利用活細胞系統方式探討Bβ2過度表現對細胞影響

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(1)國立臺灣師範大學生命科學系碩士論文. 利用活細胞系統方式探討 Bβ2 過度表現對細胞影響 Characterization of Bβ2 overexpression live cells. 研究生：簡君宇 Jiun-Yu Jian 指導教授：謝. 秀梅. 博士. Hsiu-Mei Hsieh Ph.D.. 中華民國. 九十九年ㄧ月.

(2) 【TABLE OF CONTENTS】. Acknowledgment 中文摘要…………………….....……..........……..…...……………...……….....…. 5. Abstract..………………...……..……........................……………………......…...... 6. 1.. Introduction……………………..…...……...………………………...…….... 7. 1.1. The structure and biological role of Protein Phosphatase 2A (PP2A)............... 7. 1.2.. Alternative splicing in Bβ (PPP2R2B) genes………...…………..…….......... 9. 1.3.. Apoptosis and programmed cell death…………………………….………...... 9. 1.4.. PP2A and apoptosis………………………..…….………..……………...….... 10. 1.5.. PP2A Bβ subunit splice form Bβ2 controls cell death in neurons……………........................................................................................... 11. 1.6.. Spinocerebellar ataxia type 12 (SCA 12)………………………. ………….. 12. 1.7.. Mitochondria function........................ ........................ ........................ ............ 13. 1.8.. Oxidative stress……………………………………….…………………….... 13. 1.9.. Lentivirus using in neurodegenerative disease model. ........ ............................ 15. 2.. Material and methods………………………….......…..…....…………….... 16. 2.1. Cell lines and culture conditions……......………..…………………..……... 16. 2.2. Lenti virus containing Bβ2 gene construction………..….………………..... 16. 2.3.. Transfection and transduction........................................................................ 18. 2.4.. Immunocytochemistry (ICC)………………....…..…………………….…... 18. 2.5.. Western blot analysis………………….…………………….….......………. 19. 2.6 Mitochondrial fraction extraction..…………..……………...………..…...... 20. 1.

(3) Live image capture………………………………..…… ……...………….. 20. 2.8. Data analysis………………………………………………………...……... 20. 3. Results……………………………………………………………………….. 22. 3.1.. Generation of of Bβ2 lentiviral constructs………………………..………... 22. 3.2.. Characterization of Bβ2 overexpression by image observation and western blot. 2.7.. analysis……..………………………………..………………………….… 3.3.. Characterization of oxidative stress and apoptotic molecules by western blot. analyses…..…………….....… 3.4. Time-lapsed live cell image observation of Bβ2-transfected SH-SY5Y cells…..……………....……. 22. 22. 23. 4. Discussion……………………………………….………………………..……. 24.. References…..……………………..…….……….…...…..................…………….... 25. APPENDIX………………………………………………...……………………….. 30. 2.

(4) Acknowledgment. 回首過去兩年多來的求學歷程，許多成功或失敗之後的修正，總非自身能夠完成，仍靠著許多身邊的老師、學長姐學弟妹們不吝提供寶貴意見，才能有努力的方向；而更難能可貴的我在學習的努力有許多貴人，在生活中也有數不盡的好友一同扶持協助，才有完成論文。感謝指導教授，謝秀梅老師。老師在一開始拒絕了我加入老師的實驗團隊，可是在之後老師在再次有機會參與時也第一個寫信通知我，讓我覺得很感動。老師在實驗的指導上的細心與一針見血的個別討論，協助我一步一步的往前進。除此之外，老師平時在生活上的關心也讓君宇相當感謝感謝慧貞學姐，一開始在思考實驗方向打算以立體定位手術來進行，學姐二話不說就幫我張羅所有的器械與跟台大心理系梁庚辰老師借用實驗室，雖然最後君宇沒辦法解決手術上許多不確定性而放棄了這個題目，但學姐仍然在實驗有其他困難時，仍然提供許多寶貴經驗與解決方法。而學姐偶爾在實驗室時也不斷在鼓勵君宇能更加努力。感謝雅津學姐，同是北醫人真是份外親切呀！學姐每天早早出現在實驗室忙著實驗外，還要應付我這種小毛頭突然問很多奇奇怪怪的問題，也能從容不迫的把所有事情處理好真是太厲害了。祝學姐的 paper 能早日 accept 順利畢業！感謝怡人學姐，學姐雖然在外人眼中看起來講話總是得理不饒人，但靜下心來看學姐的直言不諱才是讓君宇有所成長；學姐在我缺少什麼或有需要什麼時總是幫我翻出來，甚至還幫忙想 construct 要怎麼做才能有效的使用。祝學姐能賺大錢，也能快速的把 data 生出來瞬間畢業。感謝我碩士班的同學：孝修與峻緯。謝謝孝修在 cell culture 上的協助，讓我在 cell culture 上能很順利的上手；感謝峻緯每天跟我耍白爛，也謝謝你在我碩士生涯之間在實驗上鼎力相助，平常的生活常識上提供，改天再一起去吃海鮮呀！謝謝現在碩二的學弟妹們：謝謝天駿，你的用功真的讓我感到望塵莫及，而且你對事物的堅持真的是我很好的榜樣；謝謝興杰，謝謝你在動物行為實驗上，能提供許多很好的經驗談，你跟昭均學姐真是超級閃光彈，祝你們永遠幸福；謝謝偉毅，雖然每天也是跟我「ㄌ一ㄝ肖偎」，可是在實驗的討論上，你的眼光與方向真的是我很好的明燈；謝謝荳荳，感謝妳幫我打點了許許多多在 cell culture 的事物，也在我座位旁忍受我垃圾山的座位 XD 當然也要謝謝其他許多實驗室的大學部學生，謝謝星蓉與志謙，兩位 3+2 學程的小朋友真的很厲害呢！在一年之內真是進步神速！有你們在實驗室真是帶來很多歡樂！謝謝欣潔，妳穩紮穩打的實驗功力與不論多早多晚都會出現在實驗室的拼勁，真是讓我感到汗顏。當然畢業的偉齊、伊婷、如蕙、豐碩、妍佳、紫綾、執中……，感謝你們大家在我碩士生涯中， 3.

(5) 都因為有你們才能讓我不斷向前進。感謝 NDG 的老師、醫生與其他實驗室的大家，謝謝大家在每學期的 retreat 中互相切磋與交換意見，才能使君宇知道自己的實驗出現了多麼致命的盲點，感謝大家！除了實驗室的大家，我也有時向外求救兵：謝謝中研院基因體中心林國儀老師實驗室；謝謝老師在我大學帶我進到科學的世界，也在我大學畢業之後，有時還能回去看看；謝謝林老師實驗室的志明學長：學長在 construct 的功力已臻化境，每次我只要接不出來都去煩你，也謝謝你百忙之中幫我找出問題所在。謝謝北醫寄蟲學科范家堃老師。范老師在研究上總是有很多有趣的新點子，也因緣際會的跟老師常能交換老師許多創意，當然君宇的論文能夠完成，很多時候都是范老師有意無意的詢問才能讓君宇繼續努力，老師希望君宇能如您所願能繼續努力、精益求精！除了在實驗上的貴人，我的生活中也許多貴人協助：謝謝黃主任等等老師，要不是您的容忍我想我也沒機會能在你的班上努力。謝謝過去在大學期間認識的補習班同事，在我碩士求學期間能夠聽我胡說八道、吐苦水。最後要謝謝我的家人：我祖母我是家中唯一不反對我念碩士的！感謝她在這段時間超挺我的，很少回家好好陪陪您！謝謝我兩個姑姑，她們兩位嘴巴真的很壞，但是回頭一看都可以發現她們其實都只是為了我好。當然最後的感謝，要留給嘉玫！雖然她每次在我很忙的時候，都會有許多讓我覺得十分瘋狂的一些對話，真的 Drive me crazy！但是這兩年半來妳少了很多娛樂，因為找我我都說要去實驗室忙，讓妳整個悶翻，加上還要聽我天天抱怨實驗不順的悶！但是妳還是仍然陪在我身邊鼓勵我繼續努力，半年後就換妳要畢業了，祝福妳的研究成果有好的佳績！. 4.

(6) 中文摘要. 目前的研究指出脊髓小腦運動失調症 12 型(Spinocerebellar ataxia type 12, SCA12) 可能是 PPP2R2B 基因的 5’端未轉譯區域的 CAG 三核苷酸擴充所致。PPP2R2B 基因轉譯出具有腦特異性之 Bβ 調控次單元，這個 Bβ 調控次單元為蛋白去磷酸酶 2A(PP2A)的其中一單元。由於 5’端未轉譯區的不正常擴增，可能使得基因 PPP2R2B 產生選擇性剪切(alternative splicing)，而轉譯出其中的一個 Bβ 調節次單元 Bβ2。過去的文獻提出 Bβ2-PP2A 會有粒腺體專一性，使 Bβ2-PP2A 停留在粒腺體外膜造成類神經細胞株 PC-12 凋亡。在細胞模式上，我們利用脂質體攜帶 Bβ2 基因送入神經母細胞瘤細胞株 SH-SY5Y，並處理視網酸(retinoic acid)使其分化為似神經的成熟細胞來進行觀察過量 Bβ2 表現是否位於粒腺體上，及 Bβ2 過度表現對細胞所造成的氧化壓力。在活細胞系統的長時間連續性觀察中，我們可以發現過度表現 Bβ2 的 SH-SY5Y 細胞有較對照組不正常的細胞型態。在了解到 Bβ2 在細胞中所參與的角色，期能在未來提供一個藥物篩選的平台。. 【關鍵字】脊髓小腦運動失調症 12 型(SCA12)、粒線體、PP2A、Bβ2 過度表現. 5.

(7) Abstract. Spinocerebellar ataxia type 12 (SCA12) is associated with an expansion of a CAG repeats in the 5’region of the gene PPP2R2B, which encodes a brain-specific regulatory subunit (Bβ) of the protein phosphatase 2A (PP2A). One of the splice variants of PPP2R2B, Bβ2, has been reported to target PP2A to mitochondria to promote apoptosis in PC12 cells. In our cell model, we used liposome based transient transfection overexpressing Bβ2. The cellular localization of Bβ2 and oxidative stress stimulation by Bβ2 was characterized in retinoic acid differentiated SH-SY5Y neuroblastoma cell line. These tranfected SH-SY5Y cells were characterized on a time-lapsed live image recorded system. Further analysis using Western blot, and immunocytochemistry will be conducted to study the molecular significance. Hoping this system could provide a neurodegenerative platforms for cell model based pathological analysis and potential compound screening.. 【Keyword】 Spinocerebellar ataxia type 12 (SCA12)、mitochondria、PP2A holoenzyme、Bβ2 overexpression. 6.

(8) 1. Introduction. 1.1 The structure and physiological function of protein phosphatase 2A (PP2A). Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated and highly conserved in eukaryotes (Janssens and Goris, 2001). PP2A exists in cells in two major forms, core enzyme and holoenzyme. The core enzyme is composed of a 36 kDa catalytic C subunit and a 65 kDa regulatory A subunit. The holoenzyme is composed of the core enzyme bound to one regulatory B subunit (Janssens and Goris, 2001). The A subunit exists in two forms, Aα and Aβ, which are 86% identical (Walter et al., 1989). Both forms were found to be mutated in a variety of human cancers (Takagi et al., 2000; Ruediger et al., 2001). The C subunit also exists in two forms, Cα and Cβ, that are 96% identical. The B subunits consist four families designated B, B’, B” and B”’. The B family has four members, Bα, Bβ, Bγ, and Bδ, each with a molecular mass of approximate 55 kDa. The B’ family consists of numerous isoforms and splice variants, whose molecular masses range from 54 to 68 kDa. The B” family has four members, which have molecular masses of 48 kDa (PR48), 59 kDa (PR59), 72 kDa (PR72) and 130 kDa (PR130) respectively. The latter two are splice variants of the same gene. Striatin (PR110) and S/G2 nuclear autoantigen (SG2NA; PR93) were identified as new members of a potential B’’’ subunit family. Like the B/ PR55 family, striatin and SG2NA contain WD-40 repeats and interact with PP2A in dimer form. Both proteins also bind to calmodulin in a calcium-dependent manner. Striatin is localized to the post-synaptic densities of neuronal dendrites, whereas SG2NA is a nuclear protein. Both of 7.

(9) complexes contain several additional unidentified proteins, suggesting that striatin and SG2NA may function as scaffolding proteins involved in Ca+-dependent signaling (Moreno et al., 2000). The regulation of neuronal functions by PP2A is mediated by the interaction of the AC core dimer with different regulatory subunits. The regulatory subunits of PP2A that are exclusively expressed in brain are Bγ, a cytoskeletal associated regulatory subunit, and the Bβ splice variants. PP2A holoenzymes that contain these neuronal regulatory subunits have been shown to regulate cytoskeletal dynamics, survival, and promote neuronal differentiation. Analysis of the spatial and temporal expression patterns of Bα, Bβ and Bγ in the brain revealed that levels of PR55α are high in striatum, those of PR55γ are high in hindbrain, and those of PR55β are low in cerebellum (Strack et al., 1998; Dagda et al., 2003).The ubiquitous regulatory subunits Bα and Bδ dephosphorylate and inactivate ERKs in neurons while B’ subunits have been shown to regulate PC6-3 cells pheochromocytoma survival by modulating Akt (Van Kanegan et al., 2005). Furthermore, PP2A binds to neurofilaments where it dephosphorylates neurofilaments NF-M and NF-L to regulate the stability of neurofilaments (Saito et al., 1995; Strack et al., 1997). PP2A has also been shown to dephosphorylate microtubule associated proteins (MAP) such as MAP-2 and tau at multiple serine residues, and dephosphorylation of MAPs by PP2A results in increased assembly and stability of microtubules (Merrick et al., 1997; Sontag et al., 1999; Alexa et al., 2002). Hyperphosphorylated tau, an axonal MAP, is the principal component of neurofibrillary tangles, a pathological hallmark of Alzheimer’s disease. The PP2A holoenzyme that contains the Bα regulatory subunit has been suggested to play a role in the pathophysiology of Alzheimer’s disease (Sontag et al., 2004a; Sontag et al., 2004b). Parkinson’s disease is a common and gradual neurodegenerative disorder that leads 8.

(10) to the loss of nigrostriatal neurons leading to a substantial decline in the synthesis of dopamine by tyrosine hydroxylase (TH). Decreased dopamine synthesis in Parkinson’s patients leads to a gradual decline in locomotor activity and coordination (Bosboom et al., 2004). PP2A is a regulator of TH, which is involved in the synthesis of dopamine. Therefore, dephosphrylation of TH by PP2A has therapeutic implications for Parkinson’s disease. Dephosphorylation of CaMKII by PP2A has also been implicated in Angelman’s syndrome, a developmental cognitive disorder characterized by epilepsy and severe mental retardation (Weeber et al., 2003).. 1.2 Alternative splicing in Bβ (PPP2R2B) genes. Bβ gene, also called PPP2R2B, is located in chromosome 5q31- q32. Previous data showed that Spinocerebellar ataxia type 12 (SCA 12) is caused by a trinucleotide repeat expansion in the promoter region of the human Bβ gene (Holmes et al., 1999). By alternative promoter splicing, Bβ gene was derived into two mRNA, Bβ1 and Bβ2 (Dagda et al., 2003). Bβ2 a splice variant with a unique N-terminal, is widely expressed in most brain areas. PP2A holoenzyme containing Bβ2 is about 10-fold less abundant than those containing the Bβ1 isoform. The divergent N-terminus of Bβ2 does not affect phosphatase activity, but encodes a subcellular targeting signal that targets PP2A to the mitochondria to promote apoptosis (Dagda et al., 2005).. 1.3 Apoptosis and programmed cell death. Apoptosis, or also known as programmed cell death, is essential for pruning excess cells during embryonic development, for removing virally infected cells, cancer cells or damaged cells, and is a hallmark of many neurodegenerative diseases. 9.

(11) Programmed cell death is morphologically characterized by the formation of membrane blebs, chromatin condensation, and cell shrinkage. Apoptosis can be divided into two main categories: the extrinsic and intrinsic pathway of apoptosis (Farber, 1994; Ashe and Berry, 2003). The extrinsic pathway of apoptosis usually involves a death ligand such as Fas that binds to Fas ligand, or a lipid such as ceramide that elicits the release of intracellular calcium from calcium stores. The intrinsic pathways of apoptosis are cell death signals that are triggered within the cell due to an imbalance in cellular homeostasis and these pathways usually are converged at the mitochondria. There are a series of events that take place at the mitochondria before the cell irreversibly commits to cell death. During toxic insult, pro-apoptotic Bcl-2 family proteins are activated in the cytosol by posttranslational mechanisms and translocate to the outer mitochondria membrane (OMM) to promote homodimerization of Bax, and Bak. Homodimerized Bax and Bak insert into the mitochondrial lipid bilayer to form pore channels at the OMM, causing the release of apoptogenic molecules such as cytochrome c (Uo et al., 2005). Released cytochrome c then binds to the apoptosome activating factor-1 (Apaf-1) which then interacts and activate downstream “death executioners” such as caspase 9. Caspase 9 then activates downstream caspases 3 and 7 by proteolytic cleavage of their respective precursor procaspases. The activated caspases will then cleave and activate DNases which are the enzymes that are ultimately responsible for degrading chromosomal DNA, a hallmark of apoptosis (Ashe and Berry, 2003; Jin and El-Deiry, 2005).. 1.4 PP2A and apoptosis. PP2A has been shown to be both a negative and positive regulator of apoptosis. These opposing roles are likely carried out by different PP2A holoenzymes. For 10.

(12) example, treating cells with the PP2A inhibitor okadaic acid induces apoptosis in many cell types suggesting that PP2A is essential for survival (Herzig and Neumann, 2000). A previous study also demonstrated that intact PP2A heterotrimers containing the A, C subunits and all families of regulatory subunits are critical for survival (Strack et al., 2004). On the other hand, PP2A can promote apoptosis by dephosphorylating and inactivating antiapoptotic proteins or by activating proapoptotic Bcl-2 family proteins (Ruvolo et al., 1999). The Bcl-2 family consists of pro- and anti-apoptotic proteins localized at the mitochondrial outer membrane. Bad is one of the pro-apoptotic members and whose function is regulated by reversible phosphorylation. Bad gets phosphorylated at Ser-112, Ser-136 and/or Ser-155 by different pro-survival kinases such as PKA and PKB, which mediate binding of Bad to 14-3-3 proteins. This interaction confines Bad to the cytosol, then cannot heterodimerize with Bcl-2 and activaties the anti-apoptotic protein. In the absence of survival stimuli, Bad is dephosphorylated by PP2A and leads to inhibition of Bcl-2, leading to apoptotic cell death (Datta et al., 2000). Since PP2A activates pro-apoptotic (Bad) and inhibits anti-apoptotic (Bcl-2) proteins of the Bcl-2 family, it is generally assumed that PP2A has a positive regulatory function in apoptosis.. 1.5 PP2A Bβ subunit splice form Bβ2 controls cell death in neurons. Among the four genes coding for B-family regulatory subunits in vertebrates, the neuron-specific Bβ gene has received special attention because of its involvement in a neurodegenerative disorder. The Bβ gene gives rise to at least two N-terminal splice variants, including Bβ1 and Bβ2 (Dagda et al., 2003). The alternative N terminus of Bβ2 was found to target the PP2A heterotrimer to mitochondria in neuronal cells (Dagda et al., 2005). Furthermore, transient or stable expression of Bβ2, but not Bβ1, 11.

(13) potentiated apoptosis in growth factor-deprived PC6-3 cells (Dagda et al., 2003), a subline of PC12 pheochromocytoma cells (Pittman et al., 1993). Together, these results suggest the cryptic import sequence and unfolding-resistant β-propeller domain of Bβ2 recruits PP2A to the OMM to dephosphorylate critical apoptosis regulators (Dagda et al., 2005).. 1.6 Spinocerebellar ataxia type 12 (SCA 12). Spinocerebellar ataxias (SCAs) are progressive disorders in which the cerebellum slowly degenerates, often accompanied by degenerative changes in the brainstem and other parts of the central nervous system (and less commonly the peripheral nervous system) (Taroni and DiDonato, 2004). The number of known SCAs continues to grow. It now includes at least 31 types .(Paulson, 2009; Sato et al., 2009; Storey et al., 2009). There are three major genetic categories of SCAs (Duenas et al., 2006): (1) expanded CAG/polyQ ataxias, which result from proteins with toxic stretches of polyglutamine (SCAs 1, 2, 3, 6, 7,and 17) ;( 2) non–protein coding repeat expansion ataxias, which result from repeat expansions outside of coding regions that may quantitatively alter gene expression (SCAs 8, 10, and 12) ; and (3) ataxias caused by conventional mutations (missense, deletion, insertion, or duplication). (SCA 31) (Brusco et al., 2004; Knight et al., 2004; Schols et al., 2004; Ishikawa et al., 2005; Cagnoli et al., 2006). PR55/Bβ was implicated to be involved in SCA12. This form of autosomal dominant spinocerebellar ataxia is caused by CAG trinucleotide repeats in the 5’ region of PR55/Bβ. Onset of the disease is in the fourth decade of life and leads to loss of movement, head/extremity tremors and in a later stage to complete dementia. 12.

(14) The CAG repeat associated with SCA12 lays 133 nucleotides upstream of the predicted transcription start side for PR55/Bβ. Up to 29 CAG repeats are considered normal, whereas more than 55 repeats are considered disease causative for SCA12. It is speculated, that the CAG repeat expansion affects PR55/Bβ expression and subsequently alters the function of PP2A in the brain (Holmes et al., 1999; Holmes et al., 2001). 1.7 Mitochondria function. Mitochondria are membrane-enclosed organelle found in most eukaryotic cells. (Henze and Martin, 2003).These organelles range from 1–10 μm in size. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth (McBride et al., 2006). The most prominent roles of the mitochondrion are its production of ATP and regulation of cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. glutamate-mediated excitotoxic neuronal injury (Scanlon and Reynolds, 1998), regulation of cellular metabolism (McBride et al., 2006). A mutation in the genes regulating any of these functions can result in mitochondrial diseases.. 1.8 Oxidative stress. Oxidative stress-induced cell damage has long been implicated in both 13.

(15) physiological processes of aging and neurodegenerative disorders (Halliwell, 2006). It is mediated by reactive oxygen species (ROS), including hydrogen peroxide (H 2O2), superoxide (O2 •-) and hydroxyl radicals (OH•). It is the products of normal cellular respiration and aberrant metabolic processes that utilize molecular oxygen. Excess ROS could trigger lipid peroxidation, ion channel modification, DNA damage, and protein oxidation, thereby disrupting cellular function and integrity (Gutteridge and Halliwell, 2000; Thannickal and Fanburg, 2000). Under physiological condition, cells have developed a sophisticated antioxidant defense system to maintain intracellular redox homeostasis and protect cell against oxidative damage. For example, superoxide dismutase could reduce O2 •- to H2O2 and glutathione, thioredoxin buffering systems, catalase reduce H2O2 to H2O and there are small molecule substances such as vitamins C and E. The production of ROS and redox modifications of antioxidant defense enzymes or transcription factors are controlled by several signal transduction pathways serving important regulatory functions (Chang and Karin, 2001; Martindale and Holbrook, 2002). However, cellular antioxidant defense component may be overwhelmed during pathological conditions by the presence of excessive ROS derived from either exogenous or endogenous sources, resulting in tissue injury. Moreover, the molecular mechanisms underlying oxidative stress-induced neuronal damage are emerging and appear to involve an apoptotic mode of death. ROS-induced apoptosis requires the participation of some signaling pathways that are closely associated with both cell death and cell survival pathways. Three structurally related but biochemical and functionally distinct mitogen-activated protein kinases (MAPK) signal transduction pathways have been identified in mammalian cells under oxidative challenge in promoting both cellular life and death, including the extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase, and the p38 pathways 14.

(16) (Brunet et al., 2001; Kyriakis and Avruch, 2001). Considering evidences have showed that ROS could transactivate protein tyrosine kinases and protein serine/ threonine kinases to activate MAPK pathway which can mediate the activation of a wide variety of transcription factors and induce cell apoptosis or proliferation (Harper and LoGrasso, 2001; Ouyang and Shen, 2006). 1.9. Lentivirus using in neurodegenerative disease model.. Lentivirus (lenti-, Latin for "slow") is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period. Lentiviruses can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses (Dull et al., 1998). Since lentivus has a great spectrum of gene delivery in most cell cultures, it may use in high efficiency gene deliver and gene knock down using in the cell model. Previous data showed that lentivirus could deliver polyQ tracts into Rat striatal neurons or some rescue genes into SCA3 animal models (Alves et al., 2008; Torashima et al., 2008). Due to lentivirus have broad spectrum of infection ability, researchers have used this technique combining with microinjection for high efficiency or difficult transgenic animal production (Yang et al., 2008).. 15.

(17) 2.Material and Methods. 2.1. Cell lines and culture conditions. 293T human embryonic kidney cell transforming large T antigen was a gift from Dr. Kuo-I Lin (Genome Research Center, Academia Sinica), and SH-SY5Y human neuroblastoma cell was a gift from Dr. Ching-Jin Chang (Institute of Biochemical Sciences, National Taiwan University). 293T cells were culcured in Dulbecco's Modified Eagle's Medium with 10% FBS and 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen). SH-SY5Y cells were cultured in 50% Ham’s F12 and 50% DMEM with 10% FBS, and 100 IU penicillin/streptomycin. Both of the cells were passaged every 48 h.. 2.2. Lenti virus containing Bβ2 gene construction. 2.2.1. Virus construction. The virus construct with Rat Bβ2 cDNA was modified from The pCMV-Bβ2-Flag-EGFP plasmid (a gift from Dr. Strack, Department of Pharmacology, Carver College of Medicine, University of Iowa) and pCL-20c-MSCV-GFP provided by Dr. Arthur W. Nienhuis. St. Jude Children's Research Hospital (Hanawa et al., 2002). The Bβ2 sequence was amplified by PCR (primer pair: 5’- CTG CAG CCG CGG ACC ATG AAA TGC TTC TCT CG-3’, 5’- ATT AAC CTT GTC CTG GAA TAT A-3’ ) and ligated into pEF-myc expression vector (Invitrogen). The resulting pEF-Bβ2-myc was then digested with SacII/XbaI to acquire Bβ2 containing myc tag. 16.

(18) Next we subcloned the Bβ2-myc fragment into SacII and SmaI pIRES2-DsRED2 vector to generate Bβ2-myc-IRES2-DsRED2. Finally, we isolated the Bβ2-myc-IRES2-DsRED2 fragment by SacII and NotI digestion and ligated it into the SacII and NotI restriction enzyme sites of pCL-20c-MSCV-GFP construct, the final plasmid was denominated pCL-20c-MSCV-Bβ2-IRES2-DsRED2.. 2.2.2. Lentivirus production. VSV-G pseudotyped lentiviral vectors provided by St. Jude Children's Research Hospital (Hanawa et al., 2002) were used in this study. The virus vector was produced by cotransfection of human embryonic kidney (HEK) 293 T cells with a mixture of four plasmids using lipofectamine 2000(Invitrogen, USA) method. Briefly, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin G and 50 μg/ml streptomycin (pH 7.35) at 37 °C in a 5% CO2 atmosphere. Cells were seeded at 1 × 106 cells in a 10-cm culture dish 24 h before transfection. The four plasmid mixture consisted of 6 μg of pCAGkGP1R, 2 μg of pCAG4RTR2, 2 μg of pCAG-VSV-G, and 10 μg of vector plasmid pCL20c MSCV-DsRed2 or pCL20c MSCV-Bβ2-IRES2-DsRed2. The plasmids were mixed and diluted to a total volume of 1.5 ml with Opti-MEM(invitrogen, USA), and 36 μl of lipofectamine was added into another 1.5 ml Opti-MEM. After 10 min, 2 tubes of solution were added together and votexed vigorously and incubated for 20 min. Transfection solution was added into 293T culture after incubation. Sixteen hours after transfection, the cells were washed with phosphate-buffered saline (PBS) twice and then cultured for an additional 24 h. The medium containing vector particles was harvested 40 h after transfection. The medium samples were filtered through 0.22-μm membranes and centrifuged at 25,000 17.

(19) rpm for 90 min. The virus samples were finally suspended in 45 μl of PBS (pH 7.4), frozen in aliquots, and stored at −80 °C. The titers of virus stocks were measured by transducing A549 cells. Serial dilutions of the virus preparations were added to 105 A549 cells growing in a monolayer in six-well plates in a total volume of 2 ml of DMEM containing Polybrene (10 μg/ml). After 16 h, the medium was replaced by fresh medium, and culturing was continued for another 3 days. Titers were routinely determined using flow cytometry (Caliber, BD, USA). 2.3. transfection and transduction. Human neuroblastoma SH-SY5Y cells were grown in 50% Ham’s F12 and 50% DMEM with 10% FBS, 2.0 mM L-glutamine, and 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37 °C and 5% CO2. The medium was replaced after 48 hours and cells were subcultured after 4 days. For the cellular morphological studies, 18–24 h prior to the transfection, cells were plated at a density of 4 × 105 cells per well of a 6-well format (Falcon). Bβ2-IRES2-DsRED2 or DsRed2 genes, at the molar ratio of 2.5:1, were transfected into SH-SY5Y cells using the method of lipofectamine 2000 performed as suggested by the manufacturer (Invitrogen, USA). 4 to 8 h after incubation with DNA/ liposome complexes, cells were washed with phosphate buffered saline (PBS), and the medium was replaced. The transfected cells displaying DsRED fluorescence were observed under a fluorescence microscope 24 to 48 h after transfection.. 2.4. Immunocytochemistry (ICC) 18.

(20) Cells grown in 6-well dish with Thermanox Plastic Coverslips (Nunc), slide were fixed in 4% paraformaldehyde for 10 min. The fixed cells were rinsed 3 times in PBS containing 0.1% Triton X-100, blocked in 37℃ 1 h in PBS containing 0.1% Triton X-100, and finally incubated for 18 h at 4°C with primary antibody. They were then rinsed 3 times. in PBS and incubated for 2 h at 37℃ with the second antibody using. fluorescence conjugated secondary antibody(Jackson Immunoresearch) with DAPI (4′-6-diamidino-2′-phenylindole) at a concentration of 0.1 μg/ml as nuclear stain.. 2.5. Western blot analysis. After 48 h of transfection, cells were trypsinized and harvest for protein extraction. Total protein concentration was measured by BCA Protein Assay (Pierce, USA), and 50μg of protein lysate was used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Gel protein was then transferred onto polyvinylidene fluoride (PVDF) membrane under 350 mA for 1.5 h. Blocking membrane capability was performed using 5% skim milk for 2 h at room temperature. Primary antibodies used were, Bβ2 (1:4000 dilution; kindly provided by Dr. Fang Kang, National Taiwan Normal University), c-Myc (clone 9E-10, 1:1000 dilution; Santa cruz, USA), MnSOD (1:1000 dilution; Chemicon), Bcl-2 (1:1000 dilution; sigma), Bax (1:1000 dilution, BD) and Caspase 3(1:200 dilution; Chemicon). Antibodies were incubated with membranes overnight at 4℃. Horseradish peroxidase (HRP)-labling secondary antibodies were labeled on the membrane against which primary antibodies recognized. Observation of HRP signaling was performed using LAS 3000 image system (Fujifilm, Japan) 19.

(21) 2.6. Mitochondrial fraction extraction. The mitochondrial fraction from cells was isolated by using a mitochondrial extraction kit (Active Motif, Carlsbad, CA. USA) according to the manufacturer’s protocol. Brain tissue samples were gently homogenized with a glass–teflon homogenizer using 30-50 strokes in 1 ml of cytosolic buffer. Homogenates were centrifuged at 800 × g for 30 min at 4°C, and the supernatant fraction is cytosol fraction containing mitochondria. Supernatant was collected and centrifuged at 16000 × g for another 30 min at 4°C to pellet down the mitochondria. The supernatant thus obtained was the cytosolic fraction. The mitochondrial pellet was resuspended in 100 μl of mitochondria buffer. The purity of the mitochondrial fraction was verified by the selective expression of the mitochondrial inner membrane-specific protein prohibitin. Total protein concentration in the mitochondrial or cytosolic extracts was quantified by the BCA Protein Assay (Pierce, USA). 2.7. Live image capture After transfection for 24 h, cells were transferred into LCI Chamlide Incubator (Live cell instrument, Korea), and incubated under Leica DMI4000 Inverted Microscope. Cells were recorded for 16 to 48 h to observe the alterrtion of cell morphology. Cell status was analyzed by using Metamorph software (Leica). 2.8. Data analysis. ANOVA analysis was used to measure cell viability among RFP, Bβ2 transducing cells and control cells, In comparing the effects between the Bβ2 or vector DsRED2 transducing cells, independent samples student’s t test was used in the study. The 20.

(22) statistical results were expressed as means ± SEM.. 21.

(23) 3. Result. 3.1. Generation of of Bβ2 lentiviral constructs. The flow chart for cloning strategy is shown in Fig. 1A. First, Bβ2 cDNA was amplified from pCMV-Bβ2-flag-EGFP (a gift from Dr. Stefan Strack, Department of Pharmacology, Carver College of Medicine, University of Iowa) using PCR method. The amplified cDNA product was ligated into vector pEF-myc. Bβ2-myc fragment was further PCR amplified and subcloned into pIRES2-DsRED2. Bβ2-myc-IRES-DsRED fragment was PCR amplified and ligated to the lentiviral vector pCL-20c. The final construcs were showed in Fig 1. All of the clones were checked by DNA sequencing.. 3.2. Characterization of Bβ2 overexpression by image observation and western blot analysis. After 48 h of transfection, we could observe the RFP signals appeared in transfected 293T cells (Fig. 2A). Cells were lysed and proteins were extracted for western blot analysis. C-myc antibody was used to detect overexpressed Bβ2-myc5 protein (55 kD) and actin was used as an internal control of the western analyses. We found cells overexpressed Bβ2-myc had bands of 55kD (Fig. 2B).. 3.3. Characterization of oxidative stress and apoptotic molecules by western blot analyses 22.

(24) From the previous report, Bβ2 may locate on the mitochondria, which we suspected would affect normal cellular respiration and cause oxidative stress. 293T cells were transient transfected with Bβ2-IRES-DsRED or DsRED only vector, 100 μM of H2O2 insult was then applied to the cells after transfection for 24 h to enhance the extent of oxidative stress. As shown in Fig. 3 we found MnSOD levels were increased in H2O2 group, and in cytocol fraction (Fig. 3). This result reveals that cells might be damaged when Bβ2 overexpressed and under H2O2 insult.. 3.4. Time-lapsed live cell image observation of Bβ2-transfected SH-SY5Y cells. After Bβ2 transfection, SH-SY5Y living cells were observed on the inverted microscope (LeicaDMI4000, Leica Microsystems, Germany) with LCI Chamlide Incubator (Chamlide TC, Live cell instrument, Korea). Cells were observed for 16 h and photos were taken every 10 min. We found that transfected SH-SY5Y cells had some mophorlogical alteration compared to those non-transfect cells. Transfected cells had a more rounded shaped and less dendritic out growth in phase-contrast view (Fig 4A).Dendrite length was measured by using NeuronJ software (Biomedical Imaging Group Rotterdam, Nederland), and statistical analysis was conducted by using student t test. We observed that the dendrites length of Bβ2 overexpressed cells were significantly shorter than that of non-transfected cells (p < 0.001, Fig 4B).. 23.

(25) 4. Discussion. In this study, we try to find out what would happened when overexpressed Bβ2 protein in SH-SY5Y neuroblastoma. When using Live image system to monitor the cell morphology changes in 24 h, we observed some transduced cells had reduced dendrite length compared with non-transducing cells. In our pioneer study of in vivo transduction in rodent cerebellar, we found weight loss in lentivirus contained Bβ2 injection mice, and worse rotarod performance than vihicle group. We also found while some mice could achieve 600 sec on rods, they had abnormal steps when they ran on the rod. In fix speed (in our study, 20rpm/min) could not perform 2 groups of difference between those vihicle and virus transduction groups. We may try to correlate these methods with the mice strain and use accelerate speed.(Bohlen et al., 2009) Lentivirus transduction has used for several years, and wildly used in mediate transient gene expression in proliferating cells, stable expression in nondividing cells in vitro and in vivo, specific immune responses, RNA interference, homologous recombination (gene repair, knock-in, and knock-out), site-specific recombination, and transposition.(Wanisch and Yanez-Munoz, 2009) In our study, we use lentiviral transduction to overexpress pathological protein Bβ2 to establish a model for evaluation the effect of Bβ2. The main challenge in our study was the efficiency of tranduction efficiency. The latest study of lentivirus delivery in nervous system is using for gene therepy for neurodegenerative disease (Torashima et al., 2008; Kanninen et al., 2009).. 24.

(26) Reference. Alexa A, Schmidt G, Tompa P, Ogueta S, Vazquez J, Kulcsar P, Kovacs J, Dombradi V, Friedrich P (2002) The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c, regulates microtubule binding and stability. Biochemistry 41:12427-12435. Alves S, Regulier E, Nascimento-Ferreira I, Hassig R, Dufour N, Koeppen A, Carvalho AL, Simoes S, de Lima MC, Brouillet E, Gould VC, Deglon N, de Almeida LP (2008) Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease. Hum Mol Genet 17:2071-2083. Ashe PC, Berry MD (2003) Apoptotic signaling cascades. Prog Neuropsychopharmacol Biol Psychiatry 27:199-214. Bohlen M, Cameron A, Metten P, Crabbe JC, Wahlsten D (2009) Calibration of rotational acceleration for the rotarod test of rodent motor coordination. J Neurosci Methods 178:10-14. Bosboom JL, Stoffers D, Wolters E (2004) Cognitive dysfunction and dementia in Parkinson's disease. J Neural Transm 111:1303-1315. Brunet A, Datta SR, Greenberg ME (2001) Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 11:297-305. Brusco A, Gellera C, Cagnoli C, Saluto A, Castucci A, Michielotto C, Fetoni V, Mariotti C, Migone N, Di Donato S, Taroni F (2004) Molecular genetics of hereditary spinocerebellar ataxia: mutation analysis of spinocerebellar ataxia genes and CAG/CTG repeat expansion detection in 225 Italian families. Arch Neurol 61:727-733. Cagnoli C, Mariotti C, Taroni F, Seri M, Brussino A, Michielotto C, Grisoli M, Di Bella D, Migone N, Gellera C, Di Donato S, Brusco A (2006) SCA28, a novel form of autosomal dominant cerebellar ataxia on chromosome 18p11.22-q11.2. Brain 129:235-242. Chang L, Karin M (2001) Mammalian MAP kinase signalling cascades. Nature 410:37-40. Dagda RK, Zaucha JA, Wadzinski BE, Strack S (2003) A developmentally regulated, neuron-specific splice variant of the variable subunit Bbeta targets protein phosphatase 2A to mitochondria and modulates apoptosis. J Biol Chem 278:24976-24985. Dagda RK, Barwacz CA, Cribbs JT, Strack S (2005) Unfolding-resistant translocase targeting: a novel mechanism for outer mitochondrial membrane localization exemplified by the Bbeta2 regulatory subunit of protein phosphatase 2A. J Biol Chem 280:27375-27382. Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB, Greenberg ME (2000) 14-3-3 25.

(27) proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell 6:41-51. Duenas AM, Goold R, Giunti P (2006) Molecular pathogenesis of spinocerebellar ataxias. Brain 129:1357-1370. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72:8463-8471. Farber E (1994) Programmed cell death: necrosis versus apoptosis. Mod Pathol 7:605-609. Gutteridge JM, Halliwell B (2000) Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 899:136-147. Halliwell B (2006) Oxidative stress and neurodegeneration: where are we now? J Neurochem 97:1634-1658. Harper SJ, LoGrasso P (2001) Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38. Cell Signal 13:299-310. Henze K, Martin W (2003) Evolutionary biology: essence of mitochondria. Nature 426:127-128. Herzig S, Neumann J (2000) Effects of serine/threonine protein phosphatases on ion channels in excitable membranes. Physiol Rev 80:173-210. Holmes SE, Hearn EO, Ross CA, Margolis RL (2001) SCA12: an unusual mutation leads to an unusual spinocerebellar ataxia. Brain Res Bull 56:397-403. Holmes SE, O'Hearn EE, McInnis MG, Gorelick-Feldman DA, Kleiderlein JJ, Callahan C, Kwak NG, Ingersoll-Ashworth RG, Sherr M, Sumner AJ, Sharp AH, Ananth U, Seltzer WK, Boss MA, Vieria-Saecker AM, Epplen JT, Riess O, Ross CA, Margolis RL (1999) Expansion of a novel CAG trinucleotide repeat in the 5' region of PPP2R2B is associated with SCA12. Nat Genet 23:391-392. Ishikawa K, Toru S, Tsunemi T, Li M, Kobayashi K, Yokota T, Amino T, Owada K, Fujigasaki H, Sakamoto M, Tomimitsu H, Takashima M, Kumagai J, Noguchi Y, Kawashima Y, Ohkoshi N, Ishida G, Gomyoda M, Yoshida M, Hashizume Y, Saito Y, Murayama S, Yamanouchi H, Mizutani T, Kondo I, Toda T, Mizusawa H (2005) An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains. Am J Hum Genet 77:280-296. Janssens V, Goris J (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353:417-439. Jin Z, El-Deiry WS (2005) Overview of cell death signaling pathways. Cancer Biol Ther 4:139-163. 26.

(28) Kanninen K, Heikkinen R, Malm T, Rolova T, Kuhmonen S, Leinonen H, Yla-Herttuala S, Tanila H, Levonen AL, Koistinaho M, Koistinaho J (2009) Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A 106:16505-16510. Knight MA, Gardner RJ, Bahlo M, Matsuura T, Dixon JA, Forrest SM, Storey E (2004) Dominantly inherited ataxia and dysphonia with dentate calcification: spinocerebellar ataxia type 20. Brain 127:1172-1181. Kyriakis JM, Avruch J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807-869. Martindale JL, Holbrook NJ (2002) Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192:1-15. McBride HM, Neuspiel M, Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16:R551-560. Merrick SE, Trojanowski JQ, Lee VM (1997) Selective destruction of stable microtubules and axons by inhibitors of protein serine/threonine phosphatases in cultured human neurons. J Neurosci 17:5726-5737. Moreno CS, Park S, Nelson K, Ashby D, Hubalek F, Lane WS, Pallas DC (2000) WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A. J Biol Chem 275:5257-5263. Ouyang M, Shen X (2006) Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells. J Neurochem 97:234-244. Paulson HL (2009) The spinocerebellar ataxias. J Neuroophthalmol 29:227-237. Pittman RN, Wang S, DiBenedetto AJ, Mills JC (1993) A system for characterizing cellular and molecular events in programmed neuronal cell death. J Neurosci 13:3669-3680. Ruediger R, Pham HT, Walter G (2001) Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene 20:1892-1899. Ruvolo PP, Deng X, Ito T, Carr BK, May WS (1999) Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J Biol Chem 274:20296-20300. Saito T, Shima H, Osawa Y, Nagao M, Hemmings BA, Kishimoto T, Hisanaga S (1995) Neurofilament-associated protein phosphatase 2A: its possible role in preserving neurofilaments in filamentous states. Biochemistry 34:7376-7384. Sato N, Amino T, Kobayashi K, Asakawa S, Ishiguro T, Tsunemi T, Takahashi M, Matsuura T, Flanigan KM, Iwasaki S, Ishino F, Saito Y, Murayama S, Yoshida M, Hashizume Y, Takahashi Y, Tsuji S, Shimizu N, Toda T, Ishikawa K, Mizusawa H (2009) Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats 27.

(29) containing (TGGAA)n. Am J Hum Genet 85:544-557. Scanlon JM, Reynolds IJ (1998) Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons. J Neurochem 71:2392-2400. Schols L, Bauer P, Schmidt T, Schulte T, Riess O (2004) Autosomal dominant cerebellar ataxias: clinical features, genetics, and pathogenesis. Lancet Neurol 3:291-304. Sontag E, Hladik C, Montgomery L, Luangpirom A, Mudrak I, Ogris E, White CL, 3rd (2004a) Downregulation of protein phosphatase 2A carboxyl methylation and methyltransferase may contribute to Alzheimer disease pathogenesis. J Neuropathol Exp Neurol 63:1080-1091. Sontag E, Luangpirom A, Hladik C, Mudrak I, Ogris E, Speciale S, White CL, 3rd (2004b) Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol 63:287-301. Sontag E, Nunbhakdi-Craig V, Lee G, Brandt R, Kamibayashi C, Kuret J, White CL, 3rd, Mumby MC, Bloom GS (1999) Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies. J Biol Chem 274:25490-25498. Storey E, Bahlo M, Fahey M, Sisson O, Lueck CJ, Gardner RJ (2009) A new dominantly inherited pure cerebellar ataxia, SCA 30. J Neurol Neurosurg Psychiatry 80:408-411. Strack S, Cribbs JT, Gomez L (2004) Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival. J Biol Chem 279:47732-47739. Strack S, Westphal RS, Colbran RJ, Ebner FF, Wadzinski BE (1997) Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments. Brain Res Mol Brain Res 49:15-28. Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE (1998) Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization by B subunits. J Comp Neurol 392:515-527. Takagi Y, Futamura M, Yamaguchi K, Aoki S, Takahashi T, Saji S (2000) Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers. Gut 47:268-271. Taroni F, DiDonato S (2004) Pathways to motor incoordination: the inherited ataxias. Nat Rev Neurosci 5:641-655. Thannickal VJ, Fanburg BL (2000) Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 279:L1005-1028. Torashima T, Koyama C, Iizuka A, Mitsumura K, Takayama K, Yanagi S, Oue M, Yamaguchi H, Hirai H (2008) Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia. EMBO Rep 9:393-399. Uo T, Kinoshita Y, Morrison RS (2005) Neurons exclusively express N-Bak, a BH3 domain-only Bak isoform that promotes neuronal apoptosis. J Biol Chem 28.

(30) 280:9065-9073. Van Kanegan MJ, Adams DG, Wadzinski BE, Strack S (2005) Distinct protein phosphatase 2A heterotrimers modulate growth factor signaling to extracellular signal-regulated kinases and Akt. J Biol Chem 280:36029-36036. Walter G, Ferre F, Espiritu O, Carbone-Wiley A (1989) Molecular cloning and sequence of cDNA encoding polyoma medium tumor antigen-associated 61-kDa protein. Proc Natl Acad Sci U S A 86:8669-8672. Wanisch K, Yanez-Munoz RJ (2009) Integration-deficient lentiviral vectors: a slow coming of age. Mol Ther 17:1316-1332. Weeber EJ, Jiang YH, Elgersma Y, Varga AW, Carrasquillo Y, Brown SE, Christian JM, Mirnikjoo B, Silva A, Beaudet AL, Sweatt JD (2003) Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome. J Neurosci 23:2634-2644. Yang SH, Cheng PH, Banta H, Piotrowska-Nitsche K, Yang JJ, Cheng EC, Snyder B, Larkin K, Liu J, Orkin J, Fang ZH, Smith Y, Bachevalier J, Zola SM, Li SH, Li XJ, Chan AW (2008) Towards a transgenic model of Huntington's disease in a non-human primate. Nature 453:921-924.. 29.

(31) APPENDIX. A.. 30.

(32) B. Ampicillin. pCL20c-Bβ2 -myc-IRES2-DsRED2 98 30 bp. N otI (660 6). DsRE D2 SacII (3929). IRE S2 c-myc epitope N otI (5274). Koz ak sequence Bβ2 ORF. Fig. 1 A. Flow chart of cloning strategy. B. The final lentiviral construct.. We subcloned Bβ2–myc-IRES- DsRED2 into pCL20c vector. The Bβ2 and DsRED2 are separated by an IRES site to make 2 proteins so the DsRED2 can be used as an indicator for the transfection and expression of the construct .. 31.

(33) A.. B.. 32.

(34) Fig 2. A. Human embryonic kidney (HEK) 293T cells were transfected with DsRED control vector or Bβ2-myc-IRES-DsRED expression vector. B. The overexpressed Bβ2 was identified by western blot analysis.. 33.

(35) Fig3. The MnSOD expression levels in cells with Bβ2 overexpression.. 34.

(36) A.. 35.

(37) 36.

(38) B.. - 37 -.

(39) Fig 4 Cell morphology observed by Live image system.. A. SH-SY5Y cells transfected with Bβ2 and non-transfected cells were observed in the same view. Compare to the non-transfected cells, cells with DsRED fluorescence were transfected with Bβ2 and had more rounded cell shape, less dendritic outgrowth, and less lamellipodia. Magnified pictures were shown in the lower pannels and white arrows indicated the transfected cells (with DsRED florescence expression). B. Statistic result of dendritic length analysis. Dendrites in Bβ 2 overexpressed SH-SY5Y were significantly shorter than that of non-transfected cells (p < 0.001, using student t test). - 38 -.

(40)