臺灣博碩士論文加值系統

Disrupted in Schizophrenia 1 (DISC1)是一個精神分裂症的致病基因，最初在人類發現，後續研究發現本基因與躁鬱症、自閉症等精神疾病亦有關(Soares et al, 2011)。經由小鼠等模式生物研究發現，DISC1蛋白質參與了神經細胞新生(Singh et al, 2010)以及神經細胞遷徙(Ishizuka et al, 2011)等神經發育的過程。F-box and leucine-rich repeat protein 14 (FBXL14)是F-box蛋白家族的成員，可形成E3泛素連接酶複合體(E3 ubiquitin ligase complex)，辨認發育所需的特定蛋白質受質進入由蛋白酶體(proteasome)催化的蛋白質降解，是正常發育必須的過程(Cardozo et al, 2004)。本實驗室先前研究以免疫共沉澱法發現小鼠DISC1蛋白質(mDISC1)與小鼠FBXL14蛋白質(mFBXL14)之間有交互作用。由於mDISC1已知是調控神經發育的基因，因此本交互作用可能與調控神經發育有關。
本研究為了進一步了解mDISC1與mFBXL14的交互作用，首先製備帶有mDisc1與mFbxl14基因片段的質體，利用哺乳類細胞株(COS-b cell)為系統進行免疫共沉澱法，以定義兩蛋白質作用的結構域(interaction domains)。為檢驗兩蛋白質之交互作用是否為直接接觸，利用大腸桿菌為系統進行蛋白質結合試驗(GST pull-down assay)。為了解兩基因對小鼠胚胎大腦神經元發育之影響，以子宮內電穿孔法(in utero electroporation, IUEP)研究兩基因在小鼠胚胎腦部的功能，發現干擾mDisc1基因表現時會造成大腦皮質神經元遷徙速度減低，與先前報導相符(Kamiya et al, 2005)，並透過同一驗系統發現干擾mFbxl14基因表現時大腦皮質神經元聚集在中間帶(intermediate zone)。本研究進一步以IUEP操弄兩基因的體內表現量並定量分析神經元新生與神經元遷徙。本研究初步闡明mDISC1與mFBXL14交互作用的分子機制，為後續研究兩基因在小鼠胚胎大腦皮質發育功能之基礎。

Disrupted in Schizophrenia 1 (DISC1), first identified in human (Homo sapiens), is a disease-related gene that is associated with schizophrenia and other psychiatric disorders including bipolar disorder and autism spectrum disorders (Soares et al, 2011). DISC1 protein is known to be involved in neurodevelopment processes such as neuronal migration (Ishizuka et al, 2011) and neuronal progenitor proliferation (Singh et al, 2010). F-box and leucine-rich repeat protein 14 (FBXL14) is a subunit of E3 ubiquitin ligase complex involved in proteasome-mediated protein degradation (Cardozo et al, 2004). Preliminary data from our lab showed that mouse DISC1 (mDISC1) co-immunoprecipitates (co-IP) with mouse FBXL14 (mFBXL14), suggesting that these two proteins together may play a role in regulating neurodevelopment. To characterize the interaction of mDISC1 and mFBXL14, the deletion constructs of these two genes were prepared to define their respective interaction domains by co-IP assays. GST pull-down assay was also performed to address whether the interactions are via direct binding. Using in utero electroporation (IUEP), we found knock-down of mFbxl14 caused mouse embryonic cortical neurons gathering in the intermediate zone while knock-down of mDisc1 were reported to cause cortical neuron migration defects (Kamiya et al, 2005). How the interaction of mDISC1 and mFBXL14 may affect embryonic cortical neuronal migration and proliferation in vivo was also explored. Through these studies, the molecular basis of the interaction of mDISC1 and mFBXL14 was characterized, which provides insight into the developmental role of mDISC1 and mFBXL14 in the embryonic corticogenesis.

口試委員會審定書 i
致謝 ii
摘要 iii
Abstract iv
Content v
Introduction 1
1. Embryonic Cortical Neuronal Proliferation and Migration 1
2. Disrupted in Schizophrenia 1 as a Risk Factor in Psychiatric Disorders 3
3. DISC1 in Embryonic Mouse Cortical Neuron Proliferation 5
4. DISC1 in Embryonic Mouse Cortical Neuron Migration 6
5. The E3 Ubiquitin Ligase-mediated Protein Degradation in Cortical Development 7
6. F-box and Leucine-rich Repeat Protein 14 (Fbxl14) as an E3 Ubiquitin Ligase in Proteasome Degradation Pathway 9
Material and Methods 11
1. Pull-down Assay 11
2. SDS-PAGE and Western Blotting 11
3. Antibodies 11
4. Expression Plasmids 12
5. Short Hairpin RNA Constructs 12
6. In Utero Electroporation 12
7. Preparation of Embryonic Mouse Brain Sections 14
8. EdU Proliferation Assay 14
9. Quantification of Cortical Neuron Migration 15
Results 17
1. mDISC1 Interacts with mFBXL14 17
2. Characterization of the required interaction domains of mDISC1 and mFBXL14 18
3. mDISC1 and mFBXL14 Double Knockdown Affects Neuron Migration 19
4. Knockdown of mFBXL14 Causes Neuronal Proliferation Defects 23
Discussion 26
1. The Interaction Domains of mDISC1 and mFBXL14 Might Provide Information for the Function of Their Interaction 26
2. The Possible Signaling Pathways Controlled by the mDISC1-mFBXL14 Interaction 29
3. The Validation of mFbxl14 Knockdown Phenotypes 35
Figures 39
Figure 1. mDISC1 interacts with mFBXL14 40
Figure 2. Induction of mDISC1 and mFBXL14 in E. coli 42
Figure 3. Interaction of mDISC1 with mFBXL14 44
Figure 4. The efficiency of shRNA and shRNA-Resistant Constructs 46
Figure 5. The Migration Patterns of Cortical Neurons after IUEP 48
Figure 6. Knockdown of mFbxl14 Causes Neuronal Migration Arrest in the Intermediate Zone 50
Figure 7. Knockdown of mFbxl14 and mDisc1 Causes Combined Neuronal Migration Defects 52
Figure 8. Overexpression of mFbxl14 and mDisc1 Showed Migration Defects Similar to Knockdown 54
Figure 9. Knockdown of mFbxl14 Causes Neuronal Proliferation Defects 56
Figure 10. Model of mFBXL14-mDISC1 Interaction in Developing Cerebral Cortex 58
Tables 59
Table 1. Neuronal Migration Assay 59
Table 2. Neuronal Proliferation Assay 61
Table 3. Antibodies Used in the Study 62
Table 4. Primers Used in the Study 62
Table 5. Vectors Used in the Study 62
References 63

Austin, C.P., Ky, B., Ma, L., Morris, J.A., and Shughrue, P.J. (2004). Expression of Disrupted-In-Schizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development. Neuroscience 124, 3-10.

Baek, S.T., Kerjan, G., Bielas, S.L., Lee, J.E., Fenstermaker, A.G., Novarino, G., and Gleeson, J.G. (2014). Off-target effect of doublecortin family shRNA on neuronal migration associated with endogenous microRNA dysregulation. Neuron 82, 1255-1262.

Bradshaw, N.J., and Porteous, D.J. (2012). DISC1-binding proteins in neural development, signalling and schizophrenia. Neuropharmacology 62, 1230-1241.

Brandon, N.J., and Sawa, A. (2011). Linking neurodevelopmental and synaptic theories of mental illness through DISC1. Nature reviews Neuroscience 12, 707-722.

Cardozo, T., and Pagano, M. (2004). The SCF ubiquitin ligase: insights into a molecular machine. Nature reviews Molecular cell biology 5, 739-751.

Caviness, V.S., Jr., and Takahashi, T. (1995). Proliferative events in the cerebral ventricular zone. Brain & development 17, 159-163.

Caviness, V.S., Jr., Takahashi, T., and Nowakowski, R.S. (1995). Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model. Trends in neurosciences 18, 379-383.

Chen, S.Y., Huang, P.H., and Cheng, H.J. (2011). Disrupted-in-Schizophrenia 1-mediated axon guidance involves TRIO-RAC-PAK small GTPase pathway signaling. Proceedings of the National Academy of Sciences of the United States of America 108, 5861-5866.

Fietz, S.A., and Huttner, W.B. (2011). Cortical progenitor expansion, self-renewal and neurogenesis-a polarized perspective. Curr Opin Neurobiol 21, 23-35.

Florio, M., and Huttner, W.B. (2014). Neural progenitors, neurogenesis and the evolution of the neocortex. Development 141, 2182-2194.

Hikida, T., Gamo, N.J., and Sawa, A. (2012). DISC1 as a therapeutic target for mental illnesses. Expert opinion on therapeutic targets 16, 1151-1160.

Hindley, C.J., McDowell, G.S., Wise, H., and Philpott, A. (2011). Regulation of cell fate determination by Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligases. The International journal of developmental biology 55, 249-260.

Hirano, A., Yumimoto, K., Tsunematsu, R., Matsumoto, M., Oyama, M., Kozuka-Hata, H., Nakagawa, T., Lanjakornsiripan, D., Nakayama, K.I., and Fukada, Y. (2013). FBXL21 regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes. Cell 152, 1106-1118.

Ho, M.S., Tsai, P.I., and Chien, C.T. (2006). F-box proteins: the key to protein degradation. Journal of biomedical science 13, 181-191.

Hoeck, J.D., Jandke, A., Blake, S.M., Nye, E., Spencer-Dene, B., Brandner, S., and Behrens, A. (2010). Fbw7 controls neural stem cell differentiation and progenitor apoptosis via Notch and c-Jun. Nature neuroscience 13, 1365-1372.

Insolera, R., Shao, W., Airik, R., Hildebrandt, F., and Shi, S.H. (2014). SDCCAG8 regulates pericentriolar material recruitment and neuronal migration in the developing cortex. Neuron 83, 805-822.

Ishizuka, K., Kamiya, A., Oh, E.C., Kanki, H., Seshadri, S., Robinson, J.F., Murdoch, H., Dunlop, A.J., Kubo, K., Furukori, K., et al. (2011). DISC1-dependent switch from progenitor proliferation to migration in the developing cortex. Nature 473, 92-96.

Jin, J., Cardozo, T., Lovering, R.C., Elledge, S.J., Pagano, M., and Harper, J.W. (2004). Systematic analysis and nomenclature of mammalian F-box proteins. Genes & development 18, 2573-2580.

Kamiya, A., Kubo, K., Tomoda, T., Takaki, M., Youn, R., Ozeki, Y., Sawamura, N., Park, U., Kudo, C., Okawa, M., et al. (2005). A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nature cell biology 7, 1167-1178.

Kamiya, A., Tan, P.L., Kubo, K., Engelhard, C., Ishizuka, K., Kubo, A., Tsukita, S., Pulver, A.E., Nakajima, K., Cascella, N.G., et al. (2008). Recruitment of PCM1 to the centrosome by the cooperative action of DISC1 and BBS4: a candidate for psychiatric illnesses. Arch Gen Psychiatry 65, 996-1006.

Kawauchi, T., Shikanai, M., and Kosodo, Y. (2013). Extra-cell cycle regulatory functions of cyclin-dependent kinases (CDK) and CDK inhibitor proteins contribute to brain development and neurological disorders. Genes to cells : devoted to molecular & cellular mechanisms 18, 176-194.

Kitagawa, M. (1999). FWD1. The EMBO journal 18, 2401-2410.
Kubo, A., Sasaki, H., Yuba-Kubo, A., Tsukita, S., and Shiina, N. (1999). Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis. The Journal of cell biology 147, 969-980.

Kubo, K.-i., Tomita, K., Uto, A., Kuroda, K., Seshadri, S., Cohen, J., Kaibuchi, K., Kamiya, A., and Nakajima, K. (2010). Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi. Biochemical and biophysical research communications 400, 631-637.

Lander, R., Nordin, K., and LaBonne, C. (2011). The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1. The Journal of cell biology 194, 17-25.

Mao, Y., Ge, X., Frank, C.L., Madison, J.M., Koehler, A.N., Doud, M.K., Tassa, C., Berry, E.M., Soda, T., Singh, K.K., et al. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136, 1017-1031.

Marin, O., and Rubenstein, J.L. (2001). A long, remarkable journey: tangential migration in the telencephalon. Nature reviews Neuroscience 2, 780-790.

Metzger, M.B., Hristova, V.A., and Weissman, A.M. (2012). HECT and RING finger families of E3 ubiquitin ligases at a glance. Journal of cell science 125, 531-537.

Millar, J.K., James, R., Christie, S., and Porteous, D.J. (2005). Disrupted in schizophrenia 1 (DISC1): subcellular targeting and induction of ring mitochondria. Molecular and cellular neurosciences 30, 477-484.

Millar, J.K., Wilson-Annan, J.C., Anderson, S., Christie, S., Taylor, M.S., Semple, C.A., Devon, R.S., St Clair, D.M., Muir, W.J., Blackwood, D.H., et al. (2000). Disruption of two novel genes by a translocation co-segregating with schizophrenia. Human molecular genetics 9, 1415-1423.

Morris, J.A. (2003). DISC1 (Disrupted-In-Schizophrenia 1) is a centrosome-associated protein that interacts with MAP1A, MIPT3, ATF4/5 and NUDEL: regulation and loss of interaction with mutation. Human molecular genetics 12, 1591-1608.

Nadarajah, B., and Parnavelas, J.G. (2002). Modes of neuronal migration in the developing cerebral cortex. Nature reviews Neuroscience 3, 423-432.

Narayan, S., Nakajima, K., and Sawa, A. (2013). DISC1: a key lead in studying cortical development and associated brain disorders. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 19, 451-464.

Qu, C., Dwyer, T., Shao, Q., Yang, T., Huang, H., and Liu, G. (2013). Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance. Journal of cell science 126, 3070-3081.

Saillour, Y., Broix, L., Bruel-Jungerman, E., Lebrun, N., Muraca, G., Rucci, J., Poirier, K., Belvindrah, R., Francis, F., and Chelly, J. (2014). Beta tubulin isoforms are not interchangeable for rescuing impaired radial migration due to Tubb3 knockdown. Human molecular genetics 23, 1516-1526.

Saritas-Yildirim, B., and Silva, E.M. (2014). The role of targeted protein degradation in early neural development. Genesis 52, 287-299.

Singh, K.K., De Rienzo, G., Drane, L., Mao, Y., Flood, Z., Madison, J., Ferreira, M., Bergen, S., King, C., Sklar, P., et al. (2011). Common DISC1 polymorphisms disrupt Wnt/GSK3beta signaling and brain development. Neuron 72, 545-558.

Singh, K.K., Ge, X., Mao, Y., Drane, L., Meletis, K., Samuels, B.A., and Tsai, L.H. (2010). Dixdc1 is a critical regulator of DISC1 and embryonic cortical development. Neuron 67, 33-48.

Soares, D.C., Carlyle, B.C., Bradshaw, N.J., and Porteous, D.J. (2011). DISC1: Structure, Function, and Therapeutic Potential for Major Mental Illness. ACS chemical neuroscience 2, 609-632.

Takahashi, T., Nowakowski, R.S., and Caviness, V.S., Jr. (1995). Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall. The Journal of neuroscience : the official journal of the Society for Neuroscience 15, 6058-6068.

Tan, M.K., Lim, H.J., Bennett, E.J., Shi, Y., and Harper, J.W. (2013). Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover. Molecular cell 52, 9-24.

Vinas-Castells, R., Beltran, M., Valls, G., Gomez, I., Garcia, J.M., Montserrat-Sentis, B., Baulida, J., Bonilla, F., de Herreros, A.G., and Diaz, V.M. (2010). The hypoxia-controlled FBXL14 ubiquitin ligase targets SNAIL1 for proteasome degradation. The Journal of biological chemistry 285, 3794-3805.

Wang, L., Li, H., Chen, Q., Zhu, T., Zhu, H., and Zheng, L. (2010). Wnt signaling stabilizes the DIXDC1 protein through decreased ubiquitin-dependent degradation. Cancer science 101, 700-706.

Westbrook, T.F., Hu, G., Ang, X.L., Mulligan, P., Pavlova, N.N., Liang, A., Leng, Y., Maehr, R., Shi, Y., Harper, J.W., et al. (2008). SCFβ-TRCP controls oncogenic transformation and neural differentiation through REST degradation. Nature 452, 370-374.

Yerabham, A.S., Weiergraber, O.H., Bradshaw, N.J., and Korth, C. (2013). Revisiting disrupted-in-schizophrenia 1 as a scaffold protein. Biological chemistry 394, 1425-1437.

Yoo, S.H., Mohawk, J.A., Siepka, S.M., Shan, Y., Huh, S.K., Hong, H.K., Kornblum, I., Kumar, V., Koike, N., Xu, M., et al. (2013). Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm. Cell 152, 1091-1105.

Zheng, H., Du, Y., Hua, Y., Wu, Z., Yan, Y., and Li, Y. (2012). Essential role of Fbxl14 ubiquitin ligase in regulation of vertebrate axis formation through modulating Mkp3 level. Cell research 22, 936-940.