帕金森氏症是僅次於阿茲海默症第二常見的神經退化性疾病，主要病理特徵為中腦黑質緻密部的多巴胺神經元漸進性退化導致死亡。F-box protein 7 (FBXO7)的基因變異被認為有可能會引起體隱性早發性帕金森氏症(PARK15)。FBXO7可與SKP1、cullin組成ubiquitin E3 ligase complex，來調節細胞內蛋白質代謝。先前我們的研究發現FBXO7一改變胺基酸的Y52C變異，與帕金森氏症的低風險相關。包含Y52C變異點的FBXO7表現於細胞內時，分解速度顯著降低。人類FBXO7啟動子的調控未被探討過，因此我們以PCR增幅-1240、-694、-538、-438、-202及-56至+261的啟動子片段，置於GFP報告者前，再轉染至HEK-293T細胞表現，藉分析GFP螢光亮度，來探討調控FBXO7表現的cis因子。透過高通量影像及流式細胞儀分析，-694及-202片段的啟動子活性顯著高於-56片段。由於FBXO7表現量的上升對帕金森氏症有保護性，我們建立了-694啟動子片段驅動綠螢光蛋白的Flp-In 293細胞株作為平台，來篩檢可增強FBXO7表現的植物萃取液(工研院提供)。結果發現植物萃取液NTNU-319、379、395、439可顯著提升293及SH-SY5Y細胞中FBXO7表現。進一步檢測發現上述植物萃取液對MPP+引起的細胞毒殺性有保護性，其中植物萃取液NTNU-319、395、439並顯著減緩MPP+引起的粒線體膜電位下降。由於目前帕金森氏症沒有預防或減緩病程的藥物，本研究可能發展出有潛力的治療策略。

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is pathologically characterized by loss of dopaminergic neurons in the substantia nigra of the midbrain. Mutations in the F-box only protein 7 gene (FBXO7), the substrate-specifying subunit of Skp1-Cullin-F-Box (SCF) E3 ubiquitin ligase complex, cause PD-15 (PARK15). Previously we identified an amino acid changed variant Y52C in association with decreased risk of developing PD. Upon expression in cells, Y52C variant displayed significantly reduced rate of decay in cycloheximide chase experiment. The human FBXO7 gene promoter has not been analyzed. To investigate cis elements controlling FBXO7 expression, we cloned FBXO7 promoter fragments from -1240, -694, -538, -438, -202 and -56 to +261 by PCR amplification and placed in front of GFP reporter for transfection assay in HEK-293T cells. The expression of GFP was monitored by both high content analysis and flow cytometry. When the expressed GFP level of the -56~+261 promoter fragment was set as 100%, significantly increased FBXO7 promoter activity was observed with the -202~+261 and -694~+261 fragments by both methods. As the protective role of increased FBXO7 level in PD was implicated, Flp-In 293 cell line expressing GFP reporter driven by FBXO7 -694~+261 promoter fragment was constructed and used as a platform to screen herbal extracts provided by Industrial Technology Research Institute for enhancing FBXO7 expression. Herbal extracts NTNU-319, 379, 395 and 439 were found to increase endogenous FBXO7 protein expression in both Flp-In 293 and SH-SY5Y cells. Treatment of the above herbal extracts protected SH-SY5Y cells against MPP+-induced cell death. In addition, herbal extracts NTNU-319, 395 and 439 significantly alleviated MPP+-induced loss of mitochondrial membrane potential. As lack of treatment to prevent or slow PD progression, the proposed study may provide new insights into the therapeutic approach to PD.

Awika, J.M. and L.W. Rooney, 2004. Sorghum phytochemicals and their potential impact on human health. Phytochemistry. 65:1199-1221.
Awika, J.M., L.W. Rooney, and R.D. Waniska, 2004. Anthocycanins from black sorghum and their antioxidant properties. Food Chemistry. 90:293-301
Aher, A.N., S.C. Pal, S.K. Yadav, U.K. Patil, and S. Bhattacharya. 2009. Antioxidant activity of isolated phytoconstituents from Casuarina equisetifolia Frost (Casuarinaceae). Journal of plant sciences. 4:15-20.
Bartůnek, P., J. Králová, G. Blendinger, M. Dvorák, and M. Zenke. 2003. GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter. Oncogene. 22:1927-1935.
Bertram, C.P., M. Lemay, and G.E. Stelmach. 2005. The effect of Parkinson's disease on the control of multi-segmental coordination. Brain and cognition. 57:16-20.
Burchell, V.S., D.E. Nelson, A. Sanchez-Martinez, M. Delgado-Camprubi, R.M. Ivatt, J.H. Pogson, S.J. Randle, S. Wray, P.A. Lewis, H. Houlden, A.Y. Abramov, J. Hardy, N.W. Wood, A.J. Whitworth, H. Laman, and H. Plun-Favreau. 2013. The Parkinson's disease-linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nature neuroscience. 16:1257-1265.
Cenciarelli, C., D.S. Chiaur, D. Guardavaccaro, W. Parks, M. Vidal, and M. Pagano. 1999. Identification of a family of human F-box proteins. Current biology. 9:1177-1179.
Chang, Y.F., C.M. Cheng, L.K. Chang, Y.J. Jong, and C.Y. Yuo. 2006. The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination. Biochemical and biophysical research communications. 342:1022-1026.
Chen, C.M., I.C. Chen, Y.C. Huang, H.F. Juan, Y.L. Chen, Y.C. Chen, C.H. Lin, L.C. Lee, C.M. Lee, G.J. Lee-Chen, Y.J. Lai, and Y.R. Wu. 2014. FBXO7 Y52C polymorphism as a potential protective factor in Parkinson’s disease. PLoS one. 9:e101392.
Choi, Y.M., H.S. Jeong, and J.S. Lee. 2006. Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chemistry. 103:130-138.
Conley, S.C., and J.T. Kirchner. 1999. Parkinson's disease--the shaking palsy. Underlying factors, diagnostic considerations, and clinical course. Postgraduate medicine. 106:39-42, 45-36, 49-50 passim.
Dauer, W., and S. Przedborski. 2003. Parkinson's disease: mechanisms and models. Neuron. 39:889-909.
Di Fonzo, A., M.C. Dekker, P. Montagna, A. Baruzzi, E.H. Yonova, L. Correia Guedes, A. Szczerbinska, T. Zhao, L.O. Dubbel-Hulsman, C.H. Wouters, E. de Graaff, W.J. Oyen, E.J. Simons, G.J. Breedveld, B.A. Oostra, M.W. Horstink, and V. Bonifati. 2009. FBXO7 mutations cause autosomal recessive, early-onset parkinsonian-pyramidal syndrome. Neurology. 72:240-245.
Dykes, L., and L.W. Rooney. 2006. Sorghum and millet phenols and antioxidants. Journal of Cereal Science. 44:236-251.
El-Tantawy, W.H., S.A. Mohamed, and E.N. Abd Al Haleem. Evaluation of biochemical effects of Casuarina equisetifolia extract on gentamicin induced nephrotoxicity and oxidative stress in rats Phytochemical analysis. 2013. Journal of clinical biochemistry and nutrition. 53:158-165.
Farrer, M.J. 2006. Genetics of Parkinson disease: paradigm shifts and future prospects. Nature reviews. Genetics. 7:306-318.
Flood, P.M., L. Qian, L.J. Peterson, F. Zhang, J.S. Shi, H.M. Gao, and J.S. Hong. 2011. Transcriptional factor NF-kappaB as a target for therapy in Parkinson's disease. Parkinson’s disease. 2011:216298.
Gibb, W.R., and A.J. Lees. 1991. Anatomy, pigmentation, ventral and dorsal subpopulations of the substantia nigra, and differential cell death in Parkinson's disease. Journal of neurology, neurosurgery, and psychiatry. 54:388-396.
Ho, M.S., C. Ou, Y.R. Chan, C.T. Chien, and H. Pi. 2008. The utility F-box for protein destruction. Cellular and molecular life sciences. 65:1977-2000.
Hoehn, M.M., and M.D. Yahr. 1967. Parkinsonism: onset, progression and mortality. Neurology. 17:427-442.
Hsu, J.M., Y.C. Lee, C.T. Yu, and C.Y. Huang. 2004. Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region. Journal of biological chemistry. 279:32592-32602.
Kirk, R., H. Laman, P.P. Knowles, J. Murray-Rust, M. Lomonosov, K. Meziane el, and N.Q. McDonald. 2008. Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor. Journal of biological chemistry. 283:22325-22335.
Kuiken, H.J., D.A. Egan, H. Laman, R. Bernards, R.L. Beijersbergen, and A.M. Dirac. 2012. Identification of F-box only protein 7 as a negative regulator of NF-κB signalling. Journal of cellular and molecular medicine. 16:2140-2149.
Li, X., Z. Shuai-Nan, L. Shu-Min, and L. Fang. 2013. Recent advances in herbal medicines treating Parkinson's disease. Fitoterapia. 84:273-285.
Lin, C.H., M.L. Chen, T.T. Lai, C.H. Tai, and R.M. Wu. 2013. Mutational analysis of FBXO7 gene in Parkinson's disease in a Taiwanese population. Neurobiology of aging. 34:1713.e1-4.
Luo, L.Z., Q. Xu, J.F. Guo, L. Wang, C.H. Shi, J.H. Wei, Z.G. Long, Q. Pan, B.S. Tang, K. Xia, and X.X. Yan. 2010. FBXO7 gene mutations may be rare in Chinese early-onset Parkinsonism patients. Neuroscience letters 482:86-89.
Marjama-Lyons, J.M., and W.C. Koller. 2001. Parkinson's disease. Update in diagnosis and symptom management. Geriatrics. 56:24-25, 29-30, 33-25.
Merika, M., and S.H. Orkin. 1993. DNA-Binding specificity of GATA family transcription factors. Molecular and cellular biology. 13:3999-4010.
Montgomery, E.B. Jr. 1995. Heavy metals and the etiology of Parkinson's disease and other movement disorders. Toxicology. 97:3-9.
Munugalavadla, V., L.C. Dore, B.L. Tan, L. Hong, M. Vishnu, M.J. Weiss, and R. Kapur. 2005. Repression of c-kit and its downstream substrates by GATA-1 inhibits cell proliferation during erythroid maturation. Molecular and cellular biology. 25:6747-6759.
Nardelli, J., D. Thiesson, Y. Fujiwara, F.Y. Tsai, and S.H. Orkin. 1999. Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system. Developmental Biology. 210:305-321.
Nelson, D.E., S.J. Randle, and H. Laman. 2013. Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins. Open biology. 3:130-131.
Ogunwande, I.A., G. Flamini, A.E. Adefuye, N.O. Lawal, S. Moradeyo, and N.O. Avoseh. Chemical compositions of Casuarina equisetifolia L., Eucalyptus toreliana L. and Ficus elastica Roxb. ex Hornem cultivated in Nigeria. 2011. South African journal of botany. 77:645-649.
Paisan-Ruiz, C., R. Guevara, M. Federoff, H. Hanagasi, F. Sina, E. Elahi, S.A. Schneider, P. Schwingenschuh, N. Bajaj, M. Emre, A.B. Singleton, J. Hardy, K.P. Bhatia, S. Brandner, A.J. Lees, and H. Houlden. 2010. Early-onset L-dopa-responsive parkinsonism with pyramidal signs due to ATP13A2, PLA2G6, FBXO7 and spatacsin mutations. Movement disorders. 25:1791-1800.
Pevny, L., M.C. Simon, E. Robertson, W.H. Klein, T. Shih-Feng, V. D'Agati, S.H. Orkin, and F. Costantini. 1991. Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature. 349:257-260.
Phani, S., J.D. Loike, and S. Przedborski. 2012. Neurodegeneration and inflammation in Parkinson's disease. Parkinsonism and related disorders. 18 Suppl 1:S207-209.
Rylski, M., J.J. Welch, Y.Y. Chen, D.L. Letting, J.A. Diehl, L.A. Chodosh, G.A. Blobel, and M.J. Weiss. 2003. GATA-1-mediated proliferation arrest during erythroid maturation. Molecular and cellular biology. 23:5031-5042.
Schapira, A.H., J.M. Cooper, D. Dexter, P. Jenner, J.B. Clark, and C.D. Marsden. 1989. Mitochondrial complex I deficiency in Parkinson's disease. Lancet. 1:1269.
Schapira, A.H. 2006. Etiology of Parkinson's disease. Neurology. 66:S10-23.
Scherman, D., C. Desnos, F. Darchen, P. Pollak, F. Javoy-Agid, and Y. Agid. 1989. Striatal dopamine deficiency in Parkinson's disease: role of aging. Annals of neurology. 26:551-557.
Scherzer, C.R., J.A. Grass, Z. Liao, I. Pepivani, B. Zheng, A.C. Eklund, P.A. Ney, J. Ng, M. McGoldrick, B. Mollenhauer, E.H. Bresnick, and M.G. Schlossmacher. 2008. GATA transcription factors directly regulate theParkinson’s disease-linked gene α-synuclein. Proceedings of the National Academy of Sciences. 105: 10907-10912.
Schulz, J.B. 2008. Update on the pathogenesis of Parkinson's disease. Journal of neurology. 255 Suppl 5:3-7.
Shojaee, S., F. Sina, S.S. Banihosseini, M.H. Kazemi, R. Kalhor, G.A. Shahidi, H. Fakhrai-Rad, M. Ronaghi, and E. Elahi. 2008. Genome-wide linkage analysis of a Parkinsonian-pyramidal syndrome pedigree by 500 K SNP arrays. American journal of human genetics. 82:1375-1384.
Snyder, S.H., and R.J. D'Amato. 1986. MPTP: a neurotoxin relevant to the pathophysiology of Parkinson's disease. The 1985 George C. Cotzias lecture. Neurology. 36:250-258.
Stewart, H. L., L.G. Alfred, and E.M. William. 2006. Protein degradation by the ubiquitin–proteasome pathway in normal and disease states. Journal of the American society of nephrology. 17: 1807-1819.
Tarsy, D. 2012. Patient information: Parkinson disease treatment options - medications (Beyond the Basics). UpToDate in Waltham, MA.
Tsai, S.F., I.M. David, I. Leonard, A.D. D’Andrea, G.W. Gordon, and S.H. Orkin. 1989. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature. 339:446-451.
Zhao, T., E. De Graaff, G.J. Breedveld, A. Loda, L.A. Severijnen, C.H. Wouters, F.W. Verheijen, M.C. Dekker, P. Montagna , R. Willemsen , B.A. Oostra1, and V. Bonifati. 2011. Loss of nuclear activity of the FBXO7 protein in patients with parkinsonian-pyramidal syndrome (PARK15). PLoS one. 6:e16983.
Zhao, T., L.A. Severijnen, M. van der Weiden, P.P. Zheng, B.A. Oostra, R.K. Hukema, R. Willemsen, J.M. Kros, and V. Bonifati. 2013. FBXO7 immunoreactivity in α-synuclein-containing inclusions in Parkinson disease and multiple system atrophy. Journal of neuropathology and experimental neurology. 72:482-488.
Zheng, N., B.A. Schulman, L. Song, J.J. Miller, P.D. Jeffrey, P. Wang, C. Chu, D.M. Koepp, S.J. Elledge, M. Paganok, R.C. Conaway, J.W. Conaway, J.W. Harper, and N.P. Pavletich. 2002. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 416:703-709.