Sunday, December 22, 2024
12/22/2024
PROJECT SUMMARY/ABSTRACT. Degradative pathways maintain protein homeostasis and set signaling duration for endosomal receptors. Dendritic vesicle transport is needed for initially enabling dendrite elaboration in development and later promoting postsynaptic functions of neurons. Dendrites can reach considerable lengths and thus have to solve the “distance problem”: biosynthetic capacity and degradative capacity are highest in/near the soma. Our goal is to find novel regulatory mechanisms governing dendritic proteostasis. Our previous work showed that directional transport is needed for Rab7-mediated bulk degradation of dendritic cargos. Using the short-lived receptors NSG1/2 as “degradation sensors”, we can tackle the molecular mechanisms regulating directional dendritic transport. Rab7 is a well-known regulator of LEs and lysosomes and genetically linked to CMT2B. In fibroblasts, Rab7 is implicated in many distinct steps including endosome maturation, recruitment of retromer for retrieving receptors, and recruitment of motors for regulating endosome positioning. It does so via several effectors, many of whom are linked to human diseases. We will unravel the Rab7 effector hierarchy for two important Rab7 effectors, RILP and Vps35. Vps35 is part of the retromer complex and retrieves multiple cargos from late endosomes to escape degradation whereas RILP recruits dynein to late endosomes for transport to somatic lysosomes. Our data indicate that extrapolating from studies in fibroblasts is insufficient. Hypothesis: Rab7 “effector conversion” from Rab7-Vps35 complexes (promoting retrieval) and Rab7-RILP complexes (promoting degradation) regulates organelle positioning, degradative flux, and signaling duration of BDNF/TrkB from signaling endosomes in dendrites to change dendrite growth. Strategy: We have discovered distinct Rab7-dependent phenotypes that can be rescued with WT Rab7. We are using effector-skewed, phospho-, and CMT2B Rab7 mutants to dissect the steps at which Rab7 complexes function. These mutants will be used to test for “effector binding skew” as well as probe the functional consequences of Rab7 mutants in dendritic proteostasis (Aim 1) and BDNF-mediated dendrite growth (Aim 2). Impact: Completion of Aim 1 will advance our mechanistic, molecular understanding of where and when Rab7 effector complexes assemble to set the balance between retrieval and degradation along dendrites. Completion of Aim 2 will link BDNF-mediated dendrite growth with regulation of sorting and trafficking (“endosomal fate decisions”) via Rab7 effector cascades in dendrites. This will lead to a molecular understanding of how signal duration and functional outcomes can be regulated by Rab7 effector conversion to skew receptor fate for faster or slower degradation. This work will illuminate the cell biological mechanisms underlying signaling regulation of a critical neuronal protein, BDNF, which has many roles in developing and adult nervous systems. Discovering the molecular mechanisms by which early Rab7 complexes and late Rab7 complexes affect dendrite growth will serve to identify critical nodes as potential therapeutic targets in disease states.
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