluci- dates the potential pathway by which TGN38 is degraded in Figure 6. Inhibition of the lysosome reduced degradation of TGN38. WIF-B cells (Fig. 8) and indicates the role of Mn in this process. WIF-B hepatocytes were grown on coverslips and treated with (a and e) 0 μM MnCl , (b and f) 100 μM MnCl , (c and g) 50 nM Bafilomycin, 2 2 (d and h) 100 μM MnCl + 50 nM Bafilomycin. Cells were incubated in Bafilomycin overnight before being exposed to MnCl in standard Discussion media for 4 h (bar, 10 μm). This study is the first to identify TGN38 as a Mn-responsive protein. Extracellular Mn triggered the exit of TGN38 from resistance to Mn toxicity with no significant decrease in via- the Golgi and trafficking to the lysosome where it was bility seen over 4 h. This agrees with the findings of degraded by BAF-sensitive hydrolases. This Mn-induced Thompson et al. (2018) who described WIF-B cells resisting effect was apparent by 4 h at concentrations of 5–50 μM, intracellular change at Mn doses as high as 500 μM indicating TGN38’s sensitivity to the metal in the WIF-B cells. (Thompson et al., 2018). After 16 h of exposure we observed Our toxicology assay, however, did not identify a significant an adaptive response where viability significantly decreased at decrease in cell viability until 16 h. From these results, we can 5 μM but was similar to control at intermediate concentra- infer that over 4 h Mn induces changes to the intracellular tions. Reasons for this regression are unknown, however, we environment that protect against Mn-induced toxicity. Over hypothesize that the increase in Mn concentration caused 16 h, however, low dose Mn (5 μM) is sufficient to decrease reduced expression of Mn importers. This would prevent fur- hepatic viability. ther Mn uptake into the cell allowing a mechanism of Mn Mn, like other micronutrients, is tightly regulated by efflux to restore levels to equilibrium. homeostasis (Buchman, 2014; King, 2014; Wessling-Resnick, 2014). Substantial evidence has shown that levels are sus- Whilst characterizing the effects of Mn on WIF-B viability tained using the same metabolic pathways commonly asso- we identified a significant decrease in TGN38 levels. Given ciated with iron uptake and efflux (Kim, Buckett and that the Golgi acts as a centre for Mn storage in adrenal PC12 Wessling-Resnick, 2013; Horning et al., 2015; Seo and cells (Carmona et al., 2010) we elected to study the effect of Wessling-Resnick, 2015). The WIF-B cells displayed great this process on Golgi integrity. ............................................................................................... .................................................................. 6 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Bioscience Horizons � Volume 11 2018 Research article ............................................................................................... .................................................................. Figure 7. Treatment with BAF increased colocalization of M6PR and TGN38 when treated with Mn. WIF-B cells were exposed to (a) 0 μM MnCl , (b) 100 μM MnCl , (c) 0 μM MnCl + BAF (50 nM) or (d) 100 μM MnCl + BAF. Mander’s coefficient identified an increase in co-localization of M6PR 2 2 2 and TGN38 between control (0.296) and cells treated with Mn and BAF (0.675). Research in HeLa cells identified Mn as a key ligand for TGN38 levels were also reduced at low doses of Mn the sorting fate of another Golgi protein, GPP130 (Tewari, (5–50 μM). We identified the lysosome as the final destination Bachert and Linstedt, 2015). We observed a change in the for Mn-induced TGN38 trafficking and understand sorting fate of TGN38 when exposed to Mn in WIF-B cells TMEM165 is subject to the same sorting fate under these con- but the mechanism behind this remains unclear. GPP130 is a ditions (Potelle et al., 2017). This observation leads us to protein that normally cycles between the cis-Golgi and endo- question whether the apparent sensitivity to Mn observed in somes, but ligand binding of Mn induces conformational these studies is unique to these proteins or is instead a charac- changes of sorting signals exposed in the cytoplasm. This teristic of the TGN. alters signal-vesicle interactions, causing endocytosis 2+ The TGN acts as a cellular Mn storage compartment in (Gibbons et al., 1976; Davis, Zech and Greger, 1993; Traub yeast (Culotta, Yang and Hall, 2005). This is thought to and Bonifacino, 2013). Given that both TGN38 and GPP130 reduce Mn toxicity by decreasing cytoplasmic Mn levels. Its display a high sensitivity to Mn, we cannot exclude a func- accumulation in the Golgi then leads to Mn efflux from the tional link between their degradation. cell via exocytosis (Thines et al., 2018). A comparable mech- Recent data comparing the degradation pattern of GPP130 anism is seen in plants such as Arabidopsis thaliana. with the Golgi membrane protein TMEM165 identified Exocytosis and vesicular trafficking by Golgi based vesicles 100 μM Mn as a sufficient concentration for GPP130 degrad- prevents cytoplasmic Mn accumulation reducing Mn-induced ation (Potelle et al., 2017). In contrast TMEM165’s sensitivity toxicity (Peiter et al., 2007). Similarly, anticancer drugs have to Mn was apparent at 1–25 μM. In addition, TMEM165 been shown to accumulate in the TGN, preventing assembly loss exceeded 95% after 8 h Mn treatment whilst GPP130 in other compartments thereby decreasing their cytotoxic loss was only 40%. These results indicate differences in the Larsen, Escargueil and Skladanowski, 2000). This effects ( sensitivity of Mn-responsive proteins. Similar to TMEM165, detoxification response to Mn by the Golgi prevents ............................................................................................... .................................................................. 7 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Research article Bioscience Horizons � Volume 11 2018 ............................................................................................... .................................................................. cytoplasmic over accumulation that would otherwise result in and the Lysosomal Proteolysis pathway: the former uses the organelle damage and eventual cell death. TGN translocation protein ubiquitin as a marker for degradation. The marked to the plasma membrane (a site involved in TGN38 cycling) proteins are then trafficked to the proteasome for proteolysis. would then result in Mn being exocytosed from the cell. A Lysosomal proteolysis involves the endocytic uptake of vesicle consequence of this process would be trans-Golgi associated coated proteins that are transported to the lysosome via proteins such as TGN38 and TMEM165 trafficking to the receptor mediated endocytosis (Cooper, 2000; Wolf and lysosome. Menssen, 2018). Protein degradation is an effective method by which cells M6PR recognizes many of the lysosomal enzymes at the adapt to their environment. Eukaryotic cells control protein TGN and is involved in their sorting to the late endosome. levels via two pathways: The Ubiquitin–Proteasome pathway This cyclic process is fundamental to retaining cellular physi- ology (Altan-Bonnet, Sougrat and Lippincott-Schwartz, 2004). Whilst the relevance of Mn-induced trafficking of TGN38 within this process is unknown, an almost identical −/− observation has been made in Rab29 mutant HeLa cells. In fact, loss of functional Rab29 proved detrimental to Golgi integrity and caused fragmentation of the TGN leading to TGN46 (the human homologue of TGN38) entering the endosomal pathway (Wang et al., 2014). This observation paired with our own findings suggests two potential rational for TGN38 depletion. Rab29 may be a Mn-sensitive protein that under conditions of high Mn loses function resulting in a loss of TGN integrity. Alternatively, Rab29 plays a role in Mn detoxification by expelling Mn rich TGN compartments to the lysosome as a mechanism for Mn sequestration. Figure 8. Mn induces TGN38 trafficking through the endosomal If TGN38 degradation is involved in Mn homeostasis, pathway to the lysosome. Diagram represents the pathway by which TGN38 is trafficked from the trans-Golgi to the lysosome for then the cell must first initiate Mn TGN contact. In HeLa cells degradation. Treatment with BAF caused increased co-localization of Mn accumulates in the Golgi apparatus and is imported by 2+ 2+ TGN38 and M6PR as displayed in the late endosome. the Ca /Mn pump, SPCA1 (Micaroni et al., 2010). 2+ Figure 9. Representation of the Golgi apparatus under conditions of excess Mn . Diagram displays our hypothesized mechanism of Mn 2+ 2+ homeostasis in WIF-B cells. TGN38 cycles with Mn as vesicular cargo to the plasma membrane where Mn may be expelled from the cell via exocytosis (green). Loss of the TGN results in trafficking of TGN38 along with undesired lipids, proteins and amino acids to the lysosome via the late endosome (red). Functional Golgi continue to secrete glycosylated proteins by exocytosis (purple). ............................................................................................... .................................................................. 8 Downloaded from https://academic.oup.com/biohorizons/article/doi/10.1093/biohorizons/hzy012/5256438 by DeepDyve user on 08 August 2022 Bioscience Horizons � Volume 11 2018 Research article ............................................................................................... .................................................................. Located in tubular compartments of the TGN, SPCA1 independently organized his placement at Professor Wessling- actively transports Mn into the Golgi lumen, a mechanism Resnick’s lab and joined in September 2017. Luke’s interests that has also been observed in the WIF-B cells (Micaroni lie in public health and the role dietary metals play in cancer, et al., 2010; Leitch et al., 2011; Thompson et al., 2018). neurodegenerative and chronic diseases. After completion of Carmona et al. (2018) note an almost identical distribution of his BSc Luke aspires to complete an MB/PhD. Dr Thompson Mn and SPCA1 at the sub-Golgi level where Mn is stored pre- first observed changes to TGN38 levels in the WIF-B cells. dominantly in vesicular compartments rather than in the This observation was confirmed by follow-up experiments core-cisternae (Carmona et al., 2018). TGN38 co-localizes designed by Professor Wessling-Resnick and Luke that were with SPCA1. It is therefore plausible that as TGN38 follows conducted by Luke. As author Luke has primary responsibil- this potential pathway of Mn detoxification, its degradation ity for the paper. also contributes to this mechanism. Whether TGN38 lysosomal degradation is involved in Acknowledgements WIF-B Mn homeostasis remains an open question since its function remains to be characterized. Given that the Golgi has I would like to thank Professor Wessling-Resnick for allowing previously been identified as a compartment for excess Mn me to work in her lab and Dr Thompson for her support and storage, it seems logical that TGN38 (a Golgi trans-mem- guidance throughout this project. It has been a pleasure to brane protein) degradation plays a role in maintaining Mn work with them both. homeostasis. To achieve this the cell must expel excess Mn. Studies conducted by Thompson et al. (2018) showed that WIF-B cells release > 80% of their Mn content after just 4 h References incubation with 500 μM MnCl (Thompson et al., 2018). Our data identified a decrease in levels of TGN38 over the Adler, J. and Parmryd, I. (2010) Quantifying colocalization by correl- same time period. Given these findings, we hypothesize that ation: the Pearson correlation coefficient is superior to the TGN38 degradation is a consequence of Mn homeostasis in Mander’s overlap coefficient, Cytometry. Part A : the Journal of the the WIF-B cells. Exocytic transport of Mn by the TGN International Society for Analytical Cytology, 77, 733–742. between the Golgi and the plasma membrane may result in Altan-Bonnet, N., Sougrat, R. and Lippincott-Schwartz, J. (2004) Mn expulsion from the cell. This homeostatic process would Molecular basis for Golgi maintenance and biogenesis, Current reduce intracellular Mn whilst also resulting in depletion of Opinion in Cell Biology, 16, 364–372. the TGN and subsequent TGN38 lysosomal trafficking (Fig. 9). Further studies examining the sorting fate of other Aschner, M., Erikson, K. M., Herrero Hernandez, E. et al. (2009) TGN specific proteins are critical to determining whether our Manganese and its role in Parkinson’s disease: from transport to observation is specific to TGN38 or is a consequence of TGN neuropathology, Neuromolecular Medicine, 11, 252–266. displacement. Future studies would also look to identify co- Aschner, M., Guilarte, T. R., Schneider, J. S. et al. (2007) Manganese: localization of TGN38 and Mn efflux proteins such as recent advances in understanding its transport and neurotoxicity, ZnT10. Toxicology and Applied Pharmacology, 221, 131–147. Aydemir, T. B., Kim, M. H., Kim, J. et al. (2017) Metal Transporter Zip14 Conclusion (Slc39a14) deletion in mice increases manganese deposition and produces neurotoxic signatures and diminished motor activity, The Our results elucidate the effects of low concentrations of Mn Journal of Neuroscience, 37, 5996–6006. on WIF-B cells. The liver is responsible for Mn excretion and interruption of this process can result in Mn-induced neuro- Bakthavatsalam, S., DAS Sharma, S., Sonawane, M. et al. (2014) A zebra- degeneration in mammals. We have identified an adaptive fish model of manganism reveals reversible and treatable symp- response to Mn in the WIF-B cells. In so doing, we have cre- toms that are independent of neurotoxicity, Disease Models & ated a clearer model for Mn homeostasis in this cell line. Our Mechanisms, 7, 1239–1251. observation of significant loss of the trans-Golgi protein Barceloux, D. G. (1999) Manganese, Journal of Toxicology. Clinical TGN38 and its subsequent trafficking to the lysosome high- Toxicology, 37, 293–307. lights the intracellular sensitivity to Mn displayed by this cell type. We hope this discovery may lead to a better understand- Boycott, K. M., Beaulieu, C. L., Kernohan, K. D. et al. (2015) Autosomal- ing of intracellular Mn homeostasis and the pathobiology of recessive intellectual disability with cerebellar atrophy syndrome diseases such as manganism. caused by mutation of the manganese and zinc transporter gene SLC39A8, American Journal of Human Genetics, 97, 886–893. Buchman, A. A. (2014) Chapter 15: manganese, in Ross A. C., Cousins R. Author’s biography J., Caballero B. et al. (eds), Modern Nutrition in Health and Disease, Luke Tillman is studying for a BSc in Medical Sciences at the 11 edn, Wolters Kluwer Health, Lippincott Williams & Wilkins, University of Exeter Medical School. 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BioScience Horizons – Oxford University Press
Published: Jan 1, 2018