Proteomic Study Of Oxidative Modification In PC12 Cell Model Of PD Treated With PSI

Parkinson's disease (PD) is a slowly progressive age-related neurodegenerative disorder. The primary pathological hallmark of PD is preferential degeneration of the dopaminergic neurons of the substantia nigra pars compacta (SNc), the loss of dopaminergic neurons in SNc result in a reduction in striatal dopamine content. In addition, insoluble protein aggregates known as Lewy bodies are characteristically seen in the cytoplasm of remaining SNc dopaminergic neurons.The etiopathogenesis of PD remains elusive in most patients. Genetic and environmental factors have been implicated, but it is likely that a complex interaction between these factors underlies most sporadic cases. The pathogenesis of PD are involved in many factors. Recently, experts have paid attention to the oxidative stress and dysfunctions of the ubiquitin-proteasome system(UPS).Recent studies suggest that defects in the capacity of the UPS to degrade unwanted proteins may be a common feature in the etiopathogenesis of both familial and sporadic PD. Treatment of rat models and PC12 cells with proteasome inhibitors all lead to degeneration of dopaminergic neurons and the formation ofα-synuclein/ubiquitin-containing intracytoplasmic inclusions which simulate more closely to the primary pathological hallmarks of PD. Moreover, PC12 cells express TH and synthesize dopamine. Thus, present experiment chose PC12 cells treated with proteasomal inhibitior(PSI) as the PD cell model.Reactive oxygen species(ROS) are produced in the course of normal metabolism and they serve important physiological functions. However, because of their high reactivity, accumulation of ROS beyond the immediate needs of the cell may affect cellular structure and functional integrity, by bringing about oxidative degradation of critical molecules, such as the DNA, proteins and lipids, resulting in oxidative stress. Recently, several evidences from experiments and postmortem of PD display that the oxidative damage exists in the dopaminergic neurons of SNc. Furthermore, impairment of proteasomal function leads to free radical generation and oxidative stress, promoting us to explore the oxidative stress of PD in the cell model treated with PSI.Portein carbonyl is one of the specific marker of protein oxidation. The levels of portein carbonyls increased in dopaminergic neurons of postmortem brain tissue from patients with PD. Recently, experts have applied proteomic techniques to study the role of oxidative stress in the etiopathogenesis of neurodegenerative diseases, however, more studies of Alzheimer's disease and few studies of PD. We applied the proteomics approach to identify specifically oxidized proteins involved in the pathogenesis of PD, by coupling 2D gel electrophoresis with immunological detection of carbonyls and identification of proteins by mass spectrometry.Objective To identify the specifically oxidized proteins in the PC12 cell model of PD treated with proteasomal inhibition and explore the role of oxidative stress in the pathogenesis of PD from the point of view of oxidative proteins. Methods The cultured PC12 cells were divided into control group and 10μM PSI-treated group. MTT assay were used to test cell viability; AO-EB and HE stains were used to show apoptosis and intracytoplasmic inclusions respectively. The specifically oxidized protein spots were obtained by coupling 2D gel electrophoresis with Western blot techniques, subsequently identified by MALDI-TOF mass spectrometry(MS). Results 1. 10μM PSI treated group decreased the cell viability and increased the rate of apoptosis significantly compared with control group(p<0.05). HE stain showed that the existence of intracytoplasmic inclusions. 2. In the 10μM PSI treated group, the expressional levels of 13 specifically oxidized protein spots were up-regulated siginificantly compared with the control group, subsequently MALDI-TOF MS identified them as eEF-2, tyrosine hydroxylase catalytic and tetramerization domains, chaperonin containing TCP1(subunit 3), glucose regulated protein 58, mitochondrial aldehyde dehydrogenase precursor, eno 1 protein, HSC70 andβ-actin. Annexin A7 identified by MALDI-TOF MS in the 10μM PSI treated group expressed lower than that of control group. p60 protein and tubulinβ-15 identified by MALDI-TOF only expressed in the 10μM PSI treated group. None of the oxidative proteins only expressed in control group were identified. Conclusions 1. Treatment of the PC12 cells with 10μM PSI for 24h successfully established a cell model of PD, providing a new and reliable experimental model for further exploration of the pathogenesis of PD. 2. For the first time, we coupled Western blot techniques with 2D gel electrophoresis and MALDI-TOF MS identification to filtrate the specifically oxidized proteins in PC12 cells treated with PSI. 3. We identified a set of oxidatively modified proteins with various structures and functions, and explored their likely actions in this model. 4. The oxidative modification of CCT, p60 protein, eEF-2, ALDH2 precursor and Annexin A7 were first discovered. The oxidative modification of GRP58, tubulinβ-15,β-actin, Eno 1 protein, HSC70 and TH were first discovered in the PC12 cells treated with PSI. 5. The discovery of these oxidatively modified proteins provided powerful evidence for further explaining the pathogenesis of oxidative stress in PD, and provided new candidate target and clue for the neuroprotective treatment of PD.