Localization of RNAi Machinery to Axonal Branch Points and Growth Cones Is Facilitated by Mitochondria and Is Disrupted in ALS.

Noga Gershoni-Emek,Ariel Ionescu,Eran Perlson,Christopher J Costa,Dianna E Willis,Tal Gradus-Pery,Topaz Altman

doi:10.3389/fnmol.2018.00311

Abstract

Local protein synthesis in neuronal axons plays an important role in essential spatiotemporal signaling processes; however, the molecular basis for the post-transcriptional regulation controlling this process in axons is still not fully understood. Here we studied the axonal mechanisms underlying the transport and localization of microRNA (miRNA) and the RNAi machinery along the axon. We first identified miRNAs, Dicer, and Argonaute-2 (Ago2) in motor neuron (MN) axons. We then studied the localization of RNAi machinery and demonstrated that mitochondria associate with miR-124 and RNAi proteins in axons. Importantly, this co-localization occurs primarily at axonal branch points and growth cones. Moreover, using live cell imaging of a functional Cy3-tagged miR-124, we revealed that this miRNA is actively transported with acidic compartments in axons, and associates with stalled mitochondria at growth cones and axonal branch points. Finally, we observed enhanced retrograde transport of miR-124-Cy3, and a reduction in its localization to static mitochondria in MNs expressing the ALS causative gene hSOD1G93A. Taken together, our data suggest that mitochondria participate in the axonal localization and transport of RNAi machinery, and further imply that alterations in this mechanism may be associated with neurodegeneration in ALS.

Highlights

Neurons are highly polarized cells with well-defined structures such as axons and dendrites
We have demonstrated that the silencing machinery components, namely, Dicer, Ago2, and miRNAs, are localized in axons far from the perinuclear region and can be associated with mitochondria
A link between endosomal trafficking and RNA silencing was first established when the RNAInduced Silencing Complex (RISC) components Ago2 and GW182 were found to be enriched in their fraction of multivesicular bodies (MVBs), suggesting that these foci are distinct from Pbodies (Gibbings et al, 2009; Siomi and Siomi, 2009)

Summary

Introduction

Neurons are highly polarized cells with well-defined structures such as axons and dendrites. In order to survive and maintain its function, the neuron must be able to respond to both intracellular and extracellular cues. The spatiotemporal localization of mRNAs in distinct intracellular compartments within the neuron, together with the proteins that regulate and execute translation, is essential for the neuron’s survival and function (Holt and Schuman, 2013). MicroRNAs (miRNAs) are short, negative regulators of gene expression, which are highly conserved in multicellular organisms. Both pre- and mature miRNAs have been identified in the axonal compartment (Sasaki et al, 2014; Kim et al, 2015; Rotem et al, 2017), suggesting that

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