TBP功能喪失為聚麩醯胺神經退化性疾病之共同致病因素：與氧化壓力之關聯

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(1)國立臺灣師範大學生命科學系博士論文. TBP 功能喪失為聚麩醯胺神經退化性疾病之共同致病因素：與氧化壓力之關聯 Deactivation of TBP as a common pathogenic factor in polyglutamine induced neurodegenerations: Implication of oxidative stress. 研究生：許敦傑 Tun-Chieh Hsu. 指導教授：蘇銘燦. 博士. Dr. Ming-Tsan Su. 中華民國 104 年 2 月.

(2) Table of Content 中文摘要.....................................................................................................2 ABSTRACT ...............................................................................................4 INTRODUCTION ....................................................................................6 MATERIALS AND METHODS............................................................14 RESULTS .................................................................................................27 DISCUSSION ..........................................................................................49 ACKNOWLEDGEMENT......................................................................54 REFERENCES........................................................................................55 TABLE .....................................................................................................64 FIGURES .................................................................................................65. 1.

(3) 中文摘要在許多由聚麩醯胺擴增造成之神經退化性疾病 (polyQ diseases) 之研究中指出TATA box binding protein (TBP) 被蛋白質包涵體捕捉而失去作用。不僅如此，其中一種疾病：第十七型脊髓小腦共濟失調症 (SCA17) 正是由TBP本身的聚麩醯胺擴增所引起，進而形成具有神經毒性的蛋白質包涵體並影響下游基因。然而擴增的聚麩醯胺片段如何影響TBP功能，以及功能受到影響的TBP與SCA17的致病機制之間的關係依然不明。為深入探討此致病機制，在本研究中，我們建立新的SCA17果蠅模式，其確實展現出蛋白質聚集、運動能力降低以及壽命減短等退化症狀。此外，聚麩醯胺擴增之TBP不只會形成蛋白質聚集且其本身之DNA結合力以及轉錄活性也較低，聚麩醯胺擴增之 TBP更進一步會干擾正常TBP的功能，據此推測TBP功能缺失為第十七型小腦萎縮症之病因之一。TBP突變果蠅顯現類似SCA17模式果蠅之神經退化症狀，且第十七型小腦萎縮症果蠅模式複眼的退化性狀在 TBP功能缺失突變背景下更為突出，証實TBP功能之降低為SCA17致病機制的一環。另TBP表現降低更加劇第三型脊髓小腦萎縮症以及亨丁頓氏舞蹈症複眼感光細胞之退化。顯見TBP功能喪失可能為聚麩醯胺擴增所造成之神經退化的共同因子。TBP功能喪失在小鼠胚胎造成細胞凋亡，但原因不明。本研究發現果蠅TBP缺失不只造成神經退化 2.

(4) 亦能觀察到細胞凋亡，故以果蠅來探討TBP在神經退化與細胞凋亡之關聯。藉由微陣列基因分析篩選出能分解過氧化氫的prx2540-2基因在TBP突變的果蠅頭部中表現顯著減少。在果蠅中降低prx2540-2之表現亦產生類似TBP突變果蠅之神經退化症狀。此外，聚麩醯胺擴增所引起的退化性神經疾病果蠅模式與TBP突變之果蠅其頭部的過氧化氫濃度亦較高。故由TBP功能喪失引起prx2540-2表現下降進而造成的氧化壓力增加可能為聚麩醯胺擴增所造成之神經退化的原因之一。. 關鍵字：聚麩醯胺. 3.

(5) Abstract TATA box binding protein (TBP) has been implicated in many polygluatmine (polyQ) induced neurodegenerations as it is sequestered and inactivated in polyQ proteins containing inclusions. Unlike most polyQ mediated neuropathies, spinocerebellar ataxia 17 (SCA17) is resulted from the abnormally expanded polyQ tract of TBP itself. Previous studies have shown that polyQ expanded TBP forms neurotoxic inclusions and affects downstream genes. However, how expanded polyQ tracts affect the function of TBP and the link between dysfunction of TBP and SCA17 are not clear. In this study, we generate novel Drosophila models of SCA17 that recapitulate pathological features, including aggregate formation, mobility defects and premature death. In addition to forming neurotoxic aggregates, we showed that polyQ-expanded TBP loses its intrinsic DNA-binding and transcription abilities. Dysfunctional TBP also disrupts the function of normal TBP. Moreover, flies expressing polyQ-expanded TBP exhibited enhanced retinal degeneration and heterozygous dTbp amorph mutant flies exhibited SCA17-like phenotypes, suggesting that loss of TBP function may contribute to SCA17 pathogenesis. We also determined that the downregulation of TBP activity enhances retinal degeneration in the fly models of SCA3 and Huntington’s disease, indicating that the deactivation of TBP is likely to play a common role in polyQ-induced neurodegeneration. Moreover, inactivation of TBP has been reported to cause apoptosis in mice. Nevertheless, the mechanism by which deactivated TBP leads to apoptosis is illusive. We found that Drosophila TBP mutants exhibit both 4.

(6) neurodegeneration but also apoptosis phenotypes, indicating that Drosophila is a suitable model for unraveling the links between neurodegeneration and apoptosis. Through gene profiling experiments we have identified that prx2540-2, a peroxiredoxin (Prx) encoded gene whose gene product catalyzes the reduction of hydrogen peroxide (H2O2), is reduced greatly in the heads of dTbp mutants. Down-regulation of prx2540-2 generates similar neuropathies as seen in dTbp mutant flies. In contrast, expression of prx2540-2 reverses the above phenotypes in dTbp mutants, demonstrating that prx2540-2 acts downstream of dTbp. Additionally, the concentration of H2O2 is higher in the brains of dTbp mutants and polyQ disease models. Therefore, downregulation of prx2540-2 may mediate the neurodegeneration in polyQ disorders through increasing the oxidative stress in neuronal tissues.. Keyword: polygluatmine. 5.

(7) Introduction Heretofore, 10 neurodegenerative diseases, including the Huntington’s disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), spinocerebellar ataxia (SCA) 1, 2, 3, 6, 7, 8, and 17, have been attributed to CAG trinucleotide repeat expansion in different genes, which result in generating expanded polyglutamine tracts in encoded proteins [1-4]. These hereditary disorders are characterized by late-onset progressive neuron lost in the central nervous system, leading to a variety of clinical conditions. Common symptoms have been observed among these diseases, indicating that similar pathogenesis may exist. A hallmark of these diseases is that the expanded polyQ proteins with a gain of toxicity property, display aberrant interaction with other proteins and aggregate together in neurons to form intranuclear or cytoplasmic inclusions finally [5]. Although most proteins are prone to aggregate while aging [6], the mutant polyQ protein has stronger tendency to aggregate and cause disease [7]. Thus far, the cellular function of protein inclusion in the pathogenesis of polyQ diseases is still controversial [1, 7]. In some aspects, protein inclusions are harmful because of the protein misfolding, protein-transport interference, transcription repression, and the interference with cytoskeletal and vesicular functions [8-13]. On the contrary, some suggest that inclusions protect cells from toxicity of misfolded proteins, and the toxicity of mutant polyQ protein monomers or oligomers are more toxic than aggregate forms [14-16]. Furthermore, aggregate formation could occur in unaffected regions of patient’s brain 6.

(8) [17]. It may even protect cells by enhancing the degradation of mutant polyQ proteins [18]. For a number of aggregate prone proteins, the formation of nuclear inclusion poorly correlates with the cytotoxicity [14, 19-22]. However, the volume and location of inclusions are considered to interrupt the function of the nucleus. Expanded polyQ-containing proteins recruit and interrupt many cellular proteins, including components of the ubiquitin–proteasome system, molecular chaperones and transcription factors. Abnormal interactions among the abovementioned cellular components and disruption of their associated functions are believed to contribute to the pathogenesis of polyQ-mediated neurodegenerations. Furthermore, many disease associated polyQ containing proteins are highly related to transcription [2]. The mutant polyQ protein-binding proteins are major transcription factors including TATA box-binding protein (TBP), cAMP response element-binding protein (CREB)-binding protein (CBP), and specifity protein 1 (SP1), which are also glutamine-rich proteins [23-26]. CBP and SP1 depletion were even found to participate in the pathogenesis of polyQ-mediated neurodegeneration [27, 28]. Among the glutamine-rich transcription factors involved in polyQ-mediated neurodegenerations, TBP is noteworthy [29]. First, TBP is a eukaryotic general transcription factor that is critical in forming the transcription preinitiation complex and transcription of all RNA polymerases (pol I, II, III). Additionally, TBP is a house keeping gene, aberrant TBP activity is expected to extremely affect normal cellular 7.

(9) functions. Apart from its role in the gene transcription, TBP is also involved in embryonic development. Completely loss of TBP causes transcriptional downregulation of Pol I and Pol III dependent transcriptions, growth arrest and apoptosis in mouse embryo [30]. Secondly, TBP was observed to be trapped in several polyQ containing inclusions, such as polyQ-expanded ataxin-1 (Atx1), ataxin-3 (Atx3), Huntingtin (Htt) and atrophin-1, which respectively cause SCA1, SCA3, HD and DRPLA [25, 31-33]. Loss of TBP function has suggested to play a pivotal role in the pathomechanisms of these neuronal diseases. However, the abovementioned hypothesis has not been addressed. Finally, the expansion of polyQ tract within the N-terminal domain (NTD) of TBP has shown to cause SCA17 [34-37]. The glutamine rich domain of N-terminal of TBP exhibits transactivation capability [38]. The expanded polyQ tract of TBP may affect TBP-associated transcription and disrupt the formation of TBP–TATA box DNA complexes [39, 40]. Abnormal TBP-dependent transcription induced by the expanded polyQ tract of TBP has been assumed to be neurotoxic; however, this view has not been tested. To delineate the molecular mechanisms underlying SCA17, several disease models, including cell, Drosophila and mouse, have been established [39-47]. Overexpression of polyQ-expanded full-length TBP and polyQ-expanded truncated TBP which lacked DNA-binding domains was observed to cause the formation of inclusions, suggesting that the neurotoxicity of mutant TBP is DNA binding-independent and that the 8.

(10) process of aggregate formation or insoluble aggregates are causative factors [39, 40]. In this regard, the pathogenesis of SCA17 is similar to that of other polyQ diseases. Additionally, transcriptional dysfunction has also shown to be a causative factor of SCA17. It was reportd the expanded polyQ tracts modulate TBP dimerization and TBP–TATA box complex formation by enhancing the association of general transcription factor IIB with polyQ-expanded TBP. The aberrant binding of both proteins reduces the expression of Hspb1 [40, 45]. Therefore, downregulation of Hsbp1 is believed to be linked to the pathogenesis of SCA17 [45]. The polyQ-expanded TBP also exhibits a strong affinity for SP1 and nuclear factor-Y (NFY), which reduce the binding of the transcription factors toward the promoters of downstream genes, including Hsp70, Hsp25, HspA5, HspA8 and TrkA [42, 46]. Moreover, the Q/N-rich RBP-J/Su(H) transcription factor, a component of the Notch signaling, is particularly downregulated in one SCA17 Drosophila model, because its interaction with mutant TBP is stronger than that of wild-type TBP [41]. It was demonstrated that reduction in cellular Su(H) activity may modulate SCA17 progression [41]. Recently, it has been reported that the polyQ-expanded TBP decreases its association with XBP1s, causing the expression of the Purkinje cell-enriched mesencephalic astrocyte-derived neurotrophic factor (MANF) is specifically reduced in an inducible knock-in mouse model for SCA17 [47]. Reversibly, overexpression of HSc70 can improve the TBP-XBP1s interaction and MANF transcription, and overexpression of MANF ameliorates mutant TBP-induced 9.

(11) neurotoxicity in SCA17 mice [47]. In many neurologic disorders, apoptosis takes place in neurons through activation of caspases [48-51]. Apoptosis, a type of programmed cell death, is involved in animal development normally through tightly regulation in many defined pathways [52]. In polyQ induced diseases, a common pathogenic mechanism was suggested that the interruption of numerous transcription factors caused the death of neurons [53-55]. For example, mutant Htt can up-regulate the pro-apoptotic gene p53 in Huntington disease [9, 56, 57]. p53 plays a pivotal role in apoptosis. Interestingly, p53 interact directly with TBP [58]. However, the correlation between TBP and apoptosis is unclear. Accumulated evidences also suggest that neurodegeneration is highly associated with oxidative stress [59-63]. The facts that neurons consume 10-fold oxygen when compared to other cell types, it is conceivable that non-dividing mature neurons may suffer more oxidative stress than other cells [64]. Reactive oxygen species (ROS), such as hydrogen peroxide, not only affects cells by damaging macro-molecules, such as DNAs, proteins and lipids, but it also acts as second messengers in cell signaling [65]. Accordingly, changing the oxidative status may affect cells in various ways. In this study, we find that one of the antioxidant enzyme of Drosophila, peroxiredoxin 2540-2 (prx 2540-2), may be involved in polyQ mediated neurodegenerations. Peroxiredoxin (Prx) gene family is wildly existed in organisms ranging from bacteria to mammals. Members of Prx function as hydrogen peroxide reducers. Nevertheless, the roles of Prx remain poorly 10.

(12) understood in vivo [66, 67]. Mammalian prx2 is likely to be involved in the apoptosis pathway. It can also be released as a signaling molecule [68-70]. Mitochondrial Prx3 level was significantly decreased while the levels of prx1, 2 and 6 were markedly increased in AD patients’ brains. It was suggested that Prx is neuroprotective [71, 72]. Some studies do support the idea that Prx plays a protective role in PD, because overexpression of Prx attenuated, whereas knockdown of Prx aggravated cell damage [73-75]. Recently, Prx was proved to be neuroprotective in a Drosophila PD model overexpressing mutant LRRK2 [76]. For dissecting the pathogenesis of human neurodegenerative diseases, the Drosophila model is a preferential choice [77-79]. The numbers of Drosophila neurodegeneration models are increased substantially, because of the well-established research tools used in the model organism [80, 81]. Studies through the neurodegeneration Drosophila models, the pathogenesis of many diseases have been attributed to the aberrant expression of protein levels, in the respective neuronal impairments, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), Alzheimer’s disease (AD), and HD [82-86]. The primary reason why Drosophila is exploited as human diseases’ models is based on the conserved genetic background and cellular processes. About 75% of the disease-related human genes have at least one Drosophila homologue [87]. Secondly, it is much easier to proceed to large-scale genetic screen using Drosophila because of its small size, short lifespan and strong fertility. Particularly, numerous genetic tools are available for Drosophila. Thirdly, the Drosophila nervous system is highly organized as seen in the 11.

(13) brains of mammals which have separated specialized areas for sensing, learning and memory. In addition, the fruit fly is capable of performing highly complex behaviors like its mammalian counterparts. Although the aforementioned studies have present many valuable discoveries regarding the pathomechanism of SCA17, whether polyQ-expanded domains affect the function of TBP has yet to be understood. Besides, the link between loss of TBP function and pathogenesis of SCA17 has not been addressed. In this study, we generated novel SCA17 Drosophila models by overexpressing polyQ-expanded full-length TBP. Through studying our SCA17 model, not only the expanded polyQ tract of TBP forms neurotoxic aggregates, the disease causing TBP is also dysfunctional. We also found that the polyQ-expanded TBP sequestered wild-type TBP in the neuroblasts of Drosophila and impaired the function of wild-type TBP. We demonstrated that the loss-of-function of Dosophila TBP (dTbp) mutants displayed SCA17-like phenotypes and polyQ-expanded TBP-induced neurotoxicity was enhanced in dTbp mutant backgrounds. These results implied that the deactivation of TBP may contribute to the pathogenesis of SCA17. Especially, we demonstrated that reduced dTbp expression exacerbated retinal degeneration induced in polyQ-expanded HD and SCA3 Drosophila models. These findings imply that the loss of TBP function may play a universal role in polyQ mediated neurodegenerations. To further explore how down-regulated TBP leads to apoptosis and neurodegeneration, we conducted microarray analysis. We surprisingly 12.

(14) found that only a small number of genes were considerably affected in dTbp homozygous mutants. Among these genes, a peroxiredoxin encoded gene, prx2540-2, is profoundly decreased. Down-regulation of prx2540-2 phenocopies dTbp mutant in Drosophila. On the other hand, overexpression of prx2540-2 suppresses the phenotypes of Tbp mutation. Dwonregulation of either dTbp or prx2540-2 increases H2O2 levels in cells. Additionally, oxidative stress activates c-Jun N-terminal kinase (JNK), which up-regulates stress-responsive elements [88-92]. JNK also plays a critical role in activation of apoptosis by up-regulating pro-apoptotic genes [93]. In this study, we also showed that down-regulation of prx2540-2, caused by dysfunction of TBP, activates JNK and causes apoptosis.. 13.

(15) Materials and Methods Fly stocks and maintenance All Drosophila melanogaster were raised on standard cornmeal media supplied by Institute of Molecular Biology Academia Sinica 25 ºC unless otherwise mentioned. CO2 is utilized to anaesthetize the flies. The UAS-Gal4 system was applied for ectopic gene expression [94]. Gmr-gal4, Elav-gal4, Actin5C-gal4 and UAS-Atx3-78Q flies were obtained from the Bloomington Stock Center. The Drosophila TBP mutant allele, dTbpsI10, was a gift from S. Artavanis-Tsakonas [95]. The UAS-Htt-97Q expressing transgenic Drosophila was obtained from L. Marsh (University of California, Irvine, CA, USA). RNAi line for dTbp (109756) and prx2540-2 (Transformation no. 18708 and 109152) were obtained from the Vienna Drosophila RNAi Center (VDRC).. DNA constructs and germ-line transformations Human TBP-36Q and TBP-109Q were cloned as previous described [44]. An influenza virus hemagglutinin (HA) tag was fused in-frame to all TBP constructs then subcloned into a pUAST vector digested with EcoRI snd NotI enzymes [94]. The Drosophila dTbp cDNA clone (LD44083) was obtained from the Drosophila Genome Resource Center. To generate the UAS-dTbp construct, dTbp cDNA was PCR-amplified using a pair of primers, 5’-CACCATGGACCAAATGCTAAGCC-3’ and 5’-TGACTGCTTCTTGAACTTCTT-3’. The UAS-TBP-36QFL-Flag construct was generated using PCR with a pair of primers, 5′-CACCATGGATCAGAACAACAGCCTG-3′ and 14.

(16) 5′-CGTCGTCTTCCTGAATCCCTT-3′. Two truncated N-terminal TBP constructs, UAS-N-TBP-36Q-Myc and UAS-N-TBP-109Q-Myc, were generated using PCR using 5′-CACCATGGATCAGAACAACAGCCTG-3′ and 5′-TGGCGTGGCAGGAGTGATGG-3′ primers. All the amplified DNA fragments were gel-purified using the DNA/RNA extraction kit (Viogene) and cloned into a pENTR/TEV/D vector to generate Gateway entry clones (Invitrogen). These entry clones were then subcloned into pTWM, pTWF and pTWH vectors for C-terminal Myc, Flag and HA tagging respectively (http://www.Ciwemb.edu/labs/Murphy/Gatewayvectors.html). Before performing germ-line transformation, all the transgenic DNA constructs were confirmed using sequencing. A standard germline transformation procedure involving w1118 as the parental line was followed to generate transgenic flies. For the generation of the tr-dTbp DNA construct a pair of primers, 5′-CGCAGATCTGCGGCCATGGACCAAATGCTAAGCCCC-3′ and 5′-CCTCGAGCCGCGGCCTCACGCTGCCAGTCTGGAGTC-3′ was used in PCR reactions. The DNA fragment was cloned into pBID-UASC vector using the In-Fusion HD cloning kit (Clontech), then the plasmid was used for generation of transgenic line (GENETIC SERVICES, INC.).. Expression construct and protein purification Various polyQ repeats in N-terminal-truncated TBPs, including the DNA of N-TBP-36Q, N-TBP-54Q and N-TBP-109Q, were amplified 15.

(17) using PCR involving primers, 5′-CACCATGGATCAGAACAACAGCCTG-3′ and 5′-TTACGTCGTCTTCCTGAATCCCTT-3′. The PCR-amplified DNA was cloned into a pENTR/TEV/D vector by using TOPO cloning technology (Invitrogen). After the DNA construct was confirmed by sequencing, it was cloned into pDEST15 or pDEST17 expression vectors, which forms respective fusion proteins with of an upstream GST or His tag. The tr-dTbp was cloned into pDEST17 using In-Fusion HD cloning kit (Clontech) using primers, 5′-CAAACAAGTTTGTACAAAATGGACCAAATGCTAAG CCCC-3′ and 5′-TCAAACCACTTTGTACAATCACGCTGCCAGTCTGGAGTC-3′. All the DNA constructs were expressed in E. coli BL21 (DE3) or BL21 (DE3) pLysS. To purify the recombinant proteins, bacteria were incubated in a LB medium with 0.4 mM IPTG or an autoinducible medium at 25℃ overnight [96]. The bacteria were lysed by the ultrasonic cell disruptor and soluble proteins were purified using affinity chromatography. To express full-length or N-terminal-truncated TBP in HEK293T cells, the pSEMS1 vector, which contains a Gateway cloning cassette and an upstream CMV promoter, were used as a destination vector (Covalys Biosciences).. Filter trap assay GST-TBP proteins were purified using Glutathione Sepharose 4B (GE). Purified GST-TBP proteins were quantified using the Bradford reagent 16.

(18) (Bio-Rad). For initiating the proteolytic cleavage of the fusion proteins, equal quantity of GST-TBP-36Q or GST-TBP-109Q (final concentration = 1.5 mM/each protein) was incubated with 2.5 U of TEV protease (Invitrogen) in a reaction buffer (50 mM Tris, pH 8.0, 0.5 mM EDTA) at 30 ºC. The digestion of fusion proteins was completed in 1 hour, and stopped by adding an equal volume of stop solution [4% SDS and 100 mM dithiothreitol (DDT)] at specific time points at 95 ºC for 5 min. The reaction mixture was filtered through a nitrocellulose–acetate membrane (OE66, Schleicher & Schuell). To detect the formation of insoluble aggregates, mouse monoclonal 1C2 antibodies (Chemicon) were diluted in a ratio of 1: 100. HRP-conjugated mouse IgG secondary antibodies (Jackson ImmunoResearch) were applied at a dilution of 1: 10000. Signals were detected using chemiluminescent HRP substrate (Millipore) and captured using an imaging system (Fujifilm LAS-3000).. RNA extraction, reverse transcription PCR and real-time PCR Total RNA was extracted from the heads of 1-day-old male flies or embryos of stage 16 using the RNeasy Kit (Qiagen). Samples were then used for reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Equality quantities of cDNAs were used in Taqman gene expression assays using an StepOnePlus Real-time PCR system (Applied Biosystems). The following Taqman probes were used: hTBP (assay ID Hs000427620_m1); dTbp (assay ID Dm01821639_g1); Prx2540-2 (assay ID Dm02362204_s1); p53 (assay ID Dm02154334_m1); Edem1 (assay ID Dm01821064_g1); CG1529 17.

(19) (assay ID Dm02365051_s1); pnt (assay ID Dm02136350_g1); CG3091 (assay ID Dm01829728_g1); Rala (assay ID Dm01821323_g1); CG9616 (assay ID Dm02140263_g1); CG9518 (assay ID Dm02364655_s1); CG3699 (assay ID Dm01829358_s1); CG10962 (assay ID Dm01847441_g1); CG32553 (assay ID AJ39Q4X), HUMAN_PRE45S_RR (assay ID AILJIZM); Drosophila_U6 (assay ID AII1MM6), Drosophila_PRE-R (assay ID AIMSG5U), Drosophila_RRNAM (assay ID AIN1FB2), 18S RNA (assay ID AII1MMS) and Actin (assay ID Dm02371594_s1). Each set of experiments was performed in duplicated and was repeated at least three times. The comparative CT method was used for quantification of each gene expression. For comparing the expression of three paralogs of prx2540, RT-PCR was used. Briefly, cDNA (2.5 ng) isolated from embryos was amplified using following primers, 5′-CTGCCCAGTGTCACCGATGAC-3′ (CG12896 sense), 5′-CGAACGTAGTTTACTCCAGAT-3′ (CG12896 anti-sense), 5′-CTGCCCACTGTCACCGATGAG-3′ (prx2540-1 sense), 5′-CGGACGTAGTTTACTCCAGAG-3′ (prx2540-1 anti-sense) , 5′-CTGCCCACTGTCACCGATGAA-3′ (prx2540-2 sense), 5′-CGGACGTAGTTTACTCCAGAC-3′ (prx2540-2 sense), 5′-CGAACCAAGACGGTGAAGAAG-3′ (rps17 sense) and 5′-CCTGCAACTTGATGGAGATACC-3′ (rps17 anti-sense). For RT-PCR, the reaction was done with a denaturation step at 95 ºC for 2 min, followed by 27 cycles of denaturation at 95 ºC for 30 s, primer annealing at 46 ºC for 30 s, and primer extension at 72 ºC for 18 s. An 18.

(20) extra extension step was performed at 72 ºC for 3 min.. Microarray The RNA extraction was performed as described above. The extracted RNAs were analyzed using Affymetrix Drosophila Genome 2.0 array kit and the GeneChip scanner 3000 through the service of the core-facility at the National Health Research Institutes of Taiwan.. Immunoblotting Proteins were extracted from the heads of adult male flies in T-PER solution (Pierce). Protein concentrations were quantified using a Bradford reagent (Bio-Rad). For each sample, 20 µg of protein were mixed with an equal volume of 2× Laemmli buffer and denatured at 95 ºC for 5 min before being resolved in 12% polyacrylamide gels and blotted onto PVDF membranes (Millipore) by using a Semi-dry blotter (TE70 ECL, Amersham). Protein blots were blocked in Tris-buffered saline with 0.05% Tween 20 (Sigma) and 5% dry milk. The primary antibodies used in the study were anti-β-actin (1: 5000 dilution, GeneTex), anti-TBP antibodies (1: 200 dilution, Roche), 1TBP18 (1: 20000 dilution, QED Bioscience), 1C2 (1: 500 dilution, Millipore), JNK (FL): sc-571 (1:1000, Santa Cruz) and Anti-active JNK (1: 5000 dilution, Promega). HRP-conjugated secondary antibodies for anti-mouse (Jackson ImmunoResearch) and anti-rabbit (GeneTex) were diluted in a ratio of 1: 10000. ECL substrates were used (Millipore) and images were captured using an imaging system (GE LAS-4000) following the manufacturer’s 19.

(21) instructions.. DNA affinity assay For EMSAs, biotinylated TA-30-B6 DNA comprising a TATA box (TATAAA) and 4-nucleotide loop was generated by annealing the synthesized primers 5′-CGGTGTATAAAGCCGCGGTCC-3′ and 5′-GGACCGCCCCTTTATTGACCG-3′ [97]. TA-30-B6 DNA (final concentration = 80 fmole) was mixed with a GST-TBP fusion protein (1.5 mM) in an EMSA buffer (150 mM KCl, 4 mM MgCl2, 20 mM HEPES–KOH pH 8.0, 1 mM DTT, 50 ng/mL of polydI-dC and 5% glycerol). The DNA binding reaction was initiated by adding 2.5 U of TEV protease (Invitrogen) and incubated at 30℃. An aliquot of reaction mixture was withdrawn at each time point then frozen in liquid nitrogen until resolved on a 5% acaryamide gel buffered in 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA and pH 8.0). Biotin-labeled oligomer was electro-blotted onto a nylon membrane (Amersham Pharmacia) and detected using a chemiluminescent nucleic acid detection module kit (Pierce). The procedure recommended by the manufacturer was followed and the signal was recorded by scanning using an imager (Fujifilm, LAS-3000). In addition, liquid chemiluminescent DNA pull-down assays were adapted to determine the DNA-binding ability of TBP [98]. Purified His-TBP-36Q or His-TBP-109Q was bound to Ni-NTA magnetic beads (Taiwan Advanced Nanotech, Inc.) in binding reactions containing biotinylated TA-30-B6 DNA (1.25 pM) and EMSA buffer (150 mM KCl, 4 mM MgCl2, 20 mM HEPES–KOH, pH 8.0, 1 mM DTT and 5% 20.

(22) glycerol) at a final concentration of 0.72 mM at 25 ºC for 1 h. The bound DNA was captured using a strong magnet and washed three times with an EMSA buffer without glycerol. Streptavidin-conjugated HRP (Thermo Scientific) was added to the bound DNA in PBST (1×PBS, 0.1% Triton X-100 and 3% BSA) at 25 ºC for 1 h. After washing with 1× PBS for three times, the bound DNA was detected using an HRP substrate. Luminescence was detected using a luminescence microplate reader (BD, SpectraMax L). For inhibition of DNA binding, N-terminal-truncated TBP containing 54 glutamine residues (N-TBP-54Q) was used as the truncated TBP-109Q (N-TBP-109) was highly insoluble. Equal amounts of N-TBP-36Q and N-TBP-54Q were added to the EMSA reactions at a final concentration of 0.72 mM at 25 ºC for 1 h. The aforementioned procedure was then followed to quantify bound DNA.. Cell culture and transactivation assay The HEK293T cell line was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ºC in a humidified atmosphere with 5% CO2. The reporter vector (pG5Tluc) was a gift from Dr Y.-S. Huang (Institute of Biomedical Sciences, Academia Sinica) [99]. For transfection, equal amounts of reporter plasmid and Gal4 DBD (mock control), Gal4 N-TBP-36Q or Gal4 N-TBP-109Q (1 µg each) were cotransfected into HEK293T cells by using the lipofection method (Lipofectamine 2000 & Opti-MEM, Invitrogen). In addition, equal amounts of reporter plasmid and Gal4 N-TBP-36Q were cotransfected with either 1 μg of N-TBP-36Q or N-TBP-109Q in 21.

(23) inhibitory reactions. All transfected cells were incubated for 48 h, and cell lysates were prepared. A dual-luciferase reporter assay system was applied to measure luciferase activity according to the instructions of technical manual (Promega). Transactivation activity was expressed as the ratio of firefly luciferase to Renilla luciferase as control. All the transfection experiments were repeated three times with duplicated samples. The difference in luciferase activity was examined using the Student’s t-test.. Acridine orange(AO) staining For detection of apoptotic cells of Drosophila embryos, a previous protocol was followed with slightly modification[100]. Briefly, late-stage Drosophila embryos were collected and dechorionated in 50% bleach for 2 minutes at room temperature. The embryos were washed 3 time using distilled water, then transferred to a 20 mL glass vial containing equal volume (5 mL) of 1× PBS (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl and pH 7.2) and n-heptane supplemented with AO (Sigma) at a final concentration of 5 mg/mL. After 5 min of staining on a shaking platform, embryos were washed using 1× PBS for 2 min then mounted with paraffin oil. The embryos were observed and analyzed using a confocal microscope (Leica TCS SP2).. Caspase activity Twenty heads of 1-day-old adult flies were homogenized in 100 µL T-PER (Thermo) without adding protease-inhibitors. After centrifugation, 22.

(24) supernatant then mix with 100 µL Caspase-Glo 3/7 Assay solution (Promega), then incubated 30 min at room temperature. Luminescence signals were detected using a luminescence microplate reader (BD, SpectraMax L).. Eye morphology The eye morphology of adult male flies was captured using a Leica DMR upright microscope equipped with a digital camera (Cool-SNAP5.0, Photometrics). To increase the depth of field, 20-30 images were merged using imaging software to create montage composited images (Helicon Focus, HeliconSoft). For scanning electron microscope, adult fly heads were cut and fixed in 2% glutaraldehyde at 4 ºC overnight. The samples were briefly washed using 1× PBS and immersed in 2% osmium acid at room temperature for 5 h. Dehydration was performed using an ethanol series (50, 70, 95 and 100% of ethanol for 30 min each). Then the fly heads were transferred in pure acetone at 4 ºC overnight before critical point drying with liquid CO2 and being sputter-coated with gold. The SEM images were snapshotted using a JEOL 6400 scanning electron microscope (20 kV, 200×).. Histology The H&E staining of adult male fly brain sections was performed according to standard protocol. Briefly, male Drosophila heads were fixed in 4% paraformaldehyde and dehydrated using an ethanol series. Frontal sections of paraffin-embedded heads were sliced using a 23.

(25) microtome (Leica RM2135). A series of sections (5 μm) were collected and subjected to H&E staining. The stained samples were visualized and recorded using a microscope (Leitz wetzlar orthoplan microscope) equipped with a digital camera (Photometrics, CoolSnap5.0). The ABC and DAB (3,3’-diaminobenzidine) reactions were performed after rehydration of slides.. Immunochemistry staining Dissected eye discs, brain lobes and salivary gland from crawling third-instar larvae were fixed in 4% paraformaldehyde for 5–10 min then rinsed in PBT (1× PBS, 0.1% Triton X-100 and 3% BSA). The samples were incubated with the antibodies for 1 h at 25 ºC or overnight at 4 ºC in PBT. The primary antibodies used were: 1C2 (1: 100, Chemicon), anti-Flag (1: 100, Sigma) and anti-Myc (1: 100, Santa Cruz). FITC- and Cy5-conjugated secondary antibodies (Jackson Immunological Laboratory) were used at a dilution of 1: 200. Whole-mount immunostainings of adult retinas were performed as described previously [101]. Anti-HA antibodies (1: 200, Sigma) were used to detected protein inclusions. Rhodamine-conjugated phalloidin (1: 100, Invitrogen) was used to label F-Actin-enriched rhabdomere following the manufacturer’s instruction. All images were captured and analyzed using a confocal microscope (Leica TCS SP2).. Mobility analysis To quantify the locomotive functioning of the flies, a graded-climbing 24.

(26) assay was applied as described [102, 103]. For each trial, 10 male flies were tapped down to the bottom of the climbing apparatus and allowed 10 s to climb into each scoring areas. All trial sections were repeated 10 times and a total of 50 flies (5 groups) were assayed for each time point. The climbing index (CI) was calculated as follows: CI = Σ(nm)/10, where n is the number of flies in a given scoring area and m is the score from 1 to 5 for the given score area.. Lifespan analysis To determine the lifespan of the flies, at least 200 flies were analyzed for each genotype. A group of 10 to 20 adult flies were cultured using standard media in a vial at 25 ºC. Viable flies were calculated and transferred to a fresh food vial at 5-day intervals. The difference in lifespan was tested using the log-rank test.. Mosaic animals To produce somatic clone cells of homozygous TbpsI10 in tissues, yw; hsflp12; FRTG13 GFP/ CyO were crossed with yw;hsflp12; FRTG13 TbpsI10/ CyO . Eggs were collected with grape agar plates and the newly hatched first instar larvae were incubated for 2 h at 37 ºC to induce mosaic clones. The samples then were incubated at 25 ºC for 24 h. The above procedure was repeated once next day to ensure the generation of somatic mutant clones.. Oxidative stress detection 25.

(27) For oxidative stress detection in tissues, the CellRox Deep Red Reagent (Life Technologies) was used at a final concentration of 5 µM and the tissues were incubated 30 min at 37 ºC in dark. The stained samples were observed using a confocal microscope (Leica TCS SP2). For detecting the relative levels of reactive oxygen species (ROS) in heads of Drosophila models of polyQ diseases, 30 heads of 1-day and 10-day old male flies were decapitated then immersed in 150 µL T-PER (Thermo). The heads were homogenized (Bullet Blender, Next Advance Co.) then centrifuged at 20,000 × g for 5 min. The ROS in the supernatant was measured follows a modified chemiluminescence detection method [104, 105]. Briefly, 30 µL of supernatants were mix with 500 µL of 0.2 mM luminol (for H2O2) or 0.1 mM lucigenin (for superoxide anion). The mixture were analyzed using a Chemiluminescence Analyzer (CLA-ID3, Tohoku Electronic). The related ROS levels were normalized with the content of protein. Each assay was performed at least in trice.. 26.

(28) Results Generating Novel Drosophila models of SCA17 Although the Drosophila model for SCA17 has been generated; the truncated form mutant TBP may cause unrelated effects [41]. To create a proper fly model of SCA17, expression constructs that comprised human full-length TBP with 36 (wild-type, TBP-36Q) and 109 (mutant pathogenic allele, TBP-109Q) glutamine residues were introduced into the genome of Drosophila through germ-line transformation. The tissue-specific expression of transgenes was achieved by the UAS/gal4 expression system [94]. To exclude the difference in expression of TBP-36Q and TBP-109Q, real-time PCR and western blotting were applied. The expression levels of TBP-36Q and TBP-109Q in the heads of the 1-day-old flies were comparable (Fig. 1A–C). Retinal overexpression of TBP-36Q driven by Gmr-gal4 (Gmr>TBP-36Q) did not cause age-dependent morphological defects in the eyes of the adult flies throughout the lifespan when compared with the control Gmr-gal4 driver group (Fig. 1D). The overexpression of TBP-109Q did not cause obvious morphological defects in the eyes of the adult flies in early days (Fig. 1D, w1 and w3). However, obvious pigment-loss in the eyes of the 5-week-old adult flies expressing TBP-109Q was observed (Fig. 1D, w5). The depigmentation phenotype induced by TBP-109Q was deteriorated as age increased. Several detail phenotypes were revealed by using scanning electronic microscopy (SEM), including bristle loss and disorganization of ommatidial arrangement in TBP-109Q-expressing flies at 7 weeks of age (Fig. 1D). 27.

(29) To better understand whether Drosophila models reveal mobility defects and short lifespan, transgenes were expressed by the pan-neuronal driver, Elav-gal4. We observed that the motor function of the control Elav-gal4 flies decreased with age in negative geotaxis assays (Fig. 1E). The climbing ability of the TBP-36Q-expressing flies (Elav>TBP-36Q) also declined while aging. Statistical analysis indicated that no significant difference between the climbing index (CI) of Elav>TBP-36Q flies and control Elav-gal4 lines (p>0.05), however, the climbing ability of Elav>TBP-36Q flies aged 40 days seemed slightly lower than that of the control Elav-gal4 lines. In contrast to Elav>TBP-36Q flies, Elav>TBP-109Q flies did not exhibit climbing defects before 20 days of age. However, the climbing ability of the Elav>TBP-109Q flies was significantly lower than that of Elav>TBP-36Q flies or the control Elav-gal4 lines aged 40 days (Fig. 1E). Furthermore, the mean lifespans of the control Elav-gal4, TBP-36Q- and TBP-109Q-expressing flies were 59, 51 and 46 days, respectively (Fig. 1F). Statistical analyses showed that the neuronal overexpression of TBP-36Q reduced the lifespan of transgenic flies (log-rank test, p< 0.05). However, compared with the control Elav-gal4 lines and Elav>TBP-36Q flies, the lifespan of the Elav>TBP-109Q flies was significantly reduced (p < 0.001). Therefore, the data suggested that the toxicity of TBP is dependent on polyQ length. Remarkably, the neuronal overexpression of TBP-36Q reduced the lifespan of transgenic flies (Fig. 1F). This result seems to agree with the finding that the retinal overexpression of hTBP-34Q causes mild eye degeneration in Drosophila [41]. Because 28.

(30) TBP is a general transcription factor and is biologically active in eukaryotes, ectopic TBP activity may disrupt survival or apoptotic gene expression. The formation of intracellular inclusions is a pathological hallmark of polyQ diseases, including SCA17 [35, 36, 39, 40, 45]. Previously, we demonstrated that polyQ-expanded TBP can from insoluble inclusions both in vitro and in vivo (Supporting Information, Fig. S1). The polyQ-expanded TBP forms insoluble protein aggregates in filter trap assays (Fig. S1A and B), while TBP-36Q did not form detectable protein aggregates. In contrast, 3 hours after initial TEV digestion, aggregates of TBP-109Q were detectable and the signal increased as time increased (Fig. S1B). In addition, no inclusions was observed in eye discs of Gmr>TBP-36Q, but strong inclusion signals were observed in that of Gmr>TBP-109Q (Fig. S1C). Besides, insoluble aggregates were not detected in the control Elav-gal4 or Elav>TBP-36Q between Day 2 and Week 3. In contrast, both soluble and insoluble TBP were detectable in the heads of 2-day-old flies expressing TBP-109Q (Fig. S1D). This result shows that the formation of aggregates is polyQ- and age-dependent (Fig. S1D). Thus, our SCA17 fly models revealed several pathological hallmarks of SCA17, such as age-dependent degeneration, mobility defects a shortened lifespan, and formation of inclusions.. PolyQ expansion affects the intrinsic function of TBP In addition to forming protein aggregates that cause neurodegeneration, we suspected that the expanded-polyQ tract within TBP may affect its 29.

(31) function, thereby leading to SCA17. To test this hypothesis, electrophoretic mobility shift assays (EMSAs) were applied to examine whether the DNA-binding ability of TBP was affected by the expanded-polyQ tract. Biotin-labeled TATA DNA probes were incubated with GST-TBP recombinant proteins (Fig. S1A), and binding reactions were started by cleaving the fusion protein with TEV proteases. The binding of TBP-36Q to TATA box DNA reached a maximum within 1 h (Fig. 2A). An extended reaction time did not increase the formation of TBP–DNA complexes (Fig. 2A). Remarkably, TBP-109Q virtually lost its DNA-binding ability completely within 1 h of the initiation of the binding reaction (Fig. 2A). The DNA affinity of TBP-109Q was time-dependent and reduced gradually (Fig. 2A). As mentioned before, TBP-109Q started to form obvious protein aggregates at about 3 h in vitro (Fig. S1B) and nearly completely lost its DNA affinity within 1 h (Fig. 2A), indicating that TBP-109Q in its soluble state is dysfunctional. To quantify how expanded-polyQ affects the DNA-binding ability of TBP, liquid chemiluminescent DNA pull-down assays were conducted [98]. We found that TBP-36Q bound 15 times more DNA than the same amount of TBP-109Q did (Fig. 2B, p < 0.0001). This result is consistent with the aforementioned phenomenon in EMSA (Fig. 2A). Accordingly, we demonstrated that the expanded-polyQ tract affected the intrinsic DNA affinity of TBP. TBP comprises two functional domains: a polyQ-rich N-terminal domain (NTD) that exhibits transactivation capability and a saddle-shaped C-terminal domain (CTD) responsible for TATA box 30.

(32) DNA binding. To test whether expanded-polyQ modifies the activation capability of TBP NTD, fusion proteins consisting of the Gal4 DNA-binding domains (Gal4 DBD) and TBP-NTD-36Q (Gal4-N-TBP-36Q) or TBP-NTD-109Q (Gal4-N-TBP-109Q) were constructed (Fig. 2C). The firefly luciferase reporter plasmid containing five Gal4 DNA-binding sites (Gal4 BS) upstream of a TATA box was also constructed (Fig. 2C). In transiently transfected HEK293T cells, Gal4-N-TBP-36Q increased the signal of firefly luciferase up to 7.6-fold, compared with the control Gal4DBD construct (Fig. 2D). Gal4-N-TBP-109Q also activated the transcription of firefly luciferase; Nevertheless, its transactivation capability was significantly less than that of Gal4-N-TBP-36Q (Fig. 2D, p < 0.05). These findings indicate that the expanded-polyQ tract reduces the transactivation activity of TBP. Based on the aforementioned findings, we concluded that expanded-polyQ impairs both the TATA box DNA-binding and transactivation abilities of TBP.. PolyQ-expanded TBP interferes with normal TBP SCA17 is an inherited autosomal dominant disease; one mutant TBP allele would cause neurodegeneration. Most patients are heterozygotes that carry a pathogenic TBP allele containing expanded polyQ tracts (43– 66Q) and a normal TBP allele usually bearing 25–38 glutamine residues. The interaction of polyQ tracts is length-dependent and normal TBP alleles contain a relatively long polyQ tract compared with other Q-rich proteins. We suspected that the pathogenic TBP alleles containing 31.

(33) expanded-polyQ tracts may interfere with normal TBP. To examine this hypothesis, epitope-tagged human TBP was transgenically expressed in the neuroblasts of larval brain lobes driven by Elav-gal4. Immunocytochemistry staining showed that the control construct, full-length normal TBP fused with the C-terminal Flag tag (TBP-36QFL-Flag), was expressed in both the nuclei and cytoplasm of cells (Fig. 3A, upper panel). N-terminal-truncated wild-type TBP carrying 36 glutamine residues fused with the Myc tag (N-TBP-36Q-Myc), which was evenly distributed in most neuroblast cells (Fig. 3A, middle panel). However, nuclear inclusions (NIs) were detected unexpectedly in few neuroblasts (~28.6%) that expressed N-TBP-36Q (Fig. 3A, middle panel). The expression of proteins containing normal polyQ tracts usually does not cause the formation of inclusions in most cell types. Previous studies showed that the overexpression of short-repeat-length polyQ proteins formed insoluble aggregates on some occasions; therefore, the formation of polyQ-containing aggregates may be dose-, subcellular localization- or cell type-dependent [25, 106]. In contrast, N-terminal-truncated TBP-109Q (N-TBP-109Q-Myc) formed prominent NIs in the neuroblasts (Fig. 3A, bottom panel). Quantitative analysis showed that 70.6% of the neuroblasts contained N-TBP-109Q-positive NIs, moreover, 100% of the NIs colocalized with wild-type TBP (Fig. 3A). Because N-TBP-109Q formed more NIs with wild-type TBP (TBP-36QFL) than N-TBP-36Q, the TBP interactions are considered to depend on the length of polyQ. Notably, the interaction of 32.

(34) TBPs was not likely to be caused by dimerization because the C-terminal domain of TBP for dimerization was deleted in both N-TBP-36Q and N-TBP-109Q. In addition, we suspected that the aberrant interaction of wild-type TBP may jeopardize its own function. By using liquid chemiluminescent DNA pull-down assays, we showed that truncated TBPs decreased the DNA-binding ability of normal TBP (Fig. 3B). For example, the addition of N-TBP-36Q reduced the formation of the TBP–DNA complex by 42%, whereas that of N-TBP-54Q reduced the formation of the TBP–DNA complex by 79% (Fig. 3B). This result demonstrated that polyQ-expanded TBP negatively affected the DNA-binding ability of normal TBP. Cotransfection of N-TBP-36Q did not significantly reduce the transactivation capability of normal TBP in transactivation assays (Fig. 3C). In contrast, coexpression of N-TBP-109Q significantly reduced the transcription ability of normal TBP (Fig. 3C). Accordingly, the findings indicate that polyQ-expanded TBP reveals a strong propensity to interrupt the function of normal TBP.. Loss of TBP function causes age-associated neurodegeneration As mentioned, polyQ-expanded TBP is dysfunctional, yet it interrupts the function of normal TBP; we reasoned that polyQ-expanded TBP may act in a dominant-negative fashion. In other words, other than the gain-of-toxicity function of the polyQ-expanded TBP, the loss of normal TBP function may also contribute to SCA17 pathogenesis. Therefore, loss-of-function mutations in dTbp might show SCA17-like 33.

(35) neurodegeneration in Drosophila. Several loss-of-function dTbp mutant alleles have been recovered from a large scale genetic modifier screen [95]. Among these mutant alleles, dTbpsI10 is considered to be a protein-null allele because an A to T substitution caused a nonsense multination at codon 249, which deletes approximately half of the DNA-binding domain in dTbp protein. To test whether truncated dTbp loses its DNA affinity, the truncated dTbp (tr-dTbp) protein was purified and subjected for EMSA assays. The tr-dTbp protein encoded by the dTbpsI10 allele failed to bind TATA box DNA (Fig. 4A). The results of a quantitative real-time PCR further confirmed that the expression of functional dTbp in the heterozygous dTbpsI10 mutant was 54.6% of wildtype Drosophila (Fig. 4B). Moreover, to exclude the possibility that tr-dTbp has a gain of toxic function, we generated transgenic flies overexpressing tr-dTbp. Retinal overexpression of the tr-dTbp did not cause ether change in retinal structure (data not shown) or neurodegeration in flies, which means tr-dTbp is unlikely to possess the gain-of-toxicity function (Fig. 4C). Meanwhile, the heterozygous dTbpsI10 mutants exhibited considerably severe mobility defects (Fig. 5A). Even the newly hatched heterozygous dTbpsI10 mutant flies performed significantly poorer than wild-type flies did at the same age in climbing assays. The climbing ability defect was sustained throughout the entire lifespan of the dTbpsI10 mutants (Fig. 5A). Furthermore, the heterozygous dTbpsI10 mutants exhibited a shorter lifespan than wild-type flies (Fig. 5B). The mean lifespans of the wild-type and heterozygous dTbpsI10 flies were 50 and 39 days, 34.

(36) respectively. Although the heterozygous dTbpsI10 mutant did not reveal external morphological defects, both reduced mobility and longevity phenotypes were observed (Fig. 5A and B). Because these age-dependent phenotypes are highly associated with neurodegeneration, we suspected that the central nervous system in mutant flies may be impaired when dTbp is downregulated. To examine this, hematoxylin and eosin (H&E) staining of adult brain paraffin sections was conducted. In the brains of the newly eclosed young wild-type flies, the outer cortex, which consists of basophilic cell bodies of neurons and glia, and the inner neutrophil region, which primarily consists of eosinophilic processes, appeared normal (Fig. 5C). Similarly, no anatomic defects were observed in the nervous systems of the young heterozygous dTbpsI10 mutant flies. The morphology of the brain, such as the major projection system and brain volume, appeared intact (Fig. 5C). Few and scattered vacuoles were occasionally detected in the brains of the older wild-type flies (Fig. 5C and D). By comparison with wild-type flies of the same aged flies, more and larger vacuoles were observed in the brains of older heterozygous dTbpsI10 mutant flies (Fig. 5C and D). Because the formation of brain vacuoles is a typical feature of neurodegeneration in Drosophila, this findings suggest that downregulation of dTbp function induces age dependent neurodegeneration in flies. Because the climbing ability defect and short lifespan phenotypes as well as the degeneration phenotype found in heterozygous dTbpsI10 mutants were similar to those found in TBP-109Q-expressing flies, we concluded that a loss of TBP (dTbp) function may contribute to SCA17 35.

(37) pathogenesis in Drosophila.. TBP activity modulates the toxicity of polyQ-expanded proteins To test that the loss of TBP function is a contributing factor of SCA17, the retinal degeneration phenotypes of 7-week-old Gmr>TBP-36Q and Gmr>TBP-109Q flies in dTbpsI10 backgrounds were compared. Although the heterozygous dTbpsI10 flies exhibited short lifespan, mobility defect and brain vacuolization (Fig. 5), their external eye morphology appeared normal as seen in the Gmr-Gal4 control flies (Fig. 6, middle panels). It is possible that the neuronal tissues were more vulnerable to reduced dTbp. Similarly, reducing dTbp did not affect the eye phenotype in the Gmr>TBP-36Q flies (Fig. 6, upper and middle panels). Overexpressing TBP-109Q revealed mild depigmentation in the eyes of flies aged 7 weeks. Nevertheless, compared with Gmr>TBP-109Q flies, the external ommatidial structures were uneven and larger patches of depigmentation appeared in the eyes of the Gmr>TBP-109Q flies when dTbp was downregulated (Fig. 6, middle panel). This finding demonstrated that loss of TBP function is likely to be involved in the pathogenesis of SCA17. In previous study, TBP was recruited by polyQ-expanded Atx3 into NIs in the brains of SCA3 patients [25]. In addition, polyQ-expanded Htt inhibited the binding ability of TBP and sequestered TBP into insoluble aggregates, suggesting that deactivated TBP may also be implicated in the pathogenesis of SCA3 and HD [32, 107]. To address this we conducted a genetic analysis. We found that the eyes of the 7-week-old Gmr>Atx3-78Q flies were complete depigmented with small, scattered 36.

(38) necrotic spots (Fig. 6, upper panel). However, the eyes of the same aged Gmr>Atx3-78Q flies appeared glassy with large necrotic spots in the heterozygous dTbpsI10 mutants (Fig. 6, middle panel). Moreover, the eyes of the Gmr>Htt-97Q flies at 7 weeks exhibited mild depigmentation phenotype (Fig. 6, upper panel). However, the depigmentation phenotype was consistently more severe when dTbp was downregulated in the same-aged flies expressing Htt-97Q (Fig. 6, middle panel). Because the degenerative phenotypes of TBP-109Q, Atx3-78Q and Htt-97Q were all enhanced in dTbpsI10 mutant backgrounds, we assumed that the dysfunction of TBP might contribute to the pathogenesis of SCA17, SCA3 and HD. As mentioned, polyQ-expanded proteins induced retinal degeneration deteriorated in dTbpsI10 mutant backgrounds, and thus, we suspected increasing TBP activity might be beneficial. We found that neither Gmr>dTbp nor Gmr>TBP-36Q, dTbp flies exhibited degenerative phenotype in eyes (Fig. 6, upper and bottom panels) despite Elav>TBP-36Q flies exhibit shorter lifespan. Similarly, external ommatidial structures of Gmr>TBP-36Q were normal till old age (Fig. 6, upper panel). This finding suggested that eyes might be less sensitive than neuronal tissues to the elevated levels of normal TBP in flies. Unexpectedly, the overexpression of dTbp did not improve the depigmentation phenotype in TBP-109Q expressing flies at 7 weeks (Fig. 6, bottom panel). On the contrary, increasing dTbp expression enhanced the rough-eye phenotype in Gmr>Atx3-78Q flies in which ommatidial collapse and large necrotic areas were consistently observed (Fig. 6, 37.

(39) bottom panel). Similarly, the coexpression of dTbp and Htt-97Q induced markedly severe rough-eye phenotype in flies (Fig. 6, bottom panel). These results indicated that both the down- and up-regulation of TBP activity increased the toxicity of the polyQ-expanded proteins. To quantify the retinal phenotype, the internal ommatidial structure was revealed using confocal microscopy. Similarly, the down- or up-regulation of dTbp did not cause any obvious impairment in the internal ommatidial structure of control Gmr-gal4 line and TBP-36Q expressing flies (Fig. 7A, first and second columns from the left, and Fig. 7B). The number of rhabdomeres in the retina of the 1-week-old Gmr>TBP-109Q flies were slightly and nonsignificantly reduced (Fig. 7A and B). Unlike the round rhabdomeres of the control flies that were well arranged in an asymmetric trapezoid, the rhabdomeres in the retinas of the Gmr>TBP-109Q flies appeared oblong and organized in a slightly circular manner (Fig. 7A, arrow, first, second and third columns). In addition, immunocytochemical analysis revealed that few scattered protein inclusions which appeared as bright puncta existed in the retinas of the Gmr>TBP-109Q flies (Fig. 7A, arrow head). Although the up- or downregulation of dTbp did not substantially alter the external morphological phenotype of Gmr>TBP-109Q flies (Fig. 6), decreasing or increasing dTbp expression significantly reduced photoreceptor cells ( R cells ) and caused a rhabdomere fusion in the Gmr>TBP-109 flies (Fig. 7A, open circle, and Fig. 7B and C). Moreover, additional and large inclusions were observed when dTbp was overexpressed (Fig. 7A and C, see arrow head), indicating that the expression of TBP modulated the 38.

(40) disease progression of SCA17. In the flies expressed Atx3-78Q, the reiterative hexagonal arrangement of photoreceptor clustering totally disappeared, and numerous protein aggregates appearing as fluorescent foci were observed (Fig. 7). Moreover, increasing or decreasing the dTbp expression further reduced the number of photoreceptor cells in the Gmr>Atx3-78Q flies (Fig. 7A and B). The average number of photoreceptor cells in each ommatidium of the Gmr>Atx3-78Q and Gmr>Atx3-78Q, dTbpsI10 and Gmr>Atx3-78Q, dTbp flies were 1.13, 0.37 and 0.30, respectively (Fig. 7B). Notably, the formation of protein inclusions seemed to be correlated with the TBP expression level. Overxpression of dTbp increased Atx3-78Q aggregate formation, and decreasing the expression of dTbp significantly reduced the number of inclusions (Fig. 7A and C). The average number of inclusions in the retinas of the Gmr>Atx3-78Q and Gmr>Atx3-78Q, dTbpsI10 and Gmr>Atx3-78Q, dTbp flies were 2.54, 1.16 and 7.02 per 400 mm2, respectively (Fig. 7C). Similarly, the internal ommatidial structure of the Htt-97Q-expressing flies was completely disorganized and the R1-7 cells were significantly reduced (Fig. 7Aand B). Increasing or decreasing the expression of dTbp further reduced the number of R1-7 cells in the Htt-97Q-expressing flies (Fig. 7A and B). The average number of R1-7 cells in each ommatidium of the Gmr>Htt-97Q and Gmr>Htt-97Q, dTbpsI10 and Gmr>Htt-97Q, dTbp flies were 1.27, 0.70 and 0.97, respectively (Fig. 7B). Unlike the circumstances in the TBP-109Q and Atx3-78Q flies, the number of Htt-97Q aggregates was nonsignificantly decreased in the dTbpsI10 mutants (Fig. 7A and C). Nevertheless, the 39.

(41) overexpression of dTbp still concomitantly increased aggregate formation in Htt-97Q flies (Fig. 7A and C). These results demonstrated that the TBP expression level was highly associated with the formation of inclusion, and that increasing TBP expression may not be beneficial for polyQ mediated neurodegeneration.. Loss of TBP function causes apoptosis in Drosophila Loss of TBP causes apoptosis in TBP knockout mouse embryos [30]. As shown above, neurodegeneration and deterioration of polyQ Drosophila models caused by downregulation of dTbp could be a consequence of apoptosis (Fig. 5 and 6). To prove this, embryos at late stages (16) were collected and stained with acridine orange (AO) to detect death cells. Because apoptosis is a normal process in development, we observed few death cells in wildtype embryos (Fig. 8A). However, the number of apoptotic cells was significantly increased in homozygous dTbpsI10 embryos (Fig. 8D). By comparison, overexpression of dTbp in embryos did not increase or decrease the number of apoptotic cells (Fig. 8C). Nevertheless, overexpression of dTbp could rescue the excess apoptosis phenotype in homozygous dTbpsI10 embryos, demonstrating that loss of Tbp causes similar apoptosis phenotype as seen in TBP knockout mice (Fig. 8E, F and K). The activity of the active-caspase3 also higher in homozygous dTbpsI10 embryos as compared with wildtype embryos (Fig. 8L). Moreover, both of dominant negative p53 (p53DN) and Drosophila Inhibitor of Apoptosis protein 1 (DIAP1), can suppress the pro-apoptotic signaling, and rescue the apoptosis phenotype of 40.

(42) homozygous dTbpsI10 embryos (Fig. 8G–K). Based on these results, the neurodegeneration of polyQ disorders may be caused by reduced TBP induced apoptosis.. Transcriptome analysis indicates prx2540-2 is a downstream gene of Tbp Despite Tbp is a general transcription factor, we have demonstrated that loss of dTbp did not affect the total RNA and protein levels significantly. This suggested that the apoptosis caused by loss of Tbp may be due to the aberrant expression of only a small number of genes. To ascertain which genes were affected by loss of dTbp, microarray analysis was applied. The gene expression profile in the heads of 1-day-old heterozygous dTbpsI10 mutant was compared with those of wildtype flies. Surprisingly, there are only 59 genes had more than 2-fold changes in gene expression. Among these genes, only 19 genes had more than 3-fold changes when compared with the wildtype controls (Table 1). Of 59 genes, most of them are unknown and are not being characterized. We found one gene, prx2540-2, which had more than 6 times lower expression level in heterozygous dTbpsI10 fly heads, may be highly associated with the apoptosis and neurodegeneration phenotypes observed in dTbp mutants. To confirm the accuracy of microarray analysis, real-time PCR was performed (Fig. 9A). The fold change ratios of these genes are compatible when analyzed with microarray and real-time PCR (Fig. 9A). Because RNA polymerase I- and RNA polymerase III-dependent transcription are also affected by TBP, we 41.

(43) examined some genes transcribed by RNA polymerase I and III. We observed that RNA polymerase I- and RNA polymerase III-dependent transcriptions were also affected in dTbp mutants (Fig. 9B). The effect of these genes on Drosophila has yet to be further characterized. The 1-Cys preoxiredoxin prx2540-2, a member of Prx family in Drosophila, is a cytosolic peroxiredoxin [66]. There are three paralogue of prx2540 in the genome of Drosophila: CG12896, prx2540-1, and prx2540-2. In each paralog only 2 amino acids are different (Fig. S2A and B). The biological function of prx2540 is not clear; Apart from Prx2540, Prx6005 is also a member of 1-Cys Prx. The rest of Prxs contain active 2-Cys. In mammals, Prx6 is 1-Cys containing Prx. Sequencing comparison revealed that both Prx2540-2 and Prx6 are conservative (Fig. S2C). Despite the function of prx6 is unclear, previous studies implicated that Prx6 is involved in neurodegeneration [71, 108]. To confirm the loss of Tbp function causes down-regulation of prx2540-2 and to further manipulate the expression of prx2540-2, the real-time PCR was performed to measure the expression level of prx2540-2 in dTbp-RNAi and prx2540-2-RNAi flies (Fig. 10). Knocking down the expression of dTbp decreases the expression levels of both dTbp and prx2540-2 (Fig. 10A and B). To uncover whether simply knockdown prx2540-2 may phenocopy the neurodegeneration of dTbp mutant fly, we chose 2 RNAi lines from VDRC. Real-time PCR confirmed the efficiency of the RNAi knockdown (Fig. 10C). To clarify whether the paralogues of prx2540-2 were affected, allelic specific primers which distinguish three prx2540 paralogues were 42.

(44) used in reverse transcription PCR analysis. The microarray data and reverse transcription PCR showed no difference in expression of paralogues of prx2540, between wildtype and dTbp mutants (Fig. 11A and B). However, there are only 14 differences among the 663 nucleotides of paralogues of prx2540; there might be off-target effect in RNAi lines. The reverse transcription PCR showed v109152 knockdown both CG12896 and prx2540-2, and v18708 knockdown all three paralogues (Fig. 11C and D). This findings indicated that RNAi lines may exhibit severer phenotypes than prx2540-2 mutant flies.. Downregulation of either TBP or prx2540-2 increases cytosolic H2O2 level Because the expression of prx2540-2, a H2O2 reducer, is downregulated, we expected that the H2O2 levels in dTbp mutants or prx2540-2 knockdown flies will be higher as compared with the wide type controls. Cytosolic H2O2 levels of flies were revealed using a hydrogen peroxide sensor, CellRox. Indeed, elevated H2O2 was detected in dTbpsI10 mutant clones (Fig. 12A to C). Our findings suggest that loss of dTbp causes down-regulation of prx2540-2, thereby increasing the production of H2O2. In addition, knockdown the expression of prx2540-2 alone also can elevate the H2O2 level in eyediscs of 3rd instar larvae (Fig. 12D and E). The H2O2 level was only affected by the areas where the expressing of prx2540-2 is silenced (Fig. 12F to H).. Loss of prx2540-2 or TBP function causes similar phenotypes, and 43.

(45) overexpression of prx2540-2 can rescue the loss-of-TBP-function induced phenotypes. Although homozygous dTbp mutant does not affect many genes in our microarray analysis, loss of dTbp still causes apoptosis (Fig. 8). To understand whether apoptosis is caused by downregulation of prx2540-2, the prx2540-2 knockdown embryos were stained with acridine orange (AO), an apoptotic cell detectors. AO staining shows that excessive death cells were detected in prx2540-2 silencing embryos (Fig. 13A to D). Furthermore, the homozygous p-element insertion mutation of prx2540-2 (stock number 37474) also caused apoptosis in embryos (Fig. 13E to G). Taken together, it is very likely that apoptosis caused by loss of dTbp is mediated by prx2540-2 involved pathway. As demonstrated above, dysfunction of dTbp contributes to the polyQ induced neurodegenerations and downregulates the expression of prx2540-2. It is expected that reduced prx2540 can cause similar neurodegeneration phenotype as seen in polyQ disease fly models. We show that heterozygous mutant of prx2540-2 and knockdown of prx2540-2 driven by Elav-gal4 in neurons reduced the lifespan (Fig. 14A). However, overexpression of prx2540-2 in neuron did not extend lifespan; on the contrary, either over- or down-regulation of prx2540-2 reduced the lifespan of flies (Fig. 14A). Nevertheless, overexpression of prx2540-2 in heterozygous dTbpsI10 fly restored the lifespan in dTbp mutation (Fig. 14A). Similar results were observed in mobility assays. We found that downregulation of prx2540-2, decreased the climbing ability, and overexpression of prx2540-2 restored the climbing ability under dTbp 44.

(46) mutation background (Fig. 14B). However, overexpression of prx2540-2 in neuron did not affect the mobility (Fig. 14B). Therefore, the degenerative syndromes, including reduced lifespan and mobility defect, caused by dysfunction of dTbp could be attributed to the downregulation of prx2540-2.. Prx2540-2 activity modulates the symptoms of polyQ disease fly models To test whether the expression of prx2540-2 is reduced in polyQ disease models, real-time PCR was performed to determine the expression level. As shown in Fig.15A that the expression levels of prx2540-2 is reduced in SCA17, HD and SCA3 Drosophila models. This finding agrees with our hypothesis that polyQ protein causes loss of dTbp function, thus the prx2540-2 is reduced. To further confirm that the prx2540-2 is reduced in polyQ diseases fly models, we used luminol to detect H2O2 concentration in heads of flies. Consistent with our previous works that heterozygous dTbp mutant flies’ heads exhibited higher level of H2O2, we found that H2O2 concentrations were higher in heads of SCA17, HD and SCA3 Drosophila models when compared with the heads of Elav-Gal4 control flies (Fig. 15B). On the other hand, the level of another oxidative stress molecule, superoxide anion radical, did not altered between polyQ disease models and control flies (Fig. 15C). Therefore, the high level of H2O2 in polyQ disease models could be resulted from the reduction of prx2540-2. In climbing assay, overexpression of prx2540-2 did not improve the 45.

(47) mobility of disease fly models (Fig. 15D). However, knockdown of prx2540-2 worsen the mobility defect of disease model flies (Fig.15D). Reversibly, overexpression of prx2540-2 rescued the lifespan of SCA17, HD and SCA3 model flies (Fig. 15E). On the contrary, knockdown of prx2540-2 reduced the short lifespan of HD model flies (Fig. 15E). These results demonstrated that prx2540-2 is involved in the pathology of these polyQ disorders.. Loss of TBP or prx2540-2 function activates JNK pathway Reduced prx2540-2 and elevated oxidative stress may cause stress response JNK pathway [88-92]. Western blots demonstrated that the amount of total JNK did not change but the level of phosphorylated JNK was raised (Fig. 16A and B).. In the brains of 40 days old flies, the p-JNK was higher in the heads of heterozygous dTbpsI10 mutant flies as revealed in immunohistochemical staining using DAB as substrate (Fig. 16C and D). Besides, some condensed p-JNK signals were observed around the vacuoles of the brains of heterozygous dTbpsI10 mutant flies, which was not observed in wildtype brain (Fig. 16C and D). Unlike the natural neurodegeneration, loss of dTbp activated JNK. Because JNK mediates stress responses and loss of dTbp elevated oxidative stress, our data may indicate that oxidative stress activates JNK pathway and causes apoptosis in dTbp dysfunctional condition. To confirm that the downregulation of prx2540-2 caused by dysfunction of dTbp elevates oxidative stress thereby activating JNK pathway, the levels of p-JNK in prx2540-2 knockdown flies were determined. Knockdown of prx2540-2 increased 46.

(48) the amount of p-JNK (Fig. 16E and F). This result demonstrated that the prx2540-2 mediates the activation of JNK pathway caused by deactivation of dTbp. However, silencing the expression of prx2540-2, v109152, increased both p-JNK and total JNK protein levels (Fig. 16E and F). This difference between loss of dTbp and prx2540-2 with respect to the expression of JNK, can be attributed to the off-target effect of RNAi. To confirm the downregulation of dTbp or prx2540-2 resulted in activation of JNK pathway, bskDN, a dominant negative form of Drosophila JNK was ectopically expressed to block the activated JNK-induced apoptosis. In Drosophila embryo, overexpression of prx2540-2 did not affect the apoptotic cell number (Fig. 17A, B and I). Conversely, dTbp mutant or knockdown of prx2540-2 enhanced the apoptosis phenotype (Fig. 17D, G and I). In addition, overexpression of prx2540-2 can rescue the cell death caused by dTbp mutation as expected (Fig. 17E and I). Furthermore, overexpression of bskDN blocked apoptosis and rescued the cell death phenotype in homozygous dTbpsI10 mutants and prx2540-2 silencing flies (Fig. 17C, F, H and I). Since blocking JNK signaling can suppress the apoptosis caused by dysfunction of TBP or downregulation of prx2540-2, we concluded that dysfunction of dTbp or downregulation of prx2540-2 leads to oxidative stress which activates JNK signaling and apoptosis. In sum, our studies demonstrated that polyQ proteins interferes TBP activity. The deactivation of TBP downregulates the expression of prx2540-2. Prx2540-2 downregulation further increases oxidative stress 47.

(49) and subsequently induces apoptosis through JNK activation. The above events may provide a pathomechanism of polyQ mediated neurodegenerations.. 48.

(50) Discussion Previous studies have demonstrated that polyQ-expanded TBP forms neurotoxic aggregate that leads to SCA17 [40-46]. In this study, we first focused on the effect of polyQ expansion has on the function of TBP and its connection with SCA17, SCA3 (MJD) and HD. Next, using microarray analysis, we found that expression of prx2540-2 is decreased profoundly when TBP is dysfunctional. Finally, we tried to associate the neurodegeneration of polyQ diseases with oxidative stress induced apoptosis. For better understanding SCA17, we created new SCA17 Drosophila models. Compared with previous SCA17 fly models in which the overexpression of both normal TBP and polyQ-expanded TBP induced degeneration in retina of newly eclosed flies [41], we believed that our SCA17 Drosophila model is more representative because it showed degenerative phenotypes in old age. We proved that polyQ expansion within the NTD of TBP reduces the DNA binding ability of its CTD. In this study, TBP-109Q binds significantly less TATA box DNA in EMSA and liquid chemiluminescent DNA pull-down assay (Fig. 2A and B). These results were also in agreement with a previous study in which TBP-71Q exhibit considerably less DNA affinity [40]. Furthermore, we determined that polyQ expanded TBP loses DNA affinity before aggregate formation, suggesting that soluble polyQ expanded TBP monomer is dysfunctional. The results also support the idea that the soluble polyQ expanded proteins are neurotoxic. Moreover, we showed that TBP-109Q revealed less transcription activity 49.

(51) than TBP-36Q. Accordingly, we concluded that the function of TBP correlates its polyQ tract and that polyQ-expanded TBP is dysfunctional. Different from other glutamine rich proteins which possesses shorter glutamine residues, TBP has a longer polyQ tract [55]. Previous studies have shown that TBP usually bears 25–42 glutamines in healthy individuals, and more than 70% of people carry TBP with 35–38 glutamines [39, 109, 110]. Besides, the pathogenic TBP can form heterodimers with the normal TBP [45].The relatively long polyQ tract in TBP would make normal TBP a vulnerable target to be attacked by pathogenic TBP or other polyQ proteins, because the oligomerization of polyQ tracts is length-dependent. In this study, polyQ-expanded TBP not only exhibited more aggregate-prone trait than normal TBP, but also significantly reduced the DNA-binding and transactivation abilities of the normal TBP (Fig. 3A to C). These findings demonstrated that the polyQ-expanded TBP interfered with the function of the normal TBP. In addition to the polyQ-expanded TBP being dysfunctional, we considered that the polyQ-expanded TBP should act in a dominant-negative manner, and that the loss of TBP function probably participates in pathology of SCA17. Some common symptoms can be observed between polyQ diseases. For example, SCA17 is also called Huntington’s disease-like 4 (HDL4), because pathogenic TBP can phenocopy HD [111-113]. This phenomenon can be ascribed to the fact that the same regions of neurons are impaired in these diseases. A naïve idea that common pathomechanisms among these diseases is operated could also explain the observation. In this study, 50.

(52) we showed pathogenic TBP can interact with and deactivate normal TBP (Fig. 4). Therefore, other polyQ-expanded proteins would also cause corresponding neurodegeneration through deactivation of TBP. Because the downregulation of TBP deteriorates retinal degeneration caused by the overexpression of polyQ-expanded proteins (including TBP-109Q, Atx3-78Q, Htt-97Q), the deactivation of TBP is suggested to be a common causative factor among SCA17, SCA3 and HD (Fig. 6A). Moreover, previous studies have indicated that polyQ-expanded Htt interacts with and reduces the DNA affinity of TBP and TBP is sequestered into protein-inclusions of polyQ-expanded Atx1, Atx2, Atx3, Atrophin-1 and Htt in vivo [25, 32, 107]. Accordingly, inactivation of TBP should be a common pathogenic factor in poly-Q mediated neurodegeneration. The link between dysfunction of TBP and neurodegeneration is still poorly unclear. One previous study reported that the inactivation of the murine TBP leads to growth arrest and apoptosis at the embryonic blastocyst stage [30]. In this study, we demonstrated that the heterozygous dTbp mutant flies exhibits shorten lifespan and neurodegenerative phenotype (Fig. 5). Furthermore, we showed that the inactivation of dTbp increased the number of apoptotic cells in Drosophila embryos at late stages (Fig. 8). Therefore, inactivated TBP-induced apoptosis is probably conserved in flies and mammals. Since dysfunction of TBP involves in the pathogenesis of polyQ diseases, increasing TBP activity should be beneficial. However, the results were out of our expectation because overexpression of TBP caused more severe retinal degeneration (Fig. 6 and 7). This could be due to the fact 51.