Proteomic Studies On The Mechanism Of Cytotoxicity Induced By Rotenone In PC12 Cells

Parkinson's disease (PD) is also called"shaking palsy". PD is one of the most common neurodegenerative diseases. It is characterized by selective and progressive degeneration of dopaminergic neurons and presence of Lewy bodies in the remaining neurons of the substantia nigra. To date, the cause and pathogenesis of PD remains unclarified, and the mechanisms underlying dopaminergic neuron degeneration are obscure. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) can produce a syndrome which is clinically similar to PD. It has been hypothesized that environmental toxicants may contribute to the development of PD. Because MPTP has not been found in nature and only synthesized in limited quantities for the experimental research, it is unlikely that MPTP itself is the main cause of sporadic PD. Rotenone is a naturally occurring plant compound and a common insecticide used in vegetable gardens. It is also used to kill fish. Recently it has been demonstrated that rats administered subacute doses of rotenone develop biochemical, anatomical, and behavioral symptoms of PD. These results have renewed interest in the link between the pesticides exposure and the development of PD. Thus, rotenone treatment provides one of the best experimental models for PD research. The rotenone model may be an invaluable tool for investigating the environmental factors and the link between the cause and pathogenesis of PD. This model gives excellent experimental support to the environmental hypothesis of PD. Proteomics analysis of brain compartments may be useful to understand the complexity of PD, investigate disorders of the central nervous system and search for the corresponding early markers and screening the target of treatment in PD.In our experiment, the environmental toxin rotenone was used to create the cell model of PD. The PC12 cells are one of the best choices for the substitute of dopaminergic neuron. The aim of our studies was to further elucidate the toxic pathogenesis of rotenone by proteomics approach and provide a new insight for the cause and pathogeneis of PD.Part I The establishment of rotenone-treated PC12 cell model of Parkinson's diseasePC12 cells were treated with different concentrations of rotenone(0μM,0.1μM,0.5μM,1μM)for 24h, 48h and 72h, respectively. Cell morphological changes in PC12 cells were examined by phase–contrast microscopy. Cell viability was measured by MTT method. Cell apoptotic rate was assessed by Hoechst 33342/PI stains. The morphological results showed that the cell body became smaller and smoother, the processes of PC12 cells shortened or disappeared in a time- and concentration-dependent manner after being treated with rotenone for 24h, 48h and 72h, respectively. MTT showed cell viability decreased in 0.1μM rotenone-treated group afer 24h compared with control group ( p<0.05). The longer time past, the cell viability gradually descreased after rotenone treatment. Cell viability significantly decreased to 56.44±2.28% in 0.5μM rotenone-treated group at 72h. Hoechst33342/PI fluorescence staining showed that more apoptosis cells were found in rotenone-treated groups, the ratio of apoptosis increased significantly to 23.43±4.17% in 0.5μM rotenone-treated group at 72h. Cells shown condensed and fragmented nuclei demonstrated by fluorescent dyes, which are the characteristics of apoptosis. These results indicated that rotenone indeedly insulted PC12 cells. A widely accepted cell model of PD was established, which sets up a reliable basis for the study of rotenone mechanisms at proteomic level.Part II Proteomics study of toxic mechanism of rotenoneRotenone is a naturally occurring plant compound. As the special mitochondrial complex I inhibitor, rotenone can impair oxidation- phosphorylation of mitochondrial. The evidences from epidemiology and animal experiment indicate that rotenone is the candidate of cause factors for PD. However, since the rotenone model is a recently established model of PD, the molecular mechanisms underlying rotenone-induced insults of dopaminergic neurons are not well understood. Further exploring the toxic mechanism of rotenone at proteomic level may provide new clues to elucidate pathogenesis of PD.To explore the toxic mechanisms of rotenone, the goal of proteomics is to detect novel drug targets and diagnostic markers of PD.Proteins were extracted from control and 0.5uM rotenone-treated groups, respectively. The maps of proteins were set up by DIGE system. The altered protein spots were identified with MALDI-TOF MS and database searching.About 1740 protein spots were seen in 2D-DIGE images. The expressions of 63 proteins were significantly changed, of which 38 were up-regulated and 25 were down-regulated more than 20% in rotenone-treated PC12 cells compared with that in control. 17 protein spots were identified by mass spectrometry. The up-regulated proteins include vinculin, Grp75, TH,γ-enolase, SKRAP, ECH1, PAF-AH Ibα2, Hsp27, TPi1 and GSTpi. The down- regulated proteins include ACO2,αtubulin-2, eIF4A1, MAP-EB1 and SAE.The cytoskeleton protein,αtubulin-2, a member of the tubulin superfamily, is one of the components of microtubule and is required for the normal structure and normal fuction of microtubule. The microtubule associated protein EB1 (MAP-EB1)can regulate the assembly and functions of microtubules. Vinculin plays a pivotal role in cell adhesion, apoptosis and migration. Proteins with chaperone activity including Grp75 and Hsp27. Grp75 is a member of the heat shock/stress-response protein family. As a molecular chaperone, Hsp27 has neuroprotective effects via two actions: prevention of protein misfolding and acceleration of protein refolding and renaturation; inhibition of the apoptotic pathways within the cell. Hsp27 may be useful in the treatment of PD. ACO2 is a member of the Fe-S enzyme family. Its functions are involved TCA cycle and regulation of iron metabolism, which can contribute the pathogenesis of PD.γ-enolase and Tpi1 are both the key enzymes in glycolytic cycle. ECH1 is one of the key enzyme of the peroxisomalβ-oxidation cycle. The three enzyme (γ-enolase, Tpi1 and ECH1) regulate energy metabolism and may associate with the pathogenesis of PD. GSTpi has direct antioxidant activity and is involved in the metabolism of dopamine. GSTpi may be the key molecular of pathogenesis of PD. PAF-AH catalyzes PAF to inactivate it and has anti-inflammatory effects and anti-apoptotic actions. PAF-AH may affect the progress of neurodegeneration. eIF4A is the prototypic member of the DEAD box family of ATP-dependent RNA helicases and is involved in almost all aspects of RNA metabolism. eIF4A is capable of interacting directly with double-stranded RNA and contributes to unwind double-stranded RNA and RNA-protein complex. It is important in the initiating process of protein synthesis. SAE is a member of ubiqutin-like dynamic reversible key enzyme of posttranslational modification of proteins system. This system does not lead to degradate but instead to regulate protein-protein interactions, intracellular localization and protects some modifyed targets from ubiquitin-dependent degradation. SKRAP is associated with serine/threonine kinases receptor and regulates the cell differentiation, proliferation and apoptosis.In present study, the toxic mechanisms of rotenone were studied by DIGE and MS technology at proteomic level for the first time. It is the first time to have found the changed proteins of GSTpi, PAF-AH1bα2, hsp27, vinculin, MAP-EB1,γ-enolase, Tpi1, ECH1, eIF4A, SAE and SK-RAP in rotenone model of PD. The change of ACO2 is consistant with that from past. experiments in rotenone-treated cell model of PD. The alteration of Grp75 is different from that in other study in rotenone-treated cell model of PD. These changed protein may be invoved in the toxicity of rotenone and may be the candidate of diagnosis marker and the target of neuroprotecive treatment of PD.