Neuronal microtubules are intimately interconnected with neuronal health, yet many basic principles that control neuronal microtubule organization remain mysterious. For example, microtubules can be nucleated throughout axons and dendrites, but regulators that position nucleation sites far from the cell body have not been identified. Nucleation is upregulated throughout the neuron by axon injury and stress so it is particularly important to understand how it is controlled. If basic mechanisms that control neuronal microtubule organization are not elucidated, it will be very difficult to understand the relationships between the microtubule regulatory proteins and neurodegeneration. Hereditary spastic paraplegia is genetically one of the simplest forms of neurodegenerative disease, and mutations in the gene that encodes the microtubule severing protein spastin cause 40% of cases. However, how and where spastin functions in mature neurons has not been pinned down, and why its reduction sensitizes neurons to degeneration is also not clear. In this proposal Drosophila is used as a model system in which to efficiently identify basic mechanisms that regulate neuronal microtubules to provide a framework for future work on regeneration and degeneration.
Aim 1. An unexpected pathway controls microtubule nucleation in dendrites. Using a screening approach, Wnt signaling proteins emerged as key regulators that position microtubule nucleation sites in dendrites. Preliminary data in this aim indicates Wnt signaling proteins likely activate nucleation on endosomes. These preliminary findings will be strengthened by pairing a new nucleation assay with localization of Wnt signaling proteins and endosomes.
Aim 2. The role of severing proteins in converting minus end nucleation to minus end growth. It was recently shown that minus ends grow in dendrites and this is important for polarity control and dendrite regeneration. Preliminary data indicates spastin plays a role in generating growing minus ends. Using new assays to visualize nucleation and severing in combination with genetic tools, how and where microtubules are severed in dendrites will be investigated.
Aim 3. Control of disruptive nucleation by a checkpoint system. Microtubule nucleation has the potential to disrupt polarity by generating microtubules in random orientations. Detailed live imaging of growing microtubule plus ends determined that a checkpoint system depolymerizes microtubules nucleated in the ?wrong? orientation. Two players required for promoting growth of ?right? orientation microtubules have been identified. Their function, localization and interactions with one another will be investigated. Conclusion: We will elucidate fundamental but poorly understood mechanisms that control dendrite microtubule organization to fill in key gaps in our understanding of the neuronal cytoskeleton that are essential context for understanding neuronal regeneration and degeneration.

Public Health Relevance

Neuronal microtubules are closely linked to neuronal health; correct regulation of microtubule dynamics is critical for axon regeneration and defects in the microtubule cytoskeleton can underlie neurodegenerative disease. We use a simple model system to make maximal progress in understanding mechanisms that control microtubules in healthy cells to provide an essential framework for understanding what goes wrong in disease and what can be improved in regeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM085115-10
Application #
9971136
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Ainsztein, Alexandra M
Project Start
2010-05-01
Project End
2024-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Rao, Kavitha S; Rolls, Melissa M (2017) Two Drosophila model neurons can regenerate axons from the stump or from a converted dendrite, with feedback between the two sites. Neural Dev 12:15
Jegla, Timothy; Nguyen, Michelle M; Feng, Chengye et al. (2016) Bilaterian Giant Ankyrins Have a Common Evolutionary Origin and Play a Conserved Role in Patterning the Axon Initial Segment. PLoS Genet 12:e1006457
Chen, Li; Nye, Derek M; Stone, Michelle C et al. (2016) Mitochondria and Caspases Tune Nmnat-Mediated Stabilization to Promote Axon Regeneration. PLoS Genet 12:e1006503
Rao, Kavitha; Stone, Michelle C; Weiner, Alexis T et al. (2016) Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration. Mol Biol Cell 27:3245-3256
Tao, Juan; Feng, Chengye; Rolls, Melissa M (2016) The microtubule-severing protein fidgetin acts after dendrite injury to promote their degeneration. J Cell Sci 129:3274-81
Rolls, Melissa M; Jegla, Timothy J (2015) Neuronal polarity: an evolutionary perspective. J Exp Biol 218:572-80
Nguyen, Michelle M; McCracken, Christie J; Milner, E S et al. (2014) ?-tubulin controls neuronal microtubule polarity independently of Golgi outposts. Mol Biol Cell 25:2039-50
Stone, Michelle C; Albertson, Richard M; Chen, Li et al. (2014) Dendrite injury triggers DLK-independent regeneration. Cell Rep 6:247-53
Tai, Yilin; Janas, Justyna A; Wang, Chia-Lin et al. (2014) Regulation of chandelier cell cartridge and bouton development via DOCK7-mediated ErbB4 activation. Cell Rep 6:254-63
Hill, Sarah E; Parmar, Manpreet; Gheres, Kyle W et al. (2012) Development of dendrite polarity in Drosophila neurons. Neural Dev 7:34

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