| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Most cases of autosomal dominant form of hereditary spastic paraplegia are due to mutations in the SPG4 gene, which encodes spastin, an ATPase belonging to the AAA family. Although spastin are suggested to be involved in microtubule dynamics, we have no clue of the mechanism by which spastin mutations may lead to degeneration of corticospinal axons. Therefore, we investigated the function of wild-type spastin and the molecular mechanism of neurodegeneration due to spastin mutations.
1.Subcellular localization of wild-type and mutated spastin
Each of overexpression of wild-type spastin or spastin^<A485V> construct showed a perinuclear localization in HeLa cells. A partial co-localization of spastin^<L426V> and and α-tubulin was observed with a filamentous pattern. Spastin^<TM+> construct localized to a perinuclear area, but spastin^<TM-> construct showed a diffuse intranuclear and cytoplasmic localization. In cells highly expressing spastin, the reduction in microtubule staining and broke
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n microtubules were observed. We found a nuclear export signal (NES) in spastin.
2.Generation of polyclonal antibodies specific to spastin
We generated two polyclonal antibodies, sp-1 (amino acid residues 1-15) and sp-2 (recombinant amino acid residues 230-616), which are specific to spastin. Using these antibodies, we found that spastin showed an intranuclear localization in the interphase of HeLa cell-division cycles. In mitosis, spastin localized to the centrosomes in addition to the nuclei. Sapastin localized to the nuclei and the top of neurites in hNT2 cells.
3.Analysis of the function of wild-type spastin using siRNA
When the function of spastin was knocked down by siRNA, the cell division was disturbed in the anaphase of HeLa cell-division cycles. In hNT2 cells, the fragility of the top of neurites was observed. The gene profiling analysis was performed using two kinds of siRNA, and we found that the expression of M-Phase-Phosphoprotein 1(MPP1) is enhanced by knockdown of spastin function. Less
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