Axonal microRNAs: localization, function and regulatory mechanism during axon development

82 j Journal of Molecular Cell Biology (2017), 9(2), 82–90 doi:10.1093/jmcb/mjw050 Published online January 6, 2017 Review Axonal microRNAs: localization, function and regulatory mechanism during axon development Bin Wang1 and Lan Bao1,2,* 1 State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 2 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China * Correspondence to: Lan Bao, E-mail: Subcellular localization and translation of messenger RNAs are essential for the regulation of neuronal development and synaptic function. As post-transcriptional regulators, microRNAs (miRNAs) have been emerging as central players in the development and maturation of the nervous system. Recent discoveries reveal the critical functions of miRNAs in the axon of neurons via multiple pathways of molecular regulation. Here, we introduce methods for isolating axonal miRNAs and review recent findings on the localization and function as well as regulatory mechanism of axonal miRNAs during axon development. Keywords: axonal miRNA, compartmentalized culture, axon development, RNA-binding protein Introduction Neurons are highly polarized cells that possess dendrites with vast, complicated spines to accept information and axons extending very distally to transmit signals (Martin and Ephrussi, 2009; Jung et al., 2012). Distinct subpopulations of messenger RNAs (mRNAs) have been identified in axons by high-throughput technologies such as microarrays and RNA sequencing (Andreassi et al., 2010; Zivraj et al., 2010; Gumy et al., 2011; Briese et al., 2015). Moreover, the local translation of mRNAs in axons has been found to be essential for functions such as axon elongation, regeneration, and viability (Cox et al., 2008; Yoo et al., 2009; Jung et al., 2014; Batista and Hengst, 2016; Tasdemir-Yilmaz and Segal, 2016). As a class of small, non-coding RNA molecules, microRNAs (miRNAs) have been revealed to be involved in multiple biological processes through post-transcriptional regulation (Giraldez et al., 2005; McNeill and Van Vactor, 2012). Recent studies have demonstrated that miRNAs are distributed in the different subcellular compartment of neurons. For instance, miR-124 displays a soma-restricted pattern and regulates axonal pathfinding by Received June 27, 2016. Revised October 3, 2016. Accepted November 25, 2016. © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons. org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact targeting transcription factor (Kye et al., 2007; Baudet et al., 2012), suggesting a global and large change of gene expression mediated by miRNA in the cell body. However, some miRNAs are found to be spatially localized in the distal compartment of neurons such as the axon (Natera-Naranjo et al., 2010), allowing for the rapid and precise control of local mRNA translation without conveying signals to the soma to regulate transcription and mRNA translation as well as protein transport to axons. Compartmentalized culture system is an efficient platform to separate axons from neuronal cell bodies and obtain pure axonal samples, which is recently adopted to explore the distribution of miRNAs in the axon of neurons. Several studies detected miRNA machinery proteins and miRNAs in the axon of distinct neurons in both the central nervous system and the peripheral nervous system by using compartmentalized culture system such as Campenot chamber and microfluidic chamber (Hengst et al., 2006; Kaplan et al., 2013; Hancock et al., 2014; Sasaki et al., 2014; Phay et al., 2015). In this review, we first introduce methods for isolating axonal miRNAs (Figure 1) and discuss the advantage and disadvantage of these methods (Table 1). Then, we summarize recent studies for the localization and function as well as regulatory mechanism of axonal miRNAs during axon development. Methods for isolating axonal miRNAs Campenot chamber The Campenot chamber is the first device applied in culture for the compartmentalization of the cell body and axon (Campenot, Axonal miRNAs during axon development j 83 Figure 1 Methods for isolating axonal miRNAs. (A) The Campenot chamber includes a scaffold made of Teflon, which is tightly adhered to a glass coverslip through silicone grease. The original three-chamber system consists of a central compartment and two side compartments. The dissociated neurons are plated in the central compartment. After several days in culture, only the long axons are able to pass through the silicone grease to both side compartments. (B) The Boyden chamber consists of a hollow plastic chamber sealed with a porous membrane containing pores of various sizes, allowing the motile cells to move to the other side. Explants or dissociated neurons are plated on a glass coverslip that is placed on the top of the microporous membrane. The growing axons cross through the membrane after several days. (C) The microfluidic chamber utilizes the replica-moulded transparent polydimethylsiloxane (PDMS) to establish a multi-compartment platform for cell culture. The chamber consists of separate compartments for the cell body and the axon, with microchannels (300 μm) linking the two compartments (top view). The cell body compartment is 100 μm high and is used for plating of neurons, whereas the microchannels are 3 μm in height and used for axon growth (side view). (D) The fascicles of axoplasm are mechanically separated from dissected sciatic nerve and incubated with a hypotonic solution (0.2× phosphate-buffered saline), either retaining intact axons or destroying Schwann cells. The ‘cloudy’ fascicles are incubated with the hypotonic solution for 2 h. After washing several times with the hypotonic solution, the axoplasm is eventually obtained in the supernatant of the solution (1× phosphate-buffered saline). Table 1 Comparison of different methods for isolating axonal miRNAs. Method Advantage Disadvantage Optimal application Campenot chamber Boyden chamber Microfluidic chamber High purity, good for fluidic separation Easily manageable, high yield of RNA Easily manageable, high purity, very good for fluidic separation, good for live cell imaging High yield of axonal RNA, good in vivo system Not easily manageable, low yield of axonal RNA Not good for fluidic separation and live cell imaging Low yield of axonal RNA Cultured cells Cultured explants/cells Cultured cells (...truncated)