Distal Axonal Proteins and Their Related MiRNAs in Cultured Cortical Neurons.

Proteins and microRNAs (miRNAs) within the axon locally regulate axonal development. However, protein profiles of distal axons of cortical neurons have not been fully investigated. In particular, networks of genes encoding axonal proteins and their related miRNAs in sub compartments of neurons such as axons remain unknown. Using embryonic cortical neurons cultured in a microfluidic device and proteomic approaches, we found that distal axons contain 883 proteins. Bioinformatics analysis revealed that 94 out of these 883 proteins are related to regulating axonal growth. Of the 94 genes encoding these proteins, there were 56 candidate genes that can be putatively targeted by axon-enriched 62 miRNAs with 8mer sites that exactly match these target genes. Among them, we validated 11 proteins and 11 miRNAs, by means of western blot and RT-PCR, respectively. Treatment of distal axons with chondroitin sulfate proteoglycans (CSPGs) that inhibit axonal growth elevated miR-133b, -203a, -29a, and -92a, which were associated with reduced protein level of AKT, MTOR, PI3K, DPYSL2, MAP1B, and PPP2CA. In contrast, reduction of miR-128, -15b, -195, -26b, -34b, -376b, and -381 by CSPGs was accompanied by increased EZR, KIF5A, DCX, GSK3B, and ROCK2 proteins. In silico pathway analysis revealed an interconnected network of these miRNAs and protein coding genes that is highly related to regulating axonal growth. Our data provide new insights into networks of miRNAs and their related proteins in distal axons in mediating axonal growth.