Abstract
BackgroundRecent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells.ResultsOur analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules.ConclusionsCollectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0068-8) contains supplementary material, which is available to authorized users.
Highlights
Recent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury
One MAPKKK that emerges as a pivotal component of the mitogen-activated protein kinases (MAPKs) pathways is dual leucine zipper kinase (DLK, known as Map3k12), which was originally identified in a screen for proteins differentially expressed during the retinoic acid-induced differentiation of human NT2 teratocarcinoma cells [3]
We first silenced the expression of endogenous DLK by infecting Neuro-2a cells with lentiviral vectors expressing two different short hairpin RNAs that target mouse DLK mRNA
Summary
Recent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. The mitogen-activated protein kinases (MAPKs) are important regulators of fundamental biological processes, such as cell proliferation, differentiation, cell survival, migration and apoptosis These enzymes are activated in response to extracellular stimuli by upstream kinases termed MAPKKs, which are themselves activated by the third component of the MAPK system, the MAPKKKs [1]. It was discovered that DLK is required for axonal regeneration in adult peripheral nerves after axotomy in both vertebrate and invertebrate organisms [16, 20, 21] These findings demonstrate a key role for DLK in controlling neuronal development as well as degenerative and regenerative responses to axonal injury. JNK activation is an important and established event downstream of DLK, precisely how DLK mediates such diverse effects in neurons remains an open question
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