神經組織粒線體過度表現Bβ2之轉殖基因鼠的建立與神經病理分析

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(1)國立臺灣師範大學生命科學系碩士論文. 神經組織粒線體過度表現 Bβ2 之轉殖基因鼠的建立與神經病理分析 Neuropathological study on transgenic mice with Bβ2 overexpression in the mitochondria. 研究生：陳. 浚. 榕. Chun-Jung Chen 指導教授：謝秀. 梅. 博士. Dr. Hsiu-Mei Hsieh. 中華民國. 九十七年ㄧ月.

(2) 【TABLE OF CONTENTS】. Acknowledgment 中文摘要…………………….....……..........……..…...……………...……….....…. i. Abstract..………………...……..……........................……………………......…...... ii. 1.. Introduction……………………..…...……...………………………...…….... 1. 1.1.. The structure and biological role of Protein Phosphatase 2A (PP2A)............... 1. 1.2.. Apoptosis………...………………………………………………..…….......... 3. 1.3.. The role of PP2A in apoptosis…………………………………….………...... 4. 1.4.. Mitochondrial PP2A/Bβ2 promotes apoptosis in neurons………..……...….... 5. 1.5.. Spinocerebellar Ataxia 12…………….............................................................. 6. 2.. Material and methods………………………….......…..…....…………….... 7. 2.1.. Construct pNSE-cox8-Bβ2-flag-EGFP plasmid……......………..…………... 7. 2.2. Animals………………………………………….........……..….…………..... 7. 2.3.. 8. Mitochondria Bβ2 transgenic mice.................................................................... 2.3.1. Production of fertilized eggs for pronuclear injection....…………….…..... 8. 2.3.2. The generation of mito-Bβ2 trainsgenic mice…..…….….…..............…..... 8. 2.4.. Measurement of body weight and paw clasping………………....…..……….. 9. 2.5.. Video recording setup for HomeCageScan………………….…..…....………. 9. 2.6. Hot plate analysis…………………………………..……………...…….…..... 10. 2.7.. Measurement of locomotor activity………………………………..…...……. 10. 2.8.. Rotarod behavioral test………………………………………………...……... 10.

(3) 2.9.. Immunohistochemistry……………………………..……………………….... 11. 2.10. Mitochondria purification…………………………………………...…...……. 11. 2.11. Western blot analysis……………………………………….….……...….….... 12 2.12. Mitochondria PP2A activity…………………………………………..……..... 13 2.13. Measurement of mitochondria membrane potential…………….….…....……. 13. 2.14. Data analysis…………………………………………………………………... 14. 3.. 14. 3.1.. Results…………………………………………………………………………. Generation of transgenic mice with Bβ2 overexpression in the mitochondria. (mito-Bβ2 mice)…………………………………………………………….. 14 3.2.. Mophological phenotype of mito-Bβ2 transgenic mice……..…………….…. 14. 3.3.. Abnormal neurobehavior of mito-Bβ2 transgenic mice…..…………….....…. 15. 3.4.. PP2A activity and mitochondria membrane potential…..……………....……. 15. 3.5.. The neuropathology of mitochondria Bβ2 transgenic mice………………….. 15. 4.. Discussion……………………………………….………………………..……. 17. References…..……………………..…….……….…...…..................……………... 19 APPENDIX………………………………………………...………………………. 25.

(4) Acknowledgment 要感謝的人實在太多，首先要感謝在我碩一時候的老闆張永達老師，沒有您沒有現在的我，即使我已經沒有繼續在您研究室做研究，在遇到我時，總是關心著尚未畢業的自己是否一切順利，讓浚榕很溫暖，朝著自己的理想邁進，不辜負您的期望；現在您又當上系主任，公務繁忙的您要多多注意身體健康喔。感謝楊榮祥老師，您還記得浚榕的河馬嗎？說我拍的很生動...讓我一直很開心.. 到現在還是很想念您..希望您的身體要一直健健康康..長命百歲唷... 浚榕終於也要畢業了... 感謝桃子..可欣..一路上鼓勵著我不管我是不是還在生教組...真的很高興能遇見妳們...還好有妳們在...感動盡在不言中好啦...因為我的碩士班生涯很坎坷... 所以進入我現在的研究室... 最最感謝謝秀梅老師能夠收我這個轉組的研究生，因為一年完全沒碰到實驗全部要重新學，對老師對我真的都是一種挑戰，很感激老師的包容，自己常常因為家裡的事情，弄得自己身體很糟糕作息很不正常，老師的慰問讓浚榕很感動，雖然浚榕總是不爭氣，被許多事情打敗，還好有老師的鼓勵與刺激讓浚榕更上一層樓。感謝靜芳學姊，雖然你已經離開了，好懷念和妳一起做顯微注射的日子，還有一起辛苦彼此打氣鼓勵的日子...希望妳也能達到妳的理想目標...賺大錢啦~~ 感謝慧貞學姐，是妳在我最無助的時候拉我ㄧ把，敎我動物行為，敎我思考實驗的邏輯，敎我如何設身處地，如何當一個學生..當一個學姊...當一個女兒... 讓我學習扮演好每一個角色..這是浚榕在碩士班學到最多的地方...也讓浚榕知道溝通和彼此鼓勵的重要，真的很謝謝學姐。感謝雅津學姊，每每當我在實驗室難過的時候，都是妳陪我聊聊天，在我因為實驗四處跑辛苦的時候，體恤我、站在我這邊替我抱不平的時候，雖然身體是累的，但心卻是滿滿的溫暖，有妳，真的讓我很窩心也很開心。很慶幸自己多留這些時間，以後我也會常回來研究室找妳的啊，一起加油唷～感謝馬偕的七個小學妹，乙晴..婉真..家玲..宴瑜..家羽..映潔..如芳妳們讓我感受到人與人之間的貼心，彼此鼓勵有多重要，在妳們身上，浚榕學姊學到很多，善用時間，主動幫忙，付出不計代價，換水加飼料洗籠子換籠子都是風雨無阻.. 讓浚榕學姊無後顧之憂的努力做實驗..很感激妳們...陪我熬夜看日出..陪我睡實驗室..小小年紀..卻比許多研究生還有耐力與抗壓性，這是學姊比不上的，真的很謝謝妳們，尤其是在我弟弟出車禍那段時期，你們的主動幫忙與一聲加油，讓我真的很感動... 感謝已經離開的翎絢還有韋倫，在做顯微注射時候的指導以及 IHC 切片的幫忙浚榕很感激，積極樂觀的你們真的是實驗室的開心果。感謝雨霖還有霽雲常.

(5) 常在我研究到太晚家裡卻有事情辛苦的載我回家.. 常常在實驗室很晚的時候心情不好的時候鼓勵著自己，要我保重身體..注意身體..對啦..祝你們能夠早日找到好女孩啊... 感謝薏婷，高妹你們讓我知道當一個學姊該怎麼做才是對的，我會謹記在心，希望半年後也能聽到你們的好消息~ 感謝偉齊忍受浚榕學姊因為跑來跑去沒辦法好好教導你，卻盡量排出時間和我配合，努力幫我照顧老鼠們，量體重測 Clasping，希望你未來的一切都能平安順利心想事成啊.... 感謝君宇和峻緯..容忍我這個機車學姊常常說一些很賤的話虧你們..這也只是希望你們加油..希望以後的實驗室有你們的加入會越來越好啊..君宇很感激你常常在我最需要食物的時候奉上啊~~吃素食的我常常亂吃..你的貼心讓我很窩心.. 真的很謝謝你感謝孝修..常常用你配的 10x PBS..呵呵很好用喔~ 知道你是認真貼心的小孩要繼續保持啊.. 感謝豐碩..在我熬夜到早上..看我還在弄實驗..居然貼心的送上一瓶奶茶..讓我很感動..當時我真的還蠻餓的^^" 很謝謝你還要感謝伊婷，如蕙你們的認真讓我看了真的自嘆不如，讓我想向你們看齊，努力做實驗，實驗室有妳們真的很幸福。感謝振銘和天駿，振銘你買的早餐超好吃的，實驗室有你少了很多麻煩事，感謝天駿你的主動幫忙讓實驗室的常規工作輕鬆很多（水浴槽真的是辛苦你了），很謝謝你們.. 感謝宏斌(Apple)，玄峰，南部人的熱情我已經好久沒有感受到了，謝謝你們在我最辛苦難過的時候搞笑，其實你們比我成熟多了，謝謝你們，希望你們可以考上心目中理想的學校。還要感謝最最辛苦的管小姐幫我們動物房每個禮拜洗將近快 150 個籠子，因為我自己也下海洗過，那種辛苦可是會讓腰挺不直的，謝謝您。感謝陸泉和偉齊，有你們減輕了我們換籠換水飼料的工作，真的很謝謝你們體諒研究生們的壞習慣，很謝謝你們。感謝作豪學長，柏寬學長有你們在，讓我覺得熬夜不算什麼，辛苦是為了走更長遠的路....

(6) 感謝北醫謝榮鴻老師實驗室的所有成員，郁慧學姊，宇捷學姊很謝謝妳們在實驗上的指導... 感謝中研院李鴻老師實驗室的所有成員，文正學長，雖然你都不告訴我你的正是哪個正，但是抽的 RNA 品質是一極棒的，謝謝晶片室的書韻學姊的幫忙讓我實驗的 data 如期生出來^^ 感謝所有 NDG 的老師、醫師們，給浚榕的指導與建議，讓我在碩士班生涯過的很充實..感激不盡還要感謝我的一群大學同學流星雨，大頭凱，小仁，偉雅在我最悶的時候來實驗室陪我聊天，聊以前在長庚的事情未來的事情，讓我對於未來又有更多的衝勁與希望，一起加油喔！！還有小仁的大力幫助，讓我真的很感動。感謝奕雯，讓我在前年參加脊髓小腦萎縮症報告的時候有機會和國外演講者聊天，了解人外有人天外有天的道理，銘記在心。感謝我最可愛的瑪麻，浚榕不是個乖女兒，常常熬夜不回家睡覺，或是半夜才跑回家，現在我即將畢業了，希望未來的日子由我來照顧您，會讓你成為最幸福的瑪麻...在碩班的四年當中發生了好多事情，雖然今年的母親節您說了，第一次過沒有媽媽的母親節，往後的日子我一定會加倍補償您的... 最後感謝一直在我心裡陪伴我、支持我的所有實驗小老鼠，沒有你們我想我應該是撐不下去的，謝謝祢們的辛苦與奉獻，你們的主人有可愛的你們真的很幸福。.

(7) 中文摘要. PP2A 主要包含三個次單元分別為 A(結構次單元)，B(調控次單元)，C(催化單元)。調控次單元影響 PP2A 在細胞內存在的位置及 PP2A 受質的特異性。根據一些研究報告指出， Bβ 調控次單元基因與脊髓小腦運動失調症 12 型 (spinocerebellar ataxia type 12，SCA12)有所關聯。 Bβ 有兩種異構物，Bβ1 及 Bβ2， SCA12 是由於 Bβ1 啟動子區域 CAG 三核苷酸擴充所致，目前尚未確定這個不被轉錄的三核苷酸擴充是影響 Bβ1 轉錄活性或涉及到 Bβ1/Bβ2 啟動子間使用頻率。在之前報導指出在 PC12 細胞中 Bβ2-PP2A 會進入粒線體中，並且促使該細胞進入細胞凋亡。為了在動物模式中測試這個假設，我們建立神經組織粒線體過度表現 Bβ2 之轉殖基因小鼠。我們觀察到轉殖鼠有幾個異常的性狀，包含了體型小，毛髮稀疏，脊椎彎曲以及早期夭折等。我們也利用量化的運動行為分析以及病理分析以了解這些基因轉殖鼠於粒線體中過度表現 Bβ2 之影響，期許透過此結果能了解 Bβ2 於粒線體表現與 SCA12 之關聯。目前之研究結果顯示，因 Bβ2 於粒線體之表現的確提升了不同腦區 PP2A 之活性，也同時造成 cytochrome c 之釋出，MnSOD 氧化壓力指標及 Caspase 3 細胞自戕分子之上升，而這些傷害進一步也的確讓我們看到小鼠在 Rotarod 及 HomeCageScan 行為上的異常，由這些結果看來，Bβ2 於粒線體之表現的確造成神經退化。. 【關鍵字】脊髓小腦運動失調症 12 型(SCA12)、粒線體、PP2A、Bβ2 過度表現. -i-.

(8) Abstract. Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase which consists of scaffolding (A), catalytic (C), and variable regulatory (B) subunits. The regulatory B subunits dictate subcellular localization and substrate specificity of the PP2A holoenzyme. The Bβ regulatory subunit gene was reported to be involved in spinocerebellar ataxia type 12 (SCA12). One of the Bβ splice variants, Bβ2, was postulated to target PP2A holoenzyme to mitochondria and promote apoptosis in an in vitro PC12 cell system. To test this hypothesis in vivo, we have established the transgenic mice with Bβ2 overexpression in the mitochondria of neuronal tissues. These transgenic mice show several phenotypical abnormalities, including smaller size, sparse hair, spinal curvature and early lethality. Quantitative motor activity and neuropathological analyses are also conducted to understand the neuronal effect caused by the Bβ2 overexpression in the mitochondria. Our results show that the mitochondria PP2A activity was increased by the Bβ2 overexpression. The cytochrome c releasing, oxidative stress index MnSOD, and apoptotic marker caspase 3 were all significantly up-regulated in the transgenic mice. Behavior performances characterized by Rotarod and HomeCageScan indicate an impairment in neuron function occurred in these animals. Taken together, we suggest that Bβ2 overexpression in the mitochondria of neuronal tissues could induce neuron degeneration.. 【Keyword】 Spinocerebellar ataxia type 12 (SCA12)、mitochondria、PP2A holoenzyme、Bβ2 overexpression. - ii -.

(9) 1. Introduction 1.1. The structure and biological role of protein phosphatase 2A (PP2A) Protein phosphatase 2A (PP2A) is one of the four major classes of serine/threonine phosphatases that also include PP1, PP2B (calcineurin), and PP2C 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 subunits (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 (Ruediger et al., 2001; Takagi et al., 2000). The C subunit also exists in two forms, Cα and Cβ, that are 96% identical. The B subunits fall into 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). The latter two are splice variants of the same gene. The regulation of neuronal functions by PP2A is mediated by the interaction of the AC core dimer with 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 (Dagda et al., 2003; Strack et al., 1998). Unlike Bγ, and Bβ. -1-.

(10) which are only expressed in brain, the ubiquitous regulatory subunits Bα and Bδ dephosphorylate and inactivate ERKs in neurons while B’ subunits have been shown to regulate 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 residue, and dephosphorylation of MAPs by PP2A results in increased assembly and stability of microtubules (Alexa et al., 2002; Merrick et al., 1997; Sontag et al., 1999). 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 to the loss of nigrostriatal neurons leading to a substantial decline in the synthesis of dopamine by tyrosine hydroxylase. 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 tyrosine hydroxylase (TH), an enzyme 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). The Bβ regulatory gene of PP2A has been implicated in an autosomal dominant neurodegenerative disease termed spinocerebellar ataxia type 12 (SCA12) which is caused by a CAG repeat expansion located upstream of the presumed promoter region -2-.

(11) of the human PPP2R2B gene. SCA12 was first described in a large pedigree of German and Indian descent (Fujigasaki et al., 2001; Holmes et al., 1999). Patients afflicted with these lethal disease exhibit lethargic responses, bradykinesia, ataxic gait, cognitive decline and tremors (Holmes et al., 2003). Alternative splicing of the Bβ gene gives rise to four isoforms of the regulatory subunit that only differ in their 5’ UTR region and N-terminal tails. Since patients affected with SCA12 develop widespread neuronal degeneration, proper expression of the Bβ gene must be critical for neuronal survival. It is conceivable that the SCA12 mutation alters the transcriptional regulation and/or splicing events of the Bβ gene, which in turn leads to altered expression levels of each Bβ isoform. Bβ2, a splice variant with a unique N-terminal, is widely expressed in most brain areas. PP2A holoenzymes containing Bβ2 are 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).. 1.2. Apoptosis 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. 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 (Ashe and Berry, 2003; Farber, 1994). The extrinsic pathway of apoptosis usually involves a death ligand such as tumor necrosis factor (TNF) that binds to a death receptor (P75), or a lipid such as ceramide that elicits the release of intracellular -3-.

(12) 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 (reviewed by Ashe and Berry, 2003; Jin and El-Deiry, 2005).. 1.3. The role of PP2A in 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 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 demonstrate 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 -4-.

(13) proapoptotic Bcl-2 family proteins (Ruvolo and 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 proteins (Bcl-2) of the Bcl-2 family, it is generally assumed that PP2A has a positive regulatory function in apoptosis.. 1.4. Mitochondrial PP2A/Bβ2 promotes apoptosis 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. SCA12 is caused by a trinucleotide repeat expansion in a non-coding, presumed promoter region of the human Bβ gene, PPP2R2B (Holmes et al., 1999), indicating that proper regulation of Bβ expression is critical for neuronal survival. 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, potentiated apoptosis in growth factor-deprived PC6-3 cells (Dagda et al., 2003), a subline of PC12 pheochromocytoma cells (Pittman et al., 1993). Previous study also shows that Bβ2 is induced during postnatal brain development (Dagda et al., 2003).. -5-.

(14) 1.5. Spinocerebellar Ataxia 12 Cerebellar dysfunction is a hallmark of different neurodegenerative disorders, like Spinocerebellar Ataxias (SCAs), but many also include abnormalities in other regions of the central and/or peripheral nervous system. At present, 26 distinct genetic forms (SCA1–8, SCA10–23, SCA25–26, SCA27/FGF14 and SCA28) have been identified to date and most of them can be subclassified into three discrete groups based on pathogenesis: (1) the polyglutamine disorders, which result from proteins with toxic stretches of polyglutamine (SCAs 1, 2, 3, 6, 7,and 17); (2) the channelopathies, which result from disruption of calcium or potassium channel function (SCA6); and (3) the gene expression disorders, which result from repeat expansions outside of coding regions that may quantitatively alter gene expression (SCAs 8, 10, and 12)(Knight et al., 2004; Schols et al., 2004; Taroni and Di Donato, 2004; Ishikawa et al., 2005; Yu 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. 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., 2001; Holmes et al., 1999). However, the mechanism of PP2A/Bβ2 underlying SCA12 in vivo study remains to be elucidated. In this study, we establish a mouse model with mitochondria Bβ2 overexpression to investigate the impact of mitochondrial Bβ2 overexpression on the neurodegeneration in vivo. -6-.

(15) 2. Material and method 2.1.Construct pNSE-cox8-Bβ2-flag-EGFP plasmid The pCMV-Bβ2-Flag-EGFP plasmid (a gift from Dr. Strack, Department of Pharmacology, Carver College of Medicine, University of Iowa) was modified to generate pNSE-Bβ2-Flag-EGFP construct by replacing CMV promoter with NSE (neuron-specific-enolase) promoter. The pCMV-Bβ2-Flag-EGFP was digested by AseI/NheI and filled in the overhangs with Klenow polymerase. The NSE promoter on pGEMTeasy plasmid was digested by SphI/NsiI and filled in the overhangs with Klenow polymerase. The NSE promoter and the Bβ2-Flag-EGFP were ligated and the resulted plasmid was denominated pNSE-Bβ2-Flag-EGFP (Fig. 1A). The mouse cox-8A presequence was PCR amplified (primer pair : 5’-AGC TAG CAT GTC TGT CCT GAC GCC ACTG-3’ ; 5’-GAA GCT TAC CTT CGA GTG GAC CT G AG C-3’) (Fig. 8B) and subcloned into pNSE-Bβ2-Flag-EGFP within the cloning sites, NheI and HindIII, the resulted plasmid was denominated pNSE-cox8-Bβ2-Flag-EGFP (Fig. 1A).. 2.2. Animals FVB (3 weeks) and ICR (6-8 weeks) mice were purchased from the National Breeding Center for Laboratory Animals (Taipei, Taiwan) and group housed in a vivarium maintained at 20–25 °C with 60% relative humidity. Food and water were provided ad libitum. A light/dark cycle of 12/12 h was installed with lights on at 7:00 AM. The procedure adhered to Guidelines for Care and Use of Experimental Animals and was approved by the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan.. -7-.

(16) 2.3. Mito-Bβ2 transgenic mice 2.3.1. Production of fertilized eggs for pronuclear injection. On day 1, three week-old FVB female mice were intraperitoneal (i.p.) administration of pregnant mare's serum gonadotropin (PMSG, 5 IU) at PM12:00. After the formation of mature follicle, mice were administrated hCG (5 IU, i.p.) 48 hr after PMSG injection. After the observation of vaginal plug, female FVB mice were scarified by cervical dislocation. Embryos were isolated from the oviducts under the microscope. The cumulus cells and zygotes were separated by short treatment with hyaluronidase. The embryos were washed through several droplets of M2 medium and incubated in the 37°C CO2 incubator. 20-30 embryos were transfer into a droplet of M2 medium(M2 is modified Krebs-Ringer with HEPES buffer substituted for some of the bicarbonate)(Quinn et al., 1982) covered with silicon oil on the injection slide. Each embryo was fixed at the holding pipette by negative pressure. And 1-2-pl DNA was injected into one of the pronuclei of the 1-cell embryo (zygote). The injected embryos were incubated in a CO2 incubator for 1-2 hrs. The 1 or 2-cell embryos were transferred into the infundibulum oviduct of the foster female ICR mice via transfer capillaries. The genotyping with PCR analysis was conducted 3 weeks after pubs were born.. 2.3.2. The generation of mito-Bβ2 transgenic mice Founder mice were identified by PCR analysis of genomic DNA from mouse tail biopsy using primers: 5'-AGC TAG CAT GTC TGT CCT GAC GCC ACT G-3' (in cox-8 region) and 5'-TC ACC CCT ACG ATG AAC CTG -3' (in Bβ2 region). The procedure of PCR analysis was described as following: extract genomic DNA from. -8-.

(17) 0.5 cm mouse tail biopsy by first lysing tissue in lysis solution (50 mM EDTA, 1% SDS, 50 mM Tris-HCl, pH8.0, 100 mM NaCl, 0.35 mg/mL proteinase K) over night at 56°C with mild shaking. Tail genomic DNA was then purified with 5 M potassium acetate salt extraction and pure ethanol precipitation. The PCR amplification was performed by using a hot start followed by 35 cycles of 94°C, 30 sec, 62°C, 60 sec, and 72°C, 60 sec in a 25 μl reaction mixture containing tail genomic DNA (50–100 ng).. 2.4. Measurement of body weight and paw clasping Mouse body weight was measured in every other week until 16 weeks old. After each weighting, mice were suspended by the tail 10 cm above the cage for 20 seconds to score the presence of paw clasping. The paw clasping was scored from zero to two points. Observation of only the front legs pressed together defined as zero points. For mice pressed front paws to the stomach, one point was defined. If front paws touched hind legs, or both front and hind legs hold against the stomach, two points were scored. All clasping tests were scored by an individual who was unaware of the genotypes of the mice.. 2.5 Video recording setup for HomeCageScan JVC digital video cameras (model no. GR-D93) was mounted perpendicular to the cages. The camera inputs into a Pelco video processor connected to a computer with an ATI All-In-Wonder video card. Video data were analyzed by HomeCageScan software (Clever Systems, Reston, VA). During recording, mouse was housed in home cages, with minimal bedding to minimize mounding, which can obscure the. -9-.

(18) mouse. Each mouse was recorded for four hours during light cycle.. 2.6. Hot plate The hot-plate test is an effective and simple test for nociception. The mouse was placed on a hot plate at a controlled temperature (55.0°C; CH-100, BIOSAN, Latvia) and the latency of the first hind-paw response was recorded. The hind-paw response is defined as either shaking of the paw or a paw lick.. 2.7. Measurement of locomotor activity The test is to place one mouse in a black plexiglass box (30 x 30 x 30 cm) under a light condition. The field is continuously monitored by a computer-operated Etho Vision video tracking system (Noldus, Netherlands), which consists of eight equidistant photoreceptor beams on each side of the box, dividing the field into 64 equally sized squares.The total moving distance and average velocity of each mouse were measured during a 10 min period.. 2.8. Rotarod test The rotarod test evaluates general motor coordination and balance. The test consists of rotating drum (Ugo Basile, Comerio, Italy). The mice were trained for 5 min on two separate days with a fixed speed at 5 rpm. Testing was conducted the following week with a speed at 15 rpm. Mice were subjected to three trials, each with a maximum duration of 180 sec. The latency and the rotation speed at which the mouse falls off the drum were recorded. Mouse performance typically improves with repetition. To control for this improvement, each mouse should undergo four trials with a 30 min interval between trials.. - 10 -.

(19) 2.9. Immunohostochemistry Immediately after the rotarod test, mice were anesthetized (0.016 ml/g BW, 2.5% avertin, Sigma, Germany) and transcardiacally perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were removed and post-fixed with 4% paraformaldehyde overnight and then placed in 30% sucrose in PBS for 2 days. Brains were serially sectioned 30 µm on a cryostat. For immunohistochemistry, free-floating sections were immunostained. In brief, sections were rinsed in 0.1 M PBS three times (10 min/wash). Endogenous peroxidase activity was blocked by incubation with 3% H2O2 for 30 min. Sections were then washed in PBS for three times (10 min/wash). Nonspecific epitopes were then blocked by incubation in 5% normal goat serum and 0.1% Triton X-100 in PBS for 2 hrs. Sections were incubated. overnight at room temperature with primary. antibodies, Calbindin (Sigma, 1:1000) and caspase 3 (Chemicon, 1:40). Secondary antibodies were then applied to the sections by a linking reagent (DAKO, CA) for 1 hr. Immunostaining was highlighted using substrate-chromogen solution and diaminobenzidine oxidation. All sections were mounted on coated slides and cover-slipped for light microscopy observation.. 2.10. Mitochondria purification The mitochondrial fractions of tissues from the different regions of mouse brain (cortex, brainstem, hippocampus and cerebellum) were isolated by using a mitochondrial extraction kit (Active Motif, Carlsbad, CA. USA) according to the manufacturer protocol. In brief, tissue samples were gently homogenized with a glass–teflon homogenizer. Homogenates were centrifuged at 800 g for 10 min at 4°C, and the supernatant was collected and centrifuged at 16000 g for another 30 - 11 -.

(20) min at 4°C to pellet the mitochondria. The supernatant thus obtained was the cytosolic fraction. The mitochondrial pellet was resuspended in 100 μl of isolation medium. The purity of the mitochondrial fraction was verified by the selective expression of the mitochondrial inner membrane-specific protein prohibitin. Total protein in the mitochondrial or cytosolic extracts was quantified by the BCA Protein Assay (Pierce, USA).. 2.11. Western blot analysis Western blot analysis was conducted on proteins extracted from the frontal cortex, hippocampus and cerebellum with antibodies for Bβ2, Flag, Bcl-2, cytochrome c, prohibitin, and β-actin. The antibodies used were rabbit polyclonal anti-cytochrome c (1:1000; Santa Cruz, CA, USA); mouse monoclonal anti-Bβ2 (1:2000; a gift from Dr. Strack, Department of Pharmacology, University of Iowa Carver College of Medicine), Flag (1:1000; Sigma, USA), Bcl-2 (1:1000; Sigma, USA), prohibitin (1:1000; Labvision/NeoMarkers, Fremont, CA, USA), β-actin (1:5000; Chemicon, Temecula, CA, USA). The secondary antibody of anti-rabbit IgG HRP-linked antibody (Cell signaling, USA) was used for cytochrome c. The second antibody of anti-mouse IgG HRP-linked antibody (Cell signaling, USA) was used for Bβ2, Bcl-2, prohibitin, and β-actin. Specific antibody–antigen complex was detected by an enhanced chemiluminescence Western blot detection system (Amersham Pharmacia Biotech, USA). The intensity of Western analysis was quantified by the Fuji LAS-3000 imaging system (Fuji, Japan), and was expressed as a ratio relative to β-actin protein (for analysis of total protein or proteins in cytosolic fraction) or prohibitin (for analysis of proteins in mitochondrial fraction).. - 12 -.

(21) 2.12. Mitochondria PP2A activity PP2A activity was determined using a PP2A immunoprecipitation phosphatase assay (Upstate, USA ) that measures free phosphate with a malachite green dye. To immunoprecipitate PP2A, lysates containing 200 μg of protein were incubated with 4 μg of anti-PP2A-C subunit antibody (clone 1D6) and 40 μl of protein A-agarose slurry for 2 hrs at 4°C with constant rocking. The immunoprecipitates were washed three times in Tris-buffered saline and once with Ser/Thr assay buffer (50 mM Tris-HCl, pH 7.0, 100 μM CaCl2), and resuspended in 20 μl of Ser/Thr assay buffer. The reaction was initiated by the addition of 60 μl of phosphopeptide substrate (750 μM KRpTIRR). Following incubation for 10 min at 30°C in a shaking incubator, the reaction mixture was centrifuged briefly and the supernatant was transferred to a 96-well microtiter plate. The reaction was terminated by the addition of malachite green phosphate detection solution for 10–15 min at room temperature, and free phosphate was quantified by measuring the absorbance of the mixture at 650 nm using a microplate reader.. 2.13. Measurement of mitochondria membrane potential Mitochondrial membrane potential assay with Isolated mitochondria staining kit (Sigma, USA) is designed for a total volume of 100 μl. First, the isolated mitochondrial sample (up to 10 μl) or valinomycin( a mitochondrial membrane dissipating agent, for control experiments) treated mitochondrial sample equivalent to 5 μg of protein was added to each well. JC-1 Staining Solution 90 μl was then added to the well. If required, bring the total reaction volume to 100 μl with JC-1 Staining Solution. Fluorescence intensity was measured in a spectrofluorometer (Gemini, Molecular Devices Corp., CA, USA) using the 490 nm wavelength for excitation and the emission wavelength was set at 590 nm. - 13 -.

(22) 2.14. Data analysis To compare the Bβ2-overexpression effects between the mito-Bβ2 transgenic mice and their wild-type littermate, independent samples Student’s t tests were used in the study. The statistical results were expressed as means ± SEM.. 3. Results 3.1. Generation of mito-Bβ2 transgenic mice Total 6 transgenic founder lines (786, 790, 791, 1433, 1445 and 1461) were generated according to the results of PCR analysis (Fig. 2). However, 786 and 790 founder mice died at the age of 3 weeks. Western blot analysis also confirmed the expression of the transgene encoded protein in transgenic mouse brain (Fig. 3). The results of Western analysis indicates that line 791 and 1433 had the higher expression level among these 4 lines (Fig. 3). Therefore, lines 791 line and 1433 were selected for breeding and the generated offspring were used in the study.. 3.2. Mophological phenotype of mito-Bβ2 transgenic mice Mito-Bβ2 transgenic mice could easily be distinguished from age-matched, wild-type littermates by their abnormal appearance. For example, smaller body size, feeble, thin hair, easily got frightened, and curved spine were all conspicuous features of these transgenic mice (Fig. 4A). The mito-Bβ2 transgenic mice have a substantially reduced lifespan (Fig. 4B). They began to die at 9 weeks of age, and some transgenic mice (lines 786 and 790) even died at 3 weeks old.. From the characterization of. body weight in every other week, we found that both of the male and female - 14 -.

(23) transgenic mice grow much slowly than those of wild type mice (p < 0.05; Fig. 4C & D).. 3.3. Abnormal neurobehavior of mito-Bβ2 transgenic mice Clasping, spontaneous seizures and tremor were also identified in some of the transgenic mice. For scoring the paw clasping occurrence, we found that both male and female transgenic mice have higher frequency in showing clasping (p < 0.05; Fig. 5). In addition, transgenic male mice possess more and earlier clasping behavior than transgenic female mice (p < 0.05; Fig. 5B). We also characterized mouse behavior inside the homecage by using the HomeCageScan system (Clever Sys. Reston VA. USA). We found the number of behavior was slightly fewer in transgenic mice than in wild type littermate (Fig. 6A). However, explaratory, motor, and eating behaviors were significantly decreased in transgenic mice as compared to wild type mice (p < 0.05, Fig. 6B). Furthermore, the resting behavior was significantly prolonged in transgenic mice compared to wild type mice (p < 0.05, Fig. 6B) For the nociception test, we found that there is no difference in the length of latency on the hot plate between trangenic and wild type mice (p > 0.05, Fig.7). In addition, locomotor activity was significantly reduced in the transgenic group in comparison with the wild type group (p < 0.05, Fig. 8A & B). For the rotarod test, we found that the learning impairment of motor task was significant enhanced in transgenic mice as compared to the wild type mice (p < 0.05, Fig 9A). Furthermore, the impairment of motor coordination was also enhanced in the transgenic mice as compared to wild type mice (p<0.05, Fig. 9B).. 3.4. PP2A activity and mitochondria membrane potential. - 15 -.

(24) In order to find out whether the mitochondria function or PP2A activity altered by the overexpression of Bβ2 in the mitochondria. We first tested the PP2A activity in the mitochondria from different brain regions of transgenic and wild type mice. The results indicated that the PP2A activity in transgenic mice was significantly higher than that in wild type mice , of all the different brain regions examined, including the cerebellum, brainstem, cortex, and hippocampus (Fig. 10). The mitochondria membrane potential of transgenic mice was significantly increased in cerebellum, cortex, and hippocampus regions of transgenic mice but decreased in brain stem region as compared to wild type mice (Fig. 11).. 3.5. The neuropathology of mito-Bβ2 transgenic mice There was no obvious difference in gross brain morphology between transgenic and wild type mice. The hippocampus and cerebellum were the most affected brain regions in transgenic mice as evaluated by immunohistochemical analyses (Fig. 12). We found that the level of the MnSOD and caspase 3 were significantly enhanced in the hippocampus of the transgenic mice as compared to the wild type mice (p < 0.05, Fig. 12.1). In addition, the level of the oxdative stress was significantly enhanced in the cerebellum of the transgenic mice as compared to the wild type mice (p < 0.05, Fig. 12.2). The number of calbindin-positive cerebellar neurons was also decreased in the transgenic mice as compared to wild type mice ( p <0.05, Fig. 12.2). The result of western blot analysis also showed higher cytochrome c and Bcl-2 in. cerebellum, brainstem, cortex, and hippocampus regions of transgenic mice as compared to wild type mice ( p < 0.05, Fig. 13). - 16 -.

(25) 4. Discussion. In this study, we found that mitochondria Bβ2 overexpression induces distinctive neurological phenotypes in mice. The progressive neurological phenotype of the early onset Mito-Bβ2 mice are characterized with smaller body size, feeble, thin hair, easily got frightened, seizure, tremor, clasping, curved spine and early death. These variable phenotypes are consistent with previous reports of SCA12 case study (Holmes et al., 1999). One recent evidence also suggests that mitochondria dysfunction increases the rate of lethality (Liu et al., 2007). Consistent with the role in neuronal phenotype in Mito-Bβ2 mice, Bβ2 is implicated in a number of neurological or neurodegenerative disorders (Strack et al., 2004). The observation that the SCA12 mutation is detrimental for neurons suggests that proper regulation of the Bβ gene is critical for neuronal survival (Schmidt et al., 2002). Therefore, the phenotype of the mito-Bβ2 mice provides valuable insight into the pathogenesis of mitochondrial dysfunction or SCA12 disease. We found that mitochondria Bβ2 overexpression induced the impairment of motor function, but not nociception. A lot of studies suggest that a spectrum of myopathic and neuropathic symptoms in humans have been correlated to mitochondria dysfunction (Manfredi et al., 2005; Shukkur et al., 2006). From the results of calbindin immunohistochemistry, we also found that a significant loss of Purkinje cells was identified in the transgenic mice compared to wild type mice. Furthermore, the oxidative stress of the cerebellum was enhanced in transgenic mice but not wild type mice. Previous studies also suggest that increased oxidative stress and disruption of neuronal calcium homeostasis appear to be interrelated final common pathways that mediate the neurodegenerative process in AD (Appel et al., 2001; Cao et al., 2007; Mattson et al., 1993; McFarland et al., 2007; Rosenstein et al., 1998). Therefore, we - 17 -.

(26) suggest that the impairment in calcium homeostasis might induce the oxidative stress in Purkinje cells. Finaly, the loss of Purkinje cells induced the impairment in motor coordination. About the impairment in the learning and memory of motor in the animal model, the early degeneration presented outside of the cerebellum as hippocampus region could provide the explanation. Previous study also shows that the signs of both cerebellar and cortical are both involved in the clinical features of SCA12 patients (Holmes et al., 2001). Our study show that overexpression of Bβ2 in the mitochondria increased PP2A activity, the release of cytochrome c, and the caspase 3 expression in different mouse brain regions. However, membrane potential was reduced in most of the regions examined except for brain stem. On the other hand, the anti-apoptic molecule, Bcl-2 was also found increased in all the different regions examined. Whether some protection responses in the mitochondria induced by the Bβ2 overexpression damage in order to sustain the neuronal survival needs further investigation. In summary, we have established the animal model and evaluated some of the molecular effect of mitochondria Bβ2 overexpression in these transgenic mice.. - 18 -.

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(33) APPENDIX. Figure 1. Construct the pNSE-cox8-Bβ2-flag-EGFP plasmid. (A) The mouse cox-8 presequence was subcloned in front of Bβ2-Flag-EGFP, the plasmid was denominated pNSE-cox8-Bβ2-Flag-EGFP. (B) The mouse cox-8 presequence was PCR amplified with NheI and HindIII cloning sites within the primers. - 25 -.

(34) Figure 2. Transgenic founder mice (F0) identified by genotyping analysis. Identification of transgenic mice is performed with PCR analysis with a pair of primers designed from the loci of cox8 and Bβ2. The product size of the PCR amplification is 313 bps. M: 100 bp ladder maker; P: positive control (construct DNA + tail genomic DNA); N: negative control (without any DNA added); WT: wild type FVB mouse genomic DNA; Lane: 1-6 are DNA from lines 786, 790, 791, 1433, 1445 and 1461. β actin is used as an internal control.. - 26 -.

(35) (A). Relative percentage of protein over-expression. (B) 1400. 1200. 1000. 800. WT 791 line 1433 line 1445 line 1461 line. *. * *. 600. * 400. 200. Bβ2. Flag. Figure 3. Identification of Bβ2-flag expression in Mito-Bβ2 transgenic mouse brain.. (A) Bβ2-flag in brain of transgenic and wild type mice were characterized. by western blot analysis with anti-Bβ2 and anti-flag antibodies. (B) The densitometric ratio of Bβ2 and flag to β actin in the transgenic mice.. - 27 -.

(36) (A). (B) 100. 791 line 1433 line WT littermates. Survival (%). 80. 60. 40. 20. 0 0. 3. 6. 9. 12. 15. 18. Age (Weeks). - 28 -. 21. 24. 27. 30.

(37) (C) 35. Male TG Male WT. Body weight (g). 30. 25. 20. 15. 10. 2. 4. 6. 8. 10. 12. 14. 16. 14. 16. Age (Weeks). (D) 35. Female TG Female WT. Body weight (g). 30. 25. 20. 15. 10. 2. 4. 6. 8. 10. 12. Age (Weeks). Figure 4. Phenotype of mito-Bβ2 transgenic mice. (A) The transgenic mouse (arrow) is small relative to its wild-type littermates. (B) Survival plot showing reduced lifespan of transgenic mice compare to wild-type (WT). (n = 80 for each group). (C) The body weight of male transgenic mice was significantly lighter compared to WT mice at any age. (p < 0.05; n =10 for each group). (D) The body weight of female transgenic mice was significantly lighter compared to WT mice at any age. (p < 0.05; n = 10 for each group). - 29 -.

(38) (B). 1.8. Male TG Female TG WT. Average of Clasping Score. 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 2. 4. 6. 8. 10. 12. Age (Weeks). 14. Figure 5. Paw clasping phenotype in transgenic and wild type mice.. 16. 18. (A) The. scoring for clasping. Only the front legs pressed together defined as zero point. Mice pressed front paws to the stomach defined as one point. Front paws touched hind legs, or both front and hind legs hold against the stomach is defined as two points. (B) The average of clasping score was significantly higher in transgenic mice than in wild type mice. (p < 0.05; n = 10 for each group).. - 30 -.

(39) (A) Numbers of behavior. 40. 30. 20. 10. 0 WT. (B). TG. Relative of behavior time. 100. 80. WT TG. 60. 40. 20. 0. Self-explanatory. Motor. - 31 -. Eat.

(40) (C) Relative of behavior time. 180. 160. WT TG. 140. 120. 100. 80. Rest. Figure 6. Behavioral analysis by HomeCageScan system. (A). The number of behavior was slightly fewer in transgenic mice than in wild type mice (p > 0.05; n = 4 for each group). (B). The duration of exploratory, motor, and eating was significantly decreased in transgenic mice as compared to wild type mice (p < 0.05; n = 4 for each group). (C) In contrast, the duration of resting was significant increased in transgenic mice compared to wild type mice (p < 0.05; n = 4 for each group). Error bars indicate standard error means.. - 32 -.

(41) 60. Latencies (seconds). 50. 40. 30. 20. 10. 0. WT. TG. Figure 7. Nociceptive analysis by hot plate test. No significant of nociception was identified between transgenic and wild type mice (p > 0.05; n = 10 for each group). Error bars indicate standard error means.. - 33 -.

(42) (A) Moved distences (cm). 5000. 4000. 3000. * 2000. 1000. 0. TG. WT. (B) Moved velocity (cm/min). 20. 15. 10. *. 5. 0. TG. WT. Figure 8. Comparison of locomotor activity between transgenic and wild type mice. (A) The distances of moving were significantly decreased in transgenic mice as compared to that in wild type mice (n = 10 for each group). (B) The velocity of moving was significantly decreased in transgenic mice as compared to that in wild type mice n=10 for each group). Error bars indicate standard error means. *, p < 0.05.. - 34 -.

(43) Figure 9. Compare motor coordination between transgenic and wild type mice by rotarod test.. (A) The learning of motor task was significantly impaired in. transgenic mice when compared with wild type mice (p < 0.05; n = 10 for each group). (B) The impairment of motor coordination was significantly enhanced in transgenic mice as compared to that of wild type mcie (p < 0.05; n = 10 for each group). Error bars indicate standard error means. *, p < 0.05. - 35 -.

(44) (A) Activity of mitochondrial PP2A (pmoles). 3000 WT TG 2500. 2000. 1500. 1000. 500. 0. Cerebellum. Brain Stem. Cortex. Hippocampus. Brain regions. (B) Activity of cytosol PP2A (pmoles). 3000. WT TG. 2500. 2000. 1500. 1000. 500. 0. Cerebellum. Brain Stem. Cortex. Hippocampus. Brain regions. Figure 10. PP2A activity in mouse brain mitochondria and cytosol.. (A) The. mitochondrial PP2A activity was significantly increased in cerebellum, brainstem, cortex, and hippocampus regions of transgenic mice as compared to wild type mice ( p< 0.05, n = 4 for each group). (B) The cytosol PP2A activity was significantly decreased in brainstem and cortex regions of transgenic mice as compared to wild type mice ( p< 0.05, n = 4 for each group). Error bars indicate standard error means. - 36 -.

(45) (A) 20. RFU of JC-1 staining assay. WT-Cerebellum. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. 1000. 1200. 1400. 1000. 1200. 1400. Time (sec). (B) 20. RFU of JC-1 staining assay. TG-Cerebellum. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. Time (sec). - 37 -.

(46) (C) RFU of JC-1 staining assay. 20. WT-Brain stem. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. 1000. 1200. 1400. Time (sec). (D). RFU of JC-1 staining assay. 20. TG-Brain stem. 15. 10. 5 - Valinomycin + Valinomycin. 0 0. 200. 400. 600. 800. Time (sec). - 38 -. 1000. 1200. 1400.

(47) (E) 20. RFU of JC-1 staining assay. WT-Cortex. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. 1000. 1200. 1400. Time (sec). (F) RFU of JC-1 staining assay. 20. TG-Cortex. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. Time (sec). - 39 -. 1000. 1200. 1400.

(48) (G). 20. RFU of JC-1 staining assay. WT-Hippocampus 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. 1000. 1200. 1400. Time (sec). (H) 20. RFU of JC-1 staining assay. TG-Hippocampus. 15. 10. 5 - Valinomycin + Valinomycin 0 0. 200. 400. 600. 800. Time (sec). - 40 -. 1000. 1200. 1400.

(49) Relative percentage of mitochondrial membrane potential. (I) 140. WT TG. 120. 100. 80. Cerebellum. Brain Stem. Cortex. Hippocampus. Brain regions. Figure 11. Mitochondria membrane potential in mouse brain. (A)-(H) Time course of JC-1 stain fluorescence in cerebellum, brainstem, cortex, and hippocampus regions of wild type (A, C, E, G) and transgenic (B, D, F,H) mice. The upper lines represent the JC-1 dye uptaken by an intact mitochondrial samples and the lower lines represents the dye uptaken by the valinomycin treated mitochondrial control samples. (I) The quantitation of results of (A)-(H) The mitochondria membrane potential was significantly increased in cerebellum, cortex, and hippocampus but decreased in brain stem of transgenic mice as compared to wild type mice (p < 0.05, n = 4 for each group).. - 41 -.

(50) Figure 12.1 Immunostaining assay of hippocampus in transgenic and wild type mice. (A) The staining of MnSOD and Caspase 3 in the transgenic mice are significantly increased as shown by the presence of brown dot. Scale bar = 100 μm. (B) The relative percentages of MnSOD and caspase 3 staining area were significantly higher in transgenic mice than in wild type mice (p < 0.05).. - 42 -.

(51) Figure 12-2 Immunostaining assay of the cerebellum of the transgenic and wild type mice. (A) The positive staining of MnSOD and calbindin was significantly changed in the transgenic mice. Scale bar = 100 μm. (B) The relative percentage of MnSOD staining area was significantly enhanced in transgenic mice compared to wild type mice (p < 0.05). In contrast, the relative percentage of calbindin staining area was significanlyt decreased in transgenic mice than in wild type mice (p < 0.05). - 43 -.

(52) (A). (B). - 44 -.

(53) Relative percentage of cytochrome c release. (C). 300 WT TG 250. 200. 150. 100. 50. 0. Cerebellum. Brain Stem. Cortex. Hippocampus. Brain regions. (D) Relative percentage of Bcl-2 contents. 1000 WT TG 800. 600. 400. 200. 0. Cerebellum. Brain Stem. Cortex. Hippocampus. Brain regions. Figure 13. Apoptotic influences on mito-Bβ2 mice evaluated by the levels of cytochrome c and Bcl-2.. (A) The purity of the mitochondrial fraction was. verified by the mitochondrial inner membrane-specific proteins prohibitin and mitochondrial cytochrome c. The cytosolic fraction was verified by β-tubullin. In order to study Bβ2 distribution in brain subcellular localization and Bβ2 content on - 45 -.

(54) different WT brain region; Test of 8 weeks-old FVB WT mice (n = 4) was conducted. Left panel are protein fractions from cytoplasm and right panel are protein fractions from mitochondria of different brain regions. (B) Western blot analysis was performed with specific antibodies as indicated. The cytochrome c and Bcl2 expression are significantly increased in cerebellum, brainstem, cortex, and hippocampus regions of transgenic mice as compared to wild type mice ( p< 0.05, n = 3 for each group). (C) The densitometric ratio of cytochrome c to prohibitin in the mitochondrial fraction. (D) The densitometric ratio of Bcl-2 to β actin in the cytosol fraction, Co:cortex；Ce:cerebellum；Hippo:hippocampus；Bs:brain stem.. - 46 -.

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