The microtubule cytoskeleton acts as both the neuronal highway system and the signposts. Even small alterations in microtubule motor or organizing proteins can result in neurodegeneration as these long cells rely on transport from the cell body to axons and dendrites to function for a lifetime. While neuronal microtubules are critically important for neuronal health, many of the basic mechanisms that organize them have not yet been identified. In this proposal we focus on elucidating mechanisms that control dendrite microtubule polarity. Unlike axons, dendrites have large numbers of minus-end-out microtubules that direct dendrite-specific proteins and organelles to this compartment. As minus-end-out microtubules specify dendrite identity and are poorly understood we will maximize our impact by focusing on their generation and regulation. We previously found that dendrite branch points are hubs of microtubule control and house: 1) machinery that steers microtubules towards the cell body, and 2) nucleation sites. It was proposed that Golgi outposts localize nucleation sites to dendrites, however we have shown this is not the case. Using the microtubule steering machinery as a starting point, we have identified several sets of proteins, including heterotrimeric G proteins and branched actin regulators that play a role in branch point targeting.
In Aim 1, we will determine which of these protein sets also positions nucleation sites, and how they influence overall microtubule polarity.
In Aim 2 we will focus on the part of the dendrite beyond the branch point. Although nucleation sites are concentrated at branch points, microtubule minus ends are found even near dendrite tips. We will explore how they might get there to identify new players and mechanisms in microtubule control. For example, we can observe growing minus ends in dendrites and hypothesize these may allow population of distal dendrites with microtubules. In the last Aim of the proposal we will jump into new territory and take maximum advantage of our model system and our recent discoveries. By trying to understand how neurons can last a lifetime, even when injured, we found that microtubules are dramatically rearranged after axon severing. Microtubule dynamics is massively upregulated and dendrite polarity switches. These changes are transcription-dependent, so we will perform transcriptome analysis to identify the microtubule regulators that are up- or down-regulated by injury. We will rapidly test candidate regulators with in vivo functional assays to identify proteis that control microtubules in normal and stressed neurons. By using a simple, genetically tractable model system that allows us to examine neurons in vivo, we will make rapid progress in understanding the basic mechanisms that control neuronal microtubule organization. We will use hypothesis-driven and discovery-driven approaches to understand microtubule regulation from multiple perspectives. As microtubules support long-term neuronal health, this work should provide a foundation for deeper understanding of pathological changes that drive neurodegenerative disease.

Public Health Relevance

Unlike many cells in our bodies individual neurons must survive our entire lifetime. The critical infrastructure that allows new material to be distributedto distant regions of these long cells is the microtubule cytoskeleton, and when microtubule organization is disrupted neurodegeneration often results. We aim to identify the proteins and understand the mechanisms that organize the microtubule infrastructure in neurons.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085115-07
Application #
9265487
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Gindhart, Joseph G
Project Start
2010-05-01
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
7
Fiscal Year
2017
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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