以chaperone、proteasome為目標的多麩醯胺小腦萎縮症治療策略

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(1)國立台灣師範大學生命科學系碩士論文. 以 chaperone、proteasome 為目標的多麩醯胺小腦萎縮症治療策略 Therapeutic Strategies Targeting Chaperone and Proteasome for PolyQ-mediated SCA. 研究生：龔品睿 Pin-Jui Kung 指導教授：李桂楨博士 Guey-Jen Lee-Chen 中華民國一 0 二年二月.

(2) Index Index…………………………………………………………………. I. Abstract (Chinese)………………………………………………….... III. Abstract……………………………………………………………… IV List of figures……………………………………………………….... V. Introduction…………………………………………………………... 1. Poly glutamine expansion disease and spinocerebellar ataxia type 17………………………………………………………….... 1. PolyQ neurotoxicity-protein aggregation, misfolding and clearance......................................................................................... 2. Heat-shock proteins and related therapeutic strategies…………... 2. Ubiquitin proteasome system and related therapeutic strategies.... 4. Aims………………………………………………………………….. 6. Materials and Methods……………………………………………..... 7. I. To set up cell system for identifying lead compounds enhancing chaperone/proteasome function………………………………...... 7. Cell culture……………………………………………………..... 7. MTT assay…………………………………………………….…. 7. Generation of Flp-InTM-293 triple fluorescent reporter cells…….. 7. Characterization of triple fluorescent reporter cells…………..…. 8. Screening of compounds enhancing chaperone function………... 8. Western blotting………………………………………………...... 9. Characterization of GFPu cells…………………………….….….. 9. Screening of compounds enhancing proteasome function…..…... 10. I.

(3) II. To generateneuroblastoma SH-SY5Y lines expressing normal and expanded TBP…………………………………….….......…. 10. Cell culture…………………………………………………..…... 10. Flp-In SH-SY5Y host line…………………………………...…... 10. Stably induced SH-SY5Y isogenic TBP lines.......................…..... 11. Real time quantitative PCR (RT-PCR)…………………..………. 12. Western blotting…………………………………………..……... 13 Aggregation and neuronal phenotype examination..……….....… 13 III. To derive candidate compounds using SH-SY5Y cell model …... 14 Aggregation analysis…………………………………….…..…... 14. Western blotting………………………………………………..... 14 Results……………………………………………………………….. 15 I.. Tested compounds/herbs and cytotoxicity……..………...……... 15. II. The cell system for identifying lead compounds enhancing chaperone/proteasome function……………………....……..….. 15 III. Generation and characterization of neuroblastoma SH-SY5Y lines expressing normal and expanded TBP.................................. 17 IV. Therapeutic effects of the identified compounds/herbs in SH-SY5Y cell model…………………………………………..... 18. Discussion…………………………………………………………… 20 References…………………………………………………...………. 24. II.

(4) 摘要在多麩醯胺居間的疾病中，轉譯 CAG 三核苷酸重複的擴增造成各自的蛋白中包含一長的多麩醯胺鏈，在神經細胞之細胞核和細胞質中形成聚集。熱休克蛋白可防止蛋白錯誤摺疊及聚集。泛素蛋白酶體系統的活化亦會幫助不正常摺疊蛋白的降解。本研究目標在建立細胞系統，用來鑑定可增強熱休克蛋白/蛋白酶體功能的化合物/中草藥，來治療多麩醯胺疾病。利用螢光報告基因的細胞檢測，本研究發現 NH014-1 及 NC001-8 化合物可增強 HSF1、HSPA8 和 HSPA1A 表現， NH005 和 NH006 中草藥可增強蛋白酶體功能。另外，本研究建立誘導表現 SCA17 TBP/Q36~79 的 Flp-In SH-SY5Y 細胞株，螢光顯微鏡檢視及 Metamorph 軟體分析顯示，retinoic acid 誘導分化後，表現的 TBP/Q79 蛋白形成聚集，並伴隨神經纖維生長減緩(包含突出、分支數)。化合物/中草藥處理表現 TBP/Q79 及誘導神經分化的 SH-SY5Y 細胞後，聚集抑制的情形顯示 NC001-8、NH005、NH006 為有潛能治療策略。關鍵字：小腦萎縮症、神經退化、熱休克蛋白、蛋白酶體。. III.

(5) Abstract In polyQ-mediated disorders, the expansions of translated CAG repeats in the disease genes result in long polyQ tracts in the respective proteins, leading to intranuclear and cytoplasmic accumulation of aggregated polyQ proteins inside neurons. The molecular chaperones act in preventing protein misfolding and aggregation. Induction of ubiquitin proteasome also enhances the clearance of aggregate-prone proteins. This study set up cell systems to identify compounds/herbs enhancing chaperone/proteasome function for effective treatment of polyQ diseases. Using fluorescent reporter cell-based assay, pure compounds NH014-1 and NC001-8 were shown to enhance HSF1, HSPA8 and HSPA1A expression and Chinese herbs NH005 and NH006 were demonstrated to enhance proteasome function. In addition, Flp-In SH-SY5Y cells with SCA17 TBP/Q36~79-GFP expression in an inducible fashion were established. In retinoic acid-induced differentiated SH-SY5Y cells, fluorescent microscopy examination revealed that the expressed TBP/Q79-GFP formed aggregates, accompanying with reducing neurite outgrowth (including processes and branches) assessed by Metamorph software. With assessing aggregate suppression, the potential therapeutic strategies. can. also. be. demonstrated. upon. treatment. of. TBP/Q79-expressing differentiated SH-SY5Y cells with NC001-8, NH005 and NH006. Key words: spinocerebellar ataxias (SCA), neurodegeneration, heat shock protein, proteasome. IV.

(6) List of figures Fig. 1. Compounds and cytotoxicity……..………………...……...…. 32. Fig. 2. Triple fluorescent reporter cells……………...………..……... 34. Fig. 3. Screening lead compounds enhancing chaperone function….. 36. Fig. 4. Characterization of GFPu cells…………………………..….... 38. Fig. 5. Screening lead compounds enhancing proteasome function.... 39. Fig. 6. Flp-In SH-SY5Y TBP lines…...………..…………………….. 40. Fig. 7. SH-SY5Y cells with induced SCA17 TBP/Q79-GFP expression and neuronal phenotype…………...……………... 42. Fig. 8. Flp-In SH-SY5Y TBP cells for screening candidate compounds enhancing chaperone function…………............... 44. Fig. 9. Flp-In SH-SY5Y TBP cells for screening candidate compounds enhancing proteasome function………….…….... V. 45.

(7) Introduction. Polyglutamine expansion disease and spinocerebellar ataxia type 17. Simple sequence repeats occur throughout the human genome. An increasing number of inherited neurodegenerative diseases including huntington’s disease (HD), spinobulbar muscular atrophy (SBMA), hereditary spinocerebellar ataxias (SCAs) type 1, 2, 3, 6, 7, 17, and dentatorubral-pallidoluysian atrophy (DRPLA) are caused by the expansion of unstable trinucleotide (CAG) repeats encoding expanded polyglutamine (polyQ) tracts (Takahashi et al., 2010). Among SCAs, SCA17 is caused by an expanded polyQ in a general transcription initiation factor, the TATA-box binding protein (TBP) (Koide et al., 1999; Nakamura et al., 2001). TBP is a part of transcription factor IID (TFIID) complex which binds to the core promoter to position the polymerase properly (Roeder, 2005). A distinctive feature of TBP is a long string of glutamines in the N-terminus which modulates the DNA binding activity of the C terminus to affect the rate of transcription complex formation and initiation of transcription. In human, the polyQ tract normally contains 25-42 glutamine residues (Gostout et al., 1993). Expanded alleles ranging from 43 to 66 repeats were linked to variable phenotypic spectrum including ataxia and cognitive decline, seizure, dementia, psychiatric symptoms and/or extrapyramidal features (Koide et al., 1999; Silveira et al., 2002; Maltecca et al., 2003; Rolfs et al., 2003; Lasek et al., 2006). In our screening of SCA17 expansion mutations in various 1.

(8) neurodegenerative disorders, TBP repeat expansions of greater than 43 repeats were found in our patients with Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple system atrophy-cerebellar type (MSA-C) and psychiatric disorders (Wu et al., 2004, 2005; Chen et al., 2005; Lin et al., 2007).. PolyQ neurotoxicity - protein aggregation, misfolding and clearance. The polyQ tract may form stable β-sheets that held together by hydrogen bonds (Perutz, 1994). Abnormal expansions of the polyQ stretch in disease-causing proteins trigger misfolding of these proteins, resulting in accumulation of aggregated polyQ protein in nucleus and cytoplasm of neuronal cell. It is assumed that the common toxic gain-of-function mechanisms for the polyQ-containing protein are aggregation and deposition of misfolded proteins which sequester various molecular chaperones, ubiquitin, components of proteasome and transcription factors (Cummings et al., 1998; McCampbell et al., 2000; Schmidt et al., 2002). The clearance of misfolded expanded polyQ proteins by the proteasome is impaired and the aggregated proteins may further inhibit proteasome function through caspase-dependent cleavage of proteasome subunits (Sun et al., 2004).. Heat shock proteins and related therapeutic strategies. Accompanying increasing insights into the roles of polyQ protein aggregation, misfolding and clearance in the pathogenesis of the disease, 2.

(9) progress has been made to develop therapeutics. Heat shock proteins (HSPs) are known to function as molecular chaperones to help the new synthesized or the misfolded toxic proteins refold to their native and nontoxic formation (Arawaka et al., 2010). Experimental over-expression of molecular chaperones modulates the formation of protein aggregates in cultured cells and transgenic animals, diminishing the toxicity of glutamine expansions (Cummings et al., 1998; Kobayashi et al., 2000; Cummings et al., 2001; Kobayashi and Sobue, 2001; Bonini, 2002). Chaperones may modulate protein toxicity by stabilizing the misfolded conformation to promote inclusion body formation or by interacting with the disease protein to prevent abnormal interactions with other proteins that are causal in toxicity (Sakahira et al., 2002). Thus increasing expression level or enhancing the function of HSP chaperones will provide an avenue for the treatment of polyQ disease.. Previously decreased heat shock cognate protein (HSPA8, a constitutive HSP70) expression has been shown to underlie pathogenesis of SCA17 (Lee et al., 2009; Chen et al., 2010). HSP70 has been reported to suppress polyQ-mediated neurodegeneration in Drosophila (Warrick et al., 1999). Over-expression. of. heat-inducible. HSP70. chaperone. (HSPA1A). suppresses neuropathology and improves motor function in SCA1 mice (Cummings et al., 2001). Expression of molecular chaperones is regulated by heat shock transcription factor 1 (HSF1) and HSF1-activating compounds have been indicated as therapeutic candidates for polyQ diseases (Fujikake et al., 2008). Paeoniflorin, an herbal medicine derived from Paeonia lactiflora, activates HSF1 by enhancing phosphorylation 3.

(10) and acquisition of the DNA-binding ability of HSF1, as well as formation of characteristic HSF1 granules in the nucleus to induce HSPs expression (Yan et al., 2004). Geranylgeranylacetone (GGA), a nontoxic antiulcer drug, has also been shown to potently induce HSP expression in various tissues, including the central nervous system. In a cell model of SBMA, GGA increased the levels of Hsp70, Hsp90, and Hsp105 and inhibited cell death and the accumulation of pathogenic androgen receptor (Katsuno et al., 2005).. Ubiqutin proteasome system and related therapeutic strategies. The homeostasis of protein synthesis and degradation is crucial for cell survival. Ubiquitin proteasome system (UPS) is one of the principal routes to degrade unwanted proteins (Sorokin et al., 2010). With the assistance of molecular chaperones to target proteins to the proteasomal degradation, the UPS copes with the pathological conditions (Lehman, 2009). Protein aggregation directly impaired the function of the ubiquitin-proteasome system (Bence et al., 2001). Failure of this system might lead to an abnormal accumulation of a variety of toxic proteins, ultimately leading to neuronal dysfunction and/or cell death. The consistent findings of ubiquitin and chaperones-positive protein aggregates in neuropathological studies also evidence the relationship between SCA neurodegeneration and the UPS impairment (Ross and Poirier, 2004). Induction of ubiquitin proteasome enhances the clearance of. aggregate-prone. intracytoplasmic. proteins. that. cause. neurodegeneration and confer cytoprotective roles in cell and animal 4.

(11) models (Huang and Figueiredo-Pereira, 2010). Thus up-regulating proteasome function may be the new therapeutic approaches for the mentioned neurodegenerative diseases. Benzamil, a candidate compounds improving UPS function, decreased huntingtin-polyQ aggregation in HD mouse model (Wong et al., 2008).. Previously a clonal human embryonic kidney (HEK) 293 line stably expressing a short destabilizing sequences (CL-1) (Gilon et al., 1998)-tagged green fluorescent protein (GFPu) was isolated and used to investigate the specific relation between protein aggregation and the function of the UPS (Bence et al., 2001). In GFPu cells, the CL-1 constitutive degradation signal promotes rapid degradation of GFP under conditions of a healthy UPS. As a valid in vivo measure of UPS function, the GFPu cells may be used as a reporter to screen compounds enhancing proteasome function.. 5.

(12) Aims. In polyQ-mediated disorders, the expansions of translated CAG repeats in the disease genes result in long polyQ tracts in the respective proteins, leading to intranuclear and cytoplasmic accumulation of aggregated polyQ proteins inside neurons. Heat-shock proteins can recognize expanded polyQ proteins and suppress their aggregation. Induction of ubiquitin proteasome also enhances the clearance of aggregate-prone proteins. Thus in this study, we aim to set up a high-content analysis platform to screen synthetic and natural compounds as well as herbal medicine that may enhance chaperone or proteasome function for effective treatment of aggregates/inclusions associated neurodegenerative diseases. In addition, a neuron-based SH-SY5Y cell model expressing GFP-tagged N-terminal TBP/Q36~79 was established and used to assess the potential therapeutic strategies for enhancing chaperone or proteasome function.. 6.

(13) Materials and Methods. I. To set up cell system for identifying lead compounds enhancing chaperone/proteasome function. Cell culture Human embryonic kidney (HEK) 293FT, HEK-293-derived Flp-InTM-293 (Invitrogen) and GFPu (ATCC) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1.0 mM sodium pyruvate, 1.5 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 U/ml streptomycin, at 37˚C in an atmosphere containing 5% CO2.. MTT assay HEK293 cells (5 × 104/well in 48-well plate) were plated into 48-well dishes, grown for 24 hours and treated with different concentration of compounds. After one day, 20 µl MTT (5 mg/ml in PBS) was added to cells and incubated at 37˚C for 2 hours. After that, 200 µl of DMSO was added cells to dissolve the insoluble purple formazan. The absorbance of the formazan product was measured at 570 nm by a Bio-Tek µQuant Universal Microplate Spectrophotometer.. Generation of Flp-InTM-293 triple fluorescent reporter cells A triple fluorescent reporter plasmid was first constructed in pAmCyan1-N1 by Dr. Chih-Hsin Lin, with mCherry, ZsYellow1 and 7.

(14) AmCyan1 fluorescent reporters driven by heat shock transcription factor 1 (HSF1, -360 ~ +2, with the translation initiation A as +1), heat shock cognate protein (HSPA8, -1140 ~ +38, driving constitutively expressed HSP70) and heat-inducible HSP70 chaperone (HSPA1A, -273 ~ +215, driving heat-inducible HSP70) promoters, respectively. The fragment containing the HSF1, HSPA8 and HSPA1A driven reporters was removed with AseI and NotI restriction enzymes and used to replace an AseI-NotI fragment in pcDNA5/FRT/TO plasmid (Invitrogen). The resulting triple fluorescent reporter plasmid was used to generate triple fluorescent reporter cells by targeted insertion into Flp-InTM-293 cells, according to the supplier's instructions (Invitrogen). The cells were selected by medium containing 5 µg/ml blasticidin S and 100 µg/ml hygromycin.. Characterization of triple fluorescent reporter cells The triple fluorescent reporter cells (5 × 104/well in 96-well plate) were incubated at 42˚C for 2 hours and recovery for 2 ~ 6 hours or treated with GGA (100 nM ~ 1 mM, Sigma) for 24 hours. The three fluorescence colors were analyzed simultaneously using high-content analysis (HCA) system. (ImageXpressMICRO,. excitation/emission. wavelengths. (ZsYellow1). 587/610. and. Molecular at. nm. 453/486. (AmCyan1).. Devices), (mCherry), Three. with 531/540. independent. experiments were performed for each treatment. Difference in fluorescence was tested using the two-tailed Student’s t test.. Screening of compounds enhancing chaperone function The triple fluorescent reporter cells (5 × 104/well) were seeded in 96-well 8.

(15) plate. At day 2, GGA, NH014-1 or synthetic NC001 derivatives (NC001-2, -3, -8 and -11) (provided by Professor Ching-Fa Yao from Department of Chemistry at NTNU) was added to the medium for 24 hours. The three fluorescence colors were analyzed as described.. Western blotting HEK-293FT cells were treated with NH014-1 or NC001-8 (100 nM) for 48 hours and total proteins were prepared using lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS and 0.5% sodium deoxycholate, 1% Triton X-100, protease inhibitor cocktail (Calbiochem).. Proteins. (20. µg). were. separated. on. 10%. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to nitrocellulose membranes by reverse electrophoresis. After blocking, the membrane was probed with antibody to HSF1 (1:1000 dilution, Abnova), HSPA8 (1:500 dilution, Santa Cruz), HSPA1A (1:500 dilution, Santa Cruz), H3F3B (H3 histone, family 3B, 1:1000 dilution, GeneTex) and GAPDH (1:1000 dilution, MDBio). Then the immune complexes were detected by horseradish peroxidase-conjugated goat anti-mouse (Jackson ImmunoReseach) or donkey anti-goat (Rochland) IgG antibody and chemiluminescent substrate (Millipore).. Characterization of GFPu cells GFPu cells were characterized by treating with MG132 (proteasome inhibitor, 100 nM ~ 1 µM, Sigma) and benzamil (UPS activator, 100 nM ~ 10 µM, Sigma). After 24 hours, cells were harvested for fluorescence activated cell sorting (FACS) analysis using FACSCalibur flow cytometer 9.

(16) (Becton-Dickinson), equipped with an argon laser operating at 530 nm. 104 cells were analyzed in each sample.. Screening of compounds enhancing proteasome function Synthetic compounds tested include NC001 derivatives NC001-1, -8, -9, -10 and -11. Chinese herbs tested include NH004, NH005, NH006, NH007, NH008, NH003, NH013 and NH016 (provided by Sun Ten Pharmaceutical Corporation). The GFPu cells (1 × 105/well) were seeded in 24-well plate. At day 2, MG132, benzamil or compound/herb was added to the medium for 24 hours. The fluorescence colors were analyzed as described.. II. To generate neuroblastoma SH-SY5Y lines expressing normal and expanded TBP. Cell culture Human neuroblastoma SH-SY5Y cells (ATCC No.CRL-2266) were maintained in DMEM F12 supplemented with 10% fetal bovine serum (FBS), 1.0 mM sodium pyruvate, 100 U/ml penicillin, and 100 U/ml streptomycin, at 37˚C in an atmosphere containing 5% CO2.. Flp-In SH-SY5Y host line A Flp-In SH-SY5Y host cell line was constructed by Dr. Li-Ching Lee by transfecting pcDNA6/TR (a plasmid expressing the Tet repressor) into SH-SY5Y cells. After selection with 5 µg/ml blasticidin S, a plasmid containing FRT (Flp Recombination Target) site and neomycin resistant 10.

(17) gene (constructed by Dr. Chih-Hsin Lin) was transfected and selected with 500 µg/ml G418. Then DNA was extracted from different clones by using the DNA Extraction Kit (Stratagene) and FRT copy number was determined by PCR using primers 5’-GTAGTGAGGAGGCTTTTTTGGAGGC-3’. and. 5’-CCTTCCCGCTTCAGTGACAACG-3’. for. amplification of FRT site. One ng of template DNA was added to a 25 µl reaction containing 2.5 µl 10 × reaction buffer, 1 mM MgCl2, 25 ng sense and antisense primers, 200 µM dNTP and 0.5 U of Tag DNA polymerase (Promega). The condition of reaction was: 1 cycle of denaturation at 94˚C for 1 minute, 35 cycles of reaction comprising denaturation at 94˚C for 30 seconds, annealing at 58˚C for 30 seconds, and elongation at 72˚C for 30 seconds, followed by elongation at 72˚C for 10 minutes. The PCR products (412 bp) were resolved on a 2% agarose gel. The clone S11 with single FRT site was selected as Flp-In SH-SY5Y host cell line and maintained in medium containing 5 µg/ml blasticidin S and 500 µg/ml G418.. Stably induced SH-SY5Y isogenic TBP lines The cloned pcDNA5/FRT/TO-nTBP/Q36~79-GFP plasmids (Huang, 2011) were used to generate the isogenic TBP lines by targeted insertion into the Flp-In SH-SY5Y host cells according to the supplier's instructions. Briefly, 2.5 µg of pcDNA5/FRT/TO-nTBP/Q36~79-GFP and pOG44 (a plasmid expressing the Flprecombinase) DNA in 1:9 ratio and 4 µl of LipofectamineTM 2000 (LF2000) (Invitrogen) were separately diluted into 100 µl Opti-MEM I reduced serum medium (Invitrogen). After 5 minutes incubation at room temperature, the DNA and LF2000 were combined 11.

(18) and incubated for 20 minutes at room temperature. Then DNA-LF2000 mixture was added to the SH-SY5Y cells (5×105 in 6-well plate) in medium without antibiotics. After 6 hours incubation at 37˚C, the antibiotics free medium with DNA-LF2000 mixture was removed and fresh medium containing antibiotics was added. After one day incubation at 37˚C, medium containing 5 µg/ml blasticidin S and 100 µg/ml hygromycin was added to select cells with targeted insertion of nTBP/Q36~79-GFP. The CAG repeats in these TBP lines were examined by PCR. Doxycycline (2 µg/ml) was added to induce GFP-tagged TBP expression.. Real-time quantitative PCR (RT-PCR) Total RNA from isogenic TBP lines was extracted using Trizol reagent (Invitrogen) according to the manufacturer's specifications. Briefly, 1 ml of Trizol reagent was added to the culture dish (6-well dish) and cell suspension was collected. After incubation on ice for 5 minutes, cell suspension was mixed with 1/5 volume of chloroform and incubated on ice for 5 minutes. RNA was separated from DNA and proteins by centrifugation at 4˚C for 15 minutes. The colorless, upper aqueous phase was carefully removed to a fresh tube avoiding the material that collected at the interface, and mixed with 0.8 volume of isopropanol. The mixture was sat at -80˚C for at least 30 minutes and centrifuged at 4˚C for 15 minutes to precipitate RNA. The supernatant was discarded and RNA pellet was rinsed with 70% ethanol in DEPC-ddH2O. RNA was air dried and dissolved in DEPC-ddH2O. The RNA was DNase (Stratagene) treated, quantified, and reverse-transcribed to cDNA using High Capacity 12.

(19) cDNAReverse Transcription Kit (Applied Biosystems). Real-time quantitative PCR experiments were performed in the ABI PRISM® 7000 Sequence Detection System (Applied Biosystems). Amplification was performed on 12.5 ng cDNA with gene-specific TaqMan fluorogenic probes Hs00920494_ml for TBP and 4326321E for HPRT1 (endogenous control) (Applied Biosystems). Fold change was calculated using the formula 2ΔCt, ΔCT = CT (control) – CT (target), in which CT indicates cycle threshold.. Western blotting Total proteins from isogenic TBP lines were prepared as described. Proteins (20 µg) were separated on 10% SDS-PAGE and blotted on to nitrocellulose membranes as described. After blocking, the membrane was probed with TBP (N-12) antibody (1:500 dilution, Santa Cruz) at 4˚C overnight. The immune complexes were detected using horseradish peroxidase-conjugated. goat. anti-rabbit. IgG. antibody. and. chemiluminescent substrate as described.. Aggregate and neuronal phenotype examination Isogenic TBP lines (2 × 105/well) were seeded in 6-well plate, with all trans retinoic acid (10 μM) added at seeding time. At day 2, doxycycline (1 g/ml) was added to induce GFP-tagged TBP expression. The cells were kept in differentiation medium for 1 to 3 weeks for fluorescent microscopy examination. Three independent experiments were performed for with or without retinoic acid induced differentiation. The morphologic differentiation was assessed by analyzing neuronal phenotype including 13.

(20) total outgrowth, processes, branches and mean outgrowth intensity with Metamorph software. Differences in aggregation percentage and neurite outgrowth were tested using the two-tailed Student’s t test.. III. To derive candidate compounds using SH-SY5Y cell model. Aggregation analysis SH-SY5Y TBP lines were seeded in 6-well (2 × 105/well) plate, with all trans retinoic acid (10 µM, Sigma) added as described. At day 2, cells were treated with compounds/herbs for 8 hours. Then doxycycline was added and the cells were kept in the medium containing 10 µM trans retinoic acid, doxycycline and compounds/herbs for 7 days. After that, cells were stained with Hochest 33342 (0.1 µg/ml) and aggregation percentage was assessed by HCA system.. Western blotting SH-SY5Y TBP lines were seeded and treated with compounds/herbs for 8 hours before induced to express TBP/Q79-GFP for six days, and total proteins prepared as described. Proteins (20 µg) were separated on 10% SDS-PAGE and blotted on to nitrocellulose membranes as described. After blocking, the membrane was probed with antibody to HSF1 (1:1000 dilution, Abnova), HSPA8 (1:500 dilution, Santa Cruz), HSPA1A (1:500 dilution, Santa Cruz), ubiquitin (1:1000 dilution, Covance), and actin (1:1000 dilution, Milipore). Then the immune complexes were detected by horseradish peroxidase-conjugated goat anti-mouse or donkey anti-goat IgG antibody and chemiluminescent substrate as described. 14.

(21) Results. I. Tested compounds/herbs and cytotoxicity. The compounds/herbs tested include geranylgeranylacetone (GGA; a potent HSP inducer), benzamil (an UPS activator), NH014-1 (an active component of Chinese herb), NC001 (indole) and derivatives (NC001-1, -2, -3, -8, -9, -10, -11), NH005 and NH006 (Chinese herbs).. MTT assays were performed with HEK-293 cells after treatment with compounds/herbs (Fig. 1A) for 24 hours. The IC50 of the tested compounds/herbs were calculated using the interpolation method. As shown in Fig. 1B, the IC50 of the tested compounds/herbs were: 21.53 mM (GGA), 0.09 mM (benzamil), 16.38 mM (NH014-1), 3.22 mM (NC001), 2.61 mM (NC001-1), 0.48 mM (NC001-2), 0.06 mM (NC001-3), 3.03 mM (NC001-8), 0.67 mM (NC001-9), 0.53 mM (NC001-10), 0.12 mM (NC001-11), >30 mg/ml (NH005) and 23.51 mg/ml (NH006). Among the tested compounds/herbs, GGA, NH014-1, NC001, NC001-1, NC001-8 and NH005 had an IC50 higher than the highest concentration (1 mM for compound or 30 mg/ml for herb) tested, suggesting their low cytotoxicity.. II. The cell system for identifying lead compounds enhancing chaperone/proteasome function. 15.

(22) A fluorescent reporter cell model with mCherry, ZsYellow1 and AmCyan1 reporters downstream of HSF1, HSPA8 and HSPA1A promoters was established (Fig. 2A). The cloned promoters effectively drove the expression of red, yellow and blue fluorescent reporters (Fig. 2B). The triple fluorescent reporter cells displayed enhanced fluorescence after 2 hours at 42˚C following 2 ~ 6 hours recovery at 37˚C (Fig. 2C) (P = 0.031 ~ 0.002). GGA has been shown to potently induce HSP expression in various tissues. Treatment of GGA (100 nM ~ 1 mM) also displayed enhanced fluorescence (P = 0.025 ~ 0.000), with 200 M being the most efficient dose (Fig. 2D).. The triple fluorescent reporter cells were used to screen the potential of compounds/herbs to enhance HSF1 and HSP70 chaperone expression. NH014-1, an active component of Chinese herbal medicine, has been shown to active HSF1 and induced HSPs expression (Yan et al., 2004). Treatment of NH014-1 (100 nM ~ 1 mM) showed a dose dependent enhancement on HSF1, HSPA8 and HSPA1A promoter activities (Fig. 3A). Using GGA as a positive control, indole derivatives NC001-2, -3, -8 and -11 were tested. Both NC001-8 and -11 treatments for one day showed enhanced HSF1, HSPA8 and HSPA1A promoter activities (Fig. 3B). The enhancement of NH014-1 (100 nM) on HSF1 (158%, P = 0.027), HSPA8 (140%, P = 0.011) and HSPA1A (137%, P = 0.007) expression and NC001-8 (100 nM) on HSF1 (138%, P = 0.046), HSPA8 (144%, P = 0.042) and HSPA1A (132%, P = 0.042) expression was further confirmed by the Western blot in HEK-293 cells after two days treatment (Fig. 3C). 16.

(23) GFPu cells were used to screen compounds/herbs enhancing proteasome function. The cells were first characterized by treating with proteasome inhibitor MG132 (100 nM ~ 1 M) and UPS activator benzamil (100 nM ~ 10 M). After 24 hours, cells were prepared for fluorescence microscopy examination and fluorescence activated cell sorting (FACS) analysis. As shown in Fig. 4, both observed (A) and measured (B) fluorescence was negatively correlated with the proteasome function as predicted.. Using benzamil (1 ~ 10 M or 1/5 ~ 1/100 IC50) as a control, the GFPu cells were then used to screen synthetic compounds (1/10 ~ 1/100 IC50, Fig. 5A) and Chinese herbs (1/5 ~ 1/100 IC50, Fig. 5B) enhancing proteasome function. Among compounds/herbs tested, a significant or notable dosage dependent enhancement of proteasome activity was observed with NH005 (P = 0.013) and NH006 (P > 0.05).. III. Generation and characterization of neuroblastoma SH-SY5Y lines expressing normal and expanded TBP. Firstly SH-SY5Y-derived FIp-In host cells were generated from independent integration of plasmids pcDNA6/TR (a plasmid expressing the Tet repressor) and pFRT/lacZeo (a plasmid containing the Flp Recombination Target (FRT) site) (Fig. 6A). Then the SH-SY5Y host cells were co-transfected with pOG44 plasmid (constitutively expressed the Flp recombinase) and pcDNA5/FRT/TO-TBP/Q36~79-GFP plasmids 17.

(24) (Fig. 6B). After selection with blasticidin and hygromycin, doxycycline (2 g/ml) was added to induce GFP-tagged TBP expression (Fig. 6C). The N-terminal TBP sequence used to generate TBP-GFP fusion plasmid and PCR determination of integrated FRT site were shown in Fig. 6D-E.. The established SH-SY5Y TBP lines were characterized. As shown in Fig. 7A, real time PCR quantification of these TBP lines shows 9 ~ 11 times TBP expression after induction with doxycycline (+ Dox) for 2 days. In immunoblot, TBP antibody detected 52 ~ 62 kDa TBP/Q36~79-GFP protein, in addition to the endogenous 43 kDa TBP protein (Fig. 7B). When TBP/Q36~79 SH-SY5Y cells were differentiated for 7 to 21 days using retinoic acid, a Q length- and expression time-dependent aggregate formation was seen in 1 ~ 2% TBP/Q61~79-GFP cells, whereas no aggregate was seen in TBP/Q36-GFP cells (Fig. 7C). When neuronal phenotype was examined after 7 ~ 21 days of differentiation, significant less process and branch in TBP/Q79-GFP cells was observed at 2 ~ 3 weeks of differentiation compared to TBP/Q36-GFP cells (P = 0.000 ~ 0.002 for process; P = 0.043 ~ 0.009 for branch) (Fig. 7D).. IV. Therapeutic effects of the identified compounds/herbs in SH-SY5Y cell model. The established SH-SY5Y TBP/Q79 cells were used to examine the therapeutic effects of the identified compounds/herbs. As shown in Fig. 8A, induced expression of TBP/Q79 for 6 days attenuated the expression of HSF1, HSPA8 and HSPA1A (72 ~ 84%, P = 0.001 ~ 0.039). This 18.

(25) reduction can be rescued by the addition of GGA, indole or NC001-8 (100 nM) (96% ~ 117%, P = 0.047 ~ 0.006). The treatment of GGA, indole and NC001-8 leaded to 17% ~ 14% of aggregation reduction (P = 0.001 ~ 0.010) in TBP/Q79 expressed differentiated neuronal cells (Fig. 8B).. The above SH-SY5Y TBP/Q79-GFP cells were used to examine if NH005 and NH006 enhance proteasome activity to reduce aggregation. By immunoblotting with ubiquitin antibody, NH005 and NH006 are indeed able to enhance proteasome function in TBP/Q79 cells (Fig. 9A). Treatment of TBP/Q79 cells with benzamil (positive control, 100 nM), NH005 (10 g/ml) and NH006 (10 g/ml) for 7 days also significantly suppressed the aggregate formation (from 100% to 93, 81%, 86%, P = 0.023 ~ 0.003) (Fig. 9B).. 19.

(26) Discussion. Abnormal expansions of the polyQ proteins form aggregate in cytoplasm and nucleus of neuronal cells (Difiglia et al., 1997). Being a hallmark of neurodegenerative diseases, the inclusion of the disease-causing proteins in neuronal cell may affect molecular chaperone pathways and ubiquitin-proteasome system. Molecular chaperone not only control the assembling of native structures, but also remodel the protein which has wrong conformations. UPS plays the one of most important role in intracellular protein degradation. Over-expression of HSPs and UPS can reduce the size and number of inclusions (Cummings et al., 1998; McCampbell et al., 2000; Bence et al., 2001; Schmidt et al., 2002; Adachi et al., 2009). Thus this study aims to set up cell systems to screen compounds enhancing chaperone/proteasome function and establish neuronal SCA 17 SH-SY5Y cell model to assess the potential therapeutic strategies.. A number of evidence has indicated indole compounds as efficacious low-molecular drugs for the treatment of cancers. For example, indole-3-carbinol has been shown to suppress the proliferation, induce apoptosis and cell cycle arrest of a wide variety of cancer cells (Aggarwal and Ichikawa, 2005; Rogan, 2006). In addition, an endogenous melatonin-related indole-3-propionic acid has shown the potential to protect primary neurons and neuroblastoma cells against oxidative damage and death caused by exposure to Aβ peptide (Chyan et al., 1999). 20.

(27) Another endogenous indole derivative, indolepropionamide, was also reported to protect rodent mitochondria and extend rotifer lifespan through acting as a stabilizer of energy metabolism thereby reducing ROS production (Poeggeler et al., 2010). Our data that indole and its synthetic derivative NC001-8 displayed neuroprotective effects by enhancing the function of chaperone to reduce aggregate formation (Fig. 8) were in agreement with these reports.. In polyQ-mediated SCA, the inclusion of the disease-causing proteins affects molecular chaperone pathway (Chai et al., 1999). Our previous studies also showed that decreased heat shock cognate HSPA8 protein (a constitutive HSP70) expression could underlie pathogenesis of SCA17 (Lee et al., 2009; Chen et al., 2010). HSP70 has been reported to suppress polyQ-mediated neurodegeneration in Drosophila (Warrick et al., 1999). Over-expression. of. heat-inducible. HSP70. chaperone. (HSPA1A). suppresses neuropathology and improves motor function in SCA1 mice (Cummings et al., 2001). Expression of molecular chaperones is regulated by HSF1 (Skaggs et al., 2007) and HSF1-activating compounds have been indicated as therapeutic candidates for polyQ disorders (Fujikake et al., 2008). Our results demonstrated NC001-8 up-regulated HSF1 and HSP70 expressions to inhibit aggregate formation in TBP/Q79 SH-SY5Y (Fig. 8) cells, providing a novel mechanism of NC001-8 to decelerate the neurodegenerative process by inducing the expression of heat shock proteins.. In addition to NC001-8, NH014-1 may also perform neuroprotection via 21.

(28) enhancement of chaperone system. NH014-1 was the first reported active component of herbal medicine to induce expression of heat shock proteins in HeLa, IMR-32, and normal rat kidney cells (Yan et al., 2004). Upon stress, HSF1 is released from the chaperone complex, self-trimerizes, and then is transported into the nucleus as a transcription factor. It activates chaperones, which play an important role in preventing unwanted protein aggregation. Overexpression of HSF1 significantly improved the lifespan of R6/2 Huntington’s disease mouse (Fujimoto et al., 2005). Up-regulation of chaperone expression by HSF1 and its activating. compounds. 17-(allylamino)-17-demethoxygeldanamycin. (17-AAG) demonstrate a strong inhibitory effect on HD aggregate formation (Fujikake et al., 2008). The therapeutic potential of NH014-1 in the treatment of SCA is strongly supported by the up-regulated HSF1 and HSP70 expressions (Fig. 3).. To reduce aggregate formation, traditional Chinese herbs NH005 and NH006 were found to enhance the function of proteasome in TBP/Q79 cells (Fig. 9). Extract of NH005 has been shown to display excellent clinical effects in many cases, such as improving dizziness, headache and many symptoms, and it had been widely used for treating stroke (Zhang et al., 2012). In addition, NH005 could be a neuroprotective agent in animal models of PD (Wu and Zhu, 1999), and it could also be a cognitive enhancer in animal model of AD (Stackman et al., 2003). The pharmacological mechanisms of NH005 include modification of Ca 2+ signaling, clearance of oxygen free radical and decrease of lipid peroxidation (Zhang et al., 2012). In another previous study, NH006 had 22.

(29) been used to treat inflammations, allergy and viral infection (Garcia Prado et al., 2007). The extract of NH006 had effect upon anti-inflammatory,. cytostatic,. antioxidant,. phagocytosis-enhancing. activities and anti-mutagenic (De Martino et al., 2006; Cheng et al., 2007; Garcia Prado et al., 2007).. In. SCA17. SH-SY5Y. cells,. a. polyQ. length-. and. expression. time-dependent aggregate formation was observed with the TBP/Q61~79 proteins (Fig. 7C). In the absence of doxycycline, a small amount of TBP/Q36~79-GFP proteins was observed (Fig. 7B). The reason causing protein leaky is unclear. But in our cell line showed the same phenotype of SCA17.. There are many ways to find potential therapeutic strategies of SCA, for examples, aggregates can be suppressed by compounds activating a specific heat shock response, active UPS function to degrade unneeded or damaged proteins by proteolysis, or histone acetylation helps transcriptional activation and euchromatin. In our research, we found some candidate compounds which clear reduce aggregation. We also found the pathway of these candidate compounds whom by activate. In the end, we hope this research can push drug discovery and correlation disease of research.. 23.

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(38) Fig. 1. Compounds and cytotoxicity. (A) Structure, formula and molecular weight of the studied compounds. (B) Cytotoxicity of the compounds/herbs against HEK-293 cells using MTT viability assay. The IC50 of each compound/herb was shown under the columns. To normalize, the relative viability in untreated cells is set as 100%. The red line represents 50% viability. A. Chemical formula: Molecular weight:. NH014-1(Paeoniflorin) C23H28O11 480.47. NC001. NC001-1. C8H7N 117.15. C12H11NO 185.22. Geranylgeranylacetone C23H38O 330.55. Benzamil C13H14CIN7O∙ HCl∙ xH2O 356.21. NC001-2. NC001-3. NC001-8. NC001-9. NC001-10. NC001-11. C13H13NO2 215.25. C14H15NO 213.27. C11H9NO 171.20. C11H11NO 173.21. C21H15NO 297.35. C22H17NO2 327.38. B 數列1. Cell viability (%). 120 120. 數列2. 100. 數列3. 80 80. 數列4 數列5. 60. 數列7. 20. 數列8. Cell viability (%). 50 µM 100 µM 200 µM 1 mM. 數列9. 0. GGA IC50: 21.53 mM. untreated 100 nM 1 µM 10 µM. 數列6. 40 40. Benzamil 1 0.09 mM. NH014-1 16.38 mM. 數列10 數列11 數列12. 120 120. 數列1. 100. 數列3. 數列2. 80 80. 數列4 數列5. 60. 數列6. 40 40. 數列7. 20. 數列8 數列9. 0. NC001 IC50: 3.22 mM. NC001-1 2.61 mM. NC001-2 0.48 mM. NC001-31 0.06 mM. NC001-8 3.03 mM. NC001-9 0.67 mM. NC001-10 NC001-11 0.53 mM 0.12 mM. 數列10 數列11 數列12. 32.

(39) Cell viability (%). 數列1. 100 100. 數列2 數列3. 80. 數列4. 60 60. 數列5 數列6. 40. 數列7. 20 20. 數列8 數列9. 0. NH005. 1. IC50: > 30 mg/ml. NH006 23.51 mg/ml. 5 mg/ml 10 mg/ml 15 mg/ml. 20 mg/ml 25 mg/ml 30 mg/ml. 33. 數列10 數列11 數列12.

(40) Fig. 2. Triple fluorescent reporter cells. (A) Triple fluorescent reporter plasmid with HSF1, HSPA8 and HSPA1A promoter fragments upstream of mCherry, ZsYellow1 and AmCyan1 fluorescent reporters, respectively. (B) Microscopic images of the triple fluorescent reporter cells. (C) Effect of heat shock (42˚C for 2 hours and 2 ~ 6 hours recovery) on HSF1, HSPA8 and HSPA1A reporters. (D) Effect of GGA (100 nM ~ 1 mM) on HSF1, HSPA8 and HSPA1A reporters. To normalize, the fluorescence level in untreated cells is set as 100%. Three independent experiments were performed with P < 0.05 considered significant.. A AseI PHSF1. NheI. SspI mCherry. SV40 polyA. EcoRI PHSPA8. Eco47I ZsYellow1. SspI SV40 polyA. NotI PHSPA1A. MCS of pAmCyan1-N1. AseI. B. 34. AmCyan1.

(41) Fluorescence (%). C. P = 0.021. 120 1.2. P = 0.031 ~ 0.008. P = 0.013 ~ 0.002 Heat shock. 1. 數列1 untreated 數列2 42°C 2 hr 數列3 recovery 2 數列4 recovery 4 數列5. 0.8 80 0.6. h h recovery 6 h. 0.4 40 0.2 0 1. HSF1. D Fluorescence (%). 160 160. P = 0.025 ~ 0.000. 2. HSPA8. P = 0.024 ~ 0.000. 3. HSPA1A. P = 0.019 ~ 0.000. GGA 數列1 untreated. 140. 120 120. 數列2 100. nM 1 M 數列410 M 100 M 數列5 200 M 數列6 500 M 數列7 1 mM. 100. 數列3. 80 80 60. 40 40 20 0. 數列8 1 HSF1. 2 HSPA8. 35. 3 HSPA1A.

(42) Fig. 3. Screening lead compounds enhancing chaperone function. (A) Effect of NH014-1 (100 nM ~ 1 mM) on HSF1, HSPA8 and HSPA1A reporters. (B) Effect of GGA and indole derivatives NC001-2, -3, -8 and -11 (100 nM ~ 300 µM) on HSF1, HSPA8 and HSPA1A reporters. To normalize, the fluorescence level in untreated cells is set as 100%. Three independent experiments were performed with P < 0.05 considered significant. (C) Representative western blot image of NH014-1 and NC001-8 (100 nM)-treated 293 cells for two days using HSF1, HSPA8, HSPA1A and H3F3B antibodies. Levels of HSF1, HSPA8, HSPA1A were normalized with a loading control (H3F3B or GAPDH). Data are expressed as the mean ± SEM values from three independent experiments.. Fluorescence (%). A. P = 0.028~0.001. 140 140. P = 0.043~0.000. P = 0.030~0.000 NH014-1 untreated 數列2 100 nM 數列3 1 M 數列4 10 M 100 M 數列5 200 M 數列6 500 M 數列7 1 mM 數列1. 120. 100 100 80. 60 60 40. 20 20 0. 數列8. HSF1 1. Fluorescence (%). B. HSPA8 2. HSPA1A 3 數列1. 200 200. HSF-1. HSPA8. HSPA1A. 數列2 數列3. 150 150. 數列4 數列5. 100 100. 數列6 數列7. 50 50. 數列8 數列9. 0. Un. GGA. -2 1 -3. -8. -11. Un. GGA. NC001. -22 -3. -8. -11. NC001 untreated 100 nM GGA, NC001-2, NC001-3 1 M GGA, NC001-11 10 M GGA, NC001-11 100 M GGA 200 M GGA 30 M NC001-8 300 M NC001-8. 36. Un. GGA. -23 -3. -8 NC001. -11. 數列10 數列11 數列12.

(43) C NH014-1 (100 nM). HSF1. HSPA8. -. -. + 1.6. + 1.4. HSPA1A. -. +. NC001-8 (100 nM). HSF1. HSPA8. -. -. + 1.4. 1.4. H3F3B. GAPDH. 37. + 1.4. HSPA1A. -. + 1.3.

(44) Fig. 4. Characterization of GFPu cells. (A) Fluorescent microscopic images ofGFPu cells treated with MG132 (1 M) or benzamil (10 M). (B) Flow cytometer analysis of fluorescence in MG132 (100 nM ~ 1 M) and benzamil (100 nM ~ 10 M) treated GFPu cells.. A. DMSO. MG132. B. P = 0.005 ~ 0.000. 1250 250. Fluorescence (%). Benzamil. 數列1. 1200 200. 數列2. 150 150. P = 0.005 ~ 0.000. 數列3 數列4. 100 100. 數列5. 50 50. 數列6 數列7. 0 DMSO. MG132 100 nM. 1 M. 38. 1. Benzamil 100 nM. 1 M 10 M. 數列8.

(45) Fig. 5. Screening lead compounds enhancing proteasome function. Flow cytometer analysis of GFPu fluorescence in indole derivatives NC001-1, NC001-8, NC001-9, NC001-10, NC001-11 (1/10 IC50 ~ 1/100 IC50) (A) and Chinese herbs NH005 and NH006 (1/5 IC50 ~ 1/20 IC50) (B) treated cells.. A. 2501250. Fluorescence (%). 2001200 150150 *. 100100 50 50 0. 1/10 IC50. -9. -10. 1/100 IC50. 1 M. -8. -11. 1. 數列1. 100 100. Fluorescence (%). 10 M. 1 M. B. 100 nM. Untr. MG132 Benzamil NC001-1. P = 0.031. 數列2. P = 0.013. 數列3. 95 P = 0.031. P = 0.013. 90 90. 數列4 數列5. 1/100 IC50. 數列61/10 IC. 50. 數列71/5 IC50. 85. 數列8 數列9. 80 80 Untr.. Benzamil. 1. NH005. NH006. 數列10 數列11 數列12. 39.

(46) Fig. 6. Flp-In SH-SY5Y TBP lines. (A) Flp-In SH-SY5Y host cell line. (B) Co-transfection of pcDNA5/FRT/TO expressing vector containing GFP-tagged TBP and pOG44 into the host cell line. (C) Induced expression of TBP-GFP by the addition of doxycycline. (D) Amino acid sequences of full length (NM_003194.4) and N terminal TBP (marked in red). (E) FRT copy number determination of Flp-In SH-SY5Y host line by PCR.. A Flp-In SH-SY5Y. neomycin gene 412 bp. Neomycin. B Flp recombinase nTBP-GFP. pOG44. C + doxycycline Expression of nTBP-GFP nTBP. 40. Neomycin.

(47) D. TBP (NM_003194.4). translation="MDQNNSLPPYAQGLASPQGAMTPGIPIFSPMMPYGTGLTPQPIQNT NSLSILEEQQRQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQAVAAAAVQQS TSQQATQGTSGQAPQLFHSQTLTTAPLPGTTPLYPSPMTPMTPITPATPASESSGIVPQ LQNIVSTVNLGCKLDLKTIALRARNAEYNPKRFAAVIMRIREPRTTALIFSSGKMVCTG AKSEEQSRLAARKYARVVQKLGFPAKFLDFKIQNMVGSCDVKFPIRLEGLVLTHQQFSS YEPELFPGLIYRMIKPRIVLLIFVSGKVVLTGAKVRAEIYEAFENIYPILKGFRKTT". E. Copy no. control bp 1198 –. M. 103 102 10. 1. M s17. 517 – 350 – 192 –. s9. s11 (Flp-In T-REx SH-SY5Y). - 412 bp. 41.

(48) Fig. 7. SH-SY5Y cells with induced SCA17 TBP/Q79-GFP expression and neuronal phenotype. (A) Real time PCR quantification of TBP/Q36~79-GFP mRNA level relatively to HPRT mRNA after 2 days induction with doxycycline (+ Dox) or not (- Dox). (B) Western blot analysis of TBP/Q36~79-GFP protein level using TBP (N-12) antibody after 2 days induction with doxycycline (+ Dox) or not (- Dox). (C) Representative microscopic images (top) of TBP/Q36~79-GFP cells after induced differentiation with retinoic acid (+ RA) for 7 days and aggregate quantification (bottom) of cells with induced differentiation for 7 ~ 21 days. (D) Representative microscopic images (top) of neuronal differentiated TBP/Q36 and TBP/Q79 cells (for 14 days) and quantification of neuronal processes and branches (bottom) of cells with induced differentiation for 7 ~ 21 days.. A. B. Fold of induction. 12. 12. kDa M 95數列1 – 數列2. 10 8. 8. TBP/Q36 TBP/Q61 TBP/Q79. -. -. +. +. - +. 72 –. 數列3 數列4 數列5 數列6 數列7. 6. - TBP/Q79-GFP - TBP/Q61-GFP - TBP/Q36-GFP - Endogenous TBP. 55 –. 4. 4. 43 –. 2. Dox. 0. TBP/ Q36 Q61 Q79. - Dox C. 1. 34 –. Q36 Q61 Q79. + Dox. TBP/Q36. Ab: TBP (N-12). TBP/Q61. TBP/Q79. + RA. Aggregation (%). 50 µm. 2 2.0. P = 0.004 P = 0.028. 1.5. 1.5. P = 0.025 P = 0.044. 數列1 數列2 數列3 數列4. 1 1.0. 數列5. 0.5. 數列7. 數列6. 0.5. 數列8 數列9. 0. 7. 14 21 TBP/Q36. 7. 1 14 21 TBP/Q61. 42. 7. 14 21 TBP/Q79. days. 數列10 數列11.

(49) D TBP/Q36. TBP/Q79. 50 µm. 33. P = 0.002 P = 0.000. 22 1.5. 11 0.5 0. 7 days. 14 days 1. 1.6 1.6 數列1 1.4 數列2 1.2 1.2 數列3 1 數列4 0.8 0.8 數列5 0.6 數列6 0.4 0.4 數列7 0.2 數列8 0. P = 0.009. 21 days. 數列3 數列4 TBP/Q36 數列5 TBP/Q79 數列6. P = 0.043. 數列7 數列8. 7 days. 43. 數列1 數列2. Branches. Processes. 2.5. 14 1days. 21 days.

(50) Fig. 8. Flp-In SH-SY5Y TBP cells for screening candidate compounds enhancing chaperone function. (A) Cells were pre-treated with GGA, indole or NC001-8 (100 nM) for 8 hours and TBP/Q79-GFP expression induced for 6 days. Relative HSF1, HSPA8 and HSPA1A expressions were analyzed by immunoblot blot analysis using β-actin as a loading control. (B) Relative aggregation after treatment with GGA, indole or NC001-8 (100 nM) for 7 days. To normalize, the relative aggregation level in untreated cells is set as 100%.. Dox. -. +. +. +. +. GGA 001-8 Indole HSF1 – b-actin –. 140 140. Rel. level (%). A. 120. P = 0.047~0.017 P = 0.001. 100 100 80 數列1. 60 60 40 20 20 0 1. Rel. level (%). 140 140. HSPA8 – b-actin –. 120. 2. 3. 4. 5. P = 0.038~0.013 P = 0.019. 100 100 80 數列1. 60 60 40 20 20 0 1. Rel. level (%). 140 140. HSPA1A – b-actin –. 120. Rel. aggregation (%). P = 0.001. 1 100. 3. 4. P = 0.039 1 2 3 4 5. 80. 60 60 40. 20 20. -1. P = 0.002. +2. +3. +4. +5. GGA. 001-8. Indole. P = 0.010. 0.8 80 1. 0.6 60. 2 3. 0.4 40. 4. 20 0.2 0 1 Untreated. 2 GGA. 3 Indole. 44. 5. P = 0.010~0.006. 100 100. 0. B. 2. 4 NC001-8. Dox.

(51) Fig. 9. Flp-In SH-SY5Y TBP cells for screening candidate compounds enhancing proteasome function. (A) Cells were pre-treated with MG132 (1 M), benzamil (1/10 IC50), NH005 (1/10 IC50) or NH006 (1/10 IC50) for 8 hours and TBP/Q79-GFP expression induced for 6 days. Relative conjugated ubiquitin was analyzed by immunoblot blot analysis using ubiquitin and β-actin (as a loading control) antibodies. (B) Relative aggregation after treatment with benzamil (100 nM), NH005 or NH006 (10 g/ml) for 7 days. To normalize, the relative aggregation level in untreated cells is set as 100%.. A. M. Untr. MG132 Benza. NH005 NH006. kDa 170 – 130 –. Conjugated ubiquitin. 95 – anti-Ubiquitin. 72 – 55 – 43 –. anti-Actin. Rel. aggregation (%). B. P = 0.007. 100 1. P = 0.003. P = 0.023. 80 0.8 0.6 60 數列1. 40 0.4 20 0.2 0 1 Untreated. 2 Benzamil. 45. 3 NH005. 4 NH006.

(52) Fig. 9. Flp-In SH-SY5Y TBP cells for screening candidate compounds enhancing proteasome function. (A) Cells were pre-treated with MG132 (1 M), benzamil (1/10 IC50), NH005 (1/10 IC50) or NH006 (1/10 IC50) for 8 hours and TBP/Q79-GFP expression induced for 6 days. Relative conjugated ubiquitin was analyzed by immunoblot blot analysis using ubiquitin and β-actin (as a loading control) antibodies. (B) Relative aggregation after treatment with benzamil (100 nM), NH005 or NH006 (10 g/ml) for 7 days. To normalize, the relative aggregation level in untreated cells is set as 100%.. A. M. Untr. MG132 Benza. NH005 NH006. kDa 170 – 130 –. Conjugated ubiquitin. 95 – anti-Ubiquitin. 72 – 55 – 43 –. anti-Actin. Rel. aggregation (%). B. P = 0.007. 100 1. P = 0.003. P = 0.023. 80 0.8 0.6 60 數列1. 40 0.4 20 0.2 0 1 Untreated. 2 Benzamil. 45. 3 NH005. 4 NH006.

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