臺灣博碩士論文加值系統

第八型家族遺傳性帕金森氏症患者為體染色體顯性遺傳(autosomal dominant inheritance) 且患者表現晚發性的帕金森氏症症候群症狀。近期研究指出Leucine-rich repeat kinase 2 (第二型多白胺酸性磷酸酶; LRRK2) 的誤義突變(missense mutations) 會導致第八型遺傳型顯性帕金森氏症(PARK8)，而LRRK2 亦為導致晚發性顯性遺傳帕金森氏症最常見的基因。此外， LRRK2的突變亦在偶發性帕金森氏症佔有顯著的比例。具有LRRK2 突變的帕金森氏症病患具有與偶發性帕金森氏症患者相似的晚發性臨床特徵，顯示第八型遺傳型顯性帕金森氏症患者和偶發性帕金森氏症患者在發病過程上具有共有的分子機制。目前普遍認為第八型遺傳型顯性帕金森氏症突變是屬於一個獲得功能的突變(gain-of-function) 並且具有神經毒性的特性，導致了黑質-紋狀體多巴胺神經通路的退化以及帕金森氏症症候群症狀。為研究突變LRRK2 導致黑質-紋狀體神經系統的退化以及帕金森氏症症候群症狀的細胞及分子機轉，目前我們藉由產生野生型以及突變型(G2019S) LRRK2 基因轉殖鼠建立第八型遺傳型顯性帕金森氏症的活體動物系統。
在本實驗中我們使用PDGF-chain promoter作為表現控制以產生野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠。野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠的產生是透過原核顯微注射（Pronuclei microinjection）轉殖基因至受精卵母細胞的雄性原核中。之後以南方墨點法 ( southern blotting ) 加以篩選得到初代小鼠，且其表現大於五個表現 ( > 5 copies) 的初代小鼠將用於建立野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠表現群組。西方墨點法證實不論野生型或是突變型(G2019S) LRRK2 基因轉殖鼠在大腦皮質、黑質、海馬迴以及紋狀體等部位皆有野生型或突變型 (G2019S) LRRK2蛋白的表現。我們使用多種動物行為模式測驗，包含rotarod test， locomotor activity test 以及 pole test， 評估野生型以及突變型 (G2019S) LRRK2 基因轉殖鼠的運動功能。突變型 (G2019S) LRRK2 基因轉殖鼠於五個月大時出現運動功能障礙的症狀，並且神經學上的表徵隨著時間增加在接下來數月有明顯的進展。突變型 (G2019S) LRRK2 基因轉殖鼠表現顯著 PARK8 症狀，包含活動力低下以及動作協調以及表現障礙；相較而言，野生型 LRRK2 基因轉殖鼠並無表現出症狀以及運動功能障礙。目前研究顯示突變型 (G2019S) LRRK2 基因轉殖鼠明顯表現出 PARK8 神經學上的症狀，並且可以被用於作為 PARK8 的活體動物模式。此外野生型以及突變型(G2019S) LRRK2 基因轉殖鼠也可以用於探討LRRK2 在腦中的生理功能。

Patients with familial type 8 of Parkinson’s disease (PARK8) exhibit late-onset parkinsonism symptoms and autosomal dominant inheritance. Recent studies identified missense mutations of leucine-rich repeat kinase 2 (LRRK2) as the cause of PARK8. LRRK2 is the most frequent causative gene in late-onset autosomal dominant PD. Furthermore, LRRK2 mutations are also detected in a significant fraction of sporadic PD patients. Late-onset clinical features of PD patients with LRRK2 mutation are similar to those of sporadic PD patients, suggesting that common molecular mechanisms are involved in the pathogenesis of both PARK8 and sporadic PD. It is generally believed that PARK8 mutation confers a gain-of-function and neurotoxic property on mutant LRRK2, which causes the degeneration of nigrostriatal dopaminergic pathway and parkinsonism. To investigate the cellular and molecular mechanism of mutant LRRK2-induced degeneration of nigrostriatal system and resulting parkinsonism, in the present study we prepared in vivo animal model of PARK8 by generating transgenic mice expressing PARK8 mutant (G2019S) LRRK2.
The expression of human wild-type or mutant (G2019S) LRRK2 in transgenic mice was under the transcriptional control of PDGF-chain promoter. Transgenic mice expressing wild-type or mutant (G2019S) LRRK2 were generated by microinjecting transgene DNA into the male pronucleus of fertilized oocytes. Then, Southern blot analysis was performed to identify founder animals, and founder mice with a high copy number (> 5 copies) were used to establish stable lines of LRRK2 or (G2019S) LRRK2 transgenic mice. Western blot analysis demonstrated that wild-type or mutant (G2019S) LRRK2 protein was expressed in various brain regions of transgenic mouse, including cerebral cortex, substantia nigra , hippocampus, and striatum. Various behavioral tests, including the rotarod test, locomotor activity test and pole test, were used to evaluate the motor function of wild-type or (G2019S) LRRK2-expressing transgenic mice. Transgenic mice expressing mutant (G2019S) LRRK2 displayed the symptoms of motor dysfunction with an onset age of about 5 months, and the severity of neurological phenotypes progressively increased in the following months. (G2019S) LRRK2 transgenic mice exhibited several PARK8 symptoms, including hypoactivity, impaired motor coordination and performance. In contrast, transgenic mice expressing wild-type LRRK2 failed to display the symptom of motor dysfunction. The present study clearly shows that mutant (G2019S) LRRK2-expressing transgenic mice exhibit neurological phenotypes of PARK8 and can be used as an in vivo PARK8 animal model. Furthermore, transgenic mice expressing wild-type or mutant (G2019S) LRRK2 can also be utilized to study the physiological function(s) of LRRK2 in the brain.

Abstract (English) v
Abstract (Chinese) viii
Contents ix
I. Introduction 1
II. Specific Aim and Significance 10
III. Experimental Procedures 11
3.1 Construction of point mutant LRRK2 11
3.2 Generation of transgenic mice expressing PARK8 mutant
(G2019S) LRRK2 11
3.3 Identification of transgenic animals by Southern blotting
of tail DNA 12
3.4 Behavioral tests 12
3.5 Western blot analysis 13
3.6 Immunocytochemical staining 14
IV. Results 15
4.1 Transgene construction for transgenic mice expressing
wild-type or mutant (G2019S) LRRK2 15
4.2 Generation of transgenic mice expressing wild-type or
mutant (G2019S) LRRK2 16
4.3 Transgenic mice expressing mutant (G2019S) LRRK2
exhibit PARK8 neurological phenotypes 17
V. Discussion 19
VI. References 26
VII. Figures and Figure Legends 35

Abou-Sleiman P.M., Muqit M.M.K. and Wood N. W. (2006) Expanding insights of mitochondrial dysfunction in Parkinson’s disease. Nature Rev. Neurosci., 7, 207-219.
Beilina, A., Van Der Brug, M., Ahmad, R., Kesavapany, S., Miller, D.W., Petsko, G.A., Cookson, M.R., (2005) Mutations in PTEN-induced putative kinase 1 associated with recessive Parkinsonism have differential effects on protein stability. Proc. Natl. Acad. Sci. USA. 102, 5703-5708.
Biskup, S., Moore, D. J., Celsi, F., Higashi, S. West, A.B., Andrabi, S. A., Kurkinen, K., Yu, S.W., Savitt, J.M., Waldvogel, H.J., Faull, R.L.M., Emson, P.C., Torp, R., Ottersen, O.P., Dawson, T.M., Dawson, V.L., (2006) Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann. Neurol. 60, 557–569
Bonifati V., Rizzu P., van Baren M.J., Schaap O., Breedveld G.J., Krieger E., Dekker M.C., Squitieri F., Ibanez P., Joosse M., van Dongen J.W., Vanacore N., van Swieten J.C., Brice A., Meco G., van Duijn C.M., Oostra B.A. and Heutink P. (2003) Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science, 299, 256-259.
Bonifati V. (2007) LRRK2 low-penetrance mutations (Gly2019Ser) and risk alleles (Gly2385Arg)-linking familial and sporadic Parkinson’s disease. Neurochem. Res., In Press.
Bosgraaf L., Van Haastert P.J. (2003) Roc, a Ras/GTPase domain in complex proteins. Biochem. Biophsy. Acta., 1643, 5-10.
Canet-Aviles R.M., Wilson M.A., Miller D.W., Ahmad R., McLendon C., Bandyopadhyay S., Baptista M.J., Ringe D., Petsko G.A. amd Cooksn M.R. (2004) The Parkinson’s disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl. Acad. Sci. USA, 101, 9103-9108.
Chou A.H., Yeh T.H., Kuo Y.L., Kao Y.C., Jou M.J., Hsu C.Y., Tsai S.R., Kakizuka A. and Wang H.L. (2006) Polyglutamine-expanded ataxin-3 activates mitochondrial apoptotic pathway by upregulating Bax and downregulating Bcl-xL. Neurobiology of Disease, 21, 333-345.
Cookson M.R., Xiromerisiou G. and Singleton A. (2005) How genetics research in Parkinson’s disease is enhancing understanding of the common idiopathic forms of the disease. Current Opinion in Neurology, 18, 706-711.
Di Fonzo A, Rohe CF, Ferreira J, Chien HF, Vacca L, Stocchi F, Guedes L, Fabrizio E, Manfredi M, Vanacore N, Goldwurm S, Breedveld G, Sampaio C, Meco G, Barbosa E, Oostra BA, Bonifati V; Italian Parkinson Genetics Network. (2005) A frequent LRRK2 gene mutation associated with autosomal dominant Parkinson's disease. Lancet, 365, 412-415.
Di Fonzo A, Wu-Chou YH, Lu CS,van Doeselaar M, Simons EJ, Rohe CF, Chang HC, Chen RS, Weng YH, Vanacore N, Breedveld GJ, Oostra BA, Bonifati V. (2006) A common missense variant in the LRRK2 gene, Gly2385Arg, associated with Parkinson's disease risk in Taiwan. Neurogenetics, 7, 133-138
Fleming S.M., Salcedo J., Fernagut P.O., Rockenstein E., Masliah E., Levine M.S. and Chesselet M.F. (2004) Early and progressive sensorimotor anomalies in mice overexpressing wild-type human -synuclein. J. Neurosci., 24, 9434-9440.
Funayama, M., Hasegawa, K., Kowa, H., Saito, M., Tsuji, S., Obata, F. (2002) A new locus for Parkinson’s disease (PARK8) maps to chromosome 12p11.2-q13.1. Ann. Neurol., 51, 296-301.
Funayama, M., Hasegawa, K., Ohta, E., Kawashima, N., Komiyama, M., Kowa, H., Tsuji, S., Obata, F. (2005) An LRRK2 mutation as a cause for the parkinsonism in the original PARK8 family. Ann. Neurol., 57, 918-921.
Funayama, M., Li, Y., Tomiyama, H., Yoshino, H., Imamichi, Y., Yamamoto, M., Murata, M., Toda, T., Mizuno, Y., Hattori, N. (2007) Leucine-rich repeat kinase 2 G2385R variant is a risk factor for Parkinson disease in Asian population. Neuroreport, 18, 273-275.
Galter D,, Carmine A, Lindqvist E, Sydow O, Olson L. (2006) LRRK2 expression linked to dopamine-innervated areas. Ann. Neurol., 59, 714-719.
Gandi S. and Wood N. W. (2005) Molecular pathogenesis of Parkinson’s disease. Human Mol. Genet., 14, 2749-2755.
Gandi, S., Muqit M.M.K., Stanyer L., Healy D.G., Abou-Sleiman P.M., Hargreaves I., Heales S., Ganguly M., Parsons L., Lees A.J., Latchman D.S., Holton J.L., Wood, N.W., Revesz T. (2006) PINK1 protein in normal human brain and Parkinson’s disease. Brain 129, 1720-1731.
Gilks WP, Abou-Sleiman PM, Gandhi S, Jain S, Singleton A, Lees AJ, Shaw K, Bhatia KP,Bonifati V, Quinn NP, Lynch J, Healy DG, Holton JL, Revesz T, Wood NW (2005) A common LRRK2 mutation in idiopathic Parkinson's disease. Lancet, 365, 415-416
Gloeckner, C.J., Kinkl, N., Schumacher, A., Braun, R.J., O'Neill, E., Meitinger, T., Kolch, W., Prokisch, H., Ueffing, M., 2006. The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity. Hum. Mol. Genet. 15, 223–232.
Green D.R. (2005) Apoptotic pathways: ten minutes to dead. Cell, 121, 671-674.
Greggio E, Jain S, Kingsbury A, Bandopadhyay R, Lewis P, Kaganovich A, van der Brug MP, Beilina A, Blackinton J, Thomas KJ, Ahmad R, Miller DW, Kesavapany S, Singleton A, Lees A, Harvey RJ, Harvey K,Cookson MR. (2006) Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol. Dis., 23, 329-41.
Guo, L., Wang, W., Chen, S.G. (2006) Leucine-rich repeat kinase 2: Relevance to Parkinson’s disease. Int. J. Biochem. Cell Biol., 38, 1469–1475
Iaccarino, C., Crosio, C., Vitale C, Sanna, G., Carri, M.T., Barone, P. (2007) Apoptotic mechanisms in mutant LRRK2-mediated cell death. Hum Mol Genet., 16, 1319-1326.
Junn E., Taniguchi H., Jeong B.S., Zhao X., Ichijo H. and Mouradian M.M. (2005) Interaction of DJ-1 with Daxx inhibits apoptosis signal-regulating kinase 1 activity and cell death. Proc. Natl. Acad. Sci. USA, 102, 9691-9696.
Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y., Minoshima S., Yokochi M., Mizuno Y. and Shimizu N. (1998) Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 302, 605-608.
Kobe, B., Kajava, A.V. (2001) The leucine-rich repeat as protein recognition motif. Curr. Opin. Struc. Biol., 11, 725-732.
Kroemer G. and Reed J.C. (2000) Mitochondrial control of cell death. Nature Med., 6, 513-519.
MacLeod, D., Dowman, J., Hammond, R., Leete, T., Inoue, K., and Abeliovich, A. (2006) The familial Parkinsonism gene LRRK2 regulates neurite process morphology, Neuron, 52, 587-593.
Mata IF, Wedemeyer WJ, Farrer MJ, Taylor JP, Gallo KA. (2006) LRRK2 in Parkinson's disease: protein domains and functional insights. Trends Neurosci., 29, 286-93.
Melrose H, Lincoln S, , Dickson D,Farrer M. (2006) Anatomical localization of leucine-rich repeat kinase 2 in mouse brain. Neuroscience. 139, 791-4.
Miklossy, J., Arai, T., Guo, J.P., Klegeris, A., Yu, S., McGeer, E.G., McGeer, P.L..(2006) LRRK2 expression in normal and pathologic human brain and in human cell lines.
J. Neuropathol. Exp. Neurol., 65, 953-63.
Mitsumoto A. and Nakagawa Y. (2001) DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin. Free Radic. Res., 35, 885-893.
Moore, D.J., West, A.B., Dawson, V.L., Dawson, T.M., 2005, Molecular pathology of Parkinson’s disease. Ann. Rev. Neurosci. 28, 57-87.
Nichols WC, Pankratz N, Hernandez D, Paisan-Ruiz C, Jain S, Halter CA, Michaels VE,Reed T, Rudolph A, Shults CW, Singleton A,Foroud T; Parkinson Study Group-PROGENI investigators. (2005) Genetic screening for a single common LRRK2 mutation in familial Parkinson's disease. Lancet, 365, 410-412.
Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, van der Brug M, Lopez de Munain A, Aparicio S, Gil AM, Khan N, Johnson J, Martinez JR, Nicholl D, Carrera IM, Pena AS, de Silva R,Lees A, Marti-Masso JF, Perez-Tur J, Wood NW, Singleton AB. (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.
Neuron. 44, 595-600
Polymeropoulos M.H., Lavedan C., Leroy E, Ide S.E., Dehejia A., Dutra A., Pike B., Root H., Rubenstein J, Boyer R., Stenroos E.S., Chandrasekharappa s., Athanassiadou A, Papapetropoulos T., Johnson W.G., Lazzarini A.M., Duvoisin R.C., Di Iorio G, Golbe L.I., and Nussbaum R.L. (1997) Mutation in the -synuclein gene identified in families with Parkinson's disease. Science, 276, 2045-2047
Sasahara M, Fries JWU, Rainee EW, Gown AM, Westrum LE, Frosch MP, Bonthron DT, Ross R, Collins T. (1991) PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell, 64, 217-227.
Shimura H., Hattori N., Kobo S., Mizuno Y., Asakawa S., Minoshima S., Shimizu N., Iwai K., Chiba T., Tanaka K. and Suzuki T. (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nature Genet., 25, 302-305.
Silvestri L., Caputo V., Bellacchio E., Atorino L., Dallapiccola B., Valente E. M. and Casari G. (2005) Mitochondrial import and enzymatic activity of PINK1 mutants associated to recessive parkinsonism. Human Mol. Genet., 14, 3477-3492.
Smith, W.W., Pei, Z., Jiang, H., Moore, D.J., Liang, Y., West, A.B., Dawson, V.L., Dawson, T.M., Ross, C.A. (2005) Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration. Proc. Natl. Acad. Sci. USA, 102, 18676-18691.
Smith, W.W., Pei, Z., Jiang, H., Dawson, V.L., Dawson, T.M., Ross, C.A. (2006) Kinase activity of mutant LRRK2 mediates neuronal toxicity. Nature Neurosci., 9, 1231-1233.
Spillantini M.G., Schmidt M.L., Lee V.M., Trojanowski J.Q., Jakes R. and Goedert M. (1997) -Synuclein in Lewy bodies. Nature, 388, 839-840
Tan EK, Zhao Y, Skipper L, Tan MG, Di Fonzo A, Sun L, Fook-Chong S, Tang S, Chua E, Yuen Y, Tan L, Pavanni R, Wong MC, Kolatkar P, Lu CS, Bonifati V, Liu JJ. (2007) The LRRK2 Gly2385Arg variant is associated with Parkinson's disease: genetic and functional evidence. Hum. Genet., 120, 857-863.
Tanemura K, Murayama M, Akagi T, Hashikawa T, Tominaga T, Ichikawa M, Yamaguchi H, Takashima A. (2002) Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau. J. Neurosci., 22, 133-141.
Taylor, J.P., Mata, I.F., Farrer, M,J. (2006) LRRK2: a common pathway for parkinsonism, pathogenesis and prevention? Trends in Mol. Med., 12, 76-82
Taymans JM, Van den Haute C, Baekelandt V. (2006) Distribution of PINK1 and LRRK2 in rat and mouse brain. J. Neurochem., 98, 951-61.
Valente E. M., Abou-Sleiman P. M., Caputo V., Muqit M. M. K., Harvey K., Gispert S., Ali Z., Turco D. D., Bentivoglio A. R., Healy D. G., Albanese A., Nussbaum R., Gonzalez-Maldonado R., Deller T., Salvi S., Cortelli P., Gilks W. P., Latchman D. S., Harvey R. J., Dallapiccola B., Auburger G., and Wood N. W. (2004) Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science, 304, 1158-1160.
Wang H.L., Hsu C.Y., Huang P.C., Kuo Y.L., Li A., Yeh T.H., Tso A.S. and Chen Y.L. (2005) Heterodimerization of opioid receptor-like 1 and -opioid receptors impairs the potency of  receptor agonist J. Neurochem., 85, 345-355.
Wang H.L., Yeh T.H., Chou A.H., Kuo Y.L., Luo L.J., He C.Y., Huang P.C. and Li A.H. (2006) Polyglutamine-expanded ataxin-7 activates mitochondrial apoptotic pathway of cerebellar neurons by upregulating Bax and downregulating Bcl-xL. Cellular Signalling, 18, 541-552.
Wang H.L., Chou A.H., Yeh T.H., Li A.H., Chen Y.L., Kuo Y.L., Tsai S.R. and Yu S.T. (2007) PINK1 mutants associated with recessive Parkinson’s disease are defective in inhibiting mitochondrial release of cytochrome-c. Neurobiology of Disease, In Press
West, A.B., Moore, D.J., Biskup, S., Bugayenko, A., Smith, W.W., Ross, C.A., Dawson, V.L., Dawson, T.M., 2005. Parkinson’s disease associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc. Natl. Acad. Sci. USA 102, 16842–16847.
West, A.B., Moore, D.J., Choi, C., Andrabi, S.A., Li, X., Dikeman, D., Biskup, S., Zhang, Z., Lim, K.L., Dawson, V.L., Dawson, T.M. (2007) Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum. Mol. Genet., 16, 223-232.
Zimprich, A., Müller-Myhsok, B., Farrer, M., Leitner, P., Sharma, M., Hulihan, M., Lockhart, P., Strongosky, A., Kachergus, J., Calne, D.B., Stoessl, A.J., Uitti, R.J., Pfeiffer, R.F, Trenkwalder, C., Homann N., Ott, E., Wenzel, K., Asmus, F., Hardy, I., Wszolek, Z.K., Gasser, T., (2004a) The PARK8 locus in autosomal dominant parkinsonism: confirmation of linkage and further delineation of the disease-containing interval. Am. J. Hum.Genet., 74, 11-19.
Zimprich, A., Biskup, S., Leitner, P., Lichtner, P, Farrer, M., Lincoln, S., Kachergus, J., Hulihan, M., Uitti, R.J., Calne, D.B., Stoessl, A.J., Pfeiffer, R.F, Patenge, N., Carbajal, I.C., Vieregge, P., Asmus, F., Müller-Myhsok, B., Dickson, D.W., Meitinger, T., Strom, TM, Wszolek, Z.K., Gasser, T., (2004b) Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601-607.
Zoghbi H.Y. and Botas J. (2002) Mouse and fly models of neurodegeneration. Trends in Genet., 18, 463-471.