以小鼠及細胞模式探討ATXN8OS過量表現之影響

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(1)國立臺灣師範大學生命科學系碩士論文. 以小鼠及細胞模式探討 ATXN8OS過量表現之影響 Characterization of ATXN8OS Overexpressed-PC12 Cells and Transgenic Mice. 研究生：王薏婷 Yi-Ting Wang 指導教授：謝秀梅博士 Hsiu-Mei Hsieh-Li. 中華民國九十七年七月.

(2) Index. Abstract (chinese)……………..………….…………….…1 Abstract………………………………………...….………3 Introduction……………………………….………..……...5 Materials and methods…………………………………....14 Results…………………………………………………….25 Discussion………………………………………………...34 References…………………………………………...........41 Table……………………………………………………....50 Figures…………………………………………………….55 Appendix…………………………………………….........99.

(3) 摘要脊髓小腦萎縮症第八型是一種漸進性的神經退化疾病，其致病的原因是由於染色體 13q21 上的 ATXN8OS (又稱作 SCA8,或 KLHL1AS) 基因在 3’端外顯子的 CTG 三核苷酸不正常擴增所造成。目前對於此疾病的詳細致病機轉尚未了解，早期相關文獻指出 ATXN8OS 基因並不具有轉譯蛋白的功能，而可能是以 anti-sense RNA 影響對應股的 KLHL1 基因表現功能；然而最近文獻指出 ATXN8OS 的反譯股 ATXN8，或許會轉譯出 polyQ 蛋白質，而 polyQ 蛋白質也正是許多型脊髓小腦萎縮症的致病原因；另外，也有研究指出在 ATXN8OS 基因上 CTG 三核苷酸擴增區域前，可能具有 IRES 活性，而使得此三核苷酸擴增轉譯出 polyL 毒性蛋白質。為了更進一步去了解 ATXN8OS 致病的分子機制，我們因而建立了 ATXN8OS 的轉殖小鼠模式。利用原核胚顯微注射的方法，將人類 ATXN8OS 基因帶有正常範圍(22)及致病範圍(150)兩種 CTG 擴增的基因片段，並連接了綠螢光報導基因，藉由 NSE 啟動子表現在小鼠的神經系統，以進一步了解 CTG 擴增對於小鼠造成的影響。從實驗結果中，我們發現帶有致病 CTG 擴增的轉殖小鼠，在小腦中有神經受損及缺失的情況發生，此外藉由行為測試也觀察到小鼠的異常行為，我們同時也將相同的基因架構轉殖進大鼠嗜鉻細胞瘤細胞株(PC12)，建立離體的模式，藉此平臺可快速. 1.

(4) 觀測 ATXN8OS 過度表現會對於神經細胞所產生的影響；從實驗中我們也發現到帶有致病範圍 CTG 擴增的細胞，在低血清及神經生長因子的分化刺激下，比正常的細胞更容易死亡，且其神經分化的能力也較差，由這些研究結果，我們發現不管 ATXN8OS 過度表現在離體及活體內均造成神經傷害，這些結果將有利於在相關疾病的基因及藥物治療研究之應用。. 2.

(5) Abstract Spinocerebellar ataxia type 8 (SCA8) was reported caused by an unstable CTG repeat expansion in the 3’ terminal exon of ATXN8OS (also named SCA, or KLHL1AS) gene on chromosome 13q21. How the trinucleotide expansion causes the disease is not clear now. Some studies indicate that ATXN8OS might not encode protein and play an anti-sense regulatory role on the sense strand gene, KLHL1. However, a recent study indicates that the opposite strand of ATXN8OS, ATXN8, encodes a polyglutamine gain-of-function. expansion mutation. protein, of. which SCA8. might. explain. disease. as. the other. polyglutamine-mediated SCAs. To further investigate the molecular mechanism of SCA8, a transgenic mouse model was established. The human ATXN8OS full-length cDNA with 22 or 150 CTG repeats in-frame fused with flag-EGFP was used as the transgene and driven by a neuron-specific-enolase (NSE) promoter. Our results show that the transgenic mice with expanded CTGs have some neuropathologies in the cerebella, including the neuronal cell loss and behavior abnormality. To have a quick platform to access the ATXN8OS overexpression effect on neuronal cell level, we also transfected constructs of ATXN8OS with 22 or 150 CTG repeats into the rat pheochromocytoma (PC12) cell line. We found that cells with expanded 150 CTG repeats were more vulnerable and showed reduced neuronal differentiation under low serum and NGF condition compared to cells with normal CTG expansion. With these models, we could gain some information about the molecular effects resulted from overexpression of ATXN8OS in vivo and in vitro, which 3.

(6) should further provide more implications for the therapeutic design of SCA8 in the future.. 4.

(7) Introduction. Spinocerebellar ataxias (SCAs). The word “ataxia” derived from Greek, refers to “incoordination or without order”. In 19th century, this disease has been identified and called “Friedreich’s ataxia”, an inherited disease that causes progressive damage of the nervous system. The symptoms include gait disturbance, speech problems and heart disease. Until year 1983, Dr. Pierre Marie noticed that inherited ataxia disease was different from Friedreich's ataxia because its onset was much later then Friedreich's ataxia. According to pathogenesis, they were classified into two types: early onset-Friedreich's ataxia and late onset-Marie's ataxia. Therefore, ataxias at that time were roughly divided into Friedreich's ataxia and Marie's ataxia, based on the age of onset. The ataxias are a complex group of debilitating and often fatal neurodegenerative diseases that lead to generalized incoordination of gait, speech and limb movements (Currier, 1984; Harding, 1993). The spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of dominantly inherited neurodegenerative disorders, characterized by prominent ataxia and cerebellar atrophy and degeneration of the cerebellum and its connections (Glickstein, 2006; Izumi et al., 2003; Juvonen et al., 2002; Koob et al., 1999; Schmahmann and Caplan, 2006). Some of these disorders also affect the retina, peripheral nerves, the pyramidal and extra pyramidal motor systems, and 5.

(8) cognitive function. Spinal cord, brainstem, basal ganglia, or peripheral nervous system are also involved in some of these disorders (Glickstein, 2006; Izumi et al., 2003; Juvonen et al., 2002; Koob et al., 1999; Schmahmann and Caplan, 2006; Zeman et al., 2004). SCAs. were. initially. classified. according. to. clinical. and. neuropathological descriptions. Dr. Harding classified autosomal dominant cerebellar ataxias (ADCAs) into three types, according to the mode of inheritance and the clinical signs. ADCA type І is characterized by a progressive cerebellar syndrome, with additional but variable associated features of supranuclear ophthalmoplegia, optic atrophy, mild dementia, peripheral neuropathy or extrapyramidal dysfunction. SCA1, SCA2,. SCA3,. SCA4,. SCA8,. SCA12,. SCA17,. dentatorubral. pallidoluysian atrophy (DRPLA) and recently defined SCA27 and SCA28 belong to this type (Flanigan et al., 1996; Imbert et al., 1996; Kawaguchi et al., 1994; Orr et al., 1993; Pulst et al., 1996; Sanpei et al., 1996). ADCA type II shows cerebellar ataxia accompanied with pigmentary macular dystrophy, maybe pathogenesis before 10 years old and only SCA7 is included (Del-Favero et al., 1998; Giunti et al., 1999; Harding, 1993). ADCA type III is a pure cerebellar syndrome and comprises SCA5, SCA6, SCA10, SCA11, SCA14, SCA15, SCA22, and SCA26. SCA13 does not fall within any categories described above (Ranum et al., 1994; Worth et al., 1999; Zhuchenko et al., 1997). It has now defined 28 types of SCA. According to clinical symptoms, SCA is categorized by radiological point of view, 3 patterns of atrophy can be observed: a pure cerebellar atrophy (SCA4, SCA5, SCA6, SCA8, SCA9, SCA10, SCA11, SCA14, SCA15, SCA16, SCA18, 6.

(9) SCA21, SCA22), a pattern of olivopontocerebellar atrophy (SCA1, SCA2, SCA3, SCA7, SCA13), or a pattern of global cerebral atrophy (SCA12, SCA17, SCA19, DRPLA). Most of the SCAs start by a cerebellar atrophy which can progress subsequently to extracerebellar structures. The prevalence of SCAs is estimated to be 1-4/10000, but it can be much higher in some regions because of a founder effect (van de Warrenburg et al., 2002). This is the case for SCA2 in Cuba, SCA3 in the Azores (Orozco Diaz et al., 1990; Silveira et al., 1998), and SCA10 has been reported in Mexico. Different SCAs may sometimes have overlapping signs and it is difficult to distinguish by clinical symptoms. However, with advance of molecular biotechnology, mutation analyses afforded the genetic classification of clinical subtypes. SCAs are largely caused by the unexpected prolongation of tandem nucleotide repeats. In SCAs due to a CAG expansion (SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, and DRPLA) associated with expansions of coded CAG repeats translated into polyglutamine (polyQ) stretch that adds a toxic protein (David et al., 1997; Imbert et al., 1996; Kawaguchi et al., 1994; Koide et al., 1994; Orr et al., 1993; Pulst et al., 1996; Zhuchenko et al., 1997), and an untranslated CTG repeat being found in SCA8 (Koob et al., 1999), the promoter CAG repeat expansion in SCA12, SCA10 with expansions in intronic ATTCT (Holmes et al., 1999; Matsuura et al., 2000). In addition, SCA5, SCA14, and SCA27 have been found caused by missense mutations (Brusse et al., 2006; Ikeda et al., 2006; Yabe et al., 2003).. Spinocerebellar ataxia type 8 (SCA8) 7.

(10) SCA 8 is an autosomal dominant late-onset neurodegenerative disorder, first described in 1999, associated with CTG trinucleotide repeat lying on chromosome 13q21 (Koob et al., 1999). SCA8 also is the first example of a dominant SCA not caused by a CAG expansion translated as a polyQ tract. Dysarthria, mild aspiration and gait instability are commonly the initial symptoms. Exam findings included spastic and ataxic dysarthria, nystagmus, limb and gait ataxia, limb spasticity and diminished vibration perception, Pyramidal and sensory features have been described in around one-third of cases with cognitive features, including dementia. One report indicated mother and son of a familiy with the SCA 8 expansion, cerebellar ataxia, personality change and a dysexecutive syndrome (Stone et al., 2001). A recent study concluded that the disorder is associated with deficits of attention, executive function and information processing (Lilja et al., 2005; Zeman et al., 2004). One other research have reported highly variable rates of cognitive and behavioural features (Zeman et al., 2004), and with clinical findings, magnetic resonance imaging (MRI) showed cerebellar atrophy, and cognitive impairments especially those related to attention, information processing and executive function (Lilja et al., 2005). The dominant inheritance pattern of the large SCA8 kindred is complicated, showing reduced penetrance with an extreme maternal penetrance bias (Koob et al., 1999). The SCA8 transcript, containing the CTG expansion, has been identified in brain tissue and weakly in lung, kidney, and testis (Benzow and Koob, 2002). 8.

(11) With 110~130 CTA/CTG combined repeats in the affected individuals, a short polymorphic CTA repeat (1–21 CTAs) precedes the major CTG repeat, the sizing of SCA8 alleles has been clarified in various populations, with unrelated expanded alleles ranging from 68 to 800 repeats found in familial and sporadic ataxia patients (Day et al., 2000; Ikeda et al., 2000; Izumi et al., 2003; Juvonen et al., 2002; Moseley et al., 2000; Tazon et al., 2002; Topisirovic et al., 2002). Quoted repeat lengths are usually for the CTA/CTG composite. Over 99% of individuals in control populations have fewer than 91 CTA/CTG repeats (Zeman et al., 2004), the prevalence of repeat expansions in such populations is much higher than the prevalence of the disorder, indicating that the expansion has a low penetrance and may be best regarded as a risk factor for ataxia (Torrens et al., 2008). Recent data suggest that bidirectional transcription of ATXN8OS (SCA8) occurs, ataxin8 (ATXN8), which encodes a polyQ protein in the CAG orientation, and ATXN8OS transcribed to potentially pathogenic CUG transcripts. Because both CUG expansion transcripts and polyQ expansion proteins are both known to be toxic in other diseases SCA8 may involve both RNA and protein gain of function mechanisms (Ikeda et al., 2008; Moseley et al., 2006; Paulson, 2006).. Heredity of Spinocerebellar ataxia. “Anticipation” refers to the phenomena the disease transmitted to the next generation begins at more earlier age. This phenomena is found in trinucleotide repeat expansion disease, including myotonic dystrophy, 9.

(12) Huntington’s disease, SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA12, SCA17, SCA22, and DRPLA.. SCA8 and KLHL1. In humans, there are three different genes ATXN8, ATXN8OS (SCA8 or KLHL1AS) and Kelch-like 1 (KLHL1) located in relatively close. The genomic organization of the overlapping ATXN8 and ATXN8OS genes and KLHL1 gene is shown in Appendix Fig 3. The SCA8 expansion mutation is located within both of the overlapping ATXN8OS and ATXN8 genes. In contrast, the KLHL1 genes overlap at 5’exon of ATXN8OS, approximately 35 kb upstream of the repeat. These three genes are summarized as follows: (i) ATXN8, which contains the repeat expansion, is transcribed in the CAG direction and encodes a nearly pure polyQ expansion protein; (ii) ATXN8OS spans the repeat in the CTG direction with the repeat of this highly alternatively spliced and apparently non-protein coding gene; (iii) KLHL1 encodes an actin binding protein expressed in the central nervous system and does not overlap with the repeat (Moseley et al., 2006). Transcripts encoded by both the ATXN8 and ATXN8OS genes are expressed at low steady-state levels in the human central nervous system. Although the SCA8 repeat is not conserved in the mouse (Andres et al., 2004; Andres et al., 2003; Benzow and Koob, 2002), while there is a mouse homolog of KLHL1 (Klhl1) and a much shorter version of the human ATXN8OS gene (Klhl1as), the mouse Klhl1as gene is much simpler with only a single exon and conservation with only the 5’ end of the human ATXN8OS (Benzow and 10.

(13) Koob, 2002).. KLHL1 ( kelch like protein 1 ). KLHL1 protein is actin binding protein, and is homology to Drosophila kelch protein. This protein family contains two highly conserved signature domains: a BTB/POZ domain involved in protein dimerization (Bardwell and Treisman, 1994; Zollman et al., 1994), and the actin-binding Kelch domain (Adams et al., 2000). The mammalian KLHL1 is a brain-specific cytosolic protein that can form multimers and bind actin filaments (Nemes et al., 2000). KLHL1 can interact with the α1A subunit of the P/Q-type Ca2+ channel and modulate its function by increasing its current density and channel availability for opening, suggesting that KLHL1 can modulate calcium channel, process neuronal calcium influx and excitability. (Aromolaran et al., 2007).. IRES (internal ribosome entry site). Initiation of translation often depends on 5’ m7G cap, which interact to 43S ribosome with the cap binding protein eIF4E (Sonenberg, 1994). A cap-independent mechanism called IRES was first demonstrated in picornaviruses, which lack a 5’ m7G cap and have long structured 5’UTRs in their RNA. The presence of an internal ribosome entry site (IRES). has. been. shown. in. different. picornaviruses,. such. as. encephalomyocarditis virus (EMCV), human rhinoviruses, and hepatitis 11.

(14) A virus (Jackson et al., 1995). This mechanism requires secondary structures that allow ribosomes to bind directly next to the initiator AUG and permit translation to start without previous scanning. Recently, IRES elements have been found in several cellular mRNAs, and also found in a variety of other mammalian genes. ATXN8OS was first thought a non-coding gene (Koob et al., 1999), but a recent genetic examination revealed that ATXN8OS may encode three ORFs, ORF1, ORF2, and ORF3, through IRES mechanism. Among them ORF1 is a 102 amino acids, and ORF3 is a 41 amino acids in front of a stretch of polyleucine encoded by the CUG repeats. If ATXN8OS have the cap-independent IRES activity, these three ORFs maybe translated and their pathogenesis need to be further investigated is shown in Appendix Fig. 6 (Lin, 2007).. Complete Control® Inducible Mammalian Expression System (Stragagene). The. ecdysone. receptor. (EcR). is. a. member. of. the. retinoid-X-receptor (RXR) family of nuclear receptors. In mammalian cells, EcR heterodimerizes with RXR and binds to multiple copies of the ecdysone-responsive element (EcRE). In the absence of PonA, transcription of expression cassette will be shut down. When PonA binds to the receptor, the receptor complex activates transcription of an interested gene. To avoid pleiotropic interactions with endogenous pathways in mammalian host cells, the EcRE recognition sequence was modified and renamed as E/GRE; the EcR protein was also modified 12.

(15) (called GEcR, glucocorticoid receptor –EcR fusion proteins) that won’t transactivate any host genes, retained the ability to dimerize with RXR and activate interested genes in E/GRE cassettes. In Complete Control® Inducible Mammalian Expression System, pERV3 vector was designed for GEcR with VP16 activation domain; pEGSH vector was designed for E/GRE with PonA-inducible cassette, multiple cloning sites (MCS) for inserting the gene of interest, and FLAG epitope.. 13.

(16) Materials and methods. Transgene construction and SCA8 transgenic mouse generation. Human full length ATXN8OS cDNAs with 22 or 150 CTG repeats fused with flag tag and EGFP reporter gene were used as transgene for generation of SCA8 transgenic mice. The ATXN8OS-flag-EGFP was driven by NSE promotor, which mainly expresses transgene in neuronal cells. The transgene was separated from the plasmid backbone by digested with MluI and injected into mouse pronuclei to generate transgenic mice. One ATXN8OS-150R transgenic line with FVB genetic background was established in our lab (Chen, 2006). The remaining ATXN8OS-22R and ATXN8OS-150R with B6 genetic background were generated by core facility of Institute of Molecular biology Academia Sinica.. Mouse Genotyping. Mouse genomic DNAs were isolated from 0.5 cm length of tail biopsy. Tissues were first lysed in tail solution [10 mg/ml proteinase K, 2% SDS, 5 M NaCl, 0.5 M EDTA (pH 8.0), 1 M Tris (pH 7.4)] at 65℃ overnight. The mixture was then mixed with 5 M potassium acetate and incubated at 4℃ for 1 hour followed by centrifugation at 13400 xg at 4℃ for 30 minutes. The supernatant was transferred to a fresh tube and mixed well with ethanol. DNA was precipitated by centrifugation at 13400 xg at 14.

(17) 4℃ for 5 minutes and then rinsed with 70% ethanol. DNA pellet was air dried and resuspended with 65℃ ddH2O. Transgenic mice were identified using polymerase chain reaction (PCR) with a set of primers localized in NSE promotor (forward: 5’-AAT AAG GAG ACG CCT GCT TCC CTC-3’), and ATXN8OS cDNA (reverse: 5’-CAT GTC AGG CTC TGG GCG AAA AG-3’). The 818 bp fragment product was amplified from the PCR by condition: 95℃, 1 minute for denaturing, 58℃(touch down) 1 minute for primer annealing, 72℃1 minute for elongation, and 35 cycles for total reaction.. Semi-quantitative PCR. To distinguish the homozygous mice from heterozygous mice, we used semi-quantitative PCR to analysis mouse genotypes with a forward primer (NSE-F: 5’-AAT AAG GAG ACG CCT GCT TCC CTC-3’) and a reverse primer (SCA8-R: 5’-CAT GTC AGG CTC TGG GCG AAA AG-3’). The 818 bp product was amplified 95℃1 minute for denaturing, 58℃ (touch down) 1 minute for primer annealing, 72℃1 minute for elongation and repeated for 30 cycles. forward primer (β-actin-F: 5’-ATG GAT GAC GAT ATC GCT-3’) and a reverse primer (β-actin-R: 5’-ATG AGG TAG TCT GTC AGG T-3’). The 1100 bp product was amplified by a PCR condition, 95℃1 minute for denaturing, 54℃ 1 minute for primer annealing, 72℃1 minute for elongation and repeated for 30 cycles. The 1100 bp β-actin product was used as an internal control for the semi-quantitation. Primers of β-actin were 3 times of the genotyping primers. 15.

(18) RNA isolation from mouse cerebellum and reverse transcription. RNA isolation from mouse tissue was conducted using Trizol reagent (Invitrogen). The procedure was carried out according to the manufacturer’s instructions. Trizol reagent 300 µl was added to tissue, after pipeting mix, the mixture was centrifuged at 13400 xg at 4°C for 15 minutes. The supernatant was collected and 60 µl of chloroform was added into the supernatant. Samples were vigorously inverted by hand for 10 times. RNA was then separated form DNA and proteins by centrifugation at 13400xg for 15 minutes at 4°C. Following centrifugation, the colorless supernatant was carefully transferred to a fresh tube avoiding touch the interface. 150 µl volume of isopropyl alcohol was added to the supernatant, following a 10 minutes incubation at 4°C RNA was precipitated by centrifuged at 13400xg at 4°C for 10 minutes. The pellets were rinsed once with 75% ethanol, RNA were air dried and dissolved in 65°C RNase-free water. After DNase treatment, 2μg of RNA was reverse transcribed into cDNA using the SuperScriptTM Ш reverse transcriptase (Invitrogen). The SCA8-F1 (5’-TTG AAG ATT GCC TTT TCT GAC TCC C -3’) and EGFP-R (5’-ACT CCA GCA GGA CCA TGT G -3’) primers were used to amplify cDNA fragments to assess the expression of transgene transcripts.. Behavioral testing A. Rotarod analysis. This task was used to assess motor coordination, balance, and motor 16.

(19) learning. We used a rotrod (UGO 47600) to assess the ability of SCA8 and wild type mice remaining on a rotating rod as the speed of rotation was fixed 25 rpm for 180 seconds. An entire testing process required four days (the first two days for training and the last two days for testing), three trials per day for each mouse. In each test, the mice were subjected to the three trials with a 40 minutes of rest between trials, and the average time to fall off the rod was determined and series of testing in coming months were completed in two days per month. Series of rotarod analysis were processed on mice every four weeks.. B. Locomotor activity monitoring. Mice were placed in an open field (30 cm x 30 cm x 30 cm black box) and allowed to explore the environment for 10 minutes. Using Etho-Vision video tracking system, we evaluated distance of horizontal movement, movement velocity and rearing frequency of each mouse in 10 minutes, and collected parameters of 10 minutes for paired-samples T test analysis.. Perfusion. Mice were anesthetized with 2.5% avertin (18 μl/g mouse weights) and then perfused with 4% paraformaldehyde. We inserted a 23 3/4 gauge needle in the aorta below the renal arteries and started perfusion with 0.9% normal saline for replacing blood and then exchanged with 4% paraformaldehyde until the limbs of mice were found totally rigid. Mouse 17.

(20) brain was then removed and cerebellum was cut and postfixed in 4% paraformaldehyde in 1X PBS for 24 hours. The tissues were then dehydrated by transferred into 10% sucrose in 1X PBS for 1 hour, 20% sucrose in 1X PBS for 2 hours, 30% sucrose in 1X PBS for 48 hours, and cut into 30 µm thick in sagittal sections.. Immunohistochemistry (IHC). The cerebellum sections floating in 1X PBS were prepared for IHC. Sections were first washed with 1X PBS 10 minutes for three times. Endogenous peroxidase activity of sections was blocked by 3% H2O2 for 30 minutes. After washed with 1X PBS for twice, nonspecific epitopes of sections were then blocked by 4% BSA for 2 hours. Sections were then incubated at room temperature with primary antibody diluted with blocking solution overnight. After three times wash with 1X PBS for 10 minutes each, cerebellum sections were incubated with linking reagent (DAKO) for 1 hour, washed with 1X PBS two times, and then incubated with labeling reagent (DAKO) for 1 hour. After washed with 1X PBS two times, immunostainings of sections were performed using DAB (diaminobenzidine, DAKO) and substrate for 10 seconds to 3 minutes. After washed with 1X PBS for four times, all floating sections were mounted on coated slides. After air-dried overnight, slides were ethanol dehydrated, and cover slipped for light microscopy.. Western blot analysis. 18.

(21) Brain tissue was lysed in a RIPA buffer containing 1M Tris (pH 7.5), 5M NaCl, 0.5M EDTA (pH 8.0), 10% SDS, 10% DOS, and 10% NP40 supplemented with protease inhibitors. Samples were sonicated and determined the protein concentrations using a BCA protein assay kit (PIERCE). Samples were incubated at 95°C for 10 minutes and loaded to SDS gels. After polyacrylamide gel electrophoresis (PAGE), proteins were transferred onto PVDF membrane (Millipore), followed by blocking with 5% nonfat skim milk in 1X PBS containing 0.05% Tween20 for 2 hours. Blots were incubated with primary antibodies 4°C overnight and secondary antibodies at room temperature for 1 hour. Secondary antibodies were diluted (1:5000) with 3% nonfat skim milk in 1X PBS containing 0.05% Tween20. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) reagent (chemiluminescent HRP substrate, Millipore).. In-situ hybridization. Expression of mRNAs in cerebellum tissues was determined by in-situ hybridization. First, we cut cerebellum tissue into 30 μm sections and then incubated slices at 37℃ overnight. We used constructs pGEM-T/NSE-ATXN8OS-Flag-EGFP which was establish for generating our transgenic mice, and PCR primers, Flag-F (5’- TGA CTA CAA AGA CGA TGA CG -3’) and EGFP-R ( 5’- CTT GAA GAA GAT GGT GCG -3’) to amplify probe templates. The templates were purified with same volume of phenol-chloroform extraction and 2.5X volume 99% ethanol 19.

(22) precipitation. The RNA pellets were then rinsed once with 1 ml 70% ethanol, air-dried and dissolved in 65°C RNase-free water. A mixture of digoxigenin(DIG)-labeled RNA mix 2 μl, 10×buffer 2 μl, RNase inhibitor 1 μl, RNA polymerase 2 μl was added into purified template, than RNase-free water was added to a 20 μl of volume. After DNase treatment for 30 minutes, the mixture was heated at 65°C for 10 minutes to stop DNase activity. 2X volume of 99% ethanol precipitation, then pellets were rinsed once with 1 ml 70% ethanol, RNA were air dried and dissolved. in. 65°C. RNase-free. water.. In. vitro. transcribed. digoxigenin(DIG)-labeled RNA products used as probes were purified by phenol-chloroform extraction and ethanol precipitation. The RNA probe was produced using DNA template, RNA polymerase (Roche) and DIG RNA labeling Mix (Roche), DIG were hybridized with labeled sense or antisense RNA probes generated from PCR product using in vitro transcription. Sections on slides were first treated with 0.2N HCl for 10 minutes, washed with 1XPBS, treated with 20 mg/ml proteinase K at 37℃ for 5 minutes, acetylated with 0.25% acetic acid and 0.1M triethanolamine. (TEA). for. 10. minutes,. postfixed. with. 4%. paraformaldehyde at 4℃ for 10 minutes. After washed with 1X PBS, slides were then prehybridized at 60℃ for 2 hours. After prehybridization buffer removed, slideswere incubated with probe (400 ng/ml) in hybridization buffer (100% formamide 5 ml, 20X SSC 2.5 ml, 50% Dextrane sulphate 2 ml, 20% SDS 0.5 ml, PVP (poly-vinylpyroline) 0.1 gm, 100X Denhardt’s solution 0.1 ml, 10 mg/ml Salmon sperm 0.1 ml) at. 20.

(23) 60℃ overnight. After wash with 2X SSC at 42℃, 1X SSC (plus 0.1% Tween 20) at 37℃, 0.2X SSC (plus 0.1% Tween 20) at room temperature for 10 minutes each for twice. Slides were then washed with buffer 1 (100 mM Tris-HCl,150 mM NaCl，pH7.5 ) for 5 minutes, two times, then blocked in 1% blocking reagent for 30 minutes. After washed with buffer 1 for 5 minutes, sections were then hybridized with anti-DIG antibody (1:8000 diluted with 1% blocking reagent) at room temperature for 2 hours. After wash with buffer1 for 5 minutes for two times, slides were then washed with buffer 2 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH9.5) for 10 minutes. Color reactions were carried out in buffer 2,. using. nitroblue. tetrazolium. chloride. (NBT). and. 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Roche) as substrates, until appropriate signals were founded. Slides were washed with buffer 2 for 10 minutes, counterstained with 1% methyl green for 1 second, rinsed by RNase-free water, air dry at 37℃ overnight, and cover slipped for light microscopy.. Establishment of ATXN8OS inducible construct. ATXN8OS cDNAs with 22 or 150 CTG repeat fused with Flag tag were digested from transgenic mouse construct (Chen, 2006) by restriction NotI and SalI, and then sub-cloned into NotI/SalI site of pEGSH vector. The completed constructs were shown in Fig. 2.. 21.

(24) Cell culture and transfection of PC12 cell. PC12. cells,. originally. derived. from. rat. adrenal. gland. pheochromocytoma, were maintained in poly-L-lysine-coated plates containing 85% RPMI 1640 media (GIBCO) supplemented with 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l D-glucose, 1 M HEPES, and 1 mM sodium pyruvate, and incubated in 5% CO2，37℃ incubator. Cells were seeded on poly-L-lysine coated plate overnight prior to transient transfection. PC12 cells were transfected using Lipofectamine 2000. as. the. manufacturer’s. instructions. (Invitrogen).. Clonal. PC12/pERV3 cells were established by transfected with 2 µg pEGSH vector DNA (Wang, 2007). or pEGSH/ ATXN8OS with different (CTG) repeat length respectively 6 μl lipofectamine 2000 and 600 μl RPMI-1640 (without serum), then pipeting mixed well, and incubated for 20 minutes at room temperature. At DNA-lipofectamine 2000 complexs formation, then add 2400 μl RPMI-1640 (without serum) mixed well, then added onto 3-cm dish which PC12/pERV3 cells were already cultured last night. After transfection, we utilized 20 mg/ml G418 for pERV3 cell selection and 10 mg/ml hygromycin B for pEGSH/ATXN8OS cell selection.. Nerve Growth Factor (NGF) treatment of PC12 cells. The PC12 cells were plated at a density of 104 cells/cm2 and allowed to adhere overnight, and the medium was replaced with RPMI 22.

(25) medium supplemented with 0.5% serum medium and 50 ng/ml NGF to induce neurites sprouting for 48 hours.. MTT assay. MTT assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and form a dark blue formazan crystals which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. We first added 0.5 mg/ml MTT into cells. After 2 hours of incubation in 37℃, we then dissolved formazan crystals with dimethyl sulfoxide (DMSO), measured OD570 values with a multiwell scanning spectrophotometer (Bio-Tek Microplate autoreader EL311). Through MTT assay, we evaluated the survival rate of cells.. Immunocytochemistry (ICC). Differentiated PC12 cells grown on 6-cm culture dish were fixed in cold 4% paraformadehyde solution for 10 minutes at room temperature. After washes with 1X PBST (0.2% Tween-20 included) four times, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 minutes, followed by washing three times. The non-specific protein binding sites will be blocked with 5% fetal bovine serum (FBS) for 2-4 hours, the cells were then incubated overnight in 4℃ with primary ATXN8OS-ORF1 23.

(26) anti-serum (1:200), which was diluted with 5% FBS solution. After four times washing with 1X PBST for 10 minutes each, cells were incubated with anti-rabbit secondary antibody (green fluorescence, Alex Fluor® 488 Donkey anti-mouse IgG) for 1 hour at room temperature and light protection. The cells were then washed with 1X PBST and counterstained with 4', 6-diamidino-2-phenylindole (DAPI, sigma) for 3 minutes, and removed DAPI then add 1X PBS into dish. The stain cells were observed with Leica microscope.. 24.

(27) Results. Generation and characterization of inducible ATXN8OS cell lines The system was previously established in our labwith a Complete Control® Inducible Mammalian Expression System by transfection of pERV3 vector into PC12 cells (Wang, 2007).. We further expressed. ATXN8OS/22R, and ATXN8OS/150R in this system (Fig. 1). After treated nerve growth factor (NGF), PC12 cell was differentiated to a neuron-like cell, then we added 5 µM PonA, ATXN8OS expression will be induced. Expression plasmids were constructed by ATXN8OS cDNAs with 22 or 150 CTG repeat fused to Flag. The constructs were then transfected into PC12/pERV3 cells by lipofectamine 2000 (invitrogen). The ATXN8OS cDNAs were digested from transgenic mouse constructs (Chen, 2006) with NotI and SalI, and then sub-cloned into pEGSH vector. The completed constructs were shown in Fig. 2. In order to selected stable line, PC12/pERV3 cells with pEGSH vector, pEGSH/ATXN8OS/22R, or pEGSH/ATXN8OS/150R constructs, were selected by 20 mg/ml G418 and 10 mg/ml hygromycin for two weeks. stable cell lineswere induced by 5 µM of PonA for 24 and 48 hours And toal RNAs were isolated for RT-PCR assay to identify the expression of ATXN8OS transcripts (Fig. 3).. Identification of ATXN8OS-ORF1 expression by western blot It was previously reported that no coding open reading frame (ORF) in ATXN8OS was identified (Koob et al., 1999), however, three small 25.

(28) cap-independent IRESmediated-ORFs were noted, ORF1, 102 amino acids, ORF2, and ORF3, 41 amino acids including the polyCUG-encoded polyleucine. By western blot assay, we used ATXN8OS-ORF1 anti-serum (kindly provided by Dr. Lee-Chen, NTNU), and detected a possible ORF1. band. of. around. 24. kDa. from. cells. with. constructs. pEGSH/ATXN8OS/22R and pEGSH/ATXN8OS/150R (Fig. 4).. Viability test of inducible ATXN8OS cell lines MTT assay results reveal stable cells with ATXN8OS-150R show significantly reduced cell number after PonA induction for 48 hours and 72 hours. However, cells with pEGSH vector or ATXN8OS-22R overexpression showed no significant difference after PonA treatment (Fig. 5).. Generation of inducible PC12/ATXN8OS clonal cell lines In order to obtain a more uniform expression and phenotypes of cells, we selected clonal cells from stable cell population. Two individual clonal cell lines were selected from stables cells with pEGSH/ATXN8OS/22R (lines 2 and 12) and pEGSH/ATXN8OS/150R (lines 1 and 4) by RT-PCR assay (Fig. 6).. Viability test of ATXN8OS inducible clonal cell lines To investigate the effect of ATXN8OS expansion overexpression in PC12 cells, MTT assay was conducted and the results reveal clonal cells with ATXN8OS-150R (line-1) show significantly reduced cell number after PonA induction for 72 hours, cells with ATXN8OS-150R (line-4) 26.

(29) show significantly reduced cell number after PonA induction for both 48 and 72 hours, and cells with pEGSH vector show significantly reduced cell number after PonA induction for 24 hours. However, cells with ATXN8OS-22R overexpression showed no significant difference after PonA treatment (Fig. 7).. Identification of ATXN8OS-ORF1 expression in clonal cells by Immunocytochemistry (ICC) ICC analysis with ATXN8OS-ORF1 anti-serum, showed that after 48 hours NGF treatment and then PonA induction, clonal ATXN8OS-150R (lines 1 and 4) and ATXN8OS-22R (lines 2 and 12) cells have stronger staining than non- induced cells (Fig. 8).. Morphology of ATXN8OS inducible clonal cell lines To investigate the effect of ATXN8OS expansion overexpression in PC12 cells, we characterized cell morphological change during the PonA induction for 48 hours. We examined the cell morphology during differentiation (after NGF treatment for 48 hours) because our preliminary MTT assay results reveal significant difference between cells with vector only and clonal cell lines with ATXN8OS-150R under differentiation states (Figs. 5 and 7). In order to identify the morphology of these cells, we measured the neurite length and numbers of these cells after PonA induction 48 hours during NGF treatment. The measurement was conducted in ten individual areas of each culture dish. Both neurite length and numbers of cells with ATXN8OS-150R are significant decreased compared to cells with ATXN8OS-22R and vector only (Figs. 27.

(30) 10 and 11). In addition, we also found ATXN8OS-150R cell neurites are thinner and weaker than ATXN8OS-22R and vector only cells (Fig. 9).. Generation and characterization of ATXN8OS transgenic mouse To generate transgenic mice expressing human ATXN8OS gene. Human ATXN8OS cDNA with D, C2, C1, B and A, bearing normal CTG/CTA repeat (22R) or expanded CTG/CTA repeat (150R) fused to flag and EGFP (kindly provided by Dr. Lee-Chen, NTNU), were subcloned behind the NSE promoter (Fig. 12A). The transgen fragments were released from the constructs by restriction enzyme MluІ and the resulted 4.1- and 4.6-kb fragments were used for microinjection to generate transgenic mice (Fig. 12B).. ATXN8OS transgene caused the Purkinje cell loss in transgenic mice To observe the transgene effect in ATXN8OS150R transgenic mice, we used IHC analysis with calbindin D28k antibody (sigma) to assess the numbers and pattern of the cerebellar Purkinje cells. First, we found transgenic mouse cerebellum at the age of 7 weeks have mild loss of Purkinje cells (Fig. 13), and at the age of 64 weeks, Purkinje cell loss is significant (Fig. 14). At the age of 76 weeks, we found not only the reduced number of Purkinje cells, but also the progress cell death. We also quantified the Purkinje cells at the stage (n=3), and our results indicate Purkinje cell loss significantly in the mice at age of 76 weeks (Fig. 15).. ATXN8OS cause the neuronal loss in transgenic mouse cerebellum 28.

(31) To understand the effect of ATXN8OS on the other neurons of cerebellum, we used IHC analysis with NeuN antibody to assess the numbers of the neuron cells. At the age of 76 weeks, we observed neuron loss in deep cerebellar nuclei (DCN) of transgenic mice (Fig. 16). We also used microtubule associated protein-2 (MAP2) antibody to exam the cerebellum of transgenic mice, at 76 weeks, we found DCN of transgenic mouse have more significant signal of MAP2 (Fig. 17). There is no difference observed in GFAP positive astrocyte staining between transgenic and wild type mice (Fig. 18).. ATXN8OS encodes translable ORF3 In order to exam the existence of ORF3 in ATXN8OS transgenic mice, we used antibody against Flag-tag to detect ORF-3-flag fusion protein in transgenic mice. In DCN of transgenic mice, we did observed stronger Flag signal than that in DCN of wild type mice (Fig. 19).. Identification neuron markers in ATXN8OS-150R mouse cerebella by western blot analyses Our preliminary IHC data showed significant difference between heterozygous transgenic and wild type mice, we performed western blot to confirm these data. We used three antibodies to detect neuron status for 4 month-old mice. First, we used NeuN to detect neuronal nuclei, is significant difference between transgenic and wild type mice (upper panel, Fig. 20). Second, we used calbindin to detect Purkinje cells, we also cannot founded significant difference (middle panel, Fig 20). Finally, we used GFAP to detect glial cells, astrocytes, and gliosis, TG2 and TG3 of 29.

(32) ATXN8OS-150R transgenic mice showed reduced signals compared to wild type (lower panel, Fig. 20). Since SCA8 is a late-onset disease, so we conducted the same analysis on 64 week-old mice. We found TG1 showed reduced staining of calbindin and NeuN, and increased GFAP staining (Fig. 23).. Identification of apoptosis marker in mouse cerebella by western blot analyses Our preliminary western blot data indicate neuron loss and gliosis occurred in some transgenic mice (Fig. 23), we thus used three widely used antibodies (BAX, Bcl-2 and Cyt c) to detect apoptosis of these transgenic mice. When mice were 4-month-old, some transgenic mice showed significantly increased staining of Bcl-2, BAX and cyt c (Fig. 21). However, when mice were 64-weeks-old, no significant Bcl-2 staining was identified (Fig. 24).. Identification of ATXN8OS-ORF1 expression in mouse cerebellum by western blot analysis We used ATXN8OS-ORF1 anti-serum (kindly provided by Dr. Lee-Chen, NTNU) and detected a possible ORF1 band around 24 kDa in cells. with. constructs. pEGSH/ATXN8OS/22R. and. pEGSH/ATXN8OS/150R (Fig. 4), and mouse cerebella (Fig. 22).. Different phenotypes between 150R transgenic littermates Some transgenic mice have more obvious phenotypes, showing small size, weakness and kyphosis (curvature of the spine) (Figs. 25A and 25B). 30.

(33) Clasping phenotype was also observed with all feet holding together when the mouse tail was suspended (Fig. 25C). And the end of the disease, we found some of adult male mice showed priapism phenotype (Fig. 25D). We thus checked testis histology to understand morphology of germ cells. The result of Nissl staining reveals that transgenic mouse sperm numbers are less than that of wild type, and germ cells with abnormal morphology were also identified in transgenic mouse testis (Fig. 26).. Behavior characterization of heterozygous transgenic mice by performance on the accelerating rotarod To exam the motor performance and coordination activity, we tested mice on the rotarod with speed from 2 to 20 rpm during first 5 minutes, and maintained at 20 rpm for another 5 minutes, and to record their latency in the rod. Our results showed that ATXN8OS-150R transgenic mice could stayed longer time on rotarod than wild type littermates at 12 weeks old of age (Fig. 27). We further modified the rotarod program from 2 to 40 rpm for the first 5 minutes and maintain at 40 rpm for another 5 minutes and observed that ATXN8OS-150R mice still could stay longer on rotarod than wild type littermates at 32, 36 and 40 weeks old of age (Fig. 28).. Locomotor activity of heterozygous transgenic mice Locomotor activity is good preliminary tests to determine motor deficits and exploratory, as assessed in the open field. From the results of locomotor analysis of 64-week-old mice, we found no significant 31.

(34) difference between heterozygous transgenic mice and wild type mice in distance moved, velocity and rearing frequency (Fig. 29).. Behavior characterization of homozygous transgenic mice by performance on the rotarod In order to generate homozygous mice, we used semi-quantative PCR to identify homozygous mice from heterozygous mice. We obtained three homozygous mice further breeding of more homozygous mice for behavior test (Fig. 30). To exam the motor performance and coordination activity, we tested mice on the rotarod with fixed 25 rpm speed for 3 minutes, and to record their latency in the rod. During the 4-month period, we observed that homozygous mice showed shorter latency on rotarod than wild type mice (Fig. 31). And the behavior results were not affected by mouse body weight because there was no significant weight difference between transgene and wild type mice (Fig. 31).. Locomotor activity of homozygous transgenic mice At 8, 14 and 22 week-old, mouse locomotor activity were characterized and the results showed homozygous mice moved less distance than wild type mice (Fig. 32). In addition, significant difference in average velocity was also identified between homozygous and wild type mice at 8, 14 and 22 weeks old, with ATXN8OS-150R homozygous mice have a slower velocity (Fig. 32). At 8 and 14 week-old, the rearing frequency were higher in homozygous mice than wild type mice, however, the result was reversed at 22 weeks old (Fig. 32). 32.

(35) Establishment of ATXN8OS transgenic mice in B6 genetic background In order to generate ATXN8OS-22R normal repeat transgenic mice and more lines of ATXN8OS-150R transgenic mice for further characterization of the effect of long CTG repeat ATXN8OS gene, we have used the same transgenic constructs for pronuclear microinjection with the help of Core facility of Institute of Molecular Biology, Academia Sinica. From RT-PCR result, we chose three lines (44, 45 and 46) from ATXN8OS-22R and five lines (29, 30, 33, 34 and 38) from ATXN8OS-150R F1 transgenic mice for further breeding and analyses. We assessed the expression of transgene in the cerebellum by RT-PCR (Fig. 33). ATXN8OS expression was identified in several regions of the cerebellum of ATXN8OS-22R mouse by in situ hybridization, such as cerebellar lobe (Fig. 34J), the deep cerebellar nuclei (Fig. 34K), and the brain stem (Fig. 34L). In the cerebellar lobe, significant expression of ATXN8OS mRNA was observed in the molecular layer and Purkinje cell (Fig. 34J). ATXN8OS expression was also found in the similar regions of the cerebellum of ATXN8OS-150R mouse, such as the cerebellar lobe (Fig. 35K), the deep cerebellar nuclei (Fig. 35L), and the brain stem (Fig. 36G, 36H). In the cerebellar lobe, significant expression of ATXN8OS mRNA was observed in the molecular layer and Purkinje cell (Fig. 35K).. 33.

(36) Discussion. Cell model Inducible system of PC12/pERV3 cells with pEGSH vector was previously established in our lab (Wang, 2007). We used the system to transfect construct pEGSH/ATXN8OS/22R or pEGSH/ATXN8OS/150R, then selected by hygromycin and G418. After pEGSH/ATXN8OS/22R and pEGSH/ATXN8OS/150R was induced by PonA, we can detect ATXN8OS transcripts expression in stable and clonal cell lines. In previous report, ATXN8OS contains six exons and is a non-coding CUG expanded RNA (Koob et al., 1999). From our result, we also found ATXN8OS was transcribed successfully in our inducible system. ATXN8OS is characterized by RT-PCR with repeat expansion segments in untranslated regions (UTRs). These RNAs may recruit splicing factors and transcription factors, and resulting in ribonucleoprotein aggregates (RNA foci) (La Spada et al., 2004). The formation of RNA foci might mis-regulation of splicing signaling or regulatory pathways. The effect of the repeats on splicing mis-regulation requires an intact CUGBP1-binding site (CUG-BP1 response element) within affected pre-mRNAs (Ho et al., 2005). In our result, we observed growth rate of cells with ATXN8OS-150R is less than cells with ATXN8OS-22R or vector only\, suggesting CTG expansion exert more cytotoxicity than normal repeat, and RNA foci maybe regulate growth factor, make it mis-regulation to cause growth rate slowly. In addition, other report indicated when cells with CTG-250 treated with NGF, cytotoxicity was increased in the cells. 34.

(37) And the CTG repeat bearing mRNA showed cis-effects through the reporter gene and neuronal death after cell differentiation in vitro (Furuya et al., 2005). From our MTT assay results, we found viability in cells with long CTG expansion is reduced compared to cells with normal repeat after NGF treatment. ATXN8OS has been proven to be an intact transcript which has six exons and alternative polyadenylation sites, but ATXN8OS revealed no translatable ORFs existing of any splice isoforms (Nemes et al., 2000). Initiation of translation often depends on 5’ m7G cap, which interact to 43S ribosome with the cap binding protein eIF4E (Sonenberg, 1994). And ATXN8OS transcripts 5’UTR containing a G/C rich region which maybe serve as a recognition site of trans interacting factor (Nemes et al., 2000). In recently report, ATXN8OS was suggested to have IRES activity, and revealed the existence of three small ORFs (Lin, 2007). In our research, we used ecdysone regulatory inducible system to overexpress ATXN8OS/22R and ATXN8OS/150R in PC12 cell line. Expression ORF1 protein with different length CTG repeat were identified after cells were induction by PonA for 48 hours. And we used ICC to confirm this result, Cells were treated with NGF to induced differentiation for 48 hours, and than induce with PonA for 48 hours, we can observe ORF1 expressed in whole cell body. Although both cells with ATXN8OS/22R and ATXN8OS/150R have ORF1 expression, we suggest ORF1 may regulate ATXN8OS toxicity, only ORF1 in cells with expanded repeats will induce its toxicity. The expanded CTG90 repeats was reported to inhibit neuronal differentiation in a PC12 cell line (Quintero-Mora et al., 2002). Our result 35.

(38) also showed that ATXN8OS/150R has reduced differentiation activity compared to cells with normal repeats or vector. In summary, we have established the cell system in which ATXN8OS/22R and ATXN8OS/150R can be induced expressed. Both the cell viability and neuronal differentiation of ATXN8OS/150R were reduced than control cells with short repeats or vector only. These results suggest that RNA and/or ORF1 expression from expanded CTG repeats of ATXN8OS make the cells more vulnerable than cells with normal repeats. This model provides an in vitro system might be useful for the SCA8 therapeutic design in the future.. Mouse model Transgenic mice expressing ATXN8OS-150R transcripts were characterized by behavior testing and IHC to exploit the pathogenesis and to understand whether the expanded trinucleotide repeat mutations of ATXN8OS can involve gain of function mechanism as myotonic dystrophy (DM1) (Gatchel and Zoghbi, 2005). IHC analysis can provide a sensitive method in examing the morphology of cerebellum. Calbindin expression was high in Purkinje cell dendrite and cell body in the molecular layer; it’s also a marker of cerebellar toxicity (Haworth et al., 2006). In our result, with calbindin immunostaining, we found Purkinje cell loss and degeneration severely in the cerebella of ATXN8OS-150R transgenic mice. It has been proved that Purkinje cell loss is a major contributing factor to motor performance dysfunction in P/Q channel-related ataxias (Walter et al., 2006).. 36.

(39) In our IHC studies, we using NeuN antibody to observe the functional changes in the cerebellum of our ATXN8OS-150R mice and found neuronal cell loss in DCN. In addition, in the DCN we characterized the MAP2 (microtubule-associated protein 2) expression, MAP2 familiy is an abundant group of cytoskeletal component with functions including stabilization of microtubule, the regulation of organelle transport within axons and dendrites, and regulation of some proteins about signal transduction (Sanchez et al., 2000). In our study, we found stronger staining of MAP2 in DCN of transgenic mice, which reflect cerebellar ataxia may in part be initiated as a consequence of increase DCN (deep cerebellar nuclei) excitability secondary to loss of inhibitory input from Purkinje cell that frequently degenerate or alter function (Grusser-Cornehls and Baurle, 2001). These results represent our transgenic DCN is under hyperexcitability which maybe secondary to loss of Purkinje cells. In our westren blot studies, among the three 64-week-old transgenic mice, we can observed one mouse has neuronal loss (calbindin and NeuN decreased) and gliosis (GFAP increased), however, these results were not found at 4 month-old mice, which suggests a late-onset disease of our mouse model with neuronal-pathogenesis detected at older age. Apoptosis, also known as programmed cell death, is an active process of cell death which controls cell numbers in a variety of tissues during development and adult life (Raff et al., 1993). The Bcl-2 family of proteins plays a central role in regulating apoptosis. It has been well established that these proteins function at the mitochondria to prevent or promote the release of apoptogenic factors such as cyt c. Bcl-2, a major 37.

(40) anti-apoptotic protein located primarily in the outer mitochondrial membrane, inhibits apoptosis by preventing cyt c release from the mitochondria and inhibiting caspase activation, while Bax, which shares extensive amino acid homology with Bcl-2, acts as a functional antagonist to Bcl-2 (Buchholz et al., 2003). The neuronal cell loss of our transgenic mice maybe through a process of apoptosis. From western bloting results, we observed Bcl-2, BAX and cyt c were increased in some mouse cerebella at both 16- and 64-weeks-old stages. Bcl-2 during development can protect neurons from both physiological and induced cell death, represent in development stage Bcl-2 maybe involved in protection of neurons during the period in which half of the neurons are undergo apoptosis (Farlie et al., 1995). Whether the apoptotic prpcess indeed occurred in our transgenic mouse model will need further characterization of expression of other apoptotic effectors, eg. caspase-3. Rota-rod assay is the most widely used test to assess coordination and overall motor function in rodents. This test is affected by experimental damage to the basal ganglia and cerebellum as well as genetic manipulations and drugs that effect motor function. This test can be carried out as a single measure or multiple times to determine cerebellar learning. In our study, there is no significant difference in the rotarod performance between heterozygous and wild-type mice, suggesting the motor activity and coordination of the heterozygous mice was sustained at the testing stages. A later onset might be identified in these heterozygous mice if we continually conduct the task. In order to generate mice with more severe phenotypes, we set up heterozygous mouse breeding to obtain homozygous mice. The rota-rod assay of the young 38.

(41) homozygous mice reveals that the latency to fall in homozygous mouse was even shorter than that in wild type control animals. We anticipate to find a poorer performance in homozygous mice in later stage. In the locomotor activity examination, the total of distance, average velocity and rearing frequency are measured. Our test results indicated that total distance and average velocity in the homozygous transgenic mice are significantly less than that in wild type mice, representing our ATXN8OS-150R homozygous transgenic mice have motor activity deficits. The test is sensitive to motor dysfunction as well as hippocampal and basal ganglia damage. Further pathological examination of these area should be conducted for elucidation of this question. About progressive neurological phenotype of our transgenic mouse model, we founded some mice with severe phenotypes, such as small size, curvature of the spine and priapism. These phenotypes are similar to BAC-ATXN8OS transgenic mice (Moseley et al., 2006). Priapsim, among these progressive neurological phenotype, was happened at the end of disease, which may cause by spinal cord curvature and the blood back-up to make hematoma of the end. Reduced penetrance is associated with SCA8 patients. For our transgenic mice, whether the expanded repeat bearing ATXN8OS reduce transcription efficiency or unstable IRES activity making severe phenotypes occurred in only some of the transgenic mice remains further study. Our. ATXN8OS. transgenic. mouse. model. demonstrates. the. morphology effects caused by the ATXN8OS expansion and provides a. 39.

(42) system for further characterizing other trinucleotide disorders, such as myotonic dystrophy1 and Huntington disease like 2. For a more reasonable study, we have already established ATXN8OS transgenic mice in B6 genetic background. With comparison between mice bearing normal repeats (ATXN8OS-22R) and expanded repeats (ATXN8OS-150R). Our research results can provide more insight of the pathogenesis of SCA8 or other neurodegenerative diseases.. 40.

(43) References. 王馨慧。(2007)。Valproic acid 在多麩醯胺酸擴增細胞及動物模式之療效評估。台灣師範大學研究所碩士論文。林玄原。(2007)。脊髓小腦運動失調症之族群遺傳分析與 CTG 三核苷重複擴增的分子致病研究。台灣師範大學研究所博士論文。陳韋倫。(2006)。脊髓小腦萎縮症第八型致病基因分析及建立脊髓小腦萎縮症第八型離體及活體模式。台灣師範大學研究所碩士論文。 Adams J, Kelso R, Cooley L. The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol 2000; 10: 17-24. Andres AM, Soldevila M, Lao O, Volpini V, Saitou N, Jacobs HT, et al. Comparative genetics of functional trinucleotide tandem repeats in humans and apes. J Mol Evol 2004; 59: 329-39. Andres AM, Soldevila M, Saitou N, Volpini V, Calafell F, Bertranpetit J. Understanding the dynamics of Spinocerebellar Ataxia 8 (SCA8) locus through a comparative genetic approach in humans and apes. Neurosci Lett 2003; 336: 143-6. Aromolaran KA, Benzow KA, Koob MD, Piedras-Renteria ES. The Kelch-like protein 1 modulates P/Q-type calcium current density. Neuroscience 2007; 145: 841-50. Bardwell VJ, Treisman R. The POZ domain: a conserved protein-protein interaction motif. Genes Dev 1994; 8: 1664-77. 41.

(44) Benzow KA, Koob MD. The KLHL1-antisense transcript ( KLHL1AS) is evolutionarily conserved. Mamm Genome 2002; 13: 134-41. Brusse E, de Koning I, Maat-Kievit A, Oostra BA, Heutink P, van Swieten JC. Spinocerebellar ataxia associated with a mutation in the fibroblast growth factor 14 gene (SCA27): A new phenotype. Mov Disord 2006; 21: 396-401. Buchholz TA, Davis DW, McConkey DJ, Symmans WF, Valero V, Jhingran A, et al. Chemotherapy-induced apoptosis and Bcl-2 levels correlate with breast cancer response to chemotherapy. Cancer J 2003; 9: 33-41. David G, Abbas N, Stevanin G, Durr A, Yvert G, Cancel G, et al. Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat Genet 1997; 17: 65-70. Day JW, Schut LJ, Moseley ML, Durand AC, Ranum LP. Spinocerebellar ataxia type 8: clinical features in a large family. Neurology 2000; 55: 649-57. Del-Favero J, Krols L, Michalik A, Theuns J, Lofgren A, Goossens D, et al. Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion. Hum Mol Genet 1998; 7: 177-86. Farlie PG, Dringen R, Rees SM, Kannourakis G, Bernard O. bcl-2. transgene. expression. can. protect. neurons. against. developmental and induced cell death. Proc Natl Acad Sci U S A 1995; 92: 4397-401. Flanigan K, Gardner K, Alderson K, Galster B, Otterud B, 42.

(45) Leppert MF, et al. Autosomal dominant spinocerebellar ataxia with sensory axonal neuropathy (SCA4): clinical description and genetic localization to chromosome 16q22.1. Am J Hum Genet 1996; 59: 392-9. Furuya H, Shinnoh N, Ohyagi Y, Ikezoe K, Kikuchi H, Osoegawa M, et al. Some flavonoids and DHEA-S prevent the cis-effect of expanded CTG repeats in a stable PC12 cell transformant. Biochem Pharmacol 2005; 69: 503-16. Gatchel JR, Zoghbi HY. Diseases of unstable repeat expansion: mechanisms and common principles. Nat Rev Genet 2005; 6: 743-55. Giunti P, Stevanin G, Worth PF, David G, Brice A, Wood NW. Molecular and clinical study of 18 families with ADCA type II: evidence for genetic heterogeneity and de novo mutation. Am J Hum Genet 1999; 64: 1594-603. Glickstein M. Thinking about the cerebellum. Brain 2006; 129: 288-90. Grusser-Cornehls U, Baurle J. Mutant mice as a model for cerebellar ataxia. Prog Neurobiol 2001; 63: 489-540. Harding AE. Clinical features and classification of inherited ataxias. Adv Neurol 1993; 61: 1-14. Haworth R, McCormack N, Selway S, Pilling AM, Williams TC. Calbindin D-28 and microtubule-associated protein-2: their use as sensitive immunohistochemical markers of cerebellar neurotoxicity in a regulatory toxicity study. Exp Toxicol Pathol 2006; 57: 419-26. He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD. Targeted deletion of a single Sca8 ataxia locus allele in mice causes 43.

(46) abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 2006; 26: 9975-82. Ho TH, Savkur RS, Poulos MG, Mancini MA, Swanson MS, Cooper TA. Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy. J Cell Sci 2005; 118: 2923-33. Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, et al. Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 1999; 23: 391-2. Ikeda Y, Daughters RS, Ranum LP. Bidirectional expression of the SCA8 expansion mutation: One mutation, two genes. Cerebellum 2008. Ikeda Y, Dick KA, Weatherspoon MR, Gincel D, Armbrust KR, Dalton JC, et al. Spectrin mutations cause spinocerebellar ataxia type 5. Nat Genet 2006; 38: 184-90. Ikeda Y, Shizuka M, Watanabe M, Okamoto K, Shoji M. Molecular and clinical analyses of spinocerebellar ataxia type 8 in Japan. Neurology 2000; 54: 950-5. Imbert G, Saudou F, Yvert G, Devys D, Trottier Y, Garnier JM, et al. Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats. Nat Genet 1996; 14: 285-91. Izumi Y, Maruyama H, Oda M, Morino H, Okada T, Ito H, et al. SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients, including those with SCA6. Am J Hum 44.

(47) Genet 2003; 72: 704-9. Jackson RJ, Hunt SL, Reynolds JE, Kaminski A. Cap-dependent and cap-independent translation: operational distinctions and mechanistic interpretations. Curr Top Microbiol Immunol 1995; 203: 1-29. Juvonen V, Kairisto V, Hietala M, Savontaus ML. Calculating predictive values for the large repeat alleles at the SCA8 locus in patients with ataxia. J Med Genet 2002; 39: 935-6. Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, et al. CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet 1994; 8: 221-8. Koide R, Ikeuchi T, Onodera O, Tanaka H, Igarashi S, Endo K, et. al.. Unstable. expansion. of. CAG. repeat. in. hereditary. dentatorubral-pallidoluysian atrophy (DRPLA). Nat Genet 1994; 6: 9-13. Koob MD, Moseley ML, Schut LJ, Benzow KA, Bird TD, Day JW, et al. An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). Nat Genet 1999; 21: 379-84. La Spada AR, Richards RI, Wieringa B. Dynamic mutations on the move in Banff. Nat Genet 2004; 36: 667-70. Lilja A, Hamalainen P, Kaitaranta E, Rinne R. Cognitive impairment in spinocerebellar ataxia type 8. J Neurol Sci 2005; 237: 31-8. Matsuura T, Yamagata T, Burgess DL, Rasmussen A, Grewal RP, Watase K, et al. Large expansion of the ATTCT pentanucleotide 45.

(48) repeat in spinocerebellar ataxia type 10. Nat Genet 2000; 26: 191-4. Moseley ML, Schut LJ, Bird TD, Koob MD, Day JW, Ranum LP. SCA8. CTG. repeat:. en. masse. contractions. in. sperm. and. intergenerational sequence changes may play a role in reduced penetrance. Hum Mol Genet 2000; 9: 2125-30. Moseley ML, Zu T, Ikeda Y, Gao W, Mosemiller AK, Daughters RS, et al. Bidirectional expression of CUG and CAG expansion transcripts. and. intranuclear. polyglutamine. inclusions. in. spinocerebellar ataxia type 8. Nat Genet 2006; 38: 758-69. Nemes JP, Benzow KA, Moseley ML, Ranum LP, Koob MD. The SCA8 transcript is an antisense RNA to a brain-specific transcript encoding a novel actin-binding protein (KLHL1). Hum Mol Genet 2000; 9: 1543-51. Orozco Diaz G, Nodarse Fleites A, Cordoves Sagaz R, Auburger G. Autosomal dominant cerebellar ataxia: clinical analysis of 263 patients from a homogeneous population in Holguin, Cuba. Neurology 1990; 40: 1369-75. Orr HT, Chung MY, Banfi S, Kwiatkowski TJ, Jr., Servadio A, Beaudet AL, et al. Expansion of an unstable trinucleotide CAG repeat in spinocerebellar ataxia type 1. Nat Genet 1993; 4: 221-6. Paulson HL. If it's not one thing, it's another. Nat Genet 2006; 38: 743-4. Pulst SM, Nechiporuk A, Nechiporuk T, Gispert S, Chen XN, Lopes-Cendes I, et al. Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2. Nat Genet 1996; 14: 269-76. 46.

(49) Quintero-Mora ML, Depardon F, JamesWaring, Robert GK, Cisneros B. Expanded CTG repeats inhibit neuronal differentiation of the PC12 cell line. Biochem Biophys Res Commun 2002; 295: 289-94. Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993; 262: 695-700. Ranum LP, Schut LJ, Lundgren JK, Orr HT, Livingston DM. Spinocerebellar ataxia type 5 in a family descended from the grandparents of President Lincoln maps to chromosome 11. Nat Genet 1994; 8: 280-4. Sanchez. C,. Diaz-Nido. J,. Avila. J.. Phosphorylation. of. microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog Neurobiol 2000; 61: 133-68. Sanpei K, Takano H, Igarashi S, Sato T, Oyake M, Sasaki H, et al. Identification of the spinocerebellar ataxia type 2 gene using a direct identification of repeat expansion and cloning technique, DIRECT. Nat Genet 1996; 14: 277-84. Schmahmann JD, Caplan D. Cognition, emotion and the cerebellum. Brain 2006; 129: 290-2. Silveira I, Coutinho P, Maciel P, Gaspar C, Hayes S, Dias A, et al. Analysis of SCA1, DRPLA, MJD, SCA2, and SCA6 CAG repeats in 48 Portuguese ataxia families. Am J Med Genet 1998; 81: 134-8. Sonenberg N. Regulation of translation and cell growth by eIF-4E. Biochimie 1994; 76: 839-46. 47.

(50) Stone J, Smith L, Watt K, Barron L, Zeman A. Incoordinated thought and emotion in spinocerebellar ataxia type 8. J Neurol 2001; 248: 229-32. Tazon B, Badenas C, Jimenez L, Munoz E, Mila M. SCA8 in the Spanish population including one homozygous patient. Clin Genet 2002; 62: 404-9. Topisirovic I, Dragasevic N, Savic D, Ristic A, Keckarevic M, Keckarevic D, et al. Genetic and clinical analysis of spinocerebellar ataxia type 8 repeat expansion in Yugoslavia. Clin Genet 2002; 62: 321-4. Torrens L, Burns E, Stone J, Graham C, Wright H, Summers D, et al. Spinocerebellar ataxia type 8 in Scotland: frequency, neurological, neuropsychological and neuropsychiatric findings. Acta Neurol Scand 2008; 117: 41-8. van de Warrenburg BP, Sinke RJ, Verschuuren-Bemelmans CC, Scheffer H, Brunt ER, Ippel PF, et al. Spinocerebellar ataxias in the Netherlands: prevalence and age at onset variance analysis. Neurology 2002; 58: 702-8. Walter JT, Alvina K, Womack MD, Chevez C, Khodakhah K. Decreases in the precision of Purkinje cell pacemaking cause cerebellar dysfunction and ataxia. Nat Neurosci 2006; 9: 389-97. Worth PF, Giunti P, Gardner-Thorpe C, Dixon PH, Davis MB, Wood NW. Autosomal dominant cerebellar ataxia type III: linkage in a large British family to a 7.6-cM region on chromosome 15q14-21.3. Am J Hum Genet 1999; 65: 420-6. Yabe I, Sasaki H, Chen DH, Raskind WH, Bird TD, Yamashita I, 48.

(51) et al. Spinocerebellar ataxia type 14 caused by a mutation in protein kinase C gamma. Arch Neurol 2003; 60: 1749-51. Zeman A, Stone J, Porteous M, Burns E, Barron L, Warner J. Spinocerebellar ataxia type 8 in Scotland: genetic and clinical features in seven unrelated cases and a review of published reports. J Neurol Neurosurg Psychiatry 2004; 75: 459-65. Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stockton DW, Amos C, et al. Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 1997; 15: 62-9. Zollman S, Godt D, Prive GG, Couderc JL, Laski FA. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 1994; 91: 10717-21.. 49.

(52) Table 1. Primer sequences and annealing temperature for PCR reactions in this study. Function. Cell. Primer. pEGSH-F. Sequence. temp.. 5'-CAACTGCAACTACTGAAATCTGCC -3’. 54℃. genotyping. Cell. Annealing. pEGSH-R. 5'-AGCAATTAACCCTCACTAAAGGGC -3’. C2-F. 5'-CACACATAGTAGAAGGCAGAAGGGC-3’. 54℃ RT-PCR Mouse. C1-R. 5'-CTTGTTCCCGCTTGACTCTGAG-3’. NSE-F. 5’-AATAAGGAGACGCCTGCTTCCCTC-3’. 58-54.5℃ genotyping SCA8-R Mouse. SCA8-F1. 5’-CATGTCAGGCTCTGGGCGAAAAG-3’ 5’-TTGAAGATTGCCTTTTCTGACTCCC-3’. 58.5℃ RT-PCR. EGFP-R. 5’-ACTCCAGCAGGACCATGTG-3’. Internal. β-actin-F. 5’-ATGGATGACGATATCGCT-3’. 54℃ control. β-actin-R. 5’-ATGAGGTAGTCTGTCAGGT-3’. 50.

(53) Table 2. Primer sequences and annealing temperature for generation of probles for in situ hybridization. Gene. Primer. Flag-F ATXN8OS. Sequence. Annealing temp.. 5'-TGACTACAAAGACGATGACG-3’. 54℃. EGFP(1624 5'-CTTGAAGAAGATGGTGCG-3’. -1607)-R Calbindin. Calbindin-F 5'-GAGAACTATTCAGGATGTGTGG-3’ 56℃. (positive Calbindin-R 5'-TTTGGTGCTTCAGAGGCAG -3’ control). 51.

(54) Table 3. Clonal cell line established after selection. line Genotyping RNA level. 150R-1 + +. 150R-2 -. 150R-3 -. 150R-4 + ++. 150R-5 + -. line Genotyping RNA level. 150R-6 + -. 150R-7 -. 150R-8 -. line Genotyping RNA level. 22R-2 + +++. 22R-3 -. 22R-4 -. 22R-5 + leaky. 22R-6 + leaky. line Genotyping RNA level. 22R-8 -. 22R-9 + leaky. 22R-11 -. 22R-12 + ++. - : negative result or No RNA expression +: positive result or RNA expression level. 52.

(55) Table 4. ATXN80S transgenic mouse lines selected by RT-PCR 22R line 4842 4843 4844 4845 4846 4847 4848 4849 4850 4851 4852 4853 4854. repeat number N N N N N N N N N N N N N. RT NA +++ +++ +++ NA NA NA dead NA. 150R line 4829 4830 4831 4832 4833 4834 4835 4836 4837 4838. repeat number N+C SC N+C N + SC N N+C N N N+C N + SC. RT +++ +++ ++ +++ +++ ++ ++ + +++. NA: non- analyzed SC: Severe contraction; C: contraction; N: normal. 53.

(56) Table 5. Antibodies used for western blot analyses. Antibody Brand Clonal Titer Host Mol. weight. NeuN Chemicon Monoclonal 1:2000 Mouse 46,48, & 66 kDa. GFAP Chemicon Monoclonal 1:2000 Mouse 51 kDa. Calbindin Sigma Polyclonal 1:2000 Rabbit 28 kDa. Antibody Brand Clonal Titer Host Mol. weight. Cyt c Santa Cruz Polyclonal 1:1000 Rabbit 12 kDa. Bcl-2 Sigma Monoclonal 1:1000 Mouse 26 kDa. BAX BD Monoclonal 1:1000 Mouse 24 kDa. Antibody Brand. HSP70 Stressgen. α-tubulin Santa Cruz. Clonal Titer Host Mol. weight. Monoclonal 1:1000 Mouse 70 kDa. ORF1 Provided by Dr. Lee’s lab Anti-serum 1:200 Rabbit 24 kDa. Cyt c: Cytochrome c. 54. Monoclonal 1:800 Mouse 55 kDa.

(57) Fig 1. Inducible expression of ATXN8OS in PC12 cells by using Complete Control® Inducible Mammalian Expression System (Stratagene).. (A) In the absence of PonA (the inducer), the promoter is tightly repressed by corepressors. (B) When PonA binds to VgEcR, the corepressors are released, coactivators are recruited, and the complex becomes transcriptionally active.. 55.

(58) Fig 2. Establishment of ATXN8OS inducible expression construct.. (A). pEGSH/ATXN8OS. construction.. The. cDNA. fragments. (ATXN8OS-22R and 150R) were subcloned into the MCS of pEGSH vector. (B) Restriction map of pEGSH/ATXN8OS constructn. Lane 1, pEGSH/ATXN8OS/22R uncut; Lane 2, pEGSH/ATXN8OS/150R uncut. Lane. 3:. pEGSH/ATXN8OS/22R. NotΙ/SalΙ. pEGSH/ATXN8OS/150R NotΙ/SalΙ digestion. 56. digestion.. Lane. 4:.

(59) Fig 3.. Cell. ATXN8OS expression induced by PonA in stable cell lines.. total. RNAs. were. isolated. from. ATXN8OS-22R. and. ATXN8OS-150R stable cells after induced with PonA for 24 hours and 48 hours. RT-PCR analysis was conducted with specific primers designed for detecting the human ATXN8OS transcripts. The β-actin transcript was used as an internal control.. 57.

(60) Fig 4. ATXN8OS-ORF1 expression induced by PonA in stable cell lines.. Cell. total. proteins. were. isolated. from. ATXN8OS-22R. and. ATXN8OS-150R stable cells after induced with PonA for 48 hours. Western blot analysis was conducted using ATXN8OS-ORF1 anti-serum. The arrow indicates ORF1. α-tubulin was used as an internal control.. 58.

(61) Fig 5. Stable cell line with overexpressed ATXN8OS-150R is more vulnerable than those with ATXN8OS-22R and pEGSH vector.. (A) Schematic diagram showing the schedule of treatment and MTT assays with stable cell lines. Cell density was analyzed by MTT assays at 24 hours, 48 hours and 72 hours after PonA induction which following a 48 hours NGF treatment. (B) Cells transfected with pEGSH vector only showed no significant difference with PonA induction. (C) Cells with ATXN8OS-22R overexpression showed no significant difference with or without PonA induction. (D) Cell with ATXN8OS-150R overexpression show significant reduced cell numbers after PonA induction for 48 and 72 hours. (*, P < 0.05 ； **, P < 0.01； ***, P < 0.005).. 59.

(62) Fig 6. ATXN8OS expression induced by PonA in PC12 clonal cell lines.. Cell. total. RNAs. were. isolated. from. ATXN8OS-22R. and. ATXN8OS-150R clonal cells after induced with PonA for 24 hours. RT-PCR analysis was conducted with specific primers designed for detecting the human ATXN8OS transcripts. β-actin transcript was used as an internal control. (A) Expression of ATXN8OS-22R in clonal cell analyses by RT-PCR. (B) Expression of ATXN8OS-150R in clonal cell analyses by RT-PCR.. 60.

(63) 61.

(64) Fig 7. Clonal cell line with overexpressed ATXN8OS-150R is more vulnerable than those with ATXN8OS-22R and pEGSH vector.. (A) Schematic diagram showing the schedule of treatment and MTT assays with stable cell lines. Cell density was analyzed by MTT assays at 24 hours, 48 hours and 72 hours after PonA induction which following a 48 hours NGF treatment. (B) Cells transfected with pEGSH vector only show significant reduced cell numbers after PonA induction for 24 hours. (C) Cells with ATXN8OS-22R-2 overexpression showed no significant difference with PonA induction. (D) Cells with ATXN8OS-22R-12 overexpression showed no significant difference with PonA induction. (E) Cell with ATXN8OS-150R-1 overexpression show significant reduced cell numbers. after. PonA induction. for. 72. hours.. (F). Cell. with. ATXN8OS-150R-1 overexpression show significant reduced cell numbers after PonA induction for 48 and 72 hours. (*, P < 0.05 ； **, P < 0.01； ***, P < 0.005).. 62.

(65) Fig 8. ATXN8OS-ORF1 expression in differentiated PC12 cell after PonA induced for 48 hours. ATXN8OS-22R-2 (A) and ATXN8OS-22R-12 (B) clonal cells, in upper panel, after 48 hours NGF treatment and PonA induced for 48 hours, showed ORF1 protein in the neuron-like status. Lower panel, cells without PonA induction. Scale bar = 10 µm. 63.

(66) ATXN8OS-150R-1 (C) and ATXN8OS-150R-4 (D) clonal cell, in upper panel, after 48 hours NGF treatment then PonA induced for 48 hours, induced cells showed ORF1 protein in the neuron-like status. In lower panel without PonA induction. Scale bar = 10 µm.. 64.

(67) Fig 9. Expression of the ATXN8OS-150R inhibits neuronal outgrowth in the differentiated PC12 cells after PonA induced for 48 hours.. Phase contrast photographs of representative fields, showing typical morphologies of PC12 cell after 48 hours treatment with NGF. Upper panel, after 48 hours NGF treatment but without PonA induction, neuronal outgrowth normaly, Lower panel, after NGF trratment and then PonA induction for 48 hours, ATXN8OS-150R clonal cell showed abnormal morphology.. 65.