Retrograde transport of Akt by a neuronal Rab5-APPL1 endosome

Livia Goto-Silva,Yannis Kalaidzidis,Marisa P Mcshane,Giampietro Schiavo,Marino Zerial,Sara Salinas

doi:10.1038/s41598-019-38637-0

Abstract

Long-distance axonal trafficking plays a critical role in neuronal function and transport defects have been linked to neurodegenerative disorders. Various lines of evidence suggest that the small GTPase Rab5 plays a role in neuronal signaling via early endosomal transport. Here, we characterized the motility of Rab5 endosomes in primary cultures of mouse hippocampal pyramidal cells by live-cell imaging and showed that they exhibit bi-directional long-range motility in axons, with a strong bias toward retrograde transport. Characterization of key Rab5 effectors revealed that endogenous Rabankyrin-5, Rabenosyn-5 and APPL1 are all present in axons. Further analysis of APPL1-positive endosomes showed that, similar to Rab5-endosomes, they display more frequent long-range retrograde than anterograde movement, with the endosomal levels of APPL1 correlated with faster retrograde movement. Interestingly, APPL1-endosomes transport the neurotrophin receptor TrkB and mediate retrograde axonal transport of the kinase Akt1. FRET analysis revealed that APPL1 and Akt1 interact in an endocytosis-dependent manner. We conclude that Rab5-APPL1 endosomes exhibit the hallmarks of axonal signaling endosomes to transport Akt1 in hippocampal pyramidal cells.

Highlights

The ability of axons to transport cargo over long distances is critical for processes ranging from axon path finding and target innervation, to neuronal survival
Neurons were plated on one side of the microfluidic chamber and agarose beads containing brain-derived neurotrophic factor (BDNF) were deposited on the opposite side, in order to stimulate axonal growth
Axons emerging from cell bodies penetrate and grow along the microchannels until they reach the source of BDNF on the other side

Summary

Introduction

The ability of axons to transport cargo over long distances is critical for processes ranging from axon path finding and target innervation, to neuronal survival. Rab[5] localizes to early endosomes, which sort cargo to the recycling pathway via Rab[4] and Rab[11], or to the degradative pathway through Rab7-positive endosomes[14] In neurons, both Rab[5] and Rab[7] were shown to be important for retrograde trafficking. Www.nature.com/scientificreports non-polarized cells two distinct populations of Rab5-positive early endosomes coexist and dynamically exchange cargo over time, one containing the canonical Rab[5] effector EEA1 and the other harboring APPL115,16. The Rab[5] effector APPL1 is an adaptor protein for Akt, a central kinase regulating cell survival[20,21] and plays a role in survival signaling from endosomes[22]. The distribution of Rabenosyn-5, which plays a dual role in endocytosis and recycling[13] has not been addressed in neurons

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