• 沒有找到結果。

以SCA17小鼠小腦切片培養及基因轉殖鼠評估HDAC抑制劑對SCA17之效果

N/A
N/A
Protected

Academic year: 2021

Share "以SCA17小鼠小腦切片培養及基因轉殖鼠評估HDAC抑制劑對SCA17之效果"

Copied!
110
0
0

加載中.... (立即查看全文)

全文

(1)國立師範大學生命科學系碩士論文. 以 SCA17 小鼠小腦切片培養及基因轉殖 鼠評估 HDAC 抑制劑對 SCA17 之效果 Evaluation of effect of HDAC inhibitors on SCA17 with SCA17 mouse slice culture and transgenic mice. 研究生: 陶育晨 Yu-Chen Tao 指導教授: 謝秀梅教授 Hsiu-Mei Hsieh, Ph.D.. 中華民國一百零二年七月.

(2) 致謝 多謝這三年來大家的協助與照顧,待在師大的日子,不知不覺就 5 年 了,說長也不長,卻也不短。. 從大三開學開始進了這個實驗室,其學習過程並不是想像中的那麼順 利,也清楚知道自己的努力並不夠,也因此給很多人添了不少的麻煩 與困擾,但我真的很謝謝各位給我幫助與指導,讓我在這期間學習並 完成碩士論文。謝謝慧貞學姊一開始的引導和照顧,以及實驗室中已 畢業的執中學長和即將要離開的賀強與薇琳學姊,另外,感謝芷妍和 朧云假日的時候幫我們照顧老鼠,最後,感謝謝老師,這近 3 年來的 指導與拉拔,支持與鼓勵,縱使我表現得不是很好,卻不斷地幫助我。. 感謝老鼠們,由於你們的犧牲換來了今天的實驗結果,希望你們下輩 子能夠別再成為實驗動物,如果可以,希望能你們都早日到達西方極 樂世界,無憂無慮的生活。. 最後,希望大家之後都能夠鵬程萬里,一帆風順! 陶育晨 民國一百零二年七月.

(3) Index. 中文摘要..................................................................................................... 1 Abstract ....................................................................................................... 2 Introduction ................................................................................................. 3 Material and method ................................................................................... 9 Results ....................................................................................................... 17 Discussion ................................................................................................. 28 Reference .................................................................................................. 33 Figure and table......................................................................................... 42. 0.

(4) 摘要. 脊髓小腦萎縮症第十七型 (SCA17) 是一種多麩胺酸造成的神經退化 性疾病,是由於在 TATA-box binding 蛋白基因 (TBP 基因) 上有不正 常的 CAG 三核甘酸的擴增,造成 TBP 蛋白不正常摺疊並聚集在細胞 當中,進而產生毒性。在 SCA17 的病徵上,目前有發現到有運動能 力失調、運動功能障礙、認知功能失常等情形。TBP 的主要功能是在 細胞中啟動轉錄功能,組蛋白去乙醯化酶抑制劑 (HDACi) 可促使基 因進行轉錄,因此被引用當作治療 SCA17 的藥物有其潛力。我們測 試了多種 HDACi 化合物,當中發現 LBH589 可以在 SCA17 小腦組織 培養上有效地降低 TBP 蛋白的聚集,然而在活體的實驗上發現 LBH589 不能有效改善 SCA17 運動失調的現象。此外,小鼠的食慾 以及中樞神經系統發育可能會受到 LBH589 刺激而造成遲緩。因此, 目前我們的活體實驗證據並無法說明 LBH589 對 SCA17 之治療是有 幫助的。. 關鍵字: HDACi、SCA17、LBH589、TBP、小腦組織培養. 1.

(5) Abstract. Spinocerebellar ataxia type 17 (SCA17) is a polyQ neurodegenerative disease caused by abnormal CAG repeat expansion of TATA-box binding protein (TBP) gene. The CAG trinucleotide expansion results in the mutant polyQ TBP protein misfolded and accumulated in the cells, which further caused the neuronal lose, especially the cerebellar Purkinje cells. Ataxia, motor dysfunction, and dementia are clinical symptoms of SCA17. As the role of TBP being critical in transcription initiation, improving the transcriptional activity by histone deacetylase inhibitors (HDACi) becomes an attractive approach for SCA17 treatment. We have screened several HDACi compounds and found that LBH589 could significantly reduce the TBP aggregation on SCA17 cerebellar slice culture. In in vivo study, we found LBH589 couldn’t ameliorate the motor function and the pathology of SCA17 mice. Additionally, the appetite of mice was reduced and the central nervous system was impacted by LBH589 treatment. We could not suggest that LBH589 is potential in SCA17 treatment from our in vivo results.. Key words: HDACi、SCA17、LBH589、slice culture、TBP. 2.

(6) Introduction. Polyglutamine disease (PolyQ disease). PolyQ disease is resulted from the trinucleotide CAG/CGG repeat expansion of the disease-causing genes. The polyQ expansion could cause the protein misfolding in the cells (Duennwald, 2011). The misfolded protein would aggregate and accumulate in cells to cause cell toxicity and apoptosis. Most of the polyQ expansion cause neurodegenerative diseases, for example, Huntington disease (Yu et al., 2003; Lee et al., 2011), spinocerebellar ataxia type 1, 2, 3, 6, 7,12, and 17(Kawai et al., 2009), and Dentatorubralpallidoluysian atrophy (DRPLA) (Lee et al., 2011).. PolyQ disease could be inherited, and increased the polyQ length with the generation. The results from polyQ of neurodegenerative disease that would impact the peripheral or central nerve to be degenerated with age(Duennwald, 2011).The mechanisms the aggregation protein induce the cell apoptosis mainly affected the mitochondria and increased the apoptosis related proteins in the neurons, for example, caspase-3 (Friedlander, 2003). The insoluble protein aggregating in the nucleus and cytoplasm of cells could be cleared by the autophagy pathway (Duncan et al., 2010). However, the mechanisms of the pathway were not clear.. The abnormal polyQ protein could also interact with several 3.

(7) transcription factors, which would cause the transcription dysfunction. It has been reported that CREB-binding protein (CBP) and TATA box-binding protein (TBP) were recruited into the polyQ aggregate and reduced the transcription (McCampbell and Fischbeck, 2001; Friedman et al., 2007; Orr, 2012).The abnormal binding of polyQ domain and transcriptional factors resulted the fatal attraction and neurodegeneration (McCampbell and Fischbeck, 2001; Masino, 2004; Popiel et al., 2007).. Spinocerebellar ataxia (SCA). SCAs were neurodegenerative diseases that cause the cerebellum, brain stem, spinal cord degeneration in the patients(Kawai et al., 2009).Currently, a total of 37 types of SCAs had been identified(Serrano-Munuera et al., 2013).The atrophy of cerebellum and progressive neurodegeneration were found in the patients. The incoordination and balance are gradually lost over time. The causes of diseases include point mutation, missense mutation, and CAG repeat expansion (Kawai et al., 2009).. Spinocerebellar ataxia type 17 (SCA17). SCA 17 is an autosomal dominant neurodegenerative disease. SCA17 is caused by abnormal CAG repeat expansion on TBP gene 4.

(8) results in the polyQ-expanded domain on N-terminal of TBP protein. SCA17 patients were found having misfolded TBP accumulated in the Purkinje cells of cerebellum. Gradually, the Purkinje cells lost and led to the cerebellar atrophy.. The major symptoms of SCA17 were similar as others SCAs, including ataxia, dementia, and seizures. In animal model, the astrocytes were increased and shivered at the late stage (Friedman et al., 2007; Chang et al., 2011).. Histone deacetylase (HDAC) and histone deacetylase inhibitor (HDACi). Acetylation, methylation and phosphorylation play important roles in transcriptional activities. Histone deacetylase, regulated the transcription activities in eukaryotes. HDAC was classified into four classes, class I to class IV. There are 18 types of HDACs in human, were been classified into two families. The classical HDACs need Zn2+ as cofactors. The Sir (Sirtuin) family needed NAD+ as cofactor (Tabe et al., 2007; Chuang et al., 2009).. HDAC not only affect the transcription factors, but also acetylated the other proteins, including tubulin (Chuang et al., 2009; Matsubara et al., 2013).The regulation also affected the gene transcription activities. If 5.

(9) the -tubulin acetylated, the microtubule stability would be affected and further impaired the protein transformation and cell survivals (Chuang et al., 2009).. Histone deacetylase inhibitor (HDACi) is widely used on the cancer treatment to kill the cancer cells or inhibit the cancer cell proliferation. Recently, HDACi were found to have benefit on neurodegenerative diseases for ameliorating the symptoms of diseases. Currently, HDAC 1, 2, 3, 6, 8, and Sirtuin of class I, II, and III were proved having improvement in neurodegeneration and help in neuroprotection. It has been found that HDACi could improve the symptoms of diverse neurodegenerative disease animal models which were summarized in Table 1(Chuang et al., 2009; Mielcarek et al., 2011; Yeh et al., 2013; Yi et al., 2013). In previous studies, the HDACi could affect certain HDACs to ameliorate the symptoms of neurodegenerative diseases. For example, M344 and LBH589 could affect HDAC to elevate SMN2 levels and ameliorate the neurodegeneration of spinal muscular atrophy (SMA) (Riessland et al., 2006; Garbes et al., 2009); SAHA could impact the HDAC2 and 4 activity to reduce the Htt aggregation of Huntington’s disease mouse model (Mielcarek et al., 2011). LBH589 (Panobinostat). LBH589 is a novel hydroxamic acid-derived HDACi. LBH589 6.

(10) could induce the acetylation of histone H3 and H4. It has already been used on the numerous clinical trials of cancers (Marks et al., 2001). It was reported to have therapeutic potential in spinal muscular atrophy (SMA), a neuromuscular degenerative disease (Garbes et al., 2009). Recently, LBH589 was found to improve this disorder Niemann-Pick type C1 disease by many mechanisms. The LBH589 could reduce the cholesterol accumulation in the cells and improve the cholesterol esterification (Pipalia et al., 2011). It's interesting to see whether LBH589 could be used in the other neurodegenerative diseases to ameliorate their phenotypes.. In Garbes et al., 2009, they treated the fibrobalst of SMA patients and SMA tansgenic mice. They found that the SMN2 protein which was decreased at SMA patients was increased in various type SMA patients and SMA transgenic mouse models. It could efficiently ameliorate the symptoms of SMA neurodegeneration.. Cerebellar slice culture. Cerebellum was controlled the body balance in the animals. It is also involved in cognitive function such as attention and language. The coordination in animals is important for survival (Gao et al., 2012). There are several kinds of neurons in cerebellum, with the most important being Purkinje cell and granular cell. The Purkinje cells in cerebellum contain 7.

(11) only 0.1%, but they are important for coordinating the signal balance with motor neurons (Hashimoto and Hibi, 2012).. Slice culture is used to study the morphology and development in early stage (Miyata et al., 2005). The organotypic slice culture has been established in various study, including hippocampus, cortex, olfactory bulb, and cerebellum. (Kapfhammer, 2004; Noraberg et al., 2005; Lonchamp et al., 2006; Igelstrom et al., 2011). Recently, ex-vivo, slice culture is used on drug screen for it could be a quick platform to identify the treatment result. The experiments, the approaches about the slice tissues had been treated by drugs are easily controlled. In addition, the cell interaction within the cerebellum, including the Purkinje cells, granular cells and glial could be observed and the signaling transduction pathways could also be studied to further analyze the molecular mechanism of the treatment (Bickler and Fahlman, 2009).. 8.

(12) Materials and methods. SCA 17 transgenic mice. We had established SCA17 transgenic mouse overexpressing human TBP (hTBP) with 109Q expansions in FVB background (Chang et al., 2011). We used the heterozygous SCA17 transgenic male mice to mate with wild-type female mice (FVB/NJNarl, NLAC, Taipei, Taiwan) to maintain the mouse line. The mice are cultivated under 12 hour/12 hour light and dark cycle in individually ventilated cages (IVC) system.. Genotyping analysis. 0.5~1 mm of mouse tail was cut into pieces and incubated in Direct-PCR reagent (Sigma) with 0.5 mg/ml proteinase K (Bionovas, ON, Canada) at 55℃for 6 hours, and then inactivated the proteinase reaction at 85℃for 45 min. DNA samples were stored at -20℃for PCR genotyping.. The primers used on PCR genotyping were pL-7-F forward primer (5' - TAT GGT GAG AGC AGA GAT GG - 3') and TBP-3R backward primer (5'- CTGCT GGGAC GTTGA CTGCT G -3'). The PCR was reacted 30 cycles under 95℃1 min for denaturing, 65℃1 min with -0.1℃ 9.

(13) touch-down in each cycle for annealing, and 72℃1 min for elongation. The amplified PCR product was a 769-bp fragment.. Cerebellar slice culture. We used postnatal day 7 transgenic mice to generate cerebellar slice culture. The cerebellum was isolated and put into 1X D-PBS with 0.5 % glucose, and then the vibratome (Leica VT1200S) was used to slice the cerebellum into 350 μm. The slices were cultured on culture plate insert (Millipore) with the medium (50 % BME, GIBCO-Invitrogen, Carlsbad, CA, USA; 25 % HBSS, GIBCO-Invitrogen; 100 ng/ml Glutamax I, GIBCO-Invitrogen; 100 unit/ml Penicillin; 100 ng/ml streptomycin, GIBCO-Invitrogen; 25 % House serum, GIBCO-Invitrogen; 0.5 % D (+)-glucose, Sigma). After cultured for 7 days, the slices were immunoflourescence stained to examine the TBP aggregation. The drugs screened (Table 2) with SCA17 cerebellar slice culture was added in the culture medium since DIV1. And then the cultured slices were changed the half of medium with screened drugs on DIV4 and harvested on DIV7.. LBH589 (Panobinostat) treatment on SCA17 mice. SCA17 transgenic mice were divided into four groups: Wild type-LBH589 treatment (0.0175 mg × 60% × the body weight of mouse), 10.

(14) Wild type-vehicle (DMSO), Transgenic-LBH589 treatment (0.0175 mg × 60% × the body weight of mouse), and Transgenic-vehicle (DMSO). LBH589 was dissolved in dimethylsulfoxide (DMSO, Sigma), and intraperitoneal injected into transgenic mice every other day for 10 weeks. All the animal experiments were conducted in accordance with the guideline and approved by the National Taiwan Normal University Research Committee.. Behavioral test Rotarod Four to twelve-week-old mice treated with LBH589 or vehicle (for each group, N=11 to 12) were analyzed by rotarod testing every two weeks with Mouse ROTA-ROD (UGO BASILE, 47600). Two days before the rotarod testing, the mice were handled twice to let mice got used to the operator. At the first time, the mice were stayed on the rod for 1 min, and then at fixed speed 4 rpm for 1 min repeated 3 times to train. Between each training interval mice were rested for 10 min. At the official tests, the rotating speed was accelerated from 4 to 30 rpm in the first 300 sec, and then the speed was maintained at 30 rpm for another 300 sec. The test was ended if the mice fall down from the rod or keep 600 sec on the rod. The trial was repeated 3 times a day for 2 days. Mice were rested for 15 min between each trial.. Locomotor Five and eleven-week-old mice treated with LBH589 or vehicle (for each group, N=11 to 12) were placed in the box (30 x 30 x 30 11.

(15) cm, black box) and monitored for 10 min which was separated into 2 periods (5 min for each period). The EthoVision® XT software (Noldus) was used to analyze the behavior.. Footprint Four to twelve week-old mice treated with LBH589 or vehicle (for each group, N=11 to 12) were placed on the footprint detector to walk. The CatWalk® software (Noldus) was used to analyze the behavior.. Homecage behavior analysis Eleven week-old mice treated with LBH589 or vehicle (for each group, N=4) were placed in the cage that was video recorded the behavior of the mice for 12 hours at night using Home Cage Scan 3000 system (Clever Sys. Inc., USA).. Western blot. Mice were anesthetized with 2.5% avertin (Sigma, 18μl/g BW). The mice were perfused with 0.9% normal saline. Cerebella were dissected out and homogenized with RIPA buffer ( 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.1% SDS, 1% DOS, 1% NP40) containing protease inhibitor (Pierce, USA) and phosphatase inhibitor (Sigma, P2850). Supernatant was collected after 30 minutes at 4℃13.4g centrifugation. Proteins were quantified by BCA Protein Assay (Pierce, USA).. 12.

(16) Fifty μg of extracted total protein was resolved on SDS-PAGE and transferred to Hybond-C extra membrane (Amersham) at 400 mA for 1 hour. The epitopes on the membrane were blocked with 5% skim milk in 1×TBS.After the membrane were incubated with primary antibodies at 4 ℃ overnight, they were stained with horseradish peroxidase (HRP)-labeled secondary antibodies for 2 hours to lighten the specific signals. The membrane was observed by LAS 4000 image system (Fujifilm, Japan).. Immunoflourescence staining For cerebellar slice culture Cerebellar slice were cultured for 7 days, on DIV 7, fixed the slice with 4% paraformaldehyde (PFA) for 30 min. The fixed slice was washed with 1× PBST (containing 2% Triton X-100) 15 min for three times. The sample was blocked by blocking solution (1×PBST containing 0.2 Triton X-100) at 4℃overnight. After the slice incubated with primary antibodies (IP3R1, 1:1000, Santa Cruz; 1TBP18, 1:30000, QED) at 4℃for 36-48 hours, and stained with the fluorescent secondary antibodies (working titer 1:500, all Invitrogen) at 37℃for 2 hours, the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, 1:5000).. For mouse cerebellum Twelve-week-old mice treated with LBH589 or vehicle were sacrificed, perfused with 0.9% normal saline, and fixed by 13.

(17) 4% PFA in PBS. The cerebella were post-fixed by 4% PFA for 4 hours and dehydrated by 10% sucrose for 1 hour, 20% sucrose for 2 hours, and 30% sucrose for overnight. The samples were storageat -80℃.. The fixed cerebella were cut into 30 μmcryosections by cryostat-microtome (LEICA CM3050S). The sections of cerebella were blocked by blocking solution (10% house serum in 1 ×TBS, pH7.6) at 4 ℃ for overnight. The blocked slice was incubated with the primary antibodies (Calbindin, 1:100, Santa Cruz; GFAP, 1:1000, Millipore; S100 , 1:500, Sigma; TBP N12, 1:2000, Santa Cruz) at 4℃ overnight and then was stained with the fluorescent secondary antibodies (1:500, all Invitrogen) at 37℃for 1 hour. The cell nuclei were stained by DAPI (1:5000) at 37℃for 30 min.. The sections/slices finally were mounted on slides with mounting medium (Southern Biotech, Los Angeles, USA) and cover-slipped. The stained slice was scanned by confocal microscopy (Leica TCS SP2 confocal spectral microscope imaging system) to get the slice or the section of cerebella fluorescence image.. Immunohistochemical staining. The sections of cerebella were incubated with 3% H2O2 for 30 min 14.

(18) to eliminate endogenous peroxidase enzyme activities, then used the TBST (containing 2% Triton X-100) to wash for 10 min twice. The sections were blocked with blocking solution (5% normal horse serum with TBST containing 0.2% Triton X-100) at 37℃ for 1 hour. Then the blocked sections were incubated with primary antibodies in TBS (Calbindin, 1:1000, Sigma; GFAP, 1:1000, Millipore, Iba1, 1:1000, Wako) at room temperature for overnight. After the sections were washed in TBS for 10 min three times followed by incubation with secondary antibodies (1:200) in TBS at room temperature for 1hour and then were colored the signals by substrate-chromogen solution and diaminobenzidine (DAB) oxidation. The sections were mounted on the gelatin coated slides and cover-slipped for light microscopy observation.. Paraffin section. The mouse liver and kidney were fixed with 4% PFA overnight. The fixed organs were paraffin embedded by National Laboratory Animal Center. The embedded organs were cut into 3 μm by paraffin-microtome (Leica RM2125 RT) and then stained with hematoxylin and eosin Y. The stained slides were examined by National Laboratory Animal Center.. 15.

(19) The quentification of TBP aggregation in cerebellae slice culture. The TBP aggregations localized within the nuclei of Purjekinje cells were quantificated by MetaMorph image system (Leica). HDAC activity assay. The protein was isolated from the cerebellum of FVB/NJNarl mouse using T-PER (Thermo) with protease inhibitor cocktail tablet (Roche). The HDAC activity was detected by HDAC-Glo™ I/II Assay and Screening System (Promega).. Statistical analysis. Independent t-test was used to analyze and compare the difference between the groups (Two-tail P values < 0.05). SPSS software (version 19.0; SPSS INC., Illinois, USA) was used for analyses.. 16.

(20) Result. Evaluation of compounds/herbs on the TBP aggregation of SCA17 cerebellar slice culture. SCA17 cerebellar slice culture was established from the postnatal day 7 SCA17 transgenic mice and used to screen the effect of HDACi. Treatments were applied to the slice culture at DIV1 and the treatment effect was characterized by immunofluorescence staining at DIV7. Antibody against IP3R1 was used to stain the Purkinje neuron and TBP (QED 1TBP18) staining was used to observe the TBP aggregation.. Several chemical compounds and herbs were tested in this study (Table 2). Some of them, including NC001, NC005, NC100, NH014, NH015, and NH021were identified to be HDACi by an HDAC activity assay (HDAC-Glo™ I/II Assay and Screening System, Promega) using mouse cerebellar cell extract as HDAC enzyme source (Table 2). We also evaluated several non-HDACi compounds which were found potential in reducing polyQ aggregation in other cellular systems (Table 3). Different amounts of compounds or herbs were applied to the SCA17 slice culture. Compared to the vehicle-treated control group, the slice morphology and IP3R1 expression level were used to estimate the toxicity of each treatment; TBP protein aggregation accumulated in the Purkinje cell nuclei was quantified and normalized to the vehicle-treated control group 17.

(21) to evaluate the potential of each treatment in aggregation reduction.. NC001and NC005 series compounds were synthesized by Dr. Ching-Fa Yao in the Chemistry Department of National Taiwan Normal University (NTNU). Compounds NC001 and NC001-8 were found to reduce the polyQ aggregation in cell system by Dr. Lee-Chen’s Lab (Department of Life Science, NTNU, personal communication). NC001 and NC001-8 were identified to have HDACi activity (Fig. 1A). Our study found that both NC001 and NC001-8 treatments could significantly reduce the TBP aggregation (Fig. 1B-C) at 10 nM and 10 M. NC005 series compounds were also identified to have HDACi activity (Fig. 1A). From the slice culture analysis, NC005-1 and NC005-4 treatments were found to reduce the TBP aggregation in Purkinje neurons (Fig. 2B-C), however, the toxicity of these treatment was high (Figure 2) from the IP3R1 staining results (Fig. 2C).. NC100 series were known HDACi compounds purchased from chemical suppliers. The HDACi activities of these compounds were confirmed by the biochemical assay (Fig. 3A). Compounds NC105, 108, and 109 could significantly reduce the TBP aggregation in Purkinje cells (Fig. 3B-C). In addition, the neuromorphologies of the slice after treatment shown by the IP3R1 staining were better than vehicle-treated group (Fig. 3C).. Several Chinese herbs and their active ingredients were also found to 18.

(22) have HDACi activity in our biochemical assay. We have tested NH014, NH021, and NH015-4 on the SCA17 slice culture. NH014 and NH021 could notably reduce the TBP aggregation and improve the slice morphology (Fig. 4). NH015-4 is an active ingredient of Chinese herb NH015. Compound NH015-4 could not reduce the TBP aggregation, although there is no significant toxicity was identified in the slice (Fig. 5).. In addition to HDACi, we also evaluated several compounds whose HDACi activities are not determined yet. NC010 series were disaccharide analogs. NC010-7 could slightly reduce the TBP aggregation, but NC010-11 enhanced the aggregation formation in Purkinje cells at the concentration tested (Fig. 6).. WCt series were chemical compounds with antioxidation activity, which were synthesized by Dr. Way-Zen Lee in the Department of Chemistry, NTNU. Whether these compounds containing HDACi activity is not determined yet. All the 3 compounds could efficiently reduce the TBP aggregation in SCA17 transgenic mouse slice culture (Fig. 7). In addition, the neuromorphology of the treated slices were improved compared to the vehicle-treated slice (Fig. 7B).. NH043-1 was also a chemical compound with antioxidation activity, which was derived from herb NH043. This compound showed no effect to reduce the TBP aggregation in SCA17 transgenic mouse slice culture (Fig. 8). However, the neuromorphology of NH043-1 treated slice was 19.

(23) improved compared to the vehicle-treated slice.. LBH589 reduces the body weight of mice.. LBH589 treatment on SCA17 transgenic mice was started at the age of 3 week-old. These mice were injected LBH589 every two day by intraperitoneal injection. Mouse body weights were measured once a week. We found that the LBH589-treated mice were significantly lighter from 4 week-old (Figure 9). The LBH589-treated mice from 7 week-old, the body weight was growing steadily. We suggested that the LBH589 inhibited the growth of mice or improved the lipid metabolism as reported (Pipalia et al., 2011). Therefore, we speculate the cause that the TG male mice were significantly lighter than WT male mice at 5 week-old of age, which might be relative with the onset of the SCA17 disease.. The blood glucose was affected by LBH589. In vehicle- and LBH589-treated mice, their blood glucose was measured (Figure 10). At 6 week-old age, the blood glucose of the LBH589-treated mice was elevated. From 8 week-old of age, however, the blood glucose was reduced in LBH589-treated male and female mice. It was in accordance with the body weight changed (Figure 9). The TG 20.

(24) vehicle groups compared with WT vehicle groups, the blood glucose were lower in male mice (Figure 10A) at 6 week-old. Therefore, we also observed the cages of LBH589-treated mice were soggier than the vehicle treated mice. It was possibly connected with the metabolism of mice with LBH589.. LBH589 treatment showed no improvement in the motor balance functionof SCA17 transgenic mice.. We found 10 nM LBH589 could significantly inhibit the TBP aggregation and could improve cell morphology in the SCA17 slice culture. Comparing to the vehicle –treated group, after LBH589-treated mice showed a better coordination performance on the rotarod task at 6 week-old of age in both TG male and female mice (Figure 11). However, the performance of LBH589-treated TG male was not better than the vehicle treatment group, only in TG female. The LBH589 treatment group was better than vehicle treatment group from 8 week-old of age, and not significantly. This result suggests that LBH589 could only lightly or shortly ameliorate the impairment of motor coordination of SCA17 transgenic female mice.. 21.

(25) The mouse activity was reduced by LBH589 treatment. We tested the mice at 5 week-old of age for locomotor analysis. The moving distance of LBH589 treatment groups were longer than vehicle groups (Figure 12A, B). However, at 11 week-old, the LBH589 treatment male mice showed no difference with vehicle treatment male mice. Only the LBH589 treatment female mice were significantly reduced (Figure 12B). The velocity on TG drug treatment groups were raised in a 300 seconds recording at 5 week-old of age (Figure 12C). At 11 week-old of age, the velocity was significantly reduced in LBH589 treatment mice (Figure C, D). In mobility analysis, as a hyperactivity index, the LBH589 treatment mice, in male and female were not significantly different from vehicle treatment mice at 5 week-old.. The mobility of WT and TG mice had no difference at 11 week-old of age. However, the mobility of LBH589 treatment groups were reduced (Figure 12C-F). We suggested the physical power wasdecreasedafterLBH589 treatment.. The anxiety of SCA17 transgenic mice were ameliorated by LBH589 treatment. The anxiety of TG mice with LBH589 treatment was reduced. The locomotor analysis showed TG mice with LBH589 treatment stayed at 22.

(26) central zone or away the peripheral zone longer than TG mice with vehicle treatment (Figure 13). The behavior of mice left the peripheral zone and preferred the central zone was reported as a less anxiety-like behavior (El Idrissi et al., 2009). We suggest the LBH589 treatment could reduce the anxiety-like behavior of TG mice.. Footprint analysis showed that LBH589 could improve the coordination of SCA17 mice. Based on the previous report, the Purkinje cell lost could result the footprint behavior dysfunction (Cendelin et al., 2010). The footprint analysis showed that the mean intensity of paws was affected by LBH589 at 4 weeks (Figure 14). The mean intensity of TG-LBH589 groups was always lower comparing to TG-vehicle groups. The step sequence regularity wasn’t impacted by LBH589 and there was no difference between WT-vehicle and TG-vehicle (Figure 15).The distance of paws of the mice, the right-left paws and the front-hind paws were increased from 8 week-old age of TG mice (Figure 16). The width of right-left paws of TG-LBH589 groups comparing to TG-vehicle groups was not reduced. At 12 week-old of age, the front-hind paws of TG-LBH589 groups were shorter than TG-vehicle groups (Figure 16C, D). It suggested the step coordination of LBH589-treated TG mice was improved. The duration and speed of swing were influenced with LBH589 from 4 week-old (Figure 17). The TG-LBH589 groups were affected most seriously. The 23.

(27) standing of hind paws, we found only left-hind paws were seriously affected in TG-vehicle mice comparing to WT-vehicle mice (Figure 18). The LBH589-treated TG mice showed longer standing duration than vehicle-treated TG mice in left-hind (Figure 18) The SCA17 transgenic mice, only the distance of front-hind paws and standing duration both were rescued in footprint analysis.. Homecage analysis showed that the extending behavior of TG-LBH589 groups were not ameliorated. The relative reared up behavior duration of TG-vehicle male mice were decreased compared with WT-vehicle male mice (Figure 19). We found remain reared up and remain partial reared (Figure 19A, C) of LBH589-treated male mice were decreased comparing to vehicle-treated male mice. However, the behaviors of TG-LBH589 female mice were not affected (Figure 19B, D).. The growth of mouse central nervous system was retarded by LBH589 treatment. We found the cerebrum, cerebellum, and brain stem were not affected after LBH589 treatment (Figure 20). The cerebella of TG-LBH589 mice were not heavier than TG-vehicle mice (Figure 20C, 24.

(28) D). We suggest the growth of central nervous system was retarded by LBH589.. Purkinje loss could not be rescued in LBH589 treated SCA17 transgenic mouse cerebellum. We analyzed the LBH589-treated TG mouse cerebellum by examining the expressions of calbindin (Purkinje cell marker) and aldolase C (which could resist the Purkinje cell death) (Slemmer et al., 2007). The reduced expressions of calbindin and aldolase C in TG mice were not ameliorated by LBH589 treatment (Figure 21). In addition, we found that gliosis was not reduced in the LBH589-treated TG mice (Figures 22).The heat shock protein 70 (Hsp 70) expression was decreased significantly in TG female (Figure 22).. The morphology of SCA17 transgenic mice was improved by LBH589 treatment. The morphology of cerebellum was improved in the mice with LBH589 treatment (Figure 23). The cell body of Purkinje cells of TG-LBH589 groups was not increased comparing to TG-vehicle groups, however, the molecular layer width of cerebellum was increased.. 25.

(29) The TBP aggregation and gliosis were not reduced by LBH589. The TBP aggregation and astrocytes were significantly increased in our SCA17 transgenic mouse model (Chang et al. 2011, also shown in Figure 24). The TBP aggregation and gliosis were not rescued by LBH589 treatment (Figure 24).It suggested the TBP aggregation and gliosis of SCA17 transgenic mice were not reduced by LBH589. The Bergmann glia cells were not rescued by LBH589 treatment in TG mouse. The Bergmann glia cells could protect the impaired neuron (Lafarga et al., 1998; Lordkipanidze and Dunaevsky, 2005). The Bergmann glia cell of SCA17 transgenic mice was significantly reduced at 12 week-old (Figure 25). In LBH589-treated TG mice, the Bergmann glia cells were not increased (Figure 25).. The microglia cells were not reduced in TG-LBH589 groups. The microglia cell was significantly increased in SCA17 transgenic mouse model (Figure 26), which was not decreased by the LBH589 treatment (Figure 26). It is in accordance with western blot and 26.

(30) immunofluorescence staining results (Figure 22 and 24).It suggested the LBH589 treatment could not improve the neuroinflammation of SCA17 disease.. The growth of mice was affected by LBH589. We found the chow consuming of LBH589 treatment groups were decreased (Figure 27). It suggested that LBH589 could reduce the appetite of the mice. This result explained the body weight, blood glucose and some behavioral analysis were reduced by LBH589.. LBH589 treatment had no significant toxicity to the liver and kidney of the mice. There were no significant impairments in liver and kidney of mice after LBH589 treatment (Figure 28 andTable 4). However, we found comparing to the vehicle treatment groups, LBH589 treatment groups even showed less inflammation sites in their livers and kidneys.. 27.

(31) Discussion. We have screened a total of 20 compounds/herbs on SCA17 mouse cerebellar slice culture. We found that the HDACi series (NC001 series, NC005 series, and NC100 series) and WCt series (WCt003, WCt006, and WCt021) could inhibit the formation of TBP aggregation on SCA17 cerebellar slice culture. Among the NC100 series (NC105, NC108, and NC109) tested, NC108 (LBH589) could maintain the slice culture in a better morphology, we thus chose LBH589 to treat SCA17 transgenic mice. In neurodegeneraive diseases, there were only a few studies using LBH589 (Garbes et al., 2009; Pipalia et al., 2011) and no report for the polyQ diseases. According to the report by Lemoine and Younes, acetylation of histone H3 and H4 could be induced by LBH589 (Lemoine and Younes, 2010). In addition, SCA17 is caused by abnormal TBP aggregation that resulted in the dysfunction of transcription. From the characteristics of HDACi and the results in cerebellar slice culture of LBH589, we expected LBH589 has potential therapeutic for SCA17 disease. Therefore, we used LBH589 which was a powerful HDACi to tested weather it can ameliorate the SCA17 disease.. During the in vivo study, the mouse body weight of LBH589 treatment groups were reduced in both males and females (Figure 9). Besides, the blood glucose of TG mice treated LBH589 was higher than TG-vehicle groups at 6 week-old of age (Figure 10). However, from 8 week-old, the blood glucose of LBH589-treated groups was reduced to a 28.

(32) lower level than the vehicle-treated groups.. From the behavior characterization, we found LBH589 have no rescuing effect on SCA17 TG mice. The latency of rotarod task in TG-LBH589 groups was not ameliorated (Figure 11). Furthermore, the results of the mean intensity and swing analysis were much worse in the LBH589 treated TG mice (Figure 14 A, B; Figure 17). Only the distance of print position and standing duration were found ameliorated in TG mice (Figure 16 C, D; Figure 18).. From the locomotor analysis, the total distance, mean velocity, and mobility of LBH589 treatment groups were all decreased at 11 week-old (Figure 12). In addition, in homecage analysis, we found the extending behavior of TG mice were not ameliorated by LBH589 (Figure 19). These data reveal that LBH589 treatment might decrease the activities of TG mice.. Based on the serial behavior analyses, we also found the onset between TG male and female mice were possibly different. From 6 week-old age, the latency of TG male mice was much more reduced than TG female mice on rotarod task (Figure 11). Moreover, compared to the male WT vehicle group, the footprint intensity of the TG vehicle groups was reduced at 4 week-old age, however, female mice showed opposite performance (Figure 14).. 29.

(33) The weight of cerebellum of TG mice between vehicle-treated groups and LBH589-treated groups was no difference (Figure 20). On western blot staining, the calbindin and aldolase C protein level of TG-LBH589 groups was not significantly increased compared with TG-vehicle groups (Figure 21). However, we found the width of molecular layer of TG-LBH589 groups was increased compared with TG-vehicle groups (Figure 23). It revealed that the neurite outgrowth of TG mice could be slightly enhanced but the neuron survival of TG mice could not be risen by LBH589 (Figure 23, 24, and 25). Therefore, we suggested the partial behavioral tests of LBH589-treated TG mice rescued by LBH589 was possible the molecular layer of TG-LBH589 mice was improved.. In cerebellar slice culture, the TBP aggregation of SCA17 cerebellum slice was decreased by LBH589 (Figure 3). However, in vivo study, the TBP aggregation of TG-LBH589 mice wasn’t decreased (Figure 24). We suggested the LBH589 could only inhibit the aggregation formation, but it couldn’t reduce the aggregation.. The gliosis is increased when central nervous system is impaired (do Nascimento et al., 2012). The gliosis of SCA17 transgenic mouse model would rise with the state of the SCA17 disease (Chang et al., 2011). We used GFAP and Iba1 to identify the gliosis of SCA17 transgenic at 12 week-old. The GFAP and Iba1 of TG-LBH589 groups were not reduced compared with TG-vehicle groups (Figure 24, 26). 30.

(34) The Bergmann glia cells of SCA17 transgenic mouse were significantly reduced at 12 week-old of age (Figure 25). LBH589-treated TG mice in both male and female were not increased the Bergmann glia cells (Figure 25). It suggested the cerebellum degeneration of SCA17 transgenic mouse model were not effectively inhibited by LBH589 treatment.. Previously, according to the behavior performance and the body weight, blood glucose, the appetite, and the weight of cerebellum of mice that were impacted by LBH589 compared with the vehicle-treated mice (Figure 9-14, 19-20, 27). Therefore, we suspected the liver or kidney would be impaired by LBH589 treatment. However, from toxicity studies, we found that the liver and kidney of LBH589 treated mice were showing less inflammation ( Figure 28 and Table 4) (Dokmanovic et al., 2007).. The mechanisms of LBH589 that impacted the central nervous system, the body weight, blood glucose, and the appetite were poorly understood (Figure 9, 10, 20, 27). There are no researches about LBH589 how to impact these. However, according to the diagnosis of liver and kidney (Figure 28 and Table 4) that the impairments of LBH589-treated mice was reduced and the report by Clive et al(Clive et al., 2012).It described that the excretion of radioactivity in urine and feces about patients with LBH589 was close to 100% of the dose recovered. Therefore, we eliminated the factors about the toxicity for LBH589 that caused to the heathy affected in LBH589 treated mice. 31.

(35) In summary, the appetite and the activity of LBH589 treated mice were impacted resulting in the body weight, blood glucose, and behavioral tests. We found only the cerebellum morphology was ameliorated by LBH589 (Figure 23). According to our studies, LBH589 showed limited the therapeutic effect for SCA17 disease.. 32.

(36) Reference. Abou-Sleymane G, Chalmel F, Helmlinger D, Lardenois A, Thibault C, Weber C, Merienne K, Mandel JL, Poch O, Devys D, Trottier Y. 2006. Polyglutamine expansion causes neurodegeneration by altering the neuronal differentiation program. Hum Mol Genet 15:691-703. Bickler PE, Fahlman CS. 2009. Expression of signal transduction genes differs after hypoxic or isoflurane preconditioning of rat hippocampal slice cultures. Anesthesiology 111:258-266. Birgbauer E, Rao TS, Webb M. 2004. Lysolecithin induces demyelination in vitro in a cerebellar slice culture system. J Neurosci Res 78:157-166. Cendelin J, Voller J, Vozeh F. 2010. Ataxic gait analysis in a mouse model of the olivocerebellar degeneration. Behav Brain Res 210:8-15. Cha TL, Chuang MJ, Wu ST, Sun GH, Chang SY, Yu DS, Huang SM, Huan SK, Cheng TC, Chen TT, Fan PL, Hsiao PW. 2009. Dual degradation of aurora A and B kinases by the histone deacetylase inhibitor LBH589 induces G2-M arrest and apoptosis of renal cancer cells. Clin Cancer Res 15:840-850. Chang YC, Lin CY, Hsu CM, Lin HC, Chen YH, Lee-Chen GJ, Su MT, Ro LS, Chen CM, Hsieh-Li HM. 2011. Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17. J Neurochem 118:288-303. Chuang DM, Leng Y, Marinova Z, Kim HJ, Chiu CT. 2009. Multiple 33.

(37) roles of HDAC inhibition in neurodegenerative conditions. Trends Neurosci 32:591-601. Clive S, Woo MM, Nydam T, Kelly L, Squier M, Kagan M. 2012. Characterizing the disposition, metabolism, and excretion of an orally active pan-deacetylase inhibitor, panobinostat, via trace radiolabeled 14C material in advanced cancer patients. Cancer Chemother Pharmacol 70:513-522. Crisanti MC, Wallace AF, Kapoor V, Vandermeers F, Dowling ML, Pereira LP, Coleman K, Campling BG, Fridlender ZG, Kao GD, Albelda SM. 2009. The HDAC inhibitor panobinostat (LBH589) inhibits mesothelioma and lung cancer cells in vitro and in vivo with particular efficacy for small cell lung cancer. Mol Cancer Ther 8:2221-2231. Davids E, Hevers W, Damgen K, Zhang K, Tarazi FI, Luddens H. 2002. Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABAA receptors. Eur Neuropsychopharmacol 12:201-208. do Nascimento AL, Dos Santos NF, Campos Pelagio F, Aparecida Teixeira S, de Moraes Ferrari EA, Langone F. 2012. Neuronal degeneration and gliosis time-course in the mouse hippocampal formation after pilocarpine-induced status epilepticus. Brain Res 1470:98-110. Dokmanovic M, Clarke C, Marks PA. 2007. Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 5:981-989. Duennwald ML. 2011. Polyglutamine misfolding in yeast: toxic and 34.

(38) protective aggregation. Prion 5:285-290. Duncan C, Papanikolaou T, Ellerby LM. 2010. Autophagy: polyQ toxic fragment turnover. Autophagy 6:312-314. Dusart I, Airaksinen MS, Sotelo C. 1997. Purkinje cell survival and axonal regeneration are age dependent: an in vitro study. J Neurosci 17:3710-3726. Eilam D. 2003. Open-field behavior withstands drastic changes in arena size. Behav Brain Res 142:53-62. El Idrissi A, Boukarrou L, Heany W, Malliaros G, Sangdee C, Neuwirth L. 2009. Effects of taurine on anxiety-like and locomotor behavior of mice. Adv Exp Med Biol 643:207-215. Fischer A, Sananbenesi F, Mungenast A, Tsai LH. 2010. Targeting the correct HDAC(s) to treat cognitive disorders. Trends Pharmacol Sci 31:605-617. Friedlander RM. 2003. Apoptosis and caspases in neurodegenerative diseases. N Engl J Med 348:1365-1375. Friedman MJ, Shah AG, Fang ZH, Ward EG, Warren ST, Li S, Li XJ. 2007. Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration. Nat Neurosci 10:1519-1528. Fujita Y, Morinobu S, Takei S, Fuchikami M, Matsumoto T, Yamamoto S, Yamawaki S. 2012. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene. J Psychiatr Res 46:635-643. 35.

(39) Gao Z, van Beugen BJ, De Zeeuw CI. 2012. Distributed synergistic plasticity and cerebellar learning. Nat Rev Neurosci 13:619-635. Garbes L, Riessland M, Holker I, Heller R, Hauke J, Trankle C, Coras R, Blumcke I, Hahnen E, Wirth B. 2009. LBH589 induces up to 10-fold SMN protein levels by several independent mechanisms and is effective even in cells from SMA patients non-responsive to valproate. Hum Mol Genet 18:3645-3658. Hashimoto M, Hibi M. 2012. Development and evolution of cerebellar neural circuits. Dev Growth Differ 54:373-389. He M, Zhang B, Wei X, Wang Z, Fan B, Du P, Zhang Y, Jian W, Chen L, Wang L, Fang H, Li X, Wang PA, Yi F. 2013. HDAC4/5-HMGB1 signalling mediated by NADPH oxidase activity contributes to cerebral ischaemia/reperfusion injury. J Cell Mol Med. Igelstrom KM, Shirley CH, Heyward PM. 2011. Low-magnesium medium induces epileptiform activity in mouse olfactory bulb slices. J Neurophysiol 106:2593-2605. Jia H, Pallos J, Jacques V, Lau A, Tang B, Cooper A, Syed A, Purcell J, Chen Y, Sharma S, Sangrey GR, Darnell SB, Plasterer H, Sadri-Vakili G, Gottesfeld JM, Thompson LM, Rusche JR, Marsh JL, Thomas EA. 2012. Histone deacetylase (HDAC) inhibitors targeting HDAC3 and HDAC1 ameliorate polyglutamine-elicited phenotypes in model systems of Huntington's disease. Neurobiol Dis 46:351-361. Kapfhammer JP. 2004. Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells. Prog Histochem 36.

(40) Cytochem 39:131-182. Kawai Y, Suenaga M, Watanabe H, Sobue G. 2009. Cognitive impairment in spinocerebellar degeneration. Eur Neurol 61:257-268. Kobayashi H, Abe K, Matsuura T, Ikeda Y, Hitomi T, Akechi Y, Habu T, Liu W, Okuda H, Koizumi A. 2011. Expansion of intronic GGCCTG hexanucleotide repeat in NOP56 causes SCA36, a type of spinocerebellar ataxia accompanied by motor neuron involvement. Am J Hum Genet 89:121-130. Koide R, Kobayashi S, Shimohata T, Ikeuchi T, Maruyama M, Saito M, Yamada M, Takahashi H, Tsuji S. 1999. A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA-binding protein gene: a new polyglutamine disease? Hum Mol Genet 8:2047-2053. Lafarga M, Andres MA, Calle E, Berciano MT. 1998. Reactive gliosis of immature Bergmann glia and microglial cell activation in response to cell death of granule cell precursors induced by methylazoxymethanol treatment in developing rat cerebellum. Anat Embryol (Berl) 198:111-122. Lee SC, Cheong HJ, Kim SJ, Yoon J, Kim HJ, Kim KH, Kim SH, Kim HJ, Bae SB, Kim CK, Lee N, Lee KT, Park SK, Hong DS, Park HS, Won JH. 2011. Low-dose combinations of LBH589 and TRAIL can overcome TRAIL-resistance in colon cancer cell lines. Anticancer Res 31:3385-3394. Lee SJ, Lim HS, Masliah E, Lee HJ. 2011. Protein aggregate spreading in neurodegenerative diseases: problems and perspectives. Neurosci 37.

(41) Res 70:339-348. Lemoine M, Younes A. 2010. Histone deacetylase inhibitors in the treatment of lymphoma. Discov Med 10:462-470. Lonchamp E, Dupont JL, Beekenkamp H, Poulain B, Bossu JL. 2006. The mouse cerebellar cortex in organotypic slice cultures: an in vitro model to analyze the consequences of mutations and pathologies on neuronal survival, development, and function. Crit Rev Neurobiol 18:179-186. Lordkipanidze T, Dunaevsky A. 2005. Purkinje cell dendrites grow in alignment with Bergmann glia. Glia 51:229-234. Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. 2001. Histone deacetylases and cancer: causes and therapies. Nat Rev Cancer 1:194-202. Markvartova V, Cendelin J, Vozeh F. 2010. Changes of motor abilities during ontogenetic development in Lurcher mutant mice. Neuroscience 168:646-651. Masino L. 2004. Polyglutamine and neurodegeneration: structural aspects. Protein Pept Lett 11:239-248. McCampbell A, Fischbeck KH. 2001. Polyglutamine and CBP: fatal attraction? Nat Med 7:528-530. Michishita E, Park JY, Burneskis JM, Barrett JC, Horikawa I. 2005. Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol Biol Cell 16:4623-4635. Mielcarek M, Benn CL, Franklin SA, Smith DL, Woodman B, Marks PA, 38.

(42) Bates GP. 2011. SAHA decreases HDAC 2 and 4 levels in vivo and improves molecular phenotypes in the R6/2 mouse model of Huntington's disease. PLoS One 6:e27746. Miyata T, Saito K, Nishizawa Y, Murayama A, Masaoka M, Ogawa M. 2005. Modern slice culture for direct observation of production and migration of brain neurons. Nagoya J Med Sci 67:65-70. Narita K, Kikuchi T, Watanabe K, Takizawa T, Oguchi T, Kudo K, Matsuhara K, Abe H, Yamori T, Yoshida M, Katoh T. 2009. Total synthesis of the bicyclic depsipeptide HDAC inhibitors spiruchostatins A and B, 5''-epi-spiruchostatin B, FK228 (FR901228) and preliminary evaluation of their biological activity. Chemistry 15:11174-11186. Neri P, Bahlis NJ, Lonial S. 2012. Panobinostat for the treatment of multiple myeloma. Expert Opin Investig Drugs 21:733-747. Noraberg J, Poulsen FR, Blaabjerg M, Kristensen BW, Bonde C, Montero M, Meyer M, Gramsbergen JB, Zimmer J. 2005. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Curr Drug Targets CNS Neurol Disord 4:435-452. Orr HT. 2012. Polyglutamine neurodegeneration: expanded glutamines enhance native functions. Curr Opin Genet Dev 22:251-255. Pipalia NH, Cosner CC, Huang A, Chatterjee A, Bourbon P, Farley N, Helquist P, Wiest O, Maxfield FR. 2011. Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human fibroblasts. Proc Natl 39.

(43) Acad Sci U S A 108:5620-5625. Popiel HA, Nagai Y, Fujikake N, Toda T. 2007. Protein transduction domain-mediated delivery of QBP1 suppresses polyglutamine-induced neurodegeneration in vivo. Mol Ther 15:303-309. Prut L, Belzung C. 2003. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3-33. Riessland M, Brichta L, Hahnen E, Wirth B. 2006. The benzamide M344, a novel histone deacetylase inhibitor, significantly increases SMN2 RNA/protein levels in spinal muscular atrophy cells. Hum Genet 120:101-110. Robinson R. 2006. Polyglutamine leads to altered chromatin architecture and neurodegeneration. PLoS Biol 4:e70. Sierra C, Coca A, Schiffrin EL. 2011. Vascular mechanisms in the pathogenesis of stroke. Curr Hypertens Rep 13:200-207. Slemmer JE, Haasdijk ED, Engel DC, Plesnila N, Weber JT. 2007. Aldolase C-positive cerebellar Purkinje cells are resistant to delayed death after cerebral trauma and AMPA-mediated excitotoxicity. Eur J Neurosci 26:649-656. Tabe Y, Jin L, Contractor R, Gold D, Ruvolo P, Radke S, Xu Y, Tsutusmi-Ishii Y, Miyake K, Miyake N, Kondo S, Ohsaka A, Nagaoka I, Andreeff M, Konopleva M. 2007. Novel role of HDAC inhibitors in AML1/ETO AML cells: activation of apoptosis and phagocytosis through induction of annexin A1. Cell Death Differ 40.

(44) 14:1443-1456. Witter DJ, Harrington P, Wilson KJ, Chenard M, Fleming JC, Haines B, Kral AM, Secrist JP, Miller TA. 2008. Optimization of biaryl Selective HDAC1&2 Inhibitors (SHI-1:2). Bioorg Med Chem Lett 18:726-731. Wood TE, Dalili S, Simpson CD, Sukhai MA, Hurren R, Anyiwe K, Mao X, Suarez Saiz F, Gronda M, Eberhard Y, MacLean N, Ketela T, Reed JC, Moffat J, Minden MD, Batey RA, Schimmer AD. 2010. Selective inhibition of histone deacetylases sensitizes malignant cells to death receptor ligands. Mol Cancer Ther 9:246-256. Yeh HH, Young D, Gelovani JG, Robinson A, Davidson Y, Herholz K, Mann DM. 2013. Histone deacetylase class II and acetylated core histone immunohistochemistry in human brains with Huntington's disease. Brain Res 1504:16-24. Yi J, Zhang L, Tang B, Han W, Zhou Y, Chen Z, Jia D, Jiang H. 2013. Sodium valproate alleviates neurodegeneration in SCA3/MJD via suppressing apoptosis and rescuing the hypoacetylation levels of histone H3 and H4. PLoS One 8:e54792. Yu ZX, Li SH, Evans J, Pillarisetti A, Li H, Li XJ. 2003. Mutant huntingtin causes context-dependent neurodegeneration in mice with Huntington's disease. J Neurosci 23:2193-2202. Zhuang X, Oosting RS, Jones SR, Gainetdinov RR, Miller GW, Caron MG, Hen R. 2001. Hyperactivity and impaired response habituation in hyperdopaminergic mice. Proc Natl Acad Sci U S A 98:1982-1987. 41.

(45) Figure and Table. 42.

(46) Figure 1.Analysis of the effect of NC001 serial compounds on TBP aggregation in SCA17 cerebellar slice culture. (A) The HDAC activity of compounds were analyzed by HDAC activity assay, TSA was used as a positive control for HDAC inhibitor. (B) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compounds NC001 (10nM ) and NC001-8 (10μM) treatment. (C) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation was significantly reduced by NC001 serial compounds (independent t-test; **, p<0.005; ***, p<0.001).. 43.

(47) 44.

(48) Figure 2. Analysis of the effect of NC005 serial compounds at high concentration on TBP aggregation in SCA17 cerebellar slice culture. (A) The HDAC activity of compounds were analyzed by using TSA as a positive control for HDAC inhibitor. (B) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compounds NC005-1 (60 nM), NC005-2 (166 nM), and NC005-4 (200 nM) treatment. (C) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation was significantly reduced by high concentration of NC005 serial compounds.However, The poor morphology of Purkinje neurons implied the high toxicity of these compounds (independent t-test; *, p<0.05).. 45.

(49) 46.

(50) Figure 3. Analysis of the effect of NC100 serial compounds on TBP aggregation and the morphology of cerebellum in SCA17 cerebellar slice culture. (A) The HDAC activity of compounds were analyzed by HDAC activity assay, TSA was used as a positive control for HDAC inhibitor. (B) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compounds NC00105 (10 nM), NC108 (10 nM), and NC109 (86 nM) treatment. (C) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation was significantly reduced and Purkinje cell morphology was maintained by NC100 serial compounds (independent t-test; ***, p<0.001).. 47.

(51) 48.

(52) Figure 4.Analysis of the effect of Chinese herbs on the morphology of cerebellum in SCA17 cerebellar slice culture. (A) The HDAC activity of Chinese herbs were analyzed by HDAC activity assay, TSA was used as a positive control for HDAC inhibitor. (B) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the Chinese herbs NH014 (100 ng/ml), and NH021 (100 ng/ml) treatment. (C) The quantification of TBP aggregationon the SCA17 cerebellar slice culture. The cell morphology was maintained after treatment.. 49.

(53) 50.

(54) Figure 5.Analysis of the effect of compound NH015-4 on themorphology of cerebellum in SCA17 cerebellar slice culture. (A)The HDAC activity of compound NH015-4 was analyzed by HDAC activity assay, TSA was used as a positive control for HDAC inhibitor. (B) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compound NH015-4 (10 nM) treatment. (C) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The Purkinje neurite outgrowth was enhanced by NH015-4.. 51.

(55) 52.

(56) Figure 6. Analysis of the effect of NC010 serial compounds on TBP aggregation and the morphology of cerebellum in SCA17 cerebellar slice culture. (A)The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compounds NC010-7 (100 μM), and NC010-11 (100 μM) treatment. (B) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation was reduced by NC010-7 but enhanced by NC010-11.. 53.

(57) 54.

(58) Figure 7. Analysis of the effect of WCt serial compounds on TBP aggregation in SCA17 cerebellar slice culture. (A) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the WCt003 (10 nM), WCt006 (10 nM),and WCt021 (20 nM) treatment. (B) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation was reduced and cell morphology was maintained by WCt serial compounds (independent t-test; *, p<0.05; **, p<0.005).. 55.

(59) 56.

(60) Figure 8. Analysis of the effect of compound NH043-1 on neuromorphology in SCA17 cerebellar slice culture. (A) The immunoflouroscence staining of the SCA17 cerebellar slice culture after the compound NH043-1 treatment. (B) The quantification of TBP aggregation on the SCA17 cerebellar slice culture. The TBP aggregation and cell morphology were maintained after the treatment of NH043-1.. 57.

(61) 58.

(62) Figure 9. The body weight of mice monitored during LBH589 treatment. LBH589 treatment siginificantly reduced the body weight in both male (A) and female (B) mice (independent t-test; *, compared WT-vehicle and WT-LBH589; #, compared TG-vehicle and TG-LBH589; +, compared WT-vehicle and TG-vehicle; *, #, +, p<0.05; **, ##, p<0.005; ***, ###, p<0.001).. 59.

(63) 60.

(64) Figure 10.The blood glucose of mice monitored during LBH589 treatment. LBH589 treatment siginificantly reduced the blood sugar in both male (A) and female (B) mice (independent t-test; *, compared WT-vehicle and WT-LBH589; #, compared TG-vehicle and TG-LBH589; +, compared WT-vehicle and TG-vehicle; *, #, +, p<0.05; ***, p<0.001).. 61.

(65) 62.

(66) Figure 11.Rotarod analysis of mice performed during LBH589 treatment. Compared vehicle treatment groups and drug treatment groups, the performance was no ameliorated by LBH589 in both male (A) and female (B) mice (independent t-test; ***, p<0.001; *, compared to WT groups).. 63.

(67) 64.

(68) Figure 12.The activities of mice were impacted by LBH589 treatment. The total distance in LBH589 treated mice was reduced at 11 week-old age in both male (A) and female (B)mice.The velocity was reduced at LBH589 treatment groups (C, D). The activity of mice was reduced by LBH589 (E, F) (independent t-test; *, p<0.05; **, p<0.005; ***, p<0.001).. 65.

(69) 66.

(70) Figure 13.The anxiety characterization with open-field on mice during LBH589 treatment. The locomotor activity between LBH589 treated mice and vehicle treated mice. The duration that mice stayed at central zone (A, B) and peripheral zone (C, D) showed no difference for both male (A, C) and female (B, D) mice (independent t-test; *, p<0.05).. 67.

(71) 68.

(72) Figure 14.The total mean intensity of mouse footprinting was analyzed during LBH589 treatment. The footprint intensity of SCA17 transgenic mice was reduced by LBH589 in both male (A) and female (B) mice (independent t-test; *, p<0.05; **, p<0.005; ***, p<0.001).. 69.

(73) 70.

(74) Figure 15.The step regularity of mouse footprinting analyzed during LBH589 treatment. The step sequence regularity of female mice with LBH589 was decreased compared to WT-vehicle female (B) mice at 4 weeks and 8 weeks (independent t-test; *, p<0.05; **, p<0.005).. 71.

(75) 72.

(76) Figure 16.The distance of mouse footprint analyzed during LBH589 treatment. The distance between front and hind (A, B) and right and left (C, D) paws of SCA17 transgenic mice were not affected by LBH589 in both male (A, C) and female (B, D) mice (independent t-test; *, p<0.05; **, p<0.005; ***, p<0.001).. 73.

(77) 74.

(78) Figure 17.The swing of mouse walking was affected by LBH589 treatment. The swing (turn to next walking) speed was decreased at TG-LBH589 mice in both male (A) and female (B) mice. The swing duration was maintained at TG-LBH589 male (C), and reduced at female (D) mice (independent t-test; *, p<0.05; **, p<0.005; ***, p<0.001).. 75.

(79) A. Single stance of Left-hind paws (male). Standing duration (s). 0.14. WT-Vehicle WT-LBH589 TG-Vehicle TG-LBH589. *. *. *. 0.12 *. *. *. *. 0.10 0.08 0.06 0.04 0.02 0.00 4. 8. 12. Weeks. B. Single stance of Left-hind paws (female). Standing duration (s). 0.14 0.12. WT-Vehicle WT-LBH589 TG-Vehicle TG-LBH589. * *. * *** * *. 0.10 0.08 0.06 0.04 0.02 0.00 4. 8 Weeks 76. 12. *.

(80) Figure 18.The standing duration in mouse footprinting was analyzed during LBH589 treatment. The standing of left-hind paw of LBH589 treated SCA17 TG mice were increased compared with vehicle treated SCA17 TG mice in both male (A) and female (B) mice (independent t-test; *, p<0.05; ***, p<0.001).. 77.

(81) 78.

(82) Figure 19. The performance related to rear up behaviors was analyzed in homecage. The action time of rear up was decreased in TG mice in both male (A, C) and female (B, D) (independent t-Test; *, p<0.05).. 79.

(83) 80.

(84) Figure 20.The weight of mouse brain analyzed after the treatment. LBH589 treatment had no effect on the brain growth in both male (A, C, E) and female (B, D, F) mice (independent t-test; *, compared to WT-vehicle; #, compared to WT-LBH589; ***, ###, p<0.001).. 81.

(85) 82.

(86) Figure 21.Mouse cerebellar Purkinje cells were analyzed after LBH589 treatment. Calbindin and aldolase C expression levels in mouse Purkinje neurons were both reduced. The aldoalse C of WT-LBH589 treatment female mice was also significantly reduced (independent t-test; *, p<0.05; **, p<0.005).. 83.

(87) 84.

(88) Figure 22.The gliosis and Hsp 70 were analyzed after LBH589 treatment. GFAP wasn’t reduced by LBH589 in both TG male and female mice. The Hsp 70 was significantly reduced in TG female mice, and wasn’t elevated with LBH589 treatment (independent t-test; *, p<0.05; ***, p<0.001).. 85.

(89) 86.

(90) Figure 23.The morphologies of cerebella of SCA17 transgenic mice analyzed after LBH589 treatment. The molecular layer (ML) of TG mice was maintained with LBH589 treatment in both male (A, C, E, G) and female (B, D, F, H).. 87.

(91) 88.

(92) Figure 24.The cerebellar TBP aggregation and gliosis were analyzed by immunofluorescence staining after LBH589 treatment. The TBP aggregation (red) and astrocyte (cyan) weren’t decreased during LBH589 treatment.. 89.

(93) 90.

(94) Figure 25.The Bergmann glia cells in mouse cerebellum were analyzed after LBH589 treatment. The Bergmann glia cells (cyan) were lost in SCA17 transgenic mice. LBH589 treatment has no effect on the level of Bergmann glia in both TG male and female.. 91.

(95) 92.

(96) Figure 26.The microglia cells in mouse cerebellum analyzed after LBH589 treatment The microglia cells were increased in SCA17 transgenic mice, and not affected by LBH589 treatment.. 93.

(97) A. Chow consumption (male) 5 *. Chow (g). 4 3 2 1 0 WT-V. B. WT-L. TG-V. TG-L. Chow consumption (female) ** 5. *. Chow (g). 4 3 2 1 0 WT-V. WT-L. 94. TG-V. TG-L.

(98) Figure 27.The chow consumption was measured in mice after the treatment. The chow consumption of SCA17 transgenic mice with LBH589 treatment was affected in both male (A) and female (B) mice (independent t-Test; *, p<0.05; **, p<0.005).. 95.

(99) 96.

(100) Figure 28. The impairment of liver and kidney was affected after LBH589 treatment. The impairment level of liver (A)and kidney (B)was reduced after LBH589 treatment. The imflammation and vacoulation of liver and the imflammation of kidney were improved by LBH589.. 97.

(101) Table 1. The HDACis influence th neurodegenerative diseases.. 98.

(102) Table 2. Summary of HDACi compounds/herbs used in this study Series. Drug. HDACi1. Used con.2. of HDACi3. Morphology. NC001 series. NC001. 1.11μM. 0.01μM. 100×. NC001-8. 1000μM. 10μM. NC005-1. 6μM. NC005 series. NC005-2. NC005-3. NC005-4. NC005-5. NC005-6. NC005-8. NC002-1. 83μM. 20μM. 10μM. 25μM. 45μM. 0.09μM. 38.4μM. aggregation. P value. --. Reduce 72.3%. P=0.001. 1/100×. --. Reduce 84.4%. P<0.001. 6μM. 1×. ---. 1.2μM. 1/5×. ---. 1μM. 1/6×. ---. 0.06μM. 1/100×. --. 83μM. 1×. ---. 8.3μM. 1/10×. ---. 0.42μM. 1/20× 4. ---. 0.166μM. 1/50×. --. 20μM. 1×. ---. 10μM. 1/2×. ---. 4μM. 1/5×. ---. 0.4μM. 1/50×. ---. 5μM. 1×. ---. 2μM. 1/5×. ---. 0.2μM. 1/50×. ---. 5μM. 1/5×. ---. 2.5μM. 1/10× 5. ---. 0.5μM. 1/50× 4. ---. 9μM. 1/5×. ---. 4.5μM. 1/10×. ---. 0.9 μM. 1/50× 4. ---. 0.1μM. 1×. ---. 0.01μM. 1/10× 4. ---. 0.2μM. 1/200× 5. ---. 99. Reduce 84.7%. Reduce 39%. P= 0.03. P =0.06. P = 0.03. Reduce 73%. Reduce. Reduce.

(103) NC004 series. NC004-1. 0.13μM. 0.06μM. 1/2× 5. ---. NC004-6. 0.33μM. 0.06μM. 1/5× 5. ---. NC100 series. NC102. 1.49μM. 0.015 μM. 1/100× 5. --. NC103. 3.88μM. 0.04μM. 1/100×. --. NC104. 4.33μM. 0.04μM. 1/100× 5. --. NC105. 0.01μM. NC106. 8.52μM. NC107. 0.09 μM. +++ 1/100× 5. Reduce 88.3%. P< 0.001. --. 0.001 μM. --++. Reduce 77.3%. P< 0.001. Reduce 73.1%. P< 0.001. NC108. << 10 M. 0.01μM. NC109. 8.67μM. 0.086μM. 1/100×. -. Y-S-01-32A. 0.71μM. 0.007μM. 1/100× 5. --. Y-S-01-32B. 6.97μM. 0.07μM. 1/100×. ---. NH015 seies. NH015-1. 5μM. 0.05 μM. 1/100× 5. -. No effect. NH015-4. 3.94μM. 0.01μM. 1/400×. -. No effect. Chinese. NH014. herbs. NH021 1. 0.72 mg/ml. 100 ng/μl. +. 100 ng/μl. +. IC50, 2 concentration, 3 the fold of HDACi IC50,4 N=2,5 N=1. 100.

(104) Table 3 Summary of other compounds used in this study Series. Drug. Used con.. Morphology. Aggregation. P value. 1. WCt series. NC010 series. 1. WCt003. 0.01 μM. ++. Reduce 50.8%. WCt006. 0.01μM. ++. Reduce 69.9%. P= 0.004. WCt021. 0.01μM. ++. Reduce 62.1%. P= 0.008. NC010-7. 100 M. ++. NC010-11. 100 M. -. Increase 71.2%. NH043-1. 0.01 μM. ++. No effect. concentration. 101. P= 0.04.

(105) Table 4.The inflammation of liver and kidney were analyzed after LBH589 treatment Histopathological findings of liver. ID. WT-vehicle. WT-LBH589. TG-vehicle. TG-LBH589. Postmortem Vacuolation autolysis. Inflammation, focal or multifocal. 145. 3. -. -. 743. -. -. -. 716. -. 3. -. 749. -. 4. 1. 740. -. -. 1. 176. -. 3. -. 753. -. -. -. 767. -. -. 2. 104. -. -. -. 719. -. -. -. 799. -. -. -. 722. 2. 2. -. 785. -. -. -. 147. -. -. 1. 106. 2. -. 1. 108. -. -. 1. 763. -. -. 1. 762. -. -. -. 142. -. -. -. 101. -. -. -. 759. -. -. -. 139. -. -. 2. 144. -. -. -. 122. -. -. -. -: no significant lesions Degree of lesions was grade from one to five depending on severity: 1 = minimal (< 1%); 2 = slight (1- 25%); 3 = moderate (26 - 50%); 4 = moderately severe (51 – 75%) 102.

(106) Histopathological findings of kidney. WT-vehicle. WT-LBH589. TG-vehicle. TG-LBH589. ID. Postmortem autolysis. Inflammation, focal. 145. 2. 1. 743. -. 1. 716. -. -. 749. -. -. 740. -. 1. 176. -. -. 753. -. -. 767. -. -. 104. -. -. 719. -. -. 799. -. -. 722. -. -. 785. -. -. 147. 2. -. 106. -. -. 108. -. -. 763. -. 1. 762. -. -. 142. -. -. 101. -. -. 759. -. -. 139. -. -. 144. -. -. 122. -. -. -: no significant lesions Degree of lesions was grade from one to five depending on severity: 1 = minimal (< 1%); 2 = slight (1- 25%); 3 = moderate (26 - 50%); 4 = moderately severe (51 – 75%). 103.

(107) Animal experiment time line. 104.

(108) Primary antibody list. Protein. Manufacturer. Titer. Source. Aldolase C. SantaCruz. 1:2500. goat. Calbindin. Sigma. 1:1000. mouse. Calbindin. Sigma. 1:1000. rabbit. Calbindin. SantaCruz. 1:100. goat. IP3R1. SantaCruz. 1:1000. goat. 1TBP18. QED. 1:30000. mouse. GFAP. Millipore. 1:1000. mouse. S100. Millipore. 1:1000. mouse. Iba1. Wako Pure. 1:1000. rabbit. Chemical β-actin. Millipore. 1:1000. mouse. HSP 70. Cell signaling. 1:1000. rabbit. TBP N12. SantaCruz. 1:2000. rabbit. 105.

(109) Secondary antibody list Antibody. Application. Manufacturer. Titer. Source. anti-mouse IgG, Alexa Fluor 555. Anti-1.Ab: 1TBP18. Invitrogen. 1:500. donkey. Invitrogen. 1:500. donkey. Invitrogen. 1:500. donkey. Invitrogen. 1:500. donkey. Vector. 1:200. goat. Vector. 1:200. goat. 1:10000. goat. Cell Signaling. 1:10000. goat. GE healthcare. 1:10000. donkey. (Immunofluorescence) anti-mouse IgG, Alexa Fluor Cyn5. Anti-1。Ab: S100/GFAP (Immunofluorescence). anti-rabbit IgG, Alexa Fluor 555. Anti-1。Ab: TBP N12 (Immunofluorescence). anti-goat IgG, Alexa Fluor 488. Anti-1。Ab: Calbindin/IP3R1 (Immunofluorescence). Biotinylated Goat Anti-Mouse IgG. Anti-1。Ab: Calbindin /GFAP (Immunohistochemistry). Biotinylated Goat Anti-Rabbit IgG. Anti-1。Ab: Iba1 (Immunohistochemistry). anti-rabbit IgG, HRP-linked. Anti-1。Ab: Calbindin/Hsp 70 Cell Signaling (Western blot). anti-mouse IgG, HRP-linked. Anti-1。Ab: -actin/GFAP (Western blot). anti-goat IgG, HRP-linked. Anti-1。Ab: Aldolase C (Western blot). 106.

(110) Abbreviation list. Abbreviation. Full name. CBP. CREB-binding protein. HDAC. histone deacetylase. HDACi. histone deacetylase inhibitor. Hsp70. heat shock protein 70. ML. molecular layer. polyQ. polyglutamine. SCA17. spinocerebellar ataxia type 17. SMA. spinal muscular atrophy. TBP. TATA box-binding protein. TG. transgenic. WT. wild type. WT-L/TG-L. WT-LBH589/TG-LBH589. WT-V/TG-V. WT-vehicle/TG-vehicle. 107.

(111)

參考文獻

相關文件

A change in treatment was performed in the group of sites where the estimated diagnosis was not con firmed by CBCT (6 sites with a change in treatment), in the group of the

Its main tool is the stem cells that are seeded on the surface of biomaterials (scaffolds), in order to create a biocom- plex. Several populations of mesenchymal stem cells are found

- In another study, when PDLSCs combined with stem cells from the dental apical papilla, (SCAPs), isolated from the 3rd molar, were seeded in a scaffold and

Treatment: Extraction of the retained supernumerary teeth and associated primary teeth in order to allow the permanent teeth to erupt.. 20 teeth were extracted under

From the left, epidermal disarray, plentiful small and bright cells, and scattered larger dendritic cells are apparent in the suprabasal epithelium, indicating likely LM (C), then a

Up-and-coming mandibular reconstruction technique with autologous human bone marrow stem cells and iliac bone graft in patients with large

They could also successfully form single-cell derived colonies (Fig.. Pyogenic granuloma-derived cells. A) After the initial seeding, many cells attached to the plates in an

Analyzed by type, most of the events were Corporate Meetings (163), Association Meetings (106) and Government Meetings (69).. The average duration increased from 2.1 days in the