Endoplasmic reticulum stress and apoptosis induced by manganese trigger α-synuclein accumulation

Purpose: To explore whether α-synuclein aggregation is linked to endoplasmic reticulum (ER) stress and apoptosis induced by manganese (Mn) on CATH.a dopaminergic cell lines. Methods: Western blot analysis for the expression of 78 kDa glucose-regulated protein (GRP78), phosphorylated eukaryotic initiation factor 2α (p-eIF-2α), eIF2α, inositol requiring enzyme 1(IRE-1α), cleaved caspase-3, and C/EBP homologous protein (CHOP) was performed, including overexpression of recombinant adenovirus-mediated α-synuclein on CATH.a dopaminergic cell line. Results: It was observed that cell viability (p < 0.05) was significantly reduced by 250 μM exposed for 3 h and 1,000 μM of MnCl2 exposed for 24 h. The expression of p-elF-2α, IRE-1α, and GRP78 was especially induced by 1,000 μM of MnCl2 exposed at 3, 6, and 12 h, respectively (p < 0.05). Twenty four-hour exposure of 250 uM of MnCl2 and the 3 h exposure of 1,000 uM of MnCl2 significantly induced CHOP, active caspase 3 and α-synuclein expression (p < 0.05). α-Synuclein combined with recombinant adenoviral transduction increased GRP78, IRE-1α and eIF2a, CHOP and caspase 3 expression at longer times and at higher concentrations of manganese exposure on CATH.a dopaminergic cells. Conclusion: Based on these findings, Mn is a risk factor for diseases associated with α-synuclein accumulation. Furthermore, α-synuclein accumulation is associated with apoptosis via ER stress induced by Mn.


INTRODUCTION
Neurodegenerative diseases result in the progressive loss of structure or function of neurons, including the death of neurons.They are caused by genetic mutation or by extracellularly excreted proteins such as amyloidβ, or by damage to the membranes of organelles by monomeric or oligomeric proteins such as αsynuclein [1], or by aggregation of misfolded proteins [2].α-Synuclein is a protein that is expressed in abundant amounts in the human brain during human fetal development [3].It is found at the tips of neurons in the presynaptic terminals which release neurotransmitters from synaptic vesicles [4].In experiments with mouse models for learning and working memory, α-synuclein function has been reported as potentially playing an important role in the development of cognitive function [5].This means that α-synuclein may be strongly related to the progressing of Alzheimer's disease (AD).It has also been reported that αsynuclein precipitates predominantly in the cytoplasm of neurons in Lewy-body disorders, and has a critical role in maintaining supply of synaptic vesicles in presynaptic terminals [6] as well as regulating dopamine, a neurotransmitter related to voluntary movements [7].Movement disorders such as rigidity, tremor, bradykinesia, postural instability and dyskinesia are usually classified as related to problems in Parkinson's disease (PD) [8].
One of the risk factors for PD, is associated with exposure to heavy metals like manganese (Mn) [9].Appropriate amount of Mn can play critical roles in growth, metabolism, and antioxidant system in the human body.But excessive Mn causes Mn toxicity that induces manganism, the symptom of neurodegenerative disorders such as motor impairment and dementia.How neurodegenerative disorders are related to Mn toxicity is not well known.
In our study, the mechanism of Mn toxicity and the relationship between α-synuclein aggregation as well as ER stress and apoptosis, were investigated.

EXPERIMENTAL Materials
Roswell Park Memoria Institute (RPMI) 1640 medium was purchased from Grand Island Biological Company (GIBCO, New York, USA).Fetal bovine serum (FBS), trypsin, and other tissue culture reagents were purchased from Life Technologies Inc. (Gaithersburg, MD, U.S.A).MnCl 2 and other reagents were purchased from either Sigma or Aldrich (St. Louis, MO, U.S.A) and stored according to the manufacturer's instructions.All reagents were of analytical grade, and plastic wares such as cell culture dishes were obtained from Falcon Inc. (Franklin, NJ, U.S.A).78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), ßactin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A).Phosphorylated eukaryotic initiation factor 2α (p-eIF-2α) was provided by Cell Signaling Technology Inc. (Danvers, MA, U.S.A).CATH.a (mouse neuroblastoma) cells were obtained from the American Type Culture Collections (Manassas, VA, USA).

Cell culture and cell viability
CATH.a (mouse neuroblastoma) cells were maintained at 37 °C in a humidified atmosphere with 5 % CO 2 .The cell lines were cultured in RPMI 1640 medium which supplemented with 8 % horse serum, 4 % FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine.Cell viability was assessed with the trypan blue exclusion assay and calculated by dividing the non-stained (viable) cell count by the total cell count (3x10 6 ).

Expression of α-synuclein and induction of aggregation
For α-synuclein expression in CATH.a.cells, a recombinant adenoviral vector containing human α-synuclein cDNA (ad.α-synu), and recombinant adenoviral vector containing β-galactosidase (BD Biosciences, USA) were used as previously described [12].Recombinant adenoviral vector containing β-galactosidase was described as adenoviral non-specific vector (ad.NS) for αsynuclein.Adenoviruses kindly were provided by Dr. S.J. Lee.CATH.a. cells were infected with ad.α-synu and ad.NS for 3 days.After infection, cells were incubated in a humidified, 5 % CO 2 atmosphere at 37 °C.Incubation with adenovirus continued for 16 h.MnCl 2 was treated at a concentration of 1,000 µM for 0 (control), 3, 6, 12, 24 h.It was treated with concentration of 0 (control), 100, 250, 500, 1,000 µM of MnCl 2 for 24 h for induction of α-synuclein aggregation at the same day when the infected cells were split.

Protein determination
Protein concentrations of the homogenates were determined using the method of with bovine serum albumin (BSA) as standard.

Statistical analysis
All the data were expressed as mean ± standard error (SE) and one-way analysis of variance (ANOVA) followed by Dunnett's test using SPSS software (version 16, Chicago.IL.USA).The criterion for significance (p < 0.05) was set as stated in Figure legends.

Cell toxicity
Cells were treated with 1,000 µM of MnCl 2 for different durations (3, 6, 12 and 24 h) and with different doses: 100, 250, 500 and 1,000 µM of MnCl 2 for 24 h.The longer the time and the higher the dose of exposure of MnCl 2 induced less cell viability (Figure 1) and more toxicity.CHOP of the apoptotic markers gradually increased expression when cells exposed to 1,000 uM of Mn at over 3 h, and showed a significant expression from over 250 µM of Mn for 24 hours exposure.Another of the apoptotic markers, caspase 3 was time and dose dependent (Figure 3), suggesting that Mn causes apoptosis.

More α-synuclein was expressed by longer time and higher dose of MnCl 2 exposure
To determine whether MnCl 2 can cause the accumulation of α-synuclein, western blotting with α-synuclein protein was performed.Interestingly, expression of α-synuclein was dependent on the time and the dose of MnCl 2 exposure in the CATH.acell (Figure 4 and 5).As the concentration of MnCl 2 increased by 100, 250 and 500 µM, the amount of α-synuclein expression gradually increased and it was increased significantly at 1,000 µM of MnCl 2. The data showed the longer the exposure time to Mn, the more accumulation of α-synuclein (Figure 4).α-Synuclein also showed more oligomerization with increasing time and concentration of Mn in cells treated with recombinant adenovirus vector containing α-synuclein cDNA (Figure 5).

α-Synuclein expression is associated with apoptosis via ER Stress
ER stress markers such as GRP 78, IRE-1, p-eIF2a, eIF2 were more expressed in the cells treated with the cDNA recombinant adenovirus vector containing α-synuclein than the cells treated with the recombinant adenovirus vector containing β-galactosidase (non-specific cells, ad.NS) when cells exposed to Mn. CHOP and active caspase 3 expressed more on the cells of recombinant adenoviral vector with α-synuclein cDNA than non-specific cells.These data suggested that apoptosis induced by Mn was significantly declined under non-specific cells of α-synuclein cDNA.Apoptosis increased in cells treated by recombinant adenoviral vector with αsynuclein cDNA.These data implied that αsynuclein overexpression could play an important role in Mn induced ER stress and apoptosis (Figure 6).

DISCUSSION
Exposure to high Mn level leads progressive and irreversible symptoms such as rigidity, tremor, bradykinesia, postural instability, dyskinesia, cognitive disorder and neurologic damage [13].The neuronal damage has been known to be associated with ER stress [11].However, the exact mechanism of Mn-induced ER stress is not fully understood.
ER stress is activated by mutations in proteins, oxidative stress, calcium depletion, nutrient deprivation, altered glycosylation.And it triggers the unfolded protein response (UPR) [14].When the UPR fails to restore ER homeostasis with prolonged ER stress, cell progresses from survival condition to apoptosis due to the activated UPR.The UPR is initiated by the activation of three molecular chaperones [15]: protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring kinase 1α (IRE-1α), the mechanism of which is not yet fully clarified.However, 78 kDa glucose regulated protein (GRP 78)/binding immunoglobulin protein (BiP) located in the lumen of the ER and it is one of the most widely accepted models for the recognition of unfolded proteins.It is an important component of the translocation [16].
In this study, it was observed that exposure to MnCl 2 increased the expression of GRP78, IRE-1α, p-eIF-2 α and eIF-2α, and it suggests that ER stress plays a salient role in Mn induced toxicity (Figure 2).CCAAT/enhancer-binding protein homologous protein (CHOP) is a pro-apoptotic protein and stimulates cell death by promoting protein synthesis and oxidation in the stressed ER [17].Caspase-3 is an active effector caspase of the cysteine aspartic acid protease protein family [18] and is activated through proteolytic cleavage in the apoptotic cell death by intrinsic (mitochondria) and extrinsic (death ligand) pathways [19].The extrinsic activation of caspase-3 plays a salient role in the caspase cascade characteristic of the apoptotic pathway [20].It was further observed that the longer exposure time and the higher exposure concentration to Mn, the more expression of CHOP and caspase-3 increased and it suggested that ER stress induced by Mn elicits apoptosis in dopaminergic models (Figure 3).
Many studies have been conducted to identify whether the mechanism of α-synuclein as well as the accumulation of α-synuclein can have harmful or beneficial effects.However, the exact function of α-synuclein has not been fully demonstrated.
While previous studies have been focused on the overexpression of α-synuclein in various cell lines [21], our study is hypothesized that Mn can induce α-synuclein directly expression, and that Mn and α-synuclein are closely related to the development of PD.Recent research reported that overexpression of α-synuclein can activate ER stress through the PERK signaling pathway following apoptosis [22].However, α-synuclein function on cell damage like ER stress and apoptosis, is also still unclear.To explore how αsynuclein could play a critical role in Mn toxicity, experiment was carried out on recombinant adenoviral vector containing human α-synuclein cDNA and non-specific recombinant adenoviral vector of α-synuclein cDNA to clarify the mechanism of Mn toxicity.
To find the relationship between α-synuclein aggregation and ER stress, we explored αsynuclein aggregation induced by Mn (Figures 4,  5 and 6).Some studies have illustrated the association between pathological α-synuclein and neuron cell death remained so far correlative [16].However, the experimental results showed that recombinant adenoviral vector containing human α-synuclein cells demonstrated significantly decreased cell viability in vitro than non-specific recombinant adenoviral vector cells, suggesting α-synuclein induced cell toxicity.As the time and the dose of Mn exposure increased, α-synuclein gradually aggregated in vitro (Figure 5).Chronically exposed to Mn, alpha synuclein aggregated in neurons and glial cells in vivo [23].This suggests that α-synuclein is associated with neuron cell death during acute or chronic exposure of Mn.Whereas a previous study suggested that the molecular chaperone GRP-78 played a neuroprotective role in α-synuclein induced PD-like neurodegeneration [24].
The data would demonstrate that GRP-78 could play an anti-protective role in it, as α-synuclein increased cell toxicity followed by activating more GRP-78 expression as well as IRE-1α, p-eIF-2α, and eIF-2α, leading to the activation of ER stress (Figure 6A).CHOP is a pro-apoptotic protein and CHOP transcription is regulated by ER stress through UPR pathways [25].Caspase 3 functioned as a dominant role in the apoptotic cells via an extrinsic pathway [20].Recombinant adenoviral vector containing human α-synuclein expressed more CHOP and caspase 3 protein than non -specific recombinant adenoviral vector (Figure 6B).These data suggest that αsynuclein activated ER stress mediators associated with IRE-1α, p-eIF-2α and eIF-2α pathway, leading to apoptosis (Figure 6).

CONCLUSION
Mn is a risk factor for diseases associated with αsynuclein accumulation.Furthermore, αsynuclein accumulation is associated with apoptosis via ER stress induced by Mn, leading to Mn-induced neurotoxicity.

Figure 1 :
Figure 1: Effects of different time and different concentration of MnCl2 exposure.Cell viability was accessed by tryphan blue assay.(A) Cells were treated with 1,000 µM of MnCl2 for 3, 6, 12, 24 h and the cell viability was assessed by trypan blue assay.(B)Cells were treated 100, 250, 500, 1,000 µM of MnCl2 for 24 h and the cell viability was assessed by trypan blue assay.Data represent mean ± SE (n = 3); *p < 0.05 significantly different from control group