The Mechanism Of α-synuclein To MnCl₂-induced Apoptosis In SH-SY5Y Cells

ObjectiveChronic occupational exposure to high levels of manganese is a selective degeneration of dopaminergic cell in corpus striatum, which leads to dopamine deficiency and functional disorder of extracorticospinal tract. Experiments in vivo and vitro implicated that manganese may be a sort of dopaminergic neurotoxicant related neuron degradation disease. The mechanism of manganese has been resulted from oxidative stress, hydrosulfuryl exhaust, disorder of energy metabolism, DA autoxidation, mitochondrial dysfunction, impairment of the ubiquitin proteasome system (UPS), generation of free radical, cytochondriome damage and so on. However, recent studies indicate that chronic exposure to Mn in certain occupations may increase the risk of acquiring or accelerating PD typical signs and symptoms shown by manganism patients resemble those of PD, including tremor, rigidity, bradykinesia and posture instability. The patients may initially exhibit palpitations, headache, memory loss, hand tremor, lower limb myalgia and hypermyotonia. Furthermore, L-dopa is utility to the patients of manganism, which implying that the mechanism of manganism is concerned with the defect of dopaminergic neuron.In spite of a considerable wealth of researches, the molecular mechanisms underlying the neurotoxicity of PD are poorly understood. Reports indicate that heredity and environment are all attributed to the etiopathogenisis of PD. Epidemiologic studies have suggested that chronic occupational exposure of several metals, such as manganese, plumbum and aluminum, shoud be the risk factors of PD. Several research papers show that oxidative stress may play a cytotoxic role in the pathology of several neurodegenerative diseases. The degeneration of dopaminergic neuron and the decrease of DA neurotransmitter are important in PD and AD. Dopaminergic neurons appear to be particularly vulnerable to such insults, for a lot of free radical could be producted in the metabolic process of DA. An important source of free radical in the intracellular space is electron transport chain of cytochondriome. Reports indicate that the abnormity of electron transport chain can facilitate the generation of free radical in substantia nigra. And the inhibition of cytochondriome complex can promote the oxidative stress damage of nerve terminal.As a transition element, manganese can pass through the blood brain barrier, and accumulate in substantia nigra and corpus striatum. Moreover, manganese can affect electron transfer chain to generate virose free radicle. H₂O₂ and superoxide anion producted from DA autoxidation could be catalyzed by Fe³⁺ and generate various kinds of free radicle. The mechanisms of above all can promote the process of oxidative stress. So far, reports indicate that the development of PD is connected with the balance breakdown between the generation and elimination of free radicle.α-Synuclein, a ubiquitous protein especially abundant in the brain, has been postulated to be associated with the pathogenesis of PD, Alzheimer's disease (AD), and other neurodegenerative disorders.α-Syn is a 140-amino acid protein, was originally isolated from cholinergic vesicle preparations of the electric organ of the ray Torpedo californica in 1988 by Maroteaux, which is highly conserved among vertebrates and was identified in humans as the precursor of the non-A beta component of disease amyloid (NACP).α-Syn in vertebrate is high conservative. However, the role ofα-syn in the intracellular space is not well understood, but it is implicated in the regulation of vesicle dynamics at the presynaptic membrane, and is important in learning and neuronal plasticity. In some cells,α-syn overexpression has been found to be protective whereas in others elicited increased toxicity. But the general consensus is that the overexpression ofα-syn is ultimately toxic to dopaminergic neurons. It has been reported that apoptosis is linked to the overexpression ofα-syn. In the last five years, many studies suggest thatα-syn plays an important role in the pathogenesy of Lewy body disease and PD, but the function of it in chronical intoxication to manganese is unkown.α-Syn was found to be a possible molecular link between PD and heavy metal exposure. The overexpression ofα-syn can decrease the activity of proteasome, and depress the toleration of cells to the proteasome inhibition, and result in mitochondrial dysfunction and apoptosis. The overexpression ofα-syn can inhibit the activity of tyrosine hydroxylase in dopaminergic neuron and lead to cytotoxicity. Moreover, The overexpression ofα-syn also can induce the neuron sensitive to oxyradical, and result in apoptosis. But some reports indicate that down regulation ofα-syn is related to the pathogenesy of PD.On the basis of former experiments, we have employed a model in vitro that used human neuroblastoma SH-SY5Y cell line to test the cytotoxicity and the concentration and time dependent relationships of manganese. We also explored the characteristics ofα-syn and the relations between oxidative stress and apoptosis under the same conditions aiming to reveal the molecular mechanisms of dysfunctions in basal ganglia concentration caused by manganese or PD related environmental exposure factors. Accordingly, research efforts have been directed toward understanding the etiology and pathogenesis of PD and PD-related neurodegenerated diseases in the hope of developing a more effective therapy that will halt the neurodegenerated diseases and providing an appropriate referenced criterion of environmental exposure factors.Methods1. The viability rate of MnCl₂-treated SH-SY5Y cells was measured by MTT assay. The dose of MnCl₂ was determined by drawing cell growth curve.2. The activity of SOD and the level of MDA in MnCl₂-treated SH-SY5Y cells were measured as the indexes of oxidative stress.3. pcDNA3.1(+)-sense-α-syn plasmids and pcDNA3.1(-)-antisense-α-syn plasmids were transfected into SH-SY5Y cells to modulatingα-syn levels. 4. The expression level ofα-syn were estimated by Western blot and RT-PCR.5. Caspase-3 activity and flow cytometry were determined to assess apoptosis in SH-SY5Y cells.Results1. Effect of MnCl₂ on the growth and proliferation of SH-SY5Y cellsMTT assay showed that: MnCl₂ at different concentrations (100, 500 and 900μmol/L) could inhibit the growth and proliferation of SH-SY5Y cells in a dose- and time-dependent manner and the cell growth curves were changed accordingly.2. Effect of MnCl₂ on the level of oxidative sress in SH-SY5Y cells(1) There was a significant decline with time of SOD activities after a 6-h treatment with 500μmol/L MnCl₂ in cells.(2) During the same incubation period, an increase on the MDA levels was observed, and the MDA levels increased in MnCl2-treated cells after 6 h and remained elevated up to 18 h.3. Effect of modulatingα-syn levels on MnCl2-induced oxidative stress(1) The SOD activity and MDA formation remained unchanged in response to up-regulatedα-syn levels in MnCl₂ treated-SH-SY5Y cells.(2) The SOD activity and MDA formation remained unchanged in response to down-regulatedα-syn levels in MnCl₂ treated-SH-SY5Y cells.4. Effect of MnCl₂ on the transcriptional and translational level ofα-syn(1) RT-PCR data confirmed the increase ofα-syn mRNA levels following MnCl₂ treatment. The mRNA transcriptional level ofα-syn started to increase as early as 9 h after MnCl₂ treatment. (2) The western blot data also showed that a nearly 3-fold increase of protein expression was detected after a 12-h time period in MnCl₂-treated cells that remained elevated up to 18 h.5. Effect of regulating oxidative stress levels on MnCl₂-inducedα-syn expression(1) Compared with the only 500μmol/L MnCl₂, the addition of vitamin C (an antioxidant nutrient) 10 mmol/L dramatically inhibited manganese-inducedα-syn protein expression as well as the transcription ofα-syn mRNA.(2) Immunoblotting revealed that MnCl₂-inducedα-syn protein levels were significantly increased in cells exposed to both MnCl₂ and H₂O₂. The RT-PCR analysis demonstrated an increase inα-syn mRNA levels during the same incubation condition.6. Effect of MnCl₂ on apoptosis in SH-SY5Y cells(1) A significant increase in the caspase-3-like proteolytic activity was detected in SH-SY5Y cells treated with MnCl₂. A 2-3-fold increase in caspase-3 activities was seen after a period of 12-18 h in MnCl₂-treated cells.(2) Similar results were observed by Annexin V and PI staining assay after a period of 12-18 h in MnCl₂-treated cells.7. Effect of regulating oxidative stress levels on MnCl₂-induced apoptosis(1) MnCl₂-induced caspase-3 activity were decreased by pretreatment with vitamin C; and the caspase-3 activity was enhanced in cells treated with both MnCl₂ and H₂O₂.(2) MnCl₂-induced the number of Annexin V-positive cells, was decreased by pretreatment with vitamin C, and the number of Annexin V-positive cells was enhanced in cells treated with both MnCl₂ and H₂O₂.8. Effect of modulatingα-syn levels on MnCl₂-induced apoptosis (1) The caspase-3 activation induced by MnCl₂ was significantly inhibited in antisenseα-syn-treated cells. In contrast, the addition of MnCl₂ toα-syn-overexpressing cells enhanced the caspase-3 activity compared with the MnCl₂-treated vector group.(2) MnCl₂-induced the number of Annexin V-positive cells, was increased inα-syn-overexpressing cells, and decreased in antisenseα-syn-treated cells.Conclusion1. MnCl₂ can significantly inhibit proliferation of SH-SY5Y cells, induce oxidative stress, transcriptional and translational up-regulations ofα-syn, and promote apoptosis in SH-SY5Y cells.2. Antioxidant vitamin C preventsα-syn expression, and hydrogen peroxide treatment increases MnCl₂-inducedα-syn expression. The level of oxidative sress remained unchanged in response to modulation ofα-syn levels in MnCl₂ treated-SH-SY5Y cells. These results implicate that MnCl₂-induced oxidative stress is upstream ofα-syn expression and not vice versa.3. The apoptosis, induced by MnCl₂, was vastly reduced in SH-SY5Y cells underexpressingα-syn and significantly elevated inα-syn-overexpressing cells, these results implicate a critical role forα-syn in MnCl₂-induced neuronal apoptosis.4. MnCl₂ inducedα-syn expression promotes apoptosis downstream to oxidative stress in MnCl₂-treated SH-SY5Y cells.