| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Nigral neuron death without Levy-body formation is caused by a loss-of-function of parkin protein. The similar nature of neuron death is caused by dysfunction of SMN and NAIP proteins. Recently, in non-neuronal cultured cells, these two proteins were shown to suppress apoptosis. Under the assumption that parkin might also be such a general suppressor of apoptosis, we performed an apoptosis suppression assay using non-neuronal 293 cell.
1)Transient overexpression of Parkin did not suppress the apoptosis induced by bax and fas. This suggests that parkin does not suppress the apoptotic pathway at or downstream of bax and fas activation.
2)Parkin did not suppress the apoptosis induced by proteasome inhibition. As the inhibition causes bax activation through JNK pathway, this suggests that parkin does not suppress this activation system of bax.
3)Parkin does not suppress the apoptosis induced by ethopoxide, menadione and serum deprivation, indicating that parkin function js not related to the
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apoptosis eyents involved in these proapoptotic agents.
4) Parkin is not cleaved by activated caspases (1, 2, 3, 6, 7) in vitro. These findings indicate that parkin is not the target of caspase.
5) IP experiment by co-tranfection or yeast two-hybrid method revealed that parkin does not associate with alpha-synuclein, SODl , Bc1-2, XIAP, CIAP-1, 2, NAIP, and HSP70.
Based on these findings, we concluded that, unlike bc1-2, SMN, NAIP and XIAP, parkin is not a general suppressor of apoptosis. Although Parkin does not by itself suppress apoptosis in 293 cell, it is possible that parkin may interact with other unknown apoptosis-related proteins which are specifically abundant in nigral neurons. The similar death suppression assay using neuronal cells will answer this question. Alternatively, neuron death by defective parkin may not be exerted through apoptotic mechanism but through a yet-unknown death machinery. Discovery of a new in vitro assay od parkin function to evaluate such death mechanism will help us to elucidate true features of neuron death observed in variuos neurodegenerative diseases. Less
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