[Proteolytic stress induced by environmental toxins in dopaminergic neurons: an experimental study].
OBJECTIVE
To explore the role of proteolytic stress induced by environmental toxins in degeneration and death of dopaminergic neurons.


METHODS
Nerve growth factor-treated-rat adrenal pheochromocytoma cells of the line PC12 were co-incubated with 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium ion (MPP(+)), and rotenone for 24 hours. MTT assay was used to measure the cell viability induced by these neurotoxins with different concentrations. The expression levels of alpha-synuclein and ubiquited proteins in every group were observed with laser scanning confocal technique. The enzymatic activities of three main hydrolases in proteasome were measured by detection of the fluorophore of various cleft synthetic fluorogenic peptides.


RESULTS
6-OHDA, MPP(+), and rotenone decreased the activity of PC12 cells dose-dependently. Co-incubated with 100 micromol/L 6-OHDA, 75 micromol/L MPP(+), and 20 nmol/L rotenone, the activity of PC12 cells decreased by 52%, 44%, and 40% respectively. Immunofluorescence double labeling confirmed the overexpression of alpha-synuclein and ubiquitin and pellet accumulation in the cytoplasm induced by three toxins. Compared with those in the control group, the trypsin-like, chymotrypsin-like, and PgH-like activities of proteasome were markedly decreased in the MPP(+) and rotenone groups (all P < 0.05) and slightly decreased in the 6-OHDA 100 micromol/L group (all P > 0.05), but after the exposure to 6-OHDA 200 micromol/L for 24 h, the activities of the three enzymes decreased rapidly, the activities of the three enzymes were 7.2 +/- 0.6, 79.6 +/- 2.7 and 4.2 +/- 0.5 FU/100 microg respectively (vs 13.9 +/- 1.8, 99.3 +/- 5.2, and 6.9 +/- 0.6 FU/100 microg respectively in the control group, all P < 0.01).


CONCLUSIONS
Environmental toxins induce proteolytic stress marked by dysfunction of ubiquitin proteasome and accumulation of alpha-synuclein and ubiquited proteins.
Wang
Lan
L
Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Cao
Xue-bing
XB
Wang
Jian-ming
JM
Qu
Rui-sen
RS
Xu
Li
L
Qiao
Xian
X
Sun
Sheng-gang
SG
D015655:1-Methyl-4-phenylpyridinium / Q000633:toxicity; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D004785:Environmental Pollutants / Q000633:toxicity; D005455:Fluorescent Antibody Technique; D006540:Herbicides / Q000633:toxicity; D006868:Hydrolysis / Q000187:drug effects; D007306:Insecticides / Q000633:toxicity; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009498:Neurotoxins / Q000633:toxicity*; D016627:Oxidopamine / Q000633:toxicity; D016716:PC12 Cells; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D051381:Rats; D012402:Rotenone / Q000633:toxicity; D025801:Ubiquitin / Q000378:metabolism; D051844:alpha-Synuclein / Q000378:metabolism
D004785:Environmental Pollutants; D006540:Herbicides; D007306:Insecticides; D009498:Neurotoxins; D025801:Ubiquitin; D051844:alpha-Synuclein; D012402:Rotenone; D016627:Oxidopamine; D046988:Proteasome Endopeptidase Complex; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine