A cullin 1 based SCF e3 ligase complex directs two distinct modes of neuronal pruning in drosophila melanogaster

A CULLIN-1 BASED SCF E3 LIGASE COMPLEX DIRECTS TWO DISTINCT MODES OF NEURONAL PRUNING IN DROSOPHILA MELANOGASTER Wong Jing Lin Jack B.Sc (Hons), Nanyang Technological University A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTERGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 i To my parents and wife ii Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Wong Jing Lin Jack May 2014 iii Acknowledgement I am heartily grateful and thankful to my wonderful supervisor, Dr. Yu Fengwei, whose selfless attitude, encouragement and guidance had allowed me to explore any possibilities in my research work. His great enthusiasm in science is always stimulating to me. His kindness and patience to impart his knowledge and skill without any reservation to me allowed me to grow into who I am today. I am also thankful to Assoc. Prof. Lou Yih-Cherng for his kind willingness and great efforts to co-supervise and support me from the start of my Ph.D study. I would also like to express my gratitude to my thesis advisory committee members, Prof. Edward Manser and Assoc. Prof. Kah Leong Lim for their support and advice. I would like to show my appreciation to members of the Yu's lab for providing me a fun, motivating, enthusiastic and stimulating environment to work in, especially Dr. Gu Ying and Dr. Daniel Kirilly for showing me the ropes when I first joined the lab. I am also grateful to Dr. Wang Hongyan and Dr. Li Song for their help and sharing of ideas and comments which made this study possible. Special thanks go to Edwin Lim, Wang Yan and Zhang Heng, who had been tremendously helpful in this study. Many thanks to, Zong Wenhui, Dr. Ng Kay Siong, Tang Quan, Ye Sing, for their bright ideas and assistance in various ways. I owe my deepest gratitude to a broader fly community for their generosity in sharing reagents and flies. My gratitude also goes out to all supporting staffs and friends at Temasek Life Sciences Laboratory and NGS for their support and help. Also I am grateful to be a recipient of NGS Post Graduate Scholarship, who funded me for my studies. I am also thankful to my parents and family for their love. Lastly, I would like to express my gratitude to my lovely wife, Sylvia Zhong who has been the pillar of my strength throughout my studies. iv Summary Sculpting or remodeling of the nervous system is vital for formation and maintenance of a functional neuronal circuitry. While emerging studies had been undertaken to elucidate the mechanism governing the remodeling of the nervous system, our knowledge of it is far from complete. Drosophila melanogaster, the fruit fly, provides us with an exceptionally easy and highly manipulative platform to gain in-depth understanding of the remodeling of the nervous system. During metamorphosis, a subset of the neurons in the PNS undergoes remodeling. In particular, Class IV ddaC neurons undergo a process known as dendrite pruning, which refers to the selective removal of exuberant dendrites without causing cell death. To gain insight into the mechanism governing ddaC dendrite pruning, an RNAi screen was carried out and a Cullin-1 based SCF E3 ligase complex was identified to be essential for ddaC dendrite pruning. The Cullin-1 based SCF E3 ligase is composed of four core components—Cullin1, Roc1a, SkpA, and Slimb. Further investigation also demonstrated that the Cullin-1 based SCF E3 ligase is required for pruning of MB ϒ neurons in the CNS. This study also revealed that while EcR-B1 and Sox14 act upstream of Cullin-1 based SCF E3 ligase complex to regulate its abundance during ddaC dendrite pruning, Mical acts in parallel to the E3 ligase complex to mediate ddaC dendrite pruning. Furthermore, we demonstrated that InR/PI3K/TOR pathway is attenuated by Cullin-1 based SCF E3 ligase complex during dendrite pruning, likely through ubiquitination and degradation of key positive regulator, Akt. Consistently, hyperactivation of the InR/PI3K/TOR pathway is sufficient to hamper ddaC dendrite pruning. Therefore, this study identified a novel link between Cullin-1 based SCF E3 ligase complex and InR/PI3K/TOR pathway in regulation of ddaC dendrite pruning. It is also the first time that the insulin signaling is implicated in neuronal pruning v process, hence raising intriguing questions about how metabolic states may interplay with such developmental processes. vi Table of Contents Declaration iii Acknowledgement iv Summary v Table of Contents . vii List of Publications xi Poster and Oral Presentation xi List of Tables xii List of Figures xiii Abbreviations . xv Chapter Introduction . 1.1 Drosophila melanogaster as a model organism 1.2 Development of the nervous system 1.3 Stereotyped neuronal pruning 1.3.1 Vertebrate neuronal pruning . 1.3.1.1 Insights into vertebrate axon pruning 1.3.1.2 Axon guidance molecules in vertebrate axon pruning . 1.3.2 Neuronal pruning in Drosophila melanogaster 1.3.2.1 Transcriptional regulation of pruning in Drosophila melanogaster 12 1.3.2.2 Caspases and calcium transients in neuronal pruning 15 1.3.2.3 Ubiquitin and proteasome system in regulation of pruning . 17 1.4 Ubiquitin proteasome system . 18 1.4.1 E3 ligases and neurodegenerative diseases . 19 1.4.2 Cullin-1 based SCF E3 ligase . 20 1.4.3 F-box proteins, Beta-TrCP and Slimb . 21 1.5 Insulin signaling pathway 22 1.5.1 Insulin signaling in Drosophila 23 1.6 Aim of this study . 27 Chapter Material and Methods . 28 2.1 List of Fly strains 28 2.2 Rapamycin treatment . 29 2.3 RU486/mifepristone treatment for elav-GeneSwitch system. . 29 2.4 Microscopy and image acquisition and quantification . 29 vii 2.5 MARCM labeling for ddaC neuron mutants 31 2.6 MARCM labeling for mushroom body ϒ neuron mutants 32 2.7 Immunohistochemistry . 32 2.8 Quantitative PCR . 33 2.8.1 Laser capture microdissection and RNA isolation 33 2.8.2 Quantitative PCR 34 2.9 S2 Cell culture, transfection and ecdysone treatment . 34 2.10 Co-immunoprecipitation . 35 2.11 Double immunoprecipitation 35 2.12 In-vivo ubiquitination assay 36 2.13 SDS-PAGE and Western blotting . 37 2.14 DNA manipulation and Gateway cloning 38 2.14.1 Escherichia. coli culture and transformation . 38 2.14.2 Polymerase Chain Reaction and DNA sequencing. 38 2.14.3 Gateway Cloning 39 Chapter Results 41 3.1 Dendrite remodeling of ddaC neurons during metamorphosis 41 3.2 RNAi screen for novel players in ddaC dendrite pruning 42 3.3 A Cullin-1 based E3 ligase is required for dendrite pruning in Class IV ddaC neuron. 49 3.3.1 A Cullin-1 based E3 ligase is required cell-autonomously for dendrite pruning in Class IV ddaC neuron . 49 3.3.2 Post-translational modification, Neddylation, is required for dendrite pruning in Class IV ddaC neuron . 51 3.4 A Cullin-1 based SCF E3 ligase comprising of Roc1a, SkpA and Slimb is required for dendrite arborization neurons remodeling. . 52 3.4.1 RING domain protein, Roc1a but not Roc1b, is required for dendrite pruning in Class IV ddaC neuron . 52 3.4.2 SkpA, an adaptor protein and Slimb, an F-box protein are required for Class IV ddaC neuron dendrite pruning. 54 3.4.3 Cullin-1 based SCF E3 ligase regulates dendrite pruning independently of initial ddaC neuron dendrite development 57 3.4.4 Cullin-1 based SCF E3 ligase is required for remodeling of Class I and Class III da neurons 59 3.4.5 Cullin-1, Roc1a, SkpA and Slimb form a protein complex in vitro and in vivo. 60 viii 3.5 Cullin-1 based SCF E3 ligase components are required for dendrite and axon pruning in MB ϒ neurons. . 62 3.6 Cullin-1 based SCF E3 ligase regulates dendrite pruning downstream of EcR-B1 and Sox14 but in parallel to Mical. 65 3.6.1 Cullin-1 based SCF E3 ligase does not affect EcR-B1 and Sox14 expression. . 65 3.6.2 Cullin-1 based SCF E3 ligase works downstream of EcR-B1 and Sox14 to regulate dendrite pruning. 67 3.6.3 Cullin-1 based SCF E3 ligase does not regulate Mical expression or transcription 70 3.6.4 Cullin-1 based SCF E3 ligase works in parallel to Mical to govern dendrite pruning. . 72 3.7 The Cullin-1 based SCF E3 ligase complex antagonises insulin signaling to promote ddaC dendrite pruning. . 75 3.7.1 The Cullin-1 based SCF E3 ligase complex regulates dendrite pruning independent on known targets, Hedgehog and Wingless signaling pathway. . 75 3.7.2 The Cullin-1-based SCF E3 ligase complex antagonises the insulin signaling pathway but not other pathways to promote ddaC dendrite pruning . 78 3.7.3 The Cullin-1 based SCF E3 ligase complex suppresses PI3K/TOR signaling during ddaC dendrite pruning. 81 3.7.4 Pharmacological attenuation of insulin signaling pathway suppresses dendrite pruning defect in ddaC neurons devoid of Cullin-1 based SCF E3 ligase complex. 84 3.7.5 Specificity of insulin signaling in Cullin-1 based SCF E3 ligase mediated dendrite pruning. 86 3.7.5.1 Attenuation of insulin signaling in cullin-1 mutant does not affect normal dendrite elaboration. 86 3.7.5.2 Attenuation of insulin signaling in cullin-1 mutant promotes proximal severing of major dendrites 87 3.7.5.3 Attenuation of insulin signaling does not rescue dendrite pruning defect in mical mutant ddaC neurons 89 3.8 Cullin-1 based SCF E3 ligase negatively regulates insulin signaling through Akt ubiquitination 90 3.8.1 Compromised Cullin-1 based SCF E3 ligase activity leads to hyperactivation of insulin signaling . 91 3.8.2 Substrate recognition domain, Slimb interacts with Akt and promotes Akt ubiquitination 94 3.9 Activation of InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning 96 3.9.1 Activation of InR/PI3K/TOR pathway is sufficient to inhibit ddaC dendrite pruning. . 96 ix 3.9.2 Activation of InR/PI3K/Tor pathway enhances cul1DN mediated ddaC dendrite pruning defect. 98 3.9.3 Activation of InR/PI3K/TOR signaling is not sufficient to inhibit MB ϒ neuron axon pruning. 99 3.10 InR/PI3K/TOR signaling does not affect EcR-B1 and Sox14 expression and functions downstream of EcR-B1 and Sox14 100 3.11 InR/PI3K/TOR signaling works in parallel to Mical to regulate dendrite pruning. 103 3.12 Cullin-1 based SCF E3 ligase and insulin signaling govern dendrite pruning partially through caspase activation. 104 Chapter Discussion . 107 4.1 Insights into mechanism of dendritic pruning 107 4.2 Cullin-1 based SCF E3 ligase complex is required for both PNS and CNS remodeling 109 4.2 Regulation of Cullin-1 based SCF E3 ligase complex for dendritic pruning . 111 4.3 Inactivation of InR/PI3K/TOR pathway by Cullin-1 based SCF E3 ligase for dendritic pruning 113 4.4 Akt as a target and substrate for Cullin-1 based SCF E3 ligase complex 114 4.5 Cullin-1 based SCF E3 ligase complex and InR/PI3K/TOR pathway controls dendrite pruning in part via local caspase activation. . 116 4.6 Future directions . 116 Chapter Conclusion 119 References 121 x the insulin signaling pathway is actively kept in check by various modes of proteasomal regulation. Several studies had investigated the interplay between ecdysone and insulin signaling, and significant and complex crosstalk had been established between these two pathways at a systemic level (Colombani et al., 2005, Riddiford et al., Caldwell et al., 2005, Rusten et al., 2004). Colombani et al had demonstrated that level the of ecdysone signaling is inversely related to insulin signaling in fat body and Rusten et al had shown similar antagonism during developmental regulated autophagy. Consistent with these reports, our studies had also suggested antagonism between the two signaling pathways. However, the model which we subscribe to involves the cell automous regulation of insulin signaling by ecdysone signaling, which has not been previously reported. In particular we provided evidences that suggest that Akt is a substrate for ecdysone regulated Cullin-1 based SCF E3 ligase complex during developmental regulated pruning. 115 4.5 Cullin-1 based SCF E3 ligase complex and InR/PI3K/TOR pathway controls dendrite pruning in part via local caspase activation. Local caspase activation is required for ddaC dendrite pruning, our results had demonstrated that both Cullin-1 based SCF E3 ligase complex and insulin signaling pathway regulates caspase activation in dendrites of ddaC neurons during dendritic pruning. Consistently in axotomizd rat retianal ganglion cells, insulin signaling protects the cells from cell death through Akt phorsphorylation and inhibition of capsase activation (Kermer et al., 2000). Since, the localisation of Akt and Cullin-1 based SCF E3 ligase complex is uniform throughout the ddaC neurons, and the degradation of Akt appears to be evenly distributed in the neuron, without any specific site of action, thus it is likely that InR/PI3K/TOR pathway partake in dendrite pruning through control of another intermediate that is directly responsible for severing of the dendrites, one possible candidate would be the caspase activator required for dendrite pruning. Interestingly, in embryonic chick lens epithelial cells, insulin signaling can down regulate Inhibitor of Caspase (IAP) (Basu et al., 2012).Further investigation into how Cullin-1 based SCF E3 ligase and InR/PI3K/TOR pathway works mechanistically to regulate temporal and spatial caspase activation would be an interesting direction to follow in future studies. 4.6 Future directions Despite the identification of a Cullin-1 based SCF E3 ligase complex that is required for neuronal pruning and InR/PI3K/TOR pathway is the downstream target of Cullin-1 based SCF E3 ligase complex during ddaC dendrite pruning, several questions still remains unanswered and requires further investigation. First of all, despite the strong severing defect observed in cul1EX ddaC MARCM clones, the phenotype observed for slimb8 ddaC 116 MARCM is much weaker. Although the difference in phenotype severity can be explained by the perdurance of maternal slimb protein, we cannot rule out the possibility of the presence of yet to be identified F-box protein that works together with Cullin-1 to regulate ddaC dendrite pruning. Further investigation should be conducted to isolate other novel F-box containing protein that may be required for ddaC dendrite pruning and this could be achieved by carrying out additional RNAi or MARCM screen on putative F-box containing protein. Although we were able to achieve a significant suppression of cullin-1 mutant phenotype via the attenuation of InR/PI3K/TOR pathway, there is still a certain percentage of ddaC dendrites which remain unpruned in the suppression assay. This indicates the presence of other substrates or pathways that is jointly regulated by Cullin-1 based SCF E3 ligase complex during ddaC dendrite pruning. It is also likely that these unidentified pathways or substrate could be regulated by the previously mentioned unidentified F-box protein during ddaC dendrite pruning. More in-depth studies to identify the presence of such unidentified pathways or substrates would shed more light into the regulation of pruning by Cullin-1 based SCF E3 ligase complex. Our studies had also identified Cullin-1 based SCF E3 ligase to be involved in two distinct modes of neuronal pruning, while InR/PI3K/TOR pathway is only required and function downstream of Cullin-based SCF E3 ligase during ddaC dendrite pruning. We would like to deepen our understanding in the MB ϒ neuron axon pruning to identify the substrate that is regulated by Cullin-1 based SCF E3 ligase during MB ϒ neuron axon pruning, this can be achieved by carrying out another genetic suppression screen of cullin-1 mutant in the MB ϒ neuron. In a recent study, calcium transients and caspase activity had been proposed to work in parallel to mediate dendrite pruning (Kanamori et al., 2013). Since Cullin-1 based SCF E3 117 ligase and InR/PI3K/TOR pathway function upstream of caspase, it would be interesting to investigate if calcium transients still occur in Cullin-1 based SCF E3 ligase or InR/PI3K/TOR pathway mutants. Finally since Cullin-1 based SCF E3 ligase is evolutionary conserved and mediates two main forms of neuronal remodeling in Drosophila melanogaster, we would like to extrapolate our studies onto other neuronal remodeling models or even in neurodegenerative diseases. 118 Chapter Conclusion Our study on the understanding of the mechanism of dendritic pruning in ddaC neurons, started off with a highly efficient reverse genetic screen that yielded several putative candidate genes, which when disrupted led to dendrite pruning defect in ddaC neurons. Amongst the putative candidates, Cullin-1, a scaffold protein of a multimeric E3 ligase stood out, as it was documented that UPS system is involved in neuronal pruning, but the specific E3 ligase involved has yet to be identified. Cullin-1 forms a multimeric complex which comprises of adaptor protein SkpA, linker protein Roc1a and substrate recognition protein Slimb, all of which are required for proper remodeling of da neurons as well as MB ϒ neuron. At the onset of metamorphosis, ecdysone signaling triggers the expression of sox14, which in turn is required for the temporal up regulation of cullin-1 expression, to increase the abundance of Cullin-1 based SCF E3 ligase complex to aid dendrite pruning. Cullin-1 based SCF E3 ligase complex then works in parallel with the previously identified ecdysone inducible gene, mical, to regulate ddaC dendrite pruning. While Cullin-1 based SCF E3 ligase complex does not regulate its canonical target during ddaC dendrite pruning, it suppresses InR/PI3K/TOR pathway in order for proper ddaC dendrite pruning to occur. Furthermore, the hyperactivation of InR/PI3K/TOR pathway recapitulates the phenotype observed in ddaC neurons mutated of the Cullin-1 based SCF E3 ligase complex. Elevated expression and activity of InR/PI3K/TOR pathway's positive regulator, Akt, was also observed in cullin-1 ddaC mutant, attesting the regulation of InR/PI3K/TOR pathway by Cullin-1 based SCF E3 ligase complex. Furthermore, Slimb, the substrate recognition domain of the E3 ligase, binds and targets Akt, for polyubiquitination followed by degradation to suppress InR/PI3K/TOR pathway. Lastly, Cullin-1 based SCF E3 ligase complex and InR/PI3K/TOR functions at least in part 119 through activation of caspase to mediate ddaC dendrite pruning. The proposed model of ddaC dendrite pruning demonstrated in this study is illustrated in the following figure. Figure 31: A schematic model for the Cullin-1 based SCF E3 ligase and the InR/PI3K/TOR pathway during ddaC dendrite pruning. While the Cullin-1 based SCF E3 ligase functions downstream of EcR-B1 and Sox14, it mediates dendrite pruning in parallel to Mical during ddaC dendrite pruning. Regulation of ddaC dendrite pruning by the Cullin-1 based SCF E3 ligase complex is achieved primarily through inactivation of the InR/PI3K/TOR pathway. Hyperactivation of InR/PI3K/TOR pathway is sufficient to inhibit dendrite pruning. Unidentified F-box containing protein may also be important in ddaC dendrite pruning. Similarly, other yet to be determined molecules may be also regulated by Cullin-1 based SCF E3 ligase complex during ddaC dendrite pruning. 120 References AGGOUN-ZOUAOUI, D. & INNOCENTI, G. M. 1994. Juvenile visual callosal axons in kittens display origin- and fate-related morphology and distribution of arbors. Eur J Neurosci, 6, 1846-63. AWASAKI, T., HUANG, Y., O'CONNOR, M. B. & LEE, T. 2011. Glia instruct developmental neuronal remodeling through TGF-beta signaling. Nat Neurosci, 14, 821-3. BAAS, P. W., DEITCH, J. S., BLACK, M. M. & BANKER, G. A. 1988. Polarity orientation of microtubules in hippocampal neurons: uniformity in the axon and nonuniformity in the dendrite. Proc Natl Acad Sci U S A, 85, 8335-9. BABETTO, E., BEIROWSKI, B., RUSSLER, E. V., MILBRANDT, J. & DIANTONIO, A. 2013. The Phr1 ubiquitin ligase promotes injury-induced axon self-destruction. Cell Rep, 3, 1422-9. BADER, M., ARAMA, E. & STELLER, H. 2010. A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila. Development, 137, 1679-88. BAEHRECKE, E. H. 2003. Autophagic programmed cell death in Drosophila. Cell Death Differ, 10, 940-5. BAGRI, A., CHENG, H. J., YARON, A., PLEASURE, S. J. & TESSIER-LAVIGNE, M. 2003. Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell, 113, 285-99. BARCELO, H. & STEWART, M. J. 2002. Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth. Genesis, 34, 83-5. BASU, S., RAJAKARUNA, S. & MENKO, A. S. 2012. Insulin-like growth factor receptor-1 and nuclear factor kappaB are crucial survival signals that regulate caspase-3mediated lens epithelial cell differentiation initiation. J Biol Chem, 287, 8384-97. BECKSTEAD, R. B., LAM, G. & THUMMEL, C. S. 2005. The genomic response to 20hydroxyecdysone at the onset of Drosophila metamorphosis. Genome Biol, 6, R99. BHUTANI, S., DAS, A., MAHESHWARI, M., LAKHOTIA, S. C. & JANA, N. R. 2012. Dysregulation of core components of SCF complex in poly-glutamine disorders. Cell Death Dis, 3, e428. BISHOP, D. L., MISGELD, T., WALSH, M. K., GAN, W. B. & LICHTMAN, J. W. 2004. Axon branch removal at developing synapses by axosome shedding. Neuron, 44, 65161. BODENSTEIN 1965. The postembryonic development of Drosophila in Biology of Drosophila.(ed M. Demerc). BOHNI, R., RIESGO-ESCOVAR, J., OLDHAM, S., BROGIOLO, W., STOCKER, H., ANDRUSS, B. F., BECKINGHAM, K. & HAFEN, E. 1999. Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell, 97, 865-75. BOULANGER, A., CLOUET-REDT, C., FARGE, M., FLANDRE, A., GUIGNARD, T., FERNANDO, C., JUGE, F. & DURA, J. M. 2011. ftz-f1 and Hr39 opposing roles on EcR expression during Drosophila mushroom body neuron remodeling. Nat Neurosci, 14, 37-44. BOURGEOIS, J. P. & RAKIC, P. 1993. Changes of synaptic density in the primary visual cortex of the macaque monkey from fetal to adult stage. J Neurosci, 13, 2801-20. BRAND, A. H. & PERRIMON, N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 118, 401-15. BROGIOLO, W., STOCKER, H., IKEYA, T., RINTELEN, F., FERNANDEZ, R. & HAFEN, E. 2001. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr Biol, 11, 213-21. 121 BROUGHTON, S. J., PIPER, M. D., IKEYA, T., BASS, T. M., JACOBSON, J., DRIEGE, Y., MARTINEZ, P., HAFEN, E., WITHERS, D. J., LEEVERS, S. J. & PARTRIDGE, L. 2005. Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc Natl Acad Sci U S A, 102, 310510. BURTIS, K. C., THUMMEL, C. S., JONES, C. W., KARIM, F. D. & HOGNESS, D. S. 1990. The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell, 61, 85-99. CALDWELL, P. E., WALKIEWICZ, M. & STERN, M. 2005. Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release. Curr Biol, 15, 1785-95. CAO, Y., ESPINOLA, J. A., FOSSALE, E., MASSEY, A. C., CUERVO, A. M., MACDONALD, M. E. & COTMAN, S. L. 2006. Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofuscinosis. J Biol Chem, 281, 20483-93. CAUSING, C. G., GLOSTER, A., ALOYZ, R., BAMJI, S. X., CHANG, E., FAWCETT, J., KUCHEL, G. & MILLER, F. D. 1997. Synaptic innervation density is regulated by neuronderived BDNF. Neuron, 18, 257-67. CHELL, J. M. & BRAND, A. H. 2010. Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell, 143, 1161-73. CHEN, C., JACK, J. & GAROFALO, R. S. 1996. The Drosophila insulin receptor is required for normal growth. Endocrinology, 137, 846-56. CHENG, H. J., BAGRI, A., YARON, A., STEIN, E., PLEASURE, S. J. & TESSIER-LAVIGNE, M. 2001. Plexin-A3 mediates semaphorin signaling and regulates the development of hippocampal axonal projections. Neuron, 32, 249-63. CLANCY, D. J., GEMS, D., HARSHMAN, L. G., OLDHAM, S., STOCKER, H., HAFEN, E., LEEVERS, S. J. & PARTRIDGE, L. 2001. Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science, 292, 104-6. COLOMBANI, J., ANDERSEN, D. S. & LEOPOLD, P. 2012. Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing. Science, 336, 582-5. COLOMBANI, J., BIANCHINI, L., LAYALLE, S., PONDEVILLE, E., DAUPHIN-VILLEMANT, C., ANTONIEWSKI, C., CARRE, C., NOSELLI, S. & LEOPOLD, P. 2005. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science, 310, 667-70. COLOMBANI, J., RAISIN, S., PANTALACCI, S., RADIMERSKI, T., MONTAGNE, J. & LEOPOLD, P. 2003. A nutrient sensor mechanism controls Drosophila growth. Cell, 114, 739-49. COWAN, C. A. & HENKEMEYER, M. 2002. Ephrins in reverse, park and drive. Trends Cell Biol, 12, 339-46. DERYNCK, R., ZHANG, Y. & FENG, X. H. 1998. Smads: transcriptional activators of TGFbeta responses. Cell, 95, 737-40. DESHAIES, R. J. 1999. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu Rev Cell Dev Biol, 15, 435-67. DHANOA, N. K., KROL, K. M., JAHED, A., CRUTCHER, K. A. & KAWAJA, M. D. 2006. Null mutations for exon III and exon IV of the p75 neurotrophin receptor gene enhance sympathetic sprouting in response to elevated levels of nerve growth factor in transgenic mice. Exp Neurol, 198, 416-26. DIBELLO, P. R., WITHERS, D. A., BAYER, C. A., FRISTROM, J. W. & GUILD, G. M. 1991. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics, 129, 385-97. 122 DONALDSON, T. D., NOUREDDINE, M. A., REYNOLDS, P. J., BRADFORD, W. & DURONIO, R. J. 2004. Targeted disruption of Drosophila Roc1b reveals functional differences in the Roc subunit of Cullin-dependent E3 ubiquitin ligases. Mol Biol Cell, 15, 4892-903. DORRELLO, N. V., PESCHIAROLI, A., GUARDAVACCARO, D., COLBURN, N. H., SHERMAN, N. E. & PAGANO, M. 2006. S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science, 314, 467-71. EDERY, I., ZWIEBEL, L. J., DEMBINSKA, M. E. & ROSBASH, M. 1994. Temporal phosphorylation of the Drosophila period protein. Proc Natl Acad Sci U S A, 91, 2260-4. FLANAGAN, J. G. & VANDERHAEGHEN, P. 1998. The ephrins and Eph receptors in neural development. Annu Rev Neurosci, 21, 309-45. FRESCAS, D. & PAGANO, M. 2008. Deregulated proteolysis by the F-box proteins SKP2 and beta-TrCP: tipping the scales of cancer. Nat Rev Cancer, 8, 438-49. FROST, D. O., MOY, Y. P. & SMITH, D. C. 1990. Effects of alternating monocular occlusion on the development of visual callosal connections. Exp Brain Res, 83, 200-9. GLAUSER, L., SONNAY, S., STAFA, K. & MOORE, D. J. 2011. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1. J Neurochem, 118, 636-45. GRIMA, B., LAMOUROUX, A., CHELOT, E., PAPIN, C., LIMBOURG-BOUCHON, B. & ROUYER, F. 2002. The F-box protein slimb controls the levels of clock proteins period and timeless. Nature, 420, 178-82. GRUEBER, W. B., JAN, L. Y. & JAN, Y. N. 2002. Tiling of the Drosophila epidermis by multidendritic sensory neurons. Development, 129, 2867-78. HAASS, C. & SELKOE, D. J. 2007. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol, 8, 101-12. HALME, A., CHENG, M. & HARIHARAN, I. K. 2010. Retinoids regulate a developmental checkpoint for tissue regeneration in Drosophila. Curr Biol, 20, 458-63. HEGDE, A. N. & DIANTONIO, A. 2002. Ubiquitin and the synapse. Nat Rev Neurosci, 3, 854-61. HENNIG, K. M. & NEUFELD, T. P. 2002. Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis, 34, 107-10. HERRMANN, K. & SHATZ, C. J. 1995. Blockade of action potential activity alters initial arborization of thalamic axons within cortical layer 4. Proc Natl Acad Sci U S A, 92, 11244-8. HILL, C. E. & VIDOVIC, M. 1989. The role of competition in the refinement of the projections of sympathetic neurons to the rat eye during development. Int J Dev Neurosci, 7, 539-51. HIRANO, T. 2006. At the heart of the chromosome: SMC proteins in action. Nat Rev Mol Cell Biol, 7, 311-22. HO, M. S., TSAI, P. I. & CHIEN, C. T. 2006. F-box proteins: the key to protein degradation. J Biomed Sci, 13, 181-91. HOOPFER, E. D., PENTON, A., WATTS, R. J. & LUO, L. 2008. Genomic analysis of Drosophila neuronal remodeling: a role for the RNA-binding protein Boule as a negative regulator of axon pruning. J Neurosci, 28, 6092-103. HU, C. Z., SETHI, J. K. & HAGEN, T. The role of the cullin-5 e3 ubiquitin ligase in the regulation of insulin receptor substrate-1. Biochem Res Int, 2012, 282648. HU, J., ZACHAREK, S., HE, Y. J., LEE, H., SHUMWAY, S., DURONIO, R. J. & XIONG, Y. 2008. WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1-CUL4-ROC1 ligase. Genes Dev, 22, 866-71. 123 HUANG, H., POTTER, C. J., TAO, W., LI, D. M., BROGIOLO, W., HAFEN, E., SUN, H. & XU, T. 1999. PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development, 126, 5365-72. HUNG, R. J., PAK, C. W. & TERMAN, J. R. 2011. Direct redox regulation of F-actin assembly and disassembly by Mical. Science, 334, 1710-3. HUNG, R. J., YAZDANI, U., YOON, J., WU, H., YANG, T., GUPTA, N., HUANG, Z., VAN BERKEL, W. J. & TERMAN, J. R. 2010. Mical links semaphorins to F-actin disassembly. Nature, 463, 823-7. HWANGBO, D. S., GERSHMAN, B., TU, M. P., PALMER, M. & TATAR, M. 2004. Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature, 429, 562-6. IKEYA, T., GALIC, M., BELAWAT, P., NAIRZ, K. & HAFEN, E. 2002. Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr Biol, 12, 1293-300. INGHAM, P. W. 1995. Signalling by hedgehog family proteins in Drosophila and vertebrate development. Curr Opin Genet Dev, 5, 492-8. INNOCENTI, G. M. & CLARKE, S. 1984. The organization of immature callosal connections. J Comp Neurol, 230, 287-309. ITO, K. & HOTTA, Y. 1992. Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster. Dev Biol, 149, 134-48. JIANG, C., BAEHRECKE, E. H. & THUMMEL, C. S. 1997. Steroid regulated programmed cell death during Drosophila metamorphosis. Development, 124, 4673-83. JIANG, J. & STRUHL, G. 1998. Regulation of the Hedgehog and Wingless signalling pathways by the F-box/WD40-repeat protein Slimb. Nature, 391, 493-6. JIN, J., CARDOZO, T., LOVERING, R. C., ELLEDGE, S. J., PAGANO, M. & HARPER, J. W. 2004. Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev, 18, 2573-80. JONES, J. I. & CLEMMONS, D. R. 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev, 16, 3-34. KANAMORI, T., KANAI, M. I., DAIRYO, Y., YASUNAGA, K., MORIKAWA, R. K. & EMOTO, K. 2013. Compartmentalized calcium transients trigger dendrite pruning in Drosophila sensory neurons. Science, 340, 1475-8. KARIN, M. & GRETEN, F. R. 2005. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol, 5, 749-59. KERMER, P., KLOCKER, N., LABES, M. & BAHR, M. 2000. Insulin-like growth factor-I protects axotomized rat retinal ganglion cells from secondary death via PI3-Kdependent Akt phosphorylation and inhibition of caspase-3 In vivo. J Neurosci, 20, 2-8. KIRILLY, D., GU, Y., HUANG, Y., WU, Z., BASHIRULLAH, A., LOW, B. C., KOLODKIN, A. L., WANG, H. & YU, F. 2009. A genetic pathway composed of Sox14 and Mical governs severing of dendrites during pruning. Nat Neurosci, 12, 1497-505. KIRILLY, D., WONG, J. J., LIM, E. K., WANG, Y., ZHANG, H., WANG, C., LIAO, Q., WANG, H., LIOU, Y. C. & YU, F. 2011. Intrinsic epigenetic factors cooperate with the steroid hormone ecdysone to govern dendrite pruning in Drosophila. Neuron, 72, 86100. KITADA, T., ASAKAWA, S., HATTORI, N., MATSUMINE, H., YAMAMURA, Y., MINOSHIMA, S., YOKOCHI, M., MIZUNO, Y. & SHIMIZU, N. 1998. Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism. Nature, 392, 605-8. KORALEK, K. A. & KILLACKEY, H. P. 1990. Callosal projections in rat somatosensory cortex are altered by early removal of afferent input. Proc Natl Acad Sci U S A, 87, 1396400. 124 KULLANDER, K. & KLEIN, R. 2002. Mechanisms and functions of Eph and ephrin signalling. Nat Rev Mol Cell Biol, 3, 475-86. KUO, C. T., JAN, L. Y. & JAN, Y. N. 2005. Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling. Proc Natl Acad Sci U S A, 102, 15230-5. KUO, C. T., ZHU, S., YOUNGER, S., JAN, L. Y. & JAN, Y. N. 2006. Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning. Neuron, 51, 283-90. LEE, C. Y., CLOUGH, E. A., YELLON, P., TESLOVICH, T. M., STEPHAN, D. A. & BAEHRECKE, E. H. 2003. Genome-wide analyses of steroid- and radiation-triggered programmed cell death in Drosophila. Curr Biol, 13, 350-7. LEE, H. H., JAN, L. Y. & JAN, Y. N. 2009. Drosophila IKK-related kinase Ik2 and Katanin p60-like regulate dendrite pruning of sensory neuron during metamorphosis. Proc Natl Acad Sci U S A, 106, 6363-8. LEE, K. F., BACHMAN, K., LANDIS, S. & JAENISCH, R. 1994. Dependence on p75 for innervation of some sympathetic targets. Science, 263, 1447-9. LEE, S., WANG, J. W., YU, W. & LU, B. 2012. Phospho-dependent ubiquitination and degradation of PAR-1 regulates synaptic morphology and tau-mediated Abeta toxicity in Drosophila. Nat Commun, 3, 1312. LEE, T., LEE, A. & LUO, L. 1999. Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast. Development, 126, 4065-76. LEE, T. & LUO, L. 1999. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron, 22, 451-61. LEE, T. & LUO, L. 2001. Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci, 24, 251-4. LEE, T., MARTICKE, S., SUNG, C., ROBINOW, S. & LUO, L. 2000. Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila. Neuron, 28, 807-18. LEEVERS, S. J., WEINKOVE, D., MACDOUGALL, L. K., HAFEN, E. & WATERFIELD, M. D. 1996. The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth. EMBO J, 15, 6584-94. LI, T. R. & WHITE, K. P. 2003. Tissue-specific gene expression and ecdysone-regulated genomic networks in Drosophila. Dev Cell, 5, 59-72. LIU, S., SAWADA, T., LEE, S., YU, W., SILVERIO, G., ALAPATT, P., MILLAN, I., SHEN, A., SAXTON, W., KANAO, T., TAKAHASHI, R., HATTORI, N., IMAI, Y. & LU, B. 2012. Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria. PLoS Genet, 8, e1002537. LOSADA, A. & HIRANO, T. 2005. Dynamic molecular linkers of the genome: the first decade of SMC proteins. Genes Dev, 19, 1269-87. LU, B., AL-RAMAHI, I., VALENCIA, A., WANG, Q., BERENSHTEYN, F., YANG, H., GALLEGOFLORES, T., ICHCHO, S., LACOSTE, A., HILD, M., DIFIGLIA, M., BOTAS, J. & PALACINO, J. 2013. Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance. Nat Neurosci, 16, 562-70. LUAN, H., PEABODY, N. C., VINSON, C. R. & WHITE, B. H. 2006. Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression. Neuron, 52, 425-36. LUCKING, C. B., DURR, A., BONIFATI, V., VAUGHAN, J., DE MICHELE, G., GASSER, T., HARHANGI, B. S., MECO, G., DENEFLE, P., WOOD, N. W., AGID, Y., BRICE, A., FRENCH PARKINSON'S DISEASE GENETICS STUDY, G. & EUROPEAN CONSORTIUM ON GENETIC SUSCEPTIBILITY IN PARKINSON'S, D. 2000. Association between 125 early-onset Parkinson's disease and mutations in the parkin gene. N Engl J Med, 342, 1560-7. LUO, L. & O'LEARY, D. D. 2005. Axon retraction and degeneration in development and disease. Annu Rev Neurosci, 28, 127-56. MARGOTTIN, F., BOUR, S. P., DURAND, H., SELIG, L., BENICHOU, S., RICHARD, V., THOMAS, D., STREBEL, K. & BENAROUS, R. 1998. A novel human WD protein, hbeta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif. Mol Cell, 1, 565-74. MATA, I. F., LOCKHART, P. J. & FARRER, M. J. 2004. Parkin genetics: one model for Parkinson's disease. Hum Mol Genet, 13 Spec No 1, R127-33. MATSUMINE, H., YAMAMURA, Y., HATTORI, N., KOBAYASHI, T., KITADA, T., YORITAKA, A. & MIZUNO, Y. 1998. A microdeletion of D6S305 in a family of autosomal recessive juvenile parkinsonism (PARK2). Genomics, 49, 143-6. MCALLISTER, A. K. 2007. Dynamic aspects of CNS synapse formation. Annu Rev Neurosci, 30, 425-50. MILETICH, I. & LIMBOURG-BOUCHON, B. 2000. Drosophila null slimb clones transiently deregulate Hedgehog-independent transcription of wingless in all limb discs, and induce decapentaplegic transcription linked to imaginal disc regeneration. Mech Dev, 93, 15-26. MISHRA, A., MAHESHWARI, M., CHHANGANI, D., FUJIMORI-TONOU, N., ENDO, F., JOSHI, A. P., JANA, N. R. & YAMANAKA, K. 2013. E6-AP association promotes SOD1 aggresomes degradation and suppresses toxicity. Neurobiol Aging, 34, 1310 e1123. MOBERG, K. H., BELL, D. W., WAHRER, D. C., HABER, D. A. & HARIHARAN, I. K. 2001. Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature, 413, 311-6. MURPHY, T. D. 2003. Drosophila skpA, a component of SCF ubiquitin ligases, regulates centrosome duplication independently of cyclin E accumulation. J Cell Sci, 116, 2321-32. MYERS, M. P., WAGER-SMITH, K., ROTHENFLUH-HILFIKER, A. & YOUNG, M. W. 1996. Light-induced degradation of TIMELESS and entrainment of the Drosophila circadian clock. Science, 271, 1736-40. NAKAO, R., HIRASAKA, K., GOTO, J., ISHIDOH, K., YAMADA, C., OHNO, A., OKUMURA, Y., NONAKA, I., YASUTOMO, K., BALDWIN, K. M., KOMINAMI, E., HIGASHIBATA, A., NAGANO, K., TANAKA, K., YASUI, N., MILLS, E. M., TAKEDA, S. & NIKAWA, T. 2009. Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor signaling during muscle atrophy caused by unloading. Mol Cell Biol, 29, 4798811. NARENDRA, D., WALKER, J. E. & YOULE, R. 2012. Mitochondrial quality control mediated by PINK1 and Parkin: links to parkinsonism. Cold Spring Harb Perspect Biol, 4. NEUFELD, T. P. 2003. Body building: regulation of shape and size by PI3K/TOR signaling during development. Mech Dev, 120, 1283-96. NIKOLAEV, A., MCLAUGHLIN, T., O'LEARY, D. D. & TESSIER-LAVIGNE, M. 2009. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature, 457, 981-9. NIWA, J., ISHIGAKI, S., HISHIKAWA, N., YAMAMOTO, M., DOYU, M., MURATA, S., TANAKA, K., TANIGUCHI, N. & SOBUE, G. 2002. Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity. J Biol Chem, 277, 36793-8. NOUREDDINE, M. A., DONALDSON, T. D., THACKER, S. A. & DURONIO, R. J. 2002. Drosophila Roc1a encodes a RING-H2 protein with a unique function in 126 processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase. Dev Cell, 2, 757-70. NUSSE, R. & VARMUS, H. E. 1992. Wnt genes. Cell, 69, 1073-87. OKAMOTO, N., NAKAMORI, R., MURAI, T., YAMAUCHI, Y., MASUDA, A. & NISHIMURA, T. 2013. A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila. Genes Dev, 27, 87-97. OKAMOTO, N., YAMANAKA, N., YAGI, Y., NISHIDA, Y., KATAOKA, H., O'CONNOR, M. B. & MIZOGUCHI, A. 2009. A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila. Dev Cell, 17, 885-91. OSTERWALDER, T., YOON, K. S., WHITE, B. H. & KESHISHIAN, H. 2001. A conditional tissue-specific transgene expression system using inducible GAL4. Proc Natl Acad Sci U S A, 98, 12596-601. OU, C. Y., LIN, Y. F., CHEN, Y. J. & CHIEN, C. T. 2002. Distinct protein degradation mechanisms mediated by Cul1 and Cul3 controlling Ci stability in Drosophila eye development. Genes Dev, 16, 2403-14. PETROSKI, M. D. & DESHAIES, R. J. 2005. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol, 6, 9-20. RICCOMAGNO, M. M., HURTADO, A., WANG, H., MACOPSON, J. G., GRINER, E. M., BETZ, A., BROSE, N., KAZANIETZ, M. G. & KOLODKIN, A. L. 2012. The RacGAP beta2Chimaerin selectively mediates axonal pruning in the hippocampus. Cell, 149, 1594-606. RIDDIFORD, L. M., TRUMAN, J. W., MIRTH, C. K. & SHEN, Y. C. A role for juvenile hormone in the prepupal development of Drosophila melanogaster. Development, 137, 1117-26. RUI, L., YUAN, M., FRANTZ, D., SHOELSON, S. & WHITE, M. F. 2002. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. J Biol Chem, 277, 42394-8. RULIFSON, E. J., KIM, S. K. & NUSSE, R. 2002. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science, 296, 1118-20. RUMPF, S., LEE, S. B., JAN, L. Y. & JAN, Y. N. 2011. Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1. Development, 138, 1153-60. RUSTEN, T. E., LINDMO, K., JUHASZ, G., SASS, M., SEGLEN, P. O., BRECH, A. & STENMARK, H. 2004. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev Cell, 7, 179-92. SANES, J. R. & LICHTMAN, J. W. 1999. Development of the vertebrate neuromuscular junction. Annu Rev Neurosci, 22, 389-442. SARIKAS, A., HARTMANN, T. & PAN, Z. Q. 2011. The cullin protein family. Genome Biol, 12, 220. SAUCEDO, L. J., GAO, X., CHIARELLI, D. A., LI, L., PAN, D. & EDGAR, B. A. 2003. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat Cell Biol, 5, 566-71. SCHULDINER, O., BERDNIK, D., LEVY, J. M., WU, J. S., LUGINBUHL, D., GONTANG, A. C. & LUO, L. 2008. piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning. Dev Cell, 14, 227-38. SCOTT, R. C., SCHULDINER, O. & NEUFELD, T. P. 2004. Role and regulation of starvationinduced autophagy in the Drosophila fat body. Dev Cell, 7, 167-78. SEGRAVES, W. A. & HOGNESS, D. S. 1990. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev, 4, 204-19. 127 SHINGLETON, A. W. 2005. Body-size regulation: combining genetics and physiology. Curr Biol, 15, R825-7. SINGH, K. K., PARK, K. J., HONG, E. J., KRAMER, B. M., GREENBERG, M. E., KAPLAN, D. R. & MILLER, F. D. 2008. Developmental axon pruning mediated by BDNF-p75NTRdependent axon degeneration. Nat Neurosci, 11, 649-58. SKAAR, J. R., D'ANGIOLELLA, V., PAGAN, J. K. & PAGANO, M. 2009. SnapShot: F Box Proteins II. Cell, 137, 1358, 1358 e1. SKAAR, J. R., PAGAN, J. K. & PAGANO, M. 2013. Mechanisms and function of substrate recruitment by F-box proteins. Nat Rev Mol Cell Biol, 14, 369-81. SLAIDINA, M., DELANOUE, R., GRONKE, S., PARTRIDGE, L. & LEOPOLD, P. 2009. A Drosophila insulin-like peptide promotes growth during nonfeeding states. Dev Cell, 17, 874-84. SONG, J. W., MISGELD, T., KANG, H., KNECHT, S., LU, J., CAO, Y., COTMAN, S. L., BISHOP, D. L. & LICHTMAN, J. W. 2008. Lysosomal activity associated with developmental axon pruning. J Neurosci, 28, 8993-9001. SONG, R., PENG, W., ZHANG, Y., LV, F., WU, H. K., GUO, J., CAO, Y., PI, Y., ZHANG, X., JIN, L., ZHANG, M., JIANG, P., LIU, F., MENG, S., CAO, C. M. & XIAO, R. P. Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders. Nature, 494, 375-9. SONG, Y., ORI-MCKENNEY, K. M., ZHENG, Y., HAN, C., JAN, L. Y. & JAN, Y. N. 2012. Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam. Genes Dev, 26, 1612-25. SOUSA-NUNES, R., YEE, L. L. & GOULD, A. P. 2011. Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature, 471, 508-12. SPENCER, E., JIANG, J. & CHEN, Z. J. 1999. Signal-induced ubiquitination of IkappaBalpha by the F-box protein Slimb/beta-TrCP. Genes Dev, 13, 284-94. STANFIELD, B. B. & O'LEARY, D. D. 1985a. Fetal occipital cortical neurones transplanted to the rostral cortex can extend and maintain a pyramidal tract axon. Nature, 313, 135-7. STANFIELD, B. B. & O'LEARY, D. D. 1985b. The transient corticospinal projection from the occipital cortex during the postnatal development of the rat. J Comp Neurol, 238, 236-48. STANFIELD, B. B., O'LEARY, D. D. & FRICKS, C. 1982. Selective collateral elimination in early postnatal development restricts cortical distribution of rat pyramidal tract neurones. Nature, 298, 371-3. SUDHOF, T. C. 2008. Neuroligins and neurexins link synaptic function to cognitive disease. Nature, 455, 903-11. TAN, P., FUCHS, S. Y., CHEN, A., WU, K., GOMEZ, C., RONAI, Z. & PAN, Z. Q. 1999. Recruitment of a ROC1-CUL1 ubiquitin ligase by Skp1 and HOS to catalyze the ubiquitination of I kappa B alpha. Mol Cell, 3, 527-33. TATAR, M., KOPELMAN, A., EPSTEIN, D., TU, M. P., YIN, C. M. & GAROFALO, R. S. 2001. A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science, 292, 107-10. TELEMAN, A. A., CHEN, Y. W. & COHEN, S. M. 2005. 4E-BP functions as a metabolic brake used under stress conditions but not during normal growth. Genes Dev, 19, 1844-8. TERMAN, J. R., MAO, T., PASTERKAMP, R. J., YU, H. H. & KOLODKIN, A. L. 2002. MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell, 109, 887-900. TESSIER-LAVIGNE, M. & GOODMAN, C. S. 1996. The molecular biology of axon guidance. Science, 274, 1123-33. 128 TREMPE, J. F., SAUVE, V., GRENIER, K., SEIRAFI, M., TANG, M. Y., MENADE, M., ALABDUL-WAHID, S., KRETT, J., WONG, K., KOZLOV, G., NAGAR, B., FON, E. A. & GEHRING, K. 2013. Structure of parkin reveals mechanisms for ubiquitin ligase activation. Science, 340, 1451-5. TRUMAN, J. W. 1990. Metamorphosis of the central nervous system of Drosophila. J Neurobiol, 21, 1072-84. TRUMAN, J. W. & BATE, M. 1988. Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster. Dev Biol, 125, 145-57. TU, M. P., EPSTEIN, D. & TATAR, M. 2002. The demography of slow aging in male and female Drosophila mutant for the insulin-receptor substrate homologue chico. Aging Cell, 1, 75-80. VEENSTRA, J. A., AGRICOLA, H. J. & SELLAMI, A. 2008. Regulatory peptides in fruit fly midgut. Cell Tissue Res, 334, 499-516. VERDU, J., BURATOVICH, M. A., WILDER, E. L. & BIRNBAUM, M. J. 1999. Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat Cell Biol, 1, 500-6. VIDOVIC, M. & HILL, C. E. 1988. Withdrawal of collaterals of sympathetic axons to the rat eye during postnatal development: the role of function. J Auton Nerv Syst, 22, 57-65. VIDOVIC, M., HILL, C. E. & HENDRY, I. A. 1987. Developmental time course of the sympathetic postganglionic innervation of the rat eye. Brain Res, 429, 133-8. WANG, X., WINTER, D., ASHRAFI, G., SCHLEHE, J., WONG, Y. L., SELKOE, D., RICE, S., STEEN, J., LAVOIE, M. J. & SCHWARZ, T. L. 2011. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell, 147, 893906. WATTS, R. J., HOOPFER, E. D. & LUO, L. 2003. Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system. Neuron, 38, 871-85. WATTS, R. J., SCHULDINER, O., PERRINO, J., LARSEN, C. & LUO, L. 2004. Glia engulf degenerating axons during developmental axon pruning. Curr Biol, 14, 678-84. WEEKS, J. C. & LEVINE, R. B. 1990. Postembryonic neuronal plasticity and its hormonal control during insect metamorphosis. Annu Rev Neurosci, 13, 183-94. WEIMANN, J. M., ZHANG, Y. A., LEVIN, M. E., DEVINE, W. P., BRULET, P. & MCCONNELL, S. K. 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron, 24, 819-31. WEINKOVE, D. & LEEVERS, S. J. 2000. The genetic control of organ growth: insights from Drosophila. Curr Opin Genet Dev, 10, 75-80. WILLIAMS, D. W., KONDO, S., KRZYZANOWSKA, A., HIROMI, Y. & TRUMAN, J. W. 2006. Local caspase activity directs engulfment of dendrites during pruning. Nat Neurosci, 9, 1234-6. WILLIAMS, D. W. & TRUMAN, J. W. 2005a. Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons. Development, 132, 3631-42. WILLIAMS, D. W. & TRUMAN, J. W. 2005b. Remodeling dendrites during insect metamorphosis. J Neurobiol, 64, 24-33. WU, C. & GHOSH, S. 1999. beta-TrCP mediates the signal-induced ubiquitination of IkappaBbeta. J Biol Chem, 274, 29591-4. WU, J. T., LIN, H. C., HU, Y. C. & CHIEN, C. T. 2005. Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation. Nat Cell Biol, 7, 1014-20. 129 XIONG, X., HAO, Y., SUN, K., LI, J., LI, X., MISHRA, B., SOPPINA, P., WU, C., HUME, R. I. & COLLINS, C. A. 2012. The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein. PLoS Biol, 10, e1001440. XIONG, X., WANG, X., EWANEK, R., BHAT, P., DIANTONIO, A. & COLLINS, C. A. 2010. Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury. J Cell Biol, 191, 211-23. XU, N. J. & HENKEMEYER, M. 2009. Ephrin-B3 reverse signaling through Grb4 and cytoskeletal regulators mediates axon pruning. Nat Neurosci, 12, 268-76. XU, X., SARIKAS, A., DIAS-SANTAGATA, D. C., DOLIOS, G., LAFONTANT, P. J., TSAI, S. C., ZHU, W., NAKAJIMA, H., NAKAJIMA, H. O., FIELD, L. J., WANG, R. & PAN, Z. Q. 2008. The CUL7 E3 ubiquitin ligase targets insulin receptor substrate for ubiquitin-dependent degradation. Mol Cell, 30, 403-14. YANG, H. S., JANSEN, A. P., KOMAR, A. A., ZHENG, X., MERRICK, W. C., COSTES, S., LOCKETT, S. J., SONENBERG, N. & COLBURN, N. H. 2003. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol Cell Biol, 23, 26-37. YING, Z., WANG, H., FAN, H., ZHU, X., ZHOU, J., FEI, E. & WANG, G. 2009. Gp78, an ER associated E3, promotes SOD1 and ataxin-3 degradation. Hum Mol Genet, 18, 4268-81. YOULE, R. J. & NARENDRA, D. P. 2011. Mechanisms of mitophagy. Nat Rev Mol Cell Biol, 12, 9-14. ZENG, H., QIAN, Z., MYERS, M. P. & ROSBASH, M. 1996. A light-entrainment mechanism for the Drosophila circadian clock. Nature, 380, 129-35. ZHENG, X., WANG, J., HAERRY, T. E., WU, A. Y., MARTIN, J., O'CONNOR, M. B., LEE, C. H. & LEE, T. 2003. TGF-beta signaling activates steroid hormone receptor expression during neuronal remodeling in the Drosophila brain. Cell, 112, 30315. ZHU, C. C., BOONE, J. Q., JENSEN, P. A., HANNA, S., PODEMSKI, L., LOCKE, J., DOE, C. Q. & O'CONNOR, M. B. 2008. Drosophila Activin- and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain. Development, 135, 513-21. ZUCCHELLI, S., MARCUZZI, F., CODRICH, M., AGOSTONI, E., VILOTTI, S., BIAGIOLI, M., PINTO, M., CARNEMOLLA, A., SANTORO, C., GUSTINCICH, S. & PERSICHETTI, F. 2011. Tumor necrosis factor receptor-associated factor (TRAF6) associates with huntingtin protein and promotes its atypical ubiquitination to enhance aggregate formation. J Biol Chem, 286, 25108-17. ZUFFEREY, P. D., JIN, F., NAKAMURA, H., TETTONI, L. & INNOCENTI, G. M. 1999. The role of pattern vision in the development of cortico-cortical connections. Eur J Neurosci, 11, 2669-88. 130 [...]... dendrite and axon pruning in MB ϒ neurons 64 Figure 12 : Cullin- 1 based SCF E3 ligase does not affect EcR-B1 and Sox14 expression 67 Figure 13 : Cullin- 1 based SCF E3 ligase works downstream of EcR-B1 and Sox14 to regulate dendrite pruning 69 Figure 14 : Cullin- 1 based SCF E3 ligase does not regulate Mical expression or transcription 71 Figure 15 : Cullin- 1 based SCF E3 ligase. .. The Cullin- 1 based SCF E3 ligase complex suppresses PI3K/TOR signaling during ddaC dendrite pruning 83 Figure 19 : Pharmacological attenuation of insulin signaling pathway suppresses dendrite pruning defect in ddaC neurons devoid of Cullin- 1 based SCF E3 ligase complex .85 xiii Figure 20: Attenuation of insulin signaling in cullin- 1 mutant does not affect normal dendrite elaboration... be characterised by defining motifs E3 ligases can be classified into monomeric or modular E3 ligases Within the modular E3 ligases, they can be further classified into HECT (Homologous to E6-associated protein C-terminus) or RING (Really Interesting New Gene) domain containing E3 ligase While HECT domain E3s are involved in the direct catalysis of the substrate, RING domain E3s serve as an adaptor-like... ligase works in parallel to Mical to govern dendrite pruning .74 Figure 16 : The Cullin- 1 based SCF E3 ligase complex regulates dendrite pruning independent on known targets, Hedgehog and Wingless signaling pathways 77 Figure 17 : The Cullin- 1- based SCF E3 ligase complex antagonises the insulin signaling pathway but not other pathways to promote ddaC dendrite pruning 80 Figure 18 :... conserved in mammals, thus making the Drosophila an ideal organism to elucidate biological mechanism or pathways in various biological processes and to enhance our understanding of them 1 Figure 1: The life cycle of Drosophila melanogaster at 25°C The 1st instar larva hatches from the fertilized egg one day after egg laying Subsequently, the larva molts twice into 2nd instar (1 day) and 3rd instar (2-3 days)... involved in both axon guidance as well as cell migration (Cowan and Henkemeyer, 2002, Flanagan and Vanderhaeghen, 19 98, Kullander and Klein, 2002) In vivo gene-targeting and in vitro live cell assay of stereotyped pruning of infra-pyramidal bundle has also implicated EphrinB3 mediated (EB3) reverse signaling in pruning Tyrosine phosphorylation of Ephrin-B3 results in postnatal shortening of IPB axons,... molecules acting as ligand to stimulate EB3 reverse signaling (Xu and Henkemeyer, 2009) Furthermore, adaptor protein Grb4 acts as a molecular linker bridging activated EB3 cytoplasmic tail with Dock180 and PAK to activate guanine nucleotide exchange and hence Rac activation to mediate axon retraction and pruning (Xu and Henkemeyer, 2009) 8 1. 3.2 Neuronal pruning in Drosophila melanogaster Development of Drosophila. .. 10 5 Figure 31: A model for the Cullin- 1 based SCF E3 ligase and the InR/PI3K/TOR pathway during ddaC dendrite pruning 11 8 xiv Abbreviations 4E-BP eukaryotic translation initiation factor 4E-binding protein APF after puparium formation BDNF brain-derived neurotrophic factor BR-C broad -complex Brm brahma containing remodeler Bsk basket CA constitutive active CBP CREB binding protein Ci... Brm, as knockdown of Brm diminishes the CBP/EcR-B1 interaction 1. 3.2.2 Caspases and calcium transients in neuronal pruning Neuronal pruning shares many similar features with cell apoptosis, including the fragmentation of cellular components, formation of blebs, local degeneration and the eventual clearance of debris by phagocytes; hence it is conceivable that caspases might also play a role in neuronal. .. be detailed in the following sections 5 1. 3 .1. 1 Insights into vertebrate axon pruning A well-established model of vertebrate neuronal pruning is the refinement of layer V subcortical processes, whereby pruning of the long axon collaterals takes place (Stanfield and O'Leary, 19 85b, Stanfield and O'Leary, 19 8 5a, Stanfield et al., 19 82) Layer V axons of the motor cortex and visual cortex are initially . pruning 11 3 4.4 Akt as a target and substrate for Cullin- 1 based SCF E3 ligase complex 11 4 4.5 Cullin- 1 based SCF E3 ligase complex and InR/PI3K/TOR pathway controls dendrite pruning in part. both PNS and CNS remodeling 10 9 4.2 Regulation of Cullin- 1 based SCF E3 ligase complex for dendritic pruning 11 1 4.3 Inactivation of InR/PI3K/TOR pathway by Cullin- 1 based SCF E3 ligase for. pruning in Drosophila melanogaster 9 1. 3.2 .1 Transcriptional regulation of pruning in Drosophila melanogaster 12 1. 3.2.2 Caspases and calcium transients in neuronal pruning 15 1. 3.2.3 Ubiquitin and