| Part I Formation and Development of Inclusions in Dopaminergic PC12 Cells Treated with Proteasome Inhibitor PSIThe cytoplasmic eosinophilic inclusions are the most common pathological hallmark of a number of neurodegenerative diseases, including Parkinson's disease(PD), diffuse Lewy body disease(DLBD) and the Lewy body variant of Alzheimer's disease (LBVAD). However, it is still not very clear about the genesis and pathological effect of the inclusions.α-synuclein is the most important component of the inclusions. So, to study the formation and development of the inclusions usingα-synuclein as the marker can provide evidence of pathogenesis for a number of neurodegenerative diseases.Objective To study the formation and development of inclusions in dopaminergic PC12 cells treated with proteasome inhibitor PSI. Methods PC12 cells were incubated for 24 hours, then were treated with 10μM PSI for 3, 6, 9, 12, 24, 48, 72, 96, 120 hours, fixed with 10% formaldehyde, stained with HE method andα-synuclein immunohistochemistry. Results At 3h,α-synuclein immunoreactive granules were found throughout the cytoplasm; at 6h, α-synuclein immunoreactive granules increased; at 12h, eosinophilic materials andα-synuclein immunoreactive granules concentrated and formed inclusions close to nucleus; at 24h, inclusions were seen in almost all PC12 cells; at 120h, the nucleus and cytoplasm decomposed, only inclusions presented. Conclusions Treated PC12 cells with proteasome inhibitor can produce cytoplasmic eosinophilic andα-synuclein immunoreactive inclusions. The formation and development of inclusions in this model are consistent with those in human Parkinson's disease and diffuse Lewy body disease. This model is helpful to further explore the mechanism of neurodegenerative diseases.Part II Proteomic study of inclusions in Dopaminergic PC12 Cells Treated with Proteasome Inhibitor PSIParkinson's disease is a chronic progressive nerodegenerative disease with the pathological characteristics including death of dopaminergic neurons in the substantia nigra and inclusions formation in the survival neurons. Extensive studies have been performed about the formation, development and components of the inclusions since it was found. Proteomics is a powerful screening method for detecting unexpected alterations in protein expression those may be missed by conventional biochemical techniques. So, to reveal the components of inclusions using proteomic method will provide valuable evidence of PD pathogenesis. Objective To study the components of the inclusions using proteomic method. Method PC12 cells were incubated for 24 hours, then were treated with 10μM PSI for 96 hours. Inclusions were collected and their components were identified by proteomic approach. Result 120 protein spots seen by naked eye were selected for proteomic study. 27 protein spots were identified by mass spectrometry, including: (1) heat shock proteins and related protein: Hsp105, Hsp90, Hsc70, Hsp27, Hsp70/Hsp90-organizing protein. (2) cytoskeletal proteins and related proteins:α-tubulin-1,β-tubulin-15,γ-actin, chaperonin containing TCP1 subunit 2(β), chaperonin containing TCP1 subunit 5. (3) proteasome related proteins: 26S proteasome subunit p40.5, proteasome protein p45. (4) mitochondria related proteins: aconitase 2, dihydrolipoamide dehydrogenase, ubiquinol-cytochrome c reductase core proteinâ… , isocitrate dehydrogenase 3, ATP synthase beta subunit. (5) other proteins: calcium binding protein 1, PKM2, elongation factor 2, laminin receptor 1, annexin A7, thioredoxin-like 2. Conclusions For the first time, the conponents of inclusions were revealed by proteomic method and for the first time Hsp105, Hsp70/Hsp90-organizing protein, chaperonin containing TCP1, aconitase 2, dihydrolipoamide dehydrogenase, isocitrate dehydrogenase 3, PKM2, elongation factor 2, laminin receptor 1, annexin A7, thioredoxin-like 2 proteins were detected in inclusions. Our proteomic results provide new clues for future studies of PD.
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