亨廷頓舞蹈症為顯性遺傳的神經退化性疾病，致病原因為亨廷頓蛋白基因發生突變，導致突變型亨廷頓蛋白具有過度擴張的多聚麩醯胺，為錯誤折疊的蛋白質易形成聚集體並堆積在神經細胞中。亨廷頓蛋白與多種蛋白質交互作用以參與細胞內的調控，其中亨廷頓蛋白與亨廷頓相關蛋白40結合，可調節早期內噬體的運輸。在患者腦部發現亨廷頓相關蛋白40大量表現，其與微管有交互作用，並與促進突變型亨廷頓蛋白聚集體形成有關。細胞內的清除機制能夠將異常的蛋白質降解以維持細胞恆定，包含泛素-蛋白酶體與細胞自噬，其中泛素-蛋白酶體的組成份子可能被滯留於聚集體，因此細胞自噬的進行顯得十分關鍵，故探討亨廷頓相關蛋白40過度表現對細胞自噬的影響。結果發現亨廷頓相關蛋白40過度表現會促進細胞自噬的進行，進一步發現其作用機制為mTOR-independent，且受到PI-3 kinase的抑制影響。目前對亨廷頓相關蛋白40的研究有限，若能知其在細胞內的分布，將有助於其功能之定位，由於市售HAP40抗體(Millipore AB5872 a.a. 314-325)偵測效果不佳，因此先前製作了兩支HAP40抗體。使用這三種抗體進行特性分析，發現HAP40 (LTK a.a. 293-310)、HAP40 (LTK a.a. 29-47)的偵測較符合HAP40的實際分子量，且knockdown後能偵測到亨廷頓相關蛋白40表現量降低，而市售的HAP40 (Millipore AB5872 a.a. 314-325)抗體則否；另外為了明瞭HAP40分布在細胞核或細胞質，以生化分餾分析，發現亨廷頓相關蛋白40存在於細胞質。在神經細胞中，囊泡藉由運動蛋白在軸突的微管上得以運輸，由驅動蛋白負責正向運輸及動力蛋白負責反向運輸，已知突變型亨廷頓蛋白聚集體內包含運動蛋白，使細胞中游離的運動蛋白減少，影響物質運送。當動力蛋白功能受抑制時，會造成聚集體的堆積，因此想了解驅動蛋白是否也有類似情形。實驗上針對驅動蛋白輕鏈的兩種異構型：KLC1和KLC2進行knockdown，因為輕鏈能夠與物質結合並調控驅動蛋白的活性。結果發現KLC1和KLC2受到抑制時，突變型亨廷頓蛋白聚集體有明顯增加的現象。綜合以上結果顯示亨廷頓相關蛋白40過度表現會促進細胞自噬的進行，其機制為mTOR- independent，且受到PI-3 kinase的抑制所影響；其次分析不同HAP40抗體特性，發現LTK的兩株抗體能夠偵測到內生性HAP40，而由生化分餾結果得知HAP40存在於細胞質；另外KLC1和KLC2表現量下降會導致突變型亨廷頓蛋白聚集體數目增加。

Mutant huntingtin forms aggregates in the brains of transgenic mice and HD patients. Those mutant proteins depend primarily on autophagy for their clearance. Our previous studies showed that HAP40 associates with microtubule and promotes aggregate formation. Therefore, we investigate the effects of HAP40 on autophagy. Using a dual color LC3 reporter to monitor the autophagic process, we found that HAP40 overexpression induced autophagic flux by an mTOR-independent manner. Furthermore, due to the lack of an efficient HAP40 antibody, we have generated and validated two polyclonal HAP40 antibodies. Using those two antibodies, we found that HAP40 signal appears in cytoplasmic fraction. Previous report had shown that depletion of DHC lead to increased aggregate formation. Therefore we investigated whether KLC knockdown have similar effects. We found that there is more aggregates formation after depletion of KLC1 or KLC2.

Altar, C.A., N. Cai, T. Bliven, M. Juhasz, J.M. Conner, A.L. Acheson, R.M. Lindsay, and S.J. Wiegand. 1997. Anterograde transport of brain-derived neurotrophic factor and its role in the brain. Nature. 389:856-860.
Bennett, E.J., T.A. Shaler, B. Woodman, K.-Y. Ryu, T.S. Zaitseva, C.H. Becker, G.P. Bates, H. Schulman, and R.R. Kopito. 2007. Global changes to the ubiquitin system in Huntington's disease. Nature. 448:704-708.
Bjørkøy, G., T. Lamark, A. Brech, H. Outzen, M. Perander, A. Øvervatn, H. Stenmark, and T. Johansen. 2005. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. The Journal of cell biology. 171:603-614.
Chen, Y., and D.J. Klionsky. 2011. The regulation of autophagy–unanswered questions. Journal of cell science. 124:161-170.
Colin, E., D. Zala, G. Liot, H. Rangone, M. Borrell-Pages, X.-J. Li, F. Saudou, and S. Humbert. 2008. Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons. EMBO J. 27:2124-2134.
Corradetti, M., and K. Guan. 2006. Upstream of the mammalian target of rapamycin: do all roads pass through mTOR? Oncogene. 25:6347-6360.
Davies, S.W., M. Turmaine, B.A. Cozens, M. DiFiglia, A.H. Sharp, C.A. Ross, E. Scherzinger, E.E. Wanker, L. Mangiarini, and G.P. Bates. 1997. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell. 90:537-548.
DiFiglia, M., E. Sapp, K.O. Chase, S.W. Davies, G.P. Bates, J. Vonsattel, and N. Aronin. 1997. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science. 277:1990-1993.
Dompierre, J.P., J.D. Godin, B.C. Charrin, F.P. Cordelieres, S.J. King, S. Humbert, and F. Saudou. 2007. Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington's disease by increasing tubulin acetylation. The Journal of neuroscience. 27:3571-3583.
Gauthier, L.R., B.C. Charrin, M. Borrell-Pagès, J.P. Dompierre, H. Rangone, F.P. Cordelières, J. De Mey, M.E. MacDonald, V. Leßmann, and S. Humbert. 2004. Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules. Cell. 118:127-138.
Geeraert, C., A. Ratier, S.G. Pfisterer, D. Perdiz, I. Cantaloube, A. Rouault, S. Pattingre, T. Proikas-Cezanne, P. Codogno, and C. Poüs. 2010. Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation. Journal of Biological Chemistry. 285:24184-24194.
Gharami, K., Y. Xie, J.J. An, S. Tonegawa, and B. Xu. 2008. Brain‐derived neurotrophic factor over‐expression in the forebrain ameliorates Huntington’s disease phenotypes in mice. Journal of neurochemistry. 105:369-379.
Goldstein, L.S., and Z. Yang. 2000. Microtubule-based transport systems in neurons: the roles of kinesins and dyneins. Annual review of neuroscience. 23:39-71.
Gunawardena, S., L.-S. Her, R.G. Brusch, R.A. Laymon, I.R. Niesman, B. Gordesky-Gold, L. Sintasath, N.M. Bonini, and L.S. Goldstein. 2003. Disruption of Axonal Transport by Loss of Huntingtin or Expression of Pathogenic PolyQ Proteins in Drosophila. Neuron. 40:25-40.
Harjes, P., and E.E. Wanker. 2003. The hunt for huntingtin function: interaction partners tell many different stories. Trends in biochemical sciences. 28:425-433.
Harper, P.S. 1996. Huntington disease. Wiley Online Library.
Hosokawa, N., T. Hara, T. Kaizuka, C. Kishi, A. Takamura, Y. Miura, S.-i. Iemura, T. Natsume, K. Takehana, and N. Yamada. 2009. Nutrient-dependent mTORC1 association with the ULK1–Atg13–FIP200 complex required for autophagy. Molecular biology of the cell. 20:1981-1991.
Itakura, E., and N. Mizushima. 2010. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy. 6:764-776.
Iwata, A., B.E. Riley, J.A. Johnston, and R.R. Kopito. 2005. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. Journal of Biological Chemistry. 280:40282-40292.
Jana, N.R., M. Tanaka, G.-h. Wang, and N. Nukina. 2000. Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity. Human Molecular Genetics. 9:2009-2018.
Junco, A., B. Bhullar, H.A. Tarnasky, and F.A. van der Hoorn. 2001. Kinesin light-chain KLC3 expression in testis is restricted to spermatids. Biology of reproduction. 64:1320-1330.
Kawaguchi, Y., J.J. Kovacs, A. McLaurin, J.M. Vance, A. Ito, and T.-P. Yao. 2003. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell. 115:727-738.
Klionsky, D.J. 2005. The molecular machinery of autophagy: unanswered questions. Journal of cell science. 118:7-18.
Klionsky, D.J., F.C. Abdalla, H. Abeliovich, R.T. Abraham, A. Acevedo-Arozena, K. Adeli, L. Agholme, M. Agnello, P. Agostinis, and J.A. Aguirre-Ghiso. 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy. 8:445-544.
Lee, J.-Y., H. Koga, Y. Kawaguchi, W. Tang, E. Wong, Y.-S. Gao, U.B. Pandey, S. Kaushik, E. Tresse, and J. Lu. 2010. HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. The EMBO journal. 29:969-980.
Li, S.-H., and X.-J. Li. 2004. Huntingtin–protein interactions and the pathogenesis of Huntington's disease. TRENDS in Genetics. 20:146-154.
Möncke‐Buchner, E., S. Reich, M. Mücke, M. Reuter, W. Messer, E.E. Wanker, and D.H. Krüger. 2002. Counting CAG repeats in the Huntington’s disease gene by restriction endonuclease EcoP15I cleavage. Nucleic acids research. 30:e83-e83.
MacDonald, M.E., C.M. Ambrose, M.P. Duyao, R.H. Myers, C. Lin, L. Srinidhi, G. Barnes, S.A. Taylor, M. James, and N. Groot. 1993. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell. 72:971-983.
Majeski, A.E., and J. Fred Dice. 2004. Mechanisms of chaperone-mediated autophagy. The international journal of biochemistry & cell biology. 36:2435-2444.
Mijaljica, D., M. Prescott, and R.J. Devenish. 2011. Microautophagy in mammalian cells: revisiting a 40-year-old conundrum. Autophagy. 7:673-682.
Milman, P., and J. Woulfe. 2013. A novel variant of neuronal intranuclear rodlet immunoreactive for 40 kDa huntingtin associated protein and ubiquitin in the mouse brain. Journal of Comparative Neurology.
Mizushima, N., and M. Komatsu. 2011. Autophagy: renovation of cells and tissues. Cell. 147:728-741.
Mizushima, N., T. Yoshimori, and B. Levine. 2010. Methods in mammalian autophagy research. Cell. 140:313-326.
Nielsen, E., F. Severin, J.M. Backer, A.A. Hyman, and M. Zerial. 1999. Rab5 regulates motility of early endosomes on microtubules. Nature Cell Biology. 1:376-382.
Pal, A., F. Severin, B. Lommer, A. Shevchenko, and M. Zerial. 2006. Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease. The Journal of cell biology. 172:605-618.
Pankiv, S., T.H. Clausen, T. Lamark, A. Brech, J.-A. Bruun, H. Outzen, A. Øvervatn, G. Bjørkøy, and T. Johansen. 2007. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. Journal of Biological Chemistry. 282:24131-24145.
Peters, M.F., and C.A. Ross. 2001. Isolation of a 40-kDa Huntingtin-associated protein. Journal of Biological Chemistry. 276:3188-3194.
Qin, Z.-h., and Z.-L. Gu. 2004. Huntingtin processing in pathogenesis of Huntington disease. Acta Pharmacologica Sinica. 25:1243-1249.
Ravikumar, B., A. Acevedo-Arozena, S. Imarisio, Z. Berger, C. Vacher, C.J. O'Kane, S.D. Brown, and D.C. Rubinsztein. 2005. Dynein mutations impair autophagic clearance of aggregate-prone proteins. Nature genetics. 37:771-776.
Ravikumar, B., R. Duden, and D.C. Rubinsztein. 2002. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Human Molecular Genetics. 11:1107-1117.
Ravikumar, B., S. Imarisio, S. Sarkar, C.J. O'Kane, and D.C. Rubinsztein. 2008. Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease. Journal of cell science. 121:1649-1660.
Ravikumar, B., C. Vacher, Z. Berger, J.E. Davies, S. Luo, L.G. Oroz, F. Scaravilli, D.F. Easton, R. Duden, and C.J. O'Kane. 2004. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nature genetics. 36:585-595.
Rubinsztein, D.C. 2007. Autophagy induction rescues toxicity mediated by proteasome inhibition. Neuron. 54:854-856.
Ruocco Heloísa, H., L.-C. Iscia, L. Laurito Tiago, M. Li Li, and C. Fernando. Clinical presentation of juvenile Huntington disease. Arquivos de Neuro-Psiquiatria. 64.
Su, H., and X. Wang. 2010. The ubiquitin-proteasome system in cardiac proteinopathy: a quality control perspective. Cardiovascular research. 85:253-262.
Trüe, O., and P. Matthias. 2011. Interplay between histone deacetylases and autophagy-from cancer therapy to neurodegeneration. Immunology and cell biology. 90:78-84.
Trushina, E., R.B. Dyer, J.D. Badger, D. Ure, L. Eide, D.D. Tran, B.T. Vrieze, V. Legendre-Guillemin, P.S. McPherson, and B.S. Mandavilli. 2004. Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro. Molecular and cellular biology. 24:8195-8209.
Velier, J., M. Kim, C. Schwarz, T.W. Kim, E. Sapp, K. Chase, N. Aronin, and M. DiFiglia. 1998. Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways. Experimental neurology. 152:34-40.
Waelter, S., A. Boeddrich, R. Lurz, E. Scherzinger, G. Lueder, H. Lehrach, and E.E. Wanker. 2001. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Molecular biology of the cell. 12:1393-1407.
Wang, J., C.-E. Wang, A. Orr, S. Tydlacka, S.-H. Li, and X.-J. Li. 2008. Impaired ubiquitin–proteasome system activity in the synapses of Huntington's disease mice. The Journal of cell biology. 180:1177-1189.
Wu, Y.-T., H.-L. Tan, G. Shui, C. Bauvy, Q. Huang, M.R. Wenk, C.-N. Ong, P. Codogno, and H.-M. Shen. 2010. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. Journal of Biological Chemistry. 285:10850-10861.
Zhou, C., W. Zhong, J. Zhou, F. Sheng, Z. Fang, Y. Wei, Y. Chen, X. Deng, B. Xia, and J. Lin. 2012. Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells. Autophagy. 8:1215-1226.
Zuccato, C., M. Valenza, and E. Cattaneo. 2010. Molecular mechanisms and potential therapeutical targets in Huntington's disease. Physiological reviews. 90:905-981.