Proteomics Analysis Of Protective Effects Of α-lipoic Acid On Parkinsonian Cell Model

Parkinson’s disease (PD) is a common neurodegenerative disorder characterizedby the selective and progressive loss of dopaminergic neurons in the substantia nigra,leading to a depletion of the dopamine neurotransmitter in the striatum. While theunderlying mechanisms by which nigrostriatal dopaminergic neuron degeneration arenot completely understood, accumulating data have suggested that oxidative stressplays a crucial role in dopaminergic cell death in PD. The interaction of ROS withmitochondria triggers sequential events in mitochondrial cell death pathway, which isthought to be responsible for oxidative stress-mediated the cell death in PD. PC12cells treated with MPP~+provide a reliable model for insight into the pathogenesis ofPD. PC12cell is a rat pheochromocytoma cell line which has been shown to producedopamine (DA), and possess a DA uptake system, indicating that these cells can beused as cell culture model for studying the dopaminergic neuron degeneration.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin which selecti-vely kills dopaminergic neurons and causes irreversible parkinsonism-like symptomsin humans and primates. MPTP is lipophilic molecule and can rapidly crossblood-brain barrier. Once crossing the barrier, it is oxidized in the brain to its toxicmetabolite MPP~+by type B monoamine oxidase (MAO-B) and finally lead todopaminergic neuron death. The exact mechanism for this toxic action of MPP~+,although incompletely understood, seems to involve ROS generation and oxidativedamage, which triggering sequential events in apoptosis in dopaminergic neuronalcells.Currently, the main therapeutic approaches for treating PD are limited to thereplacement of dopamine in the brain either by levodopa or by dopamine agonists.Although these drugs can relieve the symptoms of Parkinson’s disease, thedegeneration of dopaminergic neurons is not prevented and still slowly progressive.Promising new concepts in therapy of PD should prevent or reverse the progressivedopaminergic neuron degeneration. Since neurodegenerative diseases are mainlyrelated to oxidative stress, attenuation of oxidative stress levels may result inneuroprotection in these disorders. Lipoic acid, also known as1,2-dithiolane-3-pentanoic acid or thioctic acid, exhibits effective antioxidative activities byscavenging ROS, and inhibits free radical formation by chelating various metal types, indicating that it can exert beneficial effects on various disorders correlated withoxidative stress including PD. In addition, Lipoic acid is also reported to haveanti-inflammatory property and increase intracellular glutathione (GSH) formation ina range of cell types and tissues，which may be of additional benefit in neurode-generative conditions. Importantly, Lipoic acid serving as effective antioxidant hasbeen in common clinical used in several diseases that are associated with increasedproduction of free radicals, and administrated in moderate doses with no evidence ofserious side effects.To determine the ability of α-Lipoic acid (LA) to prevent the cytotoxicity ofMPP~+in PC12cells, cell viability was assayed by MTT、AO/EB assay. Were alsoundertook to determine whether LA protects PC12cells from MPP~+-inducedapoptosis. To investigate the effects LA on ROS levels in MPP~+-treated PC12cells,we evaluated the levels of ROS by flow cytometry with2’,7’-dichlorofluorescein-diacetate (DCFH-DA). Then we investigated the changes of proteins involved in thepathogenesis of PD and the protection of LA in PD model by proteomics analysis.The measurements show that LA administered alone did not induce changes in thecell viability, while the administration of MPP~+greatly reduced the cell viability incomparison with control cells.0.01μM LA showed a significant protective effect onthe cells. Similarly results show that LA administered strongly prevented the numberof apoptotic cells induced by MPP~+, confirming the anti-apoptotic activities of LAagainst the toxicity of MPP~+. Our results also show that exposure of PC12cells toMPP~+alone induced a significant increase of2’,7’-dichlorofluorescein (DCF) fluore-scence levels, which was prevented by a pre-treatment with LA, indicating apreventive role of LA in ROS formation in PC12cells treated by MPP~+. The result ofproteomics analysis show that four protein spots were differentially expressed inlevels. They are proliferating cell nuclear antigen (PCNA), annexin A2, guaninenucleotide-bingding protein Gi subunit alpha(Giα) and O-sialoglycoprotein endopep-tidase (OSGEP). In the present study, we investigate the underlying molecularmechanisms by which LAprotect PC12cells against MPP~+-induced apoptosis，whichmay provide potential effective strategies for PD treatment.