Control SOD

DFX Catalase Mitochondria (-)

Fig. 3. Effects of SOD, deferoxamine mesylate (DFX), and catalase on DCF production in mitochondria prepared from SH-SY5Y cells. Mitochondria (30 mg protein) were incubated with or without 2.5 mg/ml NM in PBS, and the effects of SOD (1000 units), DFX (1 mM), and catalase (500 units) were examined. Generated ROS-RNS was quantitatively measured as DCF produced from H2DCFDA and expressed as pmol/hr. The column and bar represent the mean and SD of triplicate measurements of 3 independent experiments. *P < 0.01

NM inhibits 26S proteasome via iron-mediated oxidative stress ZsGFP fluorescence (Ratio to control)

Fig. 4. Iron inhibited in situ activity of 26S proteasome in SH-SY5Y cells transfected with a proteasome sensor vector. After the treatment with iron with or without deferoxamine mesylate (DFX) for 20 h, the fluorescence intensity of ZsGFP, which is coded by a proteasome sensor vector, at 505 nm with excitation at 493 nm was quantified and expressed as arbitrary fluorescence unit/mg protein. The column and bar represent mean and SD of 3 experiments. After the treatment with iron, the fluorescence intensity increased in a dose-dependent manner. *p < 0.05 compared to the control. This increase was suppressed by 25 p.M DFX significantly. #p < 0.05 compared to the cells treated with iron without DFX


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was mediated by released iron, as shown by the complete suppression of DFX. The onset of oxidative stress may deteriorate the function of mitochondria, in addition to the direct inhibition of mitochondrial respiratory chain enzymes. NM reduced the in situ activities of 26S proteasome, as shown using a green fluorescent protein homologue targeted to 26S proteasome. The mitochondrial toxins, such as rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were reported to induce neuronal death selective to dopamine neurons with formation of Lewy body-like inclusion body in in vivo models of PD (Betarbet et al., 2000; Kowall et al., 2000). The mechanism of cell death has not been elucidated, but the involvement of reduced UPS activity was suggested. Recently we found that mitochondrial dysfunction caused by rotenone, a complex I inhibitor, increased abnormal oxidative modification of proteins with acrolein, and reduced the activity of proteasome, through binding of aggregated oxidized protein to the catalytic site of 20S proteasome and direct adduction of acrolein to 20S proto-some itself (Shamoto-Nagai et al., 2003). It may be reasonable to assume that mitochon-drial dysfunction plays a central role in the pathogenesis of sporadic PD, and impairment of the UPS may be a final executor in the neural degeneration.

Iron is known to induce oxidative stress by enhancing electron transfer in a Fenton

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