Ubiquitination is a key regulatory mechanism for synaptic development, signaling, and plasticity. The covalent attachment of the 76 aa peptide ubiquitin to target proteins is a rapid and reversible modification that regulates protein stability, activity and localization. As such, it is a potent mechanism for sculpting the synapse. We have uncovered a ubiquitination complex composed of the E3 ubiquitin ligase Highwire (Hiw) and the F-Box protein DFsn that plays a central role in controlling synaptic growth and function at the Drosophila neuromuscular junction (NMJ). A highly homologous ubiquitination complex has also been identified in the mammalian brain, where it plays a critical role in regulating axon guidance and synaptogenesis. However, the molecular architecture and molecular action of this ubiquitination complex is not well understood. We propose that Hiw and DFsn form a non-SCF ubiquitin complex where Hiw functions as an E3 ligase and a scaffolding protein to facilitate multi-subunit interaction, and the combination of different co-factors and ubiquitin substrates confers time- and cell type-specific regulation of neuronal functions. Thus identifying other components and novel ubiquitin targets of this ubiquitination complex is key to understanding how the hiw-mediated ubiquitin pathway specifically regulates synaptic development. We have taken two independent approaches to address this question. Biochemically, we identified Hiw/DFsn interacting proteins through tandem affinity purification using fly brains that express affinity-tagged Hiw and DFsn proteins, respectively. Studying the role of two of the Hiw- binding proteins, NSF and Rae1, in synaptic development (aim1), and how they work together with Hiw and DFsn to modulate the ubiquitin ligase activity (aim2) will define an essential ubiquitination machinery that controls synaptic growth. Genetically, we identified 5 hiw enhancer complementation groups through a hiw enhancer screen. Studying these genetic hiw interactors will allow us to identify other molecular pathways that work together with the Hiw/DFsn ubiquitin pathway to shape the structure and strength of synaptic connections formed during development (aim3).

Public Health Relevance

The results of this project will improve our understanding of how nerve cells make connections with other nerve and muscle cells during development. If these connections do not form or function properly in children, it may lead to neurological diseases such as mental retardation, epilepsy, and autism;in addition, if we know how to stimulate these nerve cells to make new appropriate connections we may, in the future, put normal nerve cells back to our nervous system to treat traumatic spinal cord injuries and neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Thus an understanding of the molecules that control the formation and function of nerve cell connections could aid in the future development of new therapies for devastating neurological diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS070962-04
Application #
8467765
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Talley, Edmund M
Project Start
2010-07-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
4
Fiscal Year
2013
Total Cost
$293,758
Indirect Cost
$86,886
Name
Louisiana State Univ Hsc New Orleans
Department
Neurosciences
Type
Schools of Medicine
DUNS #
782627814
City
New Orleans
State
LA
Country
United States
Zip Code
70112
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Brace, E J; Wu, Chunlai; Valakh, Vera et al. (2014) SkpA restrains synaptic terminal growth during development and promotes axonal degeneration following injury. J Neurosci 34:8398-410
Li, Long; Tian, Xiaolin; Zhu, Mingwei et al. (2014) Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons. J Neurosci 34:8474-87
Yun, Jina; Puri, Rajat; Yang, Huan et al. (2014) MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin. Elife 3:e01958
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Tian, Xiaolin; Zhu, Mingwei; Li, Long et al. (2013) Identifying protein-protein interaction in Drosophila adult heads by Tandem Affinity Purification (TAP). J Vis Exp :50968
Wong, Jack Jing Lin; Li, Song; Lim, Edwin Kok Hao et al. (2013) A Cullin1-based SCF E3 ubiquitin ligase targets the InR/PI3K/TOR pathway to regulate neuronal pruning. PLoS Biol 11:e1001657
Tian, Xiaolin; Wu, Chunlai (2013) The role of ubiquitin-mediated pathways in regulating synaptic development, axonal degeneration and regeneration: insights from fly and worm. J Physiol 591:3133-43
Xiong, Xin; Hao, Yan; Sun, Kan et al. (2012) The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein. PLoS Biol 10:e1001440
Graf, Ethan R; Valakh, Vera; Wright, Christina M et al. (2012) RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction. J Neurosci 32:16586-96

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