Previous data indicated that overexpression of TBP in Aβ-42 flies improved amyloid induced toxicity in retina degeneration. In addition, flies with neuronal expression Aβ-42 and TBP have a significant increase in lifespan and climbing ability. In order to further study the TBP effects on Aβ-42 toxicity and aggregation, brains of control, Aβ-42 as well as Aβ-42 and TBP co-expressing flies were stained with
thioflavin S to detect amyloid plaque and examined with confocal microscopy. The flies were examined at 1 and 20 days of age. Aβ-42 expressing flies showed little dense, punctuate depositions of Aβ-42 at age 1d (Fig 7B). Until the day 20, the deposits of the Aβ-42 were
densely distributed in wider brain regions compared with the age-match control group (Fig 7E). In contrast, overexpression of TBP resulted in reduced Aβ-42 foci at late and early stages when compared with Aβ-42 flies (Fig 7C and F). Overexpression of TBP in Aβ-42 flies significantly reduced the formation of amyloid aggregate and deposits in adult brain.
These results suggestthat TBP upregulation ameliorates amyloid induced toxicity in the brains adult flies.
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Discussions
The phenomenological association of insoluble proteins and late onset neurodegenerative disorders has been proposed for some time. Because normal population possess normal TBP with long polyQ repeats,
aggregation prone peptide, TBP is likely to be involved in mis-folded proteins mediated neuronal diseases. We set out to determine if aberrant and dysfunctional TBP is present in AD brains. TBP is an essential transcription factor. It is expected that alteration of TBP function will have profound effects. The accumulation of insoluble protein deposits within specific region of brain is common feature of late onset
neurodegenerative disorder. Previous study have showed that the TBP accumulates in AD brains and forms insoluble protein aggregates as seen in other polyQ disease proteins (Reid et al., 2004). It has been showed that disease length polyQ tracts may form stable anti-parallel β-pleated sheet structure (Perutz, 1996) and amyloid-like inclusions in HD brain (Huang et al., 1998), suggesting that a possible pathologically interaction between polyQ and amyloid in AD. From our study, we provided
evidence that the deactivation of TBP may lead to neuronal impairment in AD.
Overexpression of human Aβ-42 with Gmr-gal4, cause age-dependent retinal degeneration while wild type and Gmr-gal4 did not displays
defects in photoreceptors. The lifespan of mutant fly were decreased, similar age-dependent behavior defect was also observed in disease fly models. These data demonstrated that amyloid induced toxicity caused late-onset locomotor defects and premature death when expressed in
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neuron. As aforementioned, TBP were co-localized with Tau-containing neurofibrillary structure and within Aβ-42 plaques in AD brains. But the mechanism and relationship between TBP and amyloid were unclear, we hypothesis that the amyloid induced toxicity may be enhanced through decreasing the fuction of TBP. So we co-overexpress Aβ-42 and TBP in central nervous system or retinal using tissue specific driver to test whether the functional TBP could ameliorate the toxicity of amyloid.
In our study, co-overexpression Aβ-42 and TBP with tissue specific driver Gmr-gal4 delay the retinal degeneration especially at old ages. On the other hand, flies co-expression of Aβ-42 and TBP in neuron
significant increases in lifespan when compared with Aβ-42 flies.
Because of these results, we investigated that the relationship of TBP dysfunction and amyloid induced toxicity both in vitro and in vivo.
And we found that Aβ-42 decreased the binding ability of TBP in EMSA analysis. Thioflavin S staining showed that the overexpression of TBP in Aβ-42 flies ameliorates the amyloid induced toxicity and TS-positive deposits number in adult brains. In sum, we demonstrated that TBP overexpression ameliorated the amyloid induced locomotor defect and lifespan. Additionally, the toxicity of Aβ-42 may enhance by
downregulation of TBP. These data indicated that the accumulated TBP is insoluble and dysfunctional. The accumulation of misfolded TBP may contribute to AD.
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Acknowledgements
First of all, I would like to appreciate my thesis supervisor, Dr. Ming-Tsan Su, for enlightening me on the world of genetics and molecular biology. Besides, he is very kind to provide a harmonious environment, let all student in our laboratory feel agreeable.
And I sincerely appreciate Dr. Hui-Yun Chang and Dr. Yun-Ju Lai for teaching me and leading me in the way of research.
Then I would like to thank all members in our laboratory. They all helped me and encouraged me a lot.
Finally, I would like dedicate this thesis to my parents who support me in research life wholeheartedly.
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Figure 1. (A-H) Expressing Aβ-42 in eyes of Drosophila by Gmr-gal4 would cause age-dependent retinal degeneration compared to control group. (I and J) Expressing Aβ-42 in neuron system by Elav-gal4 cause age-dependent locomotor defect and significantly reduce lifespan.
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Figure 2. (A) Thioflavin S (TS) staining was used to detect the Aβ-42 fibril deposits in fly brains. No deposits were found in control brains. TS-positive deposits were found after Aβ-42 expression in fly brains. (C) Western blotting indicated that Aβ-42 form oligomer in fly brain. (D) Filter trap assay showed that Aβ-42 aggregation were trapped in membrane compared to control fly.
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Figure 3. TBP colocalizes with amyloid plaque in the fly brain.
A, D and G, Thioflavin S (TS) staining of brains of 20 dae flies. B, E and H, Immunostaining of brains of 20 dae flies with anti-TBP antibody (red).
C, F and I, TBP and TS-positive merge. (G-I) TBP and TS double positive are enlarged imaged of the boxed region.
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Figure 4. (A) Synthetic Aβ-42 peptide aggregated state. SDS-PAGE analysis of Aβ-42 peptide were incubated at 37℃ for 24h. (B) Filter retardation assay. Insoluble Aβ-42 proteins trapped on cellulose acetate membrane by vacuum filtration and blotted by 6E10 antibody. (C) Electrophoretic mobility shift assay (EMSA). Aβ-42 peptide inhibits TBP/DNA binding. Loading of equal amounts of TBP was verified by comparison with the constitutive binding activity of the Aβ-42. (D) The decrease of TBP binding ability is accompanied by the increase of the concentration of Aβ-42.
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Figure 5. Co-expression of Aβ-42 and TBP ameliorates amyloid induced age-dependent retinal degeneration. E, F, G and H, Overexpression Aβ-42 cause retinal degeneration compared with control group (A, B, C and D). M, N, O and P, TBP overexpression in Aβ-42 flies rescue amyloid induced toxicity compared with age-match Aβ-42 group. I, J, K and L, Downregulation of TBP enhanced amyloid induced toxicity.
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Figure 6. TBP overexpression can ameliorate the climbing and longevity defects of Aβ-42 flies
(A) Expressing Aβ-42 in neuron system by elav-gal4 reduced the lifespan as compared with control Elav-gal4 flies. Co-expression TBP-36Q and Aβ-42 in neuron system significantly lengthened the lifespan of Aβ-42 flies. (B) Aβ-42 expressing in fly brains induced a climbing deficit as compared with control group. Overexpression of TBP could significantly increase the climbing ability of Aβ-42 flies. Reported p values are from Mantel-Cox log-rank statistical analysis.
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Figure 7. Overexpression of TBP decreases Aβ-42 fibril deposits.
(A and D) TS staining was used to detect the Aβ-42 fibril deposits in the fly brains. Little deposits were found in control brains at 1 and 20 days.
(B and E) TS-positive deposits were found after Aβ-42 expression in fly brains at both 1 and 20 days of age. (C and F) Overexpression of TBP could reduce the deposit quantities of all Aβ-42 flies.
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Figure S1. Electrophoretic mobility shift assay (EMSA) TATA box binding protein (TBP) bind DNA probe forming a complex and shift up to upper potion gel.
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Figure. S2 Electrophoretic mobility shift assay (EMSA). Aβ-42 peptide at different state inhibits TBP/DNA binding. Loading of equal amounts of TBP was verified by comparison with the constitutive binding activity of the Aβ-42. But there was no significant between unaggregated sample (0 h) and fibril sample (24 h).