In the Right Place at the Right Time: miRNAs as Key Regulators in Developing Axons

Abstract

During neuronal circuit formation, axons progressively develop into a presynaptic compartment aided by extracellular signals. Axons display a remarkably high degree of autonomy supported in part by a local translation machinery that permits the subcellular production of proteins required for their development. Here, we review the latest findings showing that microRNAs (miRNAs) are critical regulators of this machinery, orchestrating the spatiotemporal regulation of local translation in response to cues. We first survey the current efforts toward unraveling the axonal miRNA repertoire through miRNA profiling, and we reveal the presence of a putative axonal miRNA signature. We also provide an overview of the molecular underpinnings of miRNA action. Our review of the available experimental evidence delineates two broad paradigms: cue-induced relief of miRNA-mediated inhibition, leading to bursts of protein translation, and cue-induced miRNA activation, which results in reduced protein production. Overall, this review highlights how a decade of intense investigation has led to a new appreciation of miRNAs as key elements of the local translation regulatory network controlling axon development.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining

“…Whilst the standard protocol yielded a diffuse pre-miRNA-associated signal (Fig. 6C), with our optimized protocol the signal was significantly improved and appeared as discrete puncta colocalising with CD63-GFP marked vesicles (Figure 5C), consistent with previous findings in living cells (Corradi, Dalla Costa et al 2020). Second, we investigated exogenously delivered (by electroporation) synthetic cy3-labeled pre-miRNA (Supplementary Figure 14A & B).…”

“…Since the retention of siRNAs was improved by our protocol, we tested its effect on other forms of small nucleic acids. Endogenous microRNAs (miRNAs) can associate with late endosomes/lysosomes (LE/Ly) and hitchhike them for intracellular transport (Gibbings, Ciaudo et al 2009, Lee, Pressman et al 2009, Corradi and Baudet 2020, Corradi, Dalla Costa et al 2020). Therefore, we visualized miRNAs docked to LE/Ly by two strategies.…”

“…Therefore, we visualized miRNAs docked to LE/Ly by two strategies. First, we detected endogenous precursor miRNAs (pre-miRNAs) via cy3-labeled molecular beacon (MB) (Supplementary Figure 14A) (Corradi, Dalla Costa et al 2020) and assessed its colocalisation with LE/Ly marker transmembrane tetraspanin CD63-GFP (Pols and Klumperman 2009, Corradi, Dalla Costa et al 2020) in elongating axons from Xenopus laevis whole eye explant culture (Supplementary Figure 14B). Whilst the standard protocol yielded a diffuse pre-miRNA-associated signal (Fig.…”

“…cy3-BHQ2 pre-miR-181a-1 molecular beacon (Eurogentec): 5’-CAUUGC C UUUA G AUACCAAUG -3’, 2’-O-methyl ribose backbone and 2 LNA nucleotides (bold)(Corradi, Dalla Costa et al 2020). Plasmid, pCS2-CD63-eGFP (Corradi, Dalla Costa et al 2020).…”

Molecular Mechanisms of Neural Circuit Development and Regeneration

“…The latter concept, of growth-regulated changes in protein synthesis and turnover, cargo translocation, and energy supply in the different compartments of a neuron—in particular in the axon and its growth cone—is also the topic of the final two articles in this Special Issue. Relatively new areas of investigation in the neuroregeneration field are presented by Corradi and Baudet [ 14 ] and Spead and Poulain [ 15 ], who review the latest insights into the role of miRNAs in regulating local translation in the presynaptic axonal compartment and into the molecular mechanisms underlying transaxonal signaling during neural circuit formation, respectively.…”