TBP功能缺失參與阿茲海默氏症果蠅模式中之類澱粉蛋白毒性

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(1)國立臺灣師範大學生命科學系碩士論文. TBP 功能缺失參與阿茲海默氏症果蠅模式中之類澱粉蛋白毒性 TBP deactivation contributes to amyloid mediated toxicity in a Drosophila model of Alzheimer’s disease. 研究生 : 鄭建文 Jian-Wen Zheng 指導教授 : 蘇銘燦博士 Dr. Ming-Tsan Su. 中華民國 104 年一月.

(2) Table of content Abstract ...................................................................................................... 2 中文摘要..................................................................................................... 4 Introduction ............................................................................................... 6 The goal of research .................................................................................. 9 Material and Methods ............................................................................11 Results ......................................................................................................16 Discussion.................................................................................................22 Acknowledgements…………………………………………………….24 Reference .................................................................................................25 Figures ......................................................................................................28. 1.

(3) Abstract. Protein aggregation is a pathological hallmark of many neurodegenerative diseases, including Alzheimer’s disease (AD). AD is characterized by extra cellular β-amyloid deposition, Tau-containing neurofibrillary tangles (NFTs) and progressive cortical atrophy. Abnormal protein accumulation is also a common feature of other late onset neurodegenerative diseases, including the heritable polyglutamine (polyQ) disorders such as Huntington disease (HD) and the spinocerebellar ataxias (SCAs). Since TBP is insoluble in the brain AD patients, the accumulation or misfolding of this polyQ containing protein may be a contributing factor in AD. Previous study has demonstrated that wild type length TATA box binding protein(TBP), β-amyloid and Taucontaining NFTs accumulate in AD patient brains. It was hypothesized that TBP inactivation may contribute to the pathogenesis of AD, in which the transactivation activity or binding ability of TBP may be influenced by β-amyloid deposition. In my study. Overexpression of β-amyloid in certain tissue causes various phenotypes, including degeneration of photoreceptor cells, defect in mobility and shorten lifespan. The pathological phenotypes of AD is enhanced in the loss-of-function of TBP flies. In contrast, increasing the expression of TBP ameliorates the abovementioned disorders. These observations suggest that loss-offunction of TBP is involved in the pathogenesis of AD. Immunostaining results showed that TBP accumulated and co-localized with amyloidcontaining plaque. In addition, we demonstrated that the increase of Aβ42 concentration was accompanied with the decrease of TBP-DNA bind 2.

(4) ability in vitro. Furthermore, overexpression of TBP reduced and delayed deposits in adult brain. In sum, my study demonstrates that deactivation of TBP contributes to the pathogenesis of AD, which provide new insight into the pathogenesis of AD.. Key word: TBP, Aβ-42, amyloid, Alzheimer’s disease. 3.

(5) 摘要蛋白質不正常的折疊聚集是許多退化性神經疾病的指標之一，其中也包括阿茲海默症。目前已知阿茲海默症常見的致病原因有二，其一為細胞外 β 類澱粉蛋白質堆積；另一病徵則為 Tau 蛋白質堆積所形成的神經纖維狀糾結並造成漸進性的腦皮層萎縮。不正常的蛋白質堆積也是其他退化性神經疾病的常見特徵之一，包括遺傳性的多麩醯胺酸疾病亨丁頓舞蹈症和小腦萎縮症。在上述的這些退化性疾病當中皆有一定比例的不可溶性的轉錄因子 TATA box binding protein (TBP)，這些包含麩醯胺酸的不正常折疊堆積也可能是造成阿茲海默症的原因之一。先前已有資料顯示正常野生型的 TBP 和 β 類澱粉蛋白質以及 Tau 蛋白質在阿茲海默症的病人腦中堆積，根據此一證據我們進一步在果蠅模式下實驗證實 β 類澱粉蛋白質可能透過影響 TBP 的結合功能或是轉錄功能進而成阿茲海默症的成因之一。我們的研究發現大量表現 β 類澱粉蛋白質在果蠅的特定組織部位造成許多病理上的症狀，包括複眼細胞的退化、運動行為缺陷及壽命減短等現象；相反地增加 TBP 的表現則能有效的改善上述退化及行為等性狀，而抑制果蠅內生性的 TBP 蛋白則使得這些性狀更加嚴重。另外，運用 EMSA 的方式證實類澱粉蛋白濃度提高造成了 TBP 對 TATA DNA 的結合力下降。在果蠅的腦中我們也發現隨著時 4.

(6) 間上升類澱粉蛋白聚集也因共同表現 TBP 而減少了聚集的數量，顯示出正常功能的 TBP 能夠有效的減緩類澱粉蛋白造成的毒性。我們的研究結果顯示出 TBP 參與阿茲海默症果蠅致病機轉。關鍵字:類澱粉蛋白，TBP，阿茲海默氏症. 5.

(7) Introduction Alzheimer’s disease The abnormal accumulation of intra- and/or extra-cellular protein aggregates is a hallmark of many neurodegenerative diseases. Alzheimer’s disease (AD) is typified by protein cleavage, protein misfolding and protein accumulation resulting in the abnormal deposition of beta-amyloid and neurofibrillary tangles (NFTs) throughout the brain (Alzheimer, Stelzmann, Schnitzlein, & Murtagh, 1995). The major reported component of NFTs is filamentous aggregates of hyperphosphorylated Tau protein. AD is chiefly observed as an apparently sporadic disorder, however some familial cases are caused by mutations in genes associated with the processing of beta-amyloid and its precursor, the amyloid precursor protein(Alexopoulos et al., 2013; Caccamo et al., 2013; Depardon, Cisneros, Alonso-Vilatela, & Montanez, 2001; Giacobini & Gold, 2013). Beta-Amyloid (Aβ-42) The accumulation of insoluble protein deposits within specific region of the brain is a common feature of late onset neurodegenerative disorders. AD is characterized pathologically by cerebral deposition of extracellular amyloid-β (Aβ) plaques. In healthy brains, most Aβ peptides are soluble forms; however, Aβ peptides are produced from proteolytic cleavage of amyloid precursor protein (APP) which are aggregated into oligomers, fibril and plaques in AD patients. Amyloidogenic Aβ, mainly consisting of 42 residues, is released by β- and γ-secretase (Haass, 2004). 6.

(8) Aβ was first isolated from meningeal vessels of individuals with AD, and then recognized as the main component of the senile plaques observed in AD brain tissues (Glenner & Wong, 1984; Masters et al., 1985). Overexpression of human Aβ-42 peptide in Drosophila nervous system tissues results in structural as well as behavioral phenotypes with AD-like symptoms, in a dose- and age-dependent manners (Finelli, Kelkar, Song, Yang, & Konsolaki, 2004). Using Aβ-42-expressing flies, we can screen modifiers of Aβ-42-induced phenotypes, which may lead to the dissection of pathomechanisms of Aβ toxicity and potentially novel AD treatments. TATA box binding protein (TBP) TATA-binding protein (TBP) is a critical general transcription factor which plays an important role in initiation of transcription of all three RNA polymerase (Gill & Tjian, 1992; Hernandez, 1993). These general transcription factors, including TBP, form a preinitiation complex with RNA polymerase II (pol II) and bind to TATA box (Roeder, 1991). When TBP binds to the TATA box, it bends DNA and DNA bending appears to regulate transcription (TenHarmsel & Biggin, 1995). TBP was found to be aggregated in several polyglutamine (polyQ) disorders (Perez et al., 1998; Uchihara et al., 2001; van Roon-Mom et al., 2002). It was found subsequently that TBP can cause neurodegeneration itself when the polyglutamine stretch is expanded (Koide et al., 1999). Several studies have demonstrated that reducing the function of TBP due to sequestration of TBP in inclusion leads to neurodegeneration (Huang et al., 1998; Perez et al., 1998; Reid et al., 2004; Suhr et al., 2001). As described above transcription dysfunction may be a causative factor in many 7.

(9) neurodegenerations, and TBP is one of the most critical transcription factors. Previous studies have hypnotized that the long, normal polyQ tract within TBP may play a role in late onset neurodegenerative diseases and others have reported amyloid-like inclusions in HD brain, suggesting at least a pathological and potentially a mechanistic parallel between polyQ and amyloid-associated diseases like AD. (Reid et al., 2004; Reid, Whittaker, Greenwood, & Snell, 2009; van Roon-Mom et al., 2002). The self-aggregate polyQ stretche is thought to be a site for protein–protein interaction; the extended polyQ stretch in one protein increasing the binding affinity of other proteins (Kaytor & Warren, 1999; Rich et al., 1999; Yanagisawa et al., 2000). Recent data suggest that interactions between expanded polyQ tracts in proteins can play an important role in the disease process (Nucifora et al., 2001). To answer whether TBP is involved in AD pathogenesis, we generate and characterize a novel AD fly model. Using genetic approaches, we put our AD fly model in TBP mutation background. If TBP does participate in AD pathogenesis, TBP will ameliorate or enhance AD pathogenesis. Several phenotypic criteria, including retinal degeneration, mobility and life span will be used to test if our hypothesis is correct.. 8.

(10) The goal of research. Protein misfolding is involving in cause of at least 25 amyloid disease, including well-known disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (Sipe et al., 2012). Previous studies have demonstrate that polyglutamine-containing protein TBP accumulate and form protein like the other polyQ disease proteins. As abovementioned, TBP may play a critical role in studying amyloid disease. The increased rate of aggregation of longer polyQ peptides and the disease association of Aβ mutations accelerating amyloid formation and disease progression suggest a common mechanism. With these similarities in mind we investigate the possibility that TBP may have a role in AD. Drosophila has long been demonstrated to be one of the most valuable animal models for unraveling pathogenic mechanism and finding therapeutic targets of amyloid mediated neurodegeneration. Drosophila genetics and versatile technologies can help us elucidate the mechanism of pathology efficiently. In the present study, I aim at elucidating the roles of TATA-box binding protein and beta-amyloid 42 (Aβ-42) in the neurodegenerative disease by using different mutant and transgenic Drosophila lines. In Aβ42 transgenic fly model, the photoreceptor and neural cells die with age when elevated level of Aβ-42 peptide is produced in the corresponding tissues. Fly model develop similar pathological hallmarks, in which they show characteristic late onset, progressive degeneration, abnormal protein aggregation and behavior defects as noted in patient. In a parallel 9.

(11) experiment, we have also overexpressed Aβ-42 peptide in TBPoverexpression and dTbpsI10 mutation backgrounds. We suspected that the increase of TBP may reduce cytotoxicity of Aβ-42. Conversely, mutation of dTbp may enhance and/or sensitize its cytotoxicity. With this approaches, we would be able to discern if TBP play a role in the amyloid mediated neurodegeneration.. 10.

(12) Materials and Methods. Drosophila maintenance All Drosophila melanogaster stocks are raised on standard cornmeal-yeast-agar media (supplied by Institute of Molecular Biology Academia Sinica) and cultured at 25ºC.We anaesthetize flies with CO2 then observe and manipulate flies with a stereomicroscope.. UAS-GAL4 system The UAS-Gal4 system is a method for ectopic gene expression in Drosophila which allows the selective activation of any clone gene in tissue or cell specific patterns.. Measuring climbing ability and analysis The analysis for measuring climbing ability in Drosophila is modified from a novel assay and analysis. The climbing apparatus is a 30 cm long glass tube, with a diameter of 1.5 cm. The tube is held in place by a plastic funnel as a means for transferring flies into the apparatus and acts as a base for the tube. The glass tube is divided into a series of five sections, starting from the base, each 2 cm in height (scored 1-5), with an buffer zone in the upper portion of the apparatus. Flies are allowed 10 seconds to climb after being tapped down and are given a score based upon the sections reached. The flies were scored ten times (trials) per climbing section, from which a climbing index is calculated as follows: Climbing index = Ʃ(nm)/N 11.

(13) Where n = number of flies at a given level, m = the score for that level(15) and N = total number of flies climbed for that trial. Fifty flies from each genotype under investigation are collected within 24 hours of eclosion and separated into groups of ten individuals (each group has 5 males and 5 females). Starting at day one after eclosion, each group was tested for climbing ability and was continually tested every five days throughout their lifespan.. Lifespan analysis Flies were raised to adulthood at 25ºC and newly enclosed flies were placed in vials at low density(10-25 flies per vial) and incubated at 25ºC. Males and females were kept in separate vials. Flies were transferred into fresh vial every 3 days, the number of surviving flies was recorded at the same time. Survival curves were generated by calculating the percentage of surviving flies.. Electrophoretic mobility shift assay (EMSA) Double-stranded probes were prepared from complementary single-stranded oligonucleotides, by melting at 95ºC for 5 min followed by annealing for 1 h at room temperature. An oligonucleotide pair containing a TATA-box sequence (5’-GGTGTATAAAGCCGCGGTCC3’ and 5’-GGACCGCCCCTTTATTGACCG-3’) was used as a fluorescence probe after end labeling with HEX. HEX-labeled DNAbinding oligonucleotides were incubated with 12 μg of TBP-36Q at 4ºC for 1h in the presence or absence of Aβ-42 peptide to allow the formation 12.

(14) of TBP/DNA complexes. After incubation with probe and purified TBP, native gel was pre-run in 0.5× Tris/Borate/EDTA buffer (TBE) at 80V for 10 min. TBP-DNA complexes were separated from the free probes in a 10% non-denaturing gel buffered with TBE at 180V for 40 min.. Immunostaining and Thioflavin S staining Fly brain were dissected in PBST and fixed in PBST containing 4 % paraformaldehyde, and then placed under PBST containing 4 % paraformaldehyde and 0.1 % Triton X-100. After permeabilization with PBST, the brains were incubated in 50 % EtOH containing 0.1% Thioflavin S (TS) overnight. After washing in 50 % EtOH and PBST, the brains were blocked with 3% BSA at 37 ºC for 10 min then with PBST. Brains were stained with a monoclonal mouse anti-TBP antibody 1TBP18 (QED bioscience) followed by detection with goat anti-mouse IgG conjugated with orange-fluorescent Alexa Fluor 555 dye (Molecular Probes). The brains were observed using a confocal microscope (SP2, Leica Microscope Co.).. Protein purification Recombinant TBP expression constructs were transformed into E. coli BL21 strain. For purification of the expressed protein, the bacteria were incubated in LB media for 6 hours at 37ºC and then added Isopropyl β-D1-thiogalactopyranoside (IPTG) to induce protein expression. After induction overnight in 18ºC, the bacteria were harvested in a 50 mL falcon tube by centrifugation at 6000 ×g for 10 minutes. After discarding 13.

(15) the supernatant, the pellet was re-suspended in cold 5 mL of lysis buffer and the cells were lysed by sonication. The cell lysate supernatant was applied to the column after passage through a 0.45 µm filter. The columns were equilibrated before sample was loaded. After wash, the proteins were eluted and analyzed by SDS-PAGE.. Immunoblotting Proteins were extracted from heads of Drosophila raised at 25ºC using T-PER solution (Pierce Co.) on ice. For each sample, 15 µg of total proteins were resolved in 12.5% polyacrylamide gels and blotted onto PVDF membranes (Millipore). The intensity of band with the appropriate molecular mass was captured and quantified using NIH image J software.. Immunoprecipitation Fly heads were homogenized in RIPA buffer (50mM Tris-HCl, 150 mM NaCl, 1mM EDTA, 1mM EGTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and protease inhibitor cocktail. Samples were centrifuged at 15,000 ×g for 10 min, and supernatant was collected. Protein concentration was determined (Bio-Rad Protein assay) using BSA as a standard. Protein extracts were immunoprecipitated with 4G8 antibody (BioLegend) at 4 ºC with continuous mixing overnight. Next day the antibody-protein complex was captured with Protein G Magnetic Beads (GE, Healthcare) and analyzed by Western blotting or filter trap assay. 14.

(16) Filter trap assay For filter trap assay synthetic amyloid-beta 42 (Aβ-42) peptide was incubated with EMSA binding buffer in different time point (0, 2, 4, 8, 16, 24 hrs) in a total volume of 50 µL. Samples were filtrated through cellulose acetate membrane in a 96 well vacuum manifold. The resultant membrane were washed twice with 2% SDS, then blocked in 4% nonfat dry milk dissolved in PBST overnight before incubated with betaamyloid monoclonal antibody 6E10 (covance) at 1:1000 dilution for 1 h at room temperature. Membranes were washed twice, and incubated with HRP-linked secondary antibody for 1h. The signal was detected with the ECL substrates and chemiluminescence was recorded with an image scanner (GE LAS4000).. 15.

(17) Result. Overexpression of human beta-amyloid 42 causes age-dependent retinal degeneration Alzheimer’s disease is a neurodegenerative disease which is typified by protein cleavage, protein mis-folding and protein accumulation resulting in the abnormal deposition of beta-amyloid and neurofibrillary tangles (NFTs) throughout the brains. To generate the disease fly model of AD, we overexpress human beta-amyloid-42 (Aβ-42) in flies using UAS/GAL4 binary expression system. Targeted expression of the Aβ-42 recreated same pathological features of AD (Fig.1). Overexpression of Aβ-42 in fly eyes using GMR-GAL4, causes retinal degeneration while wild type flies did not displays defects in photoreceptor (Fig.1). Aβ-42 flies showed a rapid progressive retinal degeneration. Overexpression of Aβ-42 with the GMR-Gal4 driver induced a remarkable degeneration of the external surface and morphology of the eyes. And the retinal degeneration phenotype is more severe at late stages when compared with the same aged wild type flies (Fig. 1H). Similar age-dependent behavior defect was also observed in disease fly model, while express Aβ-42 in central neuron using ELAV-Gal4 the climbing ability was decline significantly (Fig.1I). The mean average lifespan of mutant fly was decreased significantly compared with control flies. (Fig. 1J). Together, these results suggest that the overexpression of Aβ-42 caused severe pathological defects and toxicity in targeted tissues of flies. 16.

(18) Overexpression of Aβ-42 forms amyloid aggregates in fly brains Previous data indicated that overexpression of Aβ-42 induced toxicity in retina degeneration, locomotor defect and shorten lifespan. Aβ-42 is an aggregate-prone peptide and its formation into high-order aggregation states is thought to be a crucial step in development of amyloid plaques in AD. To test if disease fly models exhibits similar pathology, Thioflavin-S (TS) staining of the whole brains was used to visualize the Aβ-42 fibril deposits. We show that many Aβ-42 deposits were found in Aβ-42 fly brains compared to the brains of control flies. In addition, fly brains lysate were used for Western blotting analysis and filter retardation assay. Western blotting result demonstrated that Aβ-42 formed medium oligomer in fly brain. And Aβ-42 aggregation were detected in nitrocellulose membrane using filter trap assay.. Human TBP-36Q and Aβ-42 deposits were co-localized in fly brains TBP is a general transcription factor that is required for the transcription of all cells. Previous studies have present that polyglutamine-containing protein TBP accumulate and form aggregate in AD patient brain as seen in other polyQ disease proteins (Reid et al., 2004). Since TBP may play a critical role in the pathogenesis of amyloid diseases. To address, we first conducted immunofluorescence staining experiments. We show that human TBP-36Q and Thioflavin S staining deposits were colocalized in the adult aging brain (Fig 3I enlarged image). In contrast, both control and Aβ-42 flies have no TBP containing deposits or aggregate in brains (Fig 3B and 3E). As mentioned 17.

(19) previously, TBP is a crucial transcription factor and assemble with RNA polymerases to form transcription complex. Then, we asked whether the TBP-containing aggregation in brain could activate and bind the TATA box DNA because we suspect that the dysfunction of TBP could enhance the toxicity of amyloid.. Aβ-42 peptide decreases binding ability of TBP in vitro Aβ-42 is an aggregate-prone peptide and its formation into high-order aggregation states is thought to a crucial step in development of amyloid plaques in AD. To examine the relationship between dysfunctional TBP and Aβ-42 amyloid-like aggregation, we examed Aβ-42 aggregation state using filter trap assay and SDS-PAGE. In SDS-PAGE, equal amount ofsynthetic Aβ-42 peptides were labeled with FAM in N-terminal in TBP binding buffers. Samples were withdraw at different time points and analyzed in SDS-PAGE. As shown in Fig. 4A, Aβ-42 forms monomer, dimer, tetramer, and hexamer at (0 h) samples incubated in binding buffer (Fig 4A lane 1). Additionally, there was an increase in dimer, tetramer and hexamer after 2 h, 4 h, 8 h incubated in EMSA buffer (Fig 4A lane 2, 3 and 4). Larger oligomeric assemblies were detected after incubation for 16 h (Fig 4A lane 5). This indicates that interconversion between these Aβ oligomers. After 24 h, a reduction of small molecular weight oligmer and an increase of large oligomer were observed. In addition, SDSinsoluble Aβ-42 aggregated forms remained in the well (Fig 4A lane 6). For filter retardation assay, Equal amount of each sample were vacuum filtered through a 0.2-µm nitrocellulose membrane. Aβ-42 trapped protein 18.

(20) was then probed with anti-amyloid 6E10 antibodies (Fig 4B). Base on the previously results, we suspected that the binding ability of TBP may be interfered by Aβ-42 peptide. To test this hypothesis, we conducted electrophoretic mobility shift assays (EMSAs) to examine whether the DNA-binding ability of TBP was affected by the Aβ-42 peptide. Labeled TATA DNA probes were incubated with GST-TBP recombinant proteins, and binding reactions were initiated by cleaving with TEV proteases (Fig S1). The binding of TBP-36Q to TATA box DNA attained a maximum degree within 1 h at 4ºC. The result showed that TBP lost nearly half DNA affinity when TBP were incubated with Aβ-42. Soluble or oligomeric, but not insoluble aggregated, forms of Aβ42 can affect the formation of TBP-TATA complex, because the amount of TBP-DNA complex are equally in EMSA assay, when Aβ-42 peptide aggregated at different time point were incubated with TATA box DNA and TBP in EMSA reaction (Fig. S2). To quantify how amyloid affects the DNA-binding ability of TBP, we adjust the concentration of Aβ-42 to define the amount of Aβ-42 that inhibits the formation DNA TBP-TATA box complex at 50% (IC50) (Fig. 4C). We observed that an increase of Aβ-42 decreases the formation of TBP-DNA complex. Moreover, TBP lost about 50% of DNA binding ability when the concentration of Aβ-42 reaches 0.12 μg/μl (Fig 4D). These data suggested that the DNA-binding ability of TBP were altered by soluble amyloids.. Overexpression of TBP increases the climbing ability and lifespan of Aβ-42 flies 19.

(21) To confirm that the deactivation of TBP is a contributing factor of amyloid-mediated toxicity, we co-expressed Aβ-42 and TBP in eyes using Gmr-gal4 driver. Consistent with the previous data, retinal overexpression of Aβ-42 cause age-dependent morphological defects in the eyes of the adult flies when compared with the control Gmr-gal4 driver group (Fig 5E-H). In contrast, co-expression of Aβ-42 and TBP ameliorates and delays Aβ-42-induced age-dependent retinal degeneration (Fig 5M-P). In addition, Aβ-42 expressing flies displays locomotor dysfunction after 20 days of age (Fig 6B), and their lifespan is significantly reduced compared to Elav-gal4 flies without expressed Aβ42 (Fig 6A). Climbing assay demonstrated that Aβ-42 flies exhibit locomotor defect at 20 days of age compared to Elav-gal4 driver flies, whereas expression of Aβ-42 flies with TBP exibits locomotor defects at 30 days of age (Fig 6B). Consistently, the lifespan of Aβ-42 flies was prolonged by the overexpression of TBP (Fig 6A). The result indicated that an increase of TBP expression improves locomotor function and lifespan of Aβ-42 expressing flies. To further confirm the beneficial effects TBP expression, We have expressed Aβ-42 in loss-of-function of TBP mutant flies. The dTbpsI10 is considered to be a protein-null allele because a non-sense mutant was introduced at codon 249, which deletes approximately one-half of the DNA-binding C-terminal domain failed to binding TATA box DNA. As a negative control, reducing dTbp enhanced the rough-eye phenotype in the Gmr>Aβ-42 files (Fig 5 I-L). The eyes of 4-week-old dTbp mutant flies expressing Aβ-42 exhibited little depigmentation with small, scattered necrotic spots (Fig 5P). 20.

(22) Furthermore, downregulation of dTbp enhanced climbing deficit compared with age-matched Aβ-42 flies (Fig 6B). Taken together, we conclude that TBP expression levels can modulate A through changes in TBP expression levels.. TBP reduces Aβ-42 deposition in the fly brains 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 suggest that TBP upregulation ameliorates amyloid induced toxicity in the brains adult flies.. 21.

(23) 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 22.

(24) 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.. 23.

(25) Acknowledgements First of all, I would like to appreciate my thesis supervisor, Dr. MingTsan 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.. 24.

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(29) 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. 28.

(30) 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. TSpositive 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.. 29.

(31) 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.. 30.

(32) 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.. 31.

(33) Figure 5. Co-expression of Aβ-42 and TBP ameliorates amyloid induced age-dependent retinal degeneration. E, F, G and H, Overexpression Aβ42 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. 32.

(34) 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.. 33.

(35) 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.. 34.

(36) 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.. 35.

(37) 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).. 36.

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