. Since neurons are post-mitotic, long-lived, and extraordinarily large, maintaining the proteome is of particular importance. Not surprisingly, disturbances in protein homeostasis (?proteostasis?) have been associated with numerous neurodegenerative disorders, as well as aging. How protein turnover, is regulated in time and space is thus an essential cell biological question with profound implications for neuronal function and disease. Endosome transport in dendrites is not well understood.
Our specific aims are to:
Aim 1) discover how dendritic degradative flux (i.e. transport, acidification, fusion with lysosomes) is coordinated along dendrites by two small GTPases localized to late endosomes and lysosomes, Rab7 and Arl8b, Aim 2) identify the roles of two Rab7 effectors in regulating proteostasis, and Aim 3) discover functional consequences of disrupted endosomal maturation for neuronal health, (including degradation of membrane receptors, of aggregated proteins, maintenance of dendrites, and cell survival). Endosome maturation involves recruitment of small GTPases (Rab7 and Arl8b) and their effectors to regulate acidification, motility, and fusion events. Our recent work has implicated Rab7 in spatial regulation of degradative flux. Rab7 binds to multiple effectors, but how they spatially regulate endosome behavior in dendrites is poorly understood. Notably, Rab7 itself is mutated in Charcot-Marie-Tooth disease 2B and many Rab7 effectors are linked to diseases, highlighting the centrality of endosomal maturation pathways for normal neuronal function. Current barriers to progress include missing ?molecular handles? for studying the heterogeneity of late endosomes and lysosomes, lack of endogenous trackable cargos that can be used to follow degradation, and ignorance of the many endosomal effectors which regulate endosomal flux. The premise for this application rests on our own recent data: 1) Contrary to common belief, several endogenous dendritic receptors are not degraded in dendrites, but instead are retrogradely transported to the soma/proximal dendrite in a Rab7-dependent manner. Degradation thus does not take place in dendrites, but in somatic lysosomes. 2) LAMP1, the most commonly used lysosome marker in fibroblasts, is present in many compartments which are not degradative lysosomes, especially more distally in dendrites, highlighting neural-specific mechanisms. 3) Most Rab7-containing late endosomes in distal dendrites contain no LAMP1, and are thus different from late endosomes in fibroblasts which overwhelmingly contain both Rab7 and LAMP1. We refer to them as ?early? late endosomes to distinguish them from the conventional Rab7+/LAMP1+ late endosomes. These new discoveries allow us to now address a significant overarching question: How does the expansive dendritic arbor coordinate endosome maturation and degradation, and how do failures of proteostasis affect cellular function of neurons? Our long-term goal is to unravel the mechanisms of proteostasis in dendrites.

Public Health Relevance

Since neurons do not divide and need to be kept healthy for the whole life span of the organism, maintaining the correct protein composition and regulating degradation of proteins is of particular importance for neurons. Disturbances in protein balance (?proteostasis?) have been associated with numerous neurodegenerative disorders, as they lead to accumulation of toxic aggregates, disruption of neurotransmission, and neuron death. This proposal will determine how neurons regulate proteostasis in time and space, a fundamental cell biological question with profound implications for neuronal function and disease. The proposed research is significant because the new insights gained will not only lead to fundamental advances in understanding the regulation of neuronal degradative trafficking, but also raise the possibility of new targets for tailoring translational strategies in the future.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Riddle, Robert D
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University of Virginia
Schools of Medicine
United States
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Winckler, Bettina; Faundez, Victor; Maday, Sandra et al. (2018) The Endolysosomal System and Proteostasis: From Development to Degeneration. J Neurosci 38:9364-9374
Barford, Kelly; Keeler, Austin; McMahon, Lloyd et al. (2018) Transcytosis of TrkA leads to diversification of dendritic signaling endosomes. Sci Rep 8:4715
Zhang, Huaye; Winckler, Bettina; Cai, Qian (2018) Introduction to the special issue on membrane trafficking in neurons. Dev Neurobiol 78:167-169
Yap, Chan Choo; Digilio, Laura; McMahon, Lloyd P et al. (2018) Degradation of dendritic cargos requires Rab7-dependent transport to somatic lysosomes. J Cell Biol 217:3141-3159
Casanova, James E; Winckler, Bettina (2017) A new Rab7 effector controls phosphoinositide conversion in endosome maturation. J Cell Biol 216:2995-2997
Barford, Kelly; Deppmann, Christopher; Winckler, Bettina (2017) The neurotrophin receptor signaling endosome: Where trafficking meets signaling. Dev Neurobiol 77:405-418
Yap, Chan Choo; Digilio, Laura; McMahon, Lloyd et al. (2017) The endosomal neuronal proteins Nsg1/NEEP21 and Nsg2/P19 are itinerant, not resident proteins of dendritic endosomes. Sci Rep 7:10481
Barford, Kelly; Keeler, Austin; Deppmann, Christopher et al. (2017) TrkA Bumps into Its Future Self. Dev Cell 42:557-558
Martorella, M; Barford, K; Winkler, B et al. (2017) Emergent Role of Coronin-1a in Neuronal Signaling. Vitam Horm 104:113-131
Barford, Kelly; Yap, Chan Choo; Dwyer, Noelle D et al. (2017) The related neuronal endosomal proteins NEEP21 (Nsg1) and P19 (Nsg2) have divergent expression profiles in vivo. J Comp Neurol 525:1861-1878

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