Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells
© The Author(s). 2016
Received: 22 February 2016
Accepted: 25 July 2016
Published: 28 July 2016
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. 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.
Our 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.
Collectively, 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.
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 . Based on their structural and biochemical features, three major subgroups of MAPKs have been described in mammals, including extracellular signal-regulated kinases (ERKs), p38 kinases, and c-Jun N-terminal kinases (JNKs) [1, 2].
One MAPKKK that emerges as a pivotal component of the MAPK pathways is dual leucine zipper kinase (DLK, also 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 . DLK preferentially activates JNK, although a role for DLK in activation of ERK and p38 MAPKs has also been proposed [4–6]. Based on Northern blot analysis, in situ hybridization and immunolocalization, DLK has a tissue-specific expression pattern in both mouse and human, being mainly expressed in brain, kidney and skin [3, 7–10]. Previous studies have suggested a fundamental role for DLK in vivo since targeted deletion of the DLK gene in mice results in perinatal death . Embryos lacking DLK display abnormal brain development, characterized by defects in axon growth, neuron migration, apoptosis and axon degeneration [11–15]. Apart from its role during development, DLK has also been shown to regulate axonal damage signaling in mature neurons [13, 15–17]. For instance, as demonstrated by studies in mice and rats, loss of DLK protects neurons from somal and axonal degeneration in response to mechanical injury, growth factor deprivation and glutamate-induced excitotoxicity [16–19]. Recently, it was also 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. Although JNK activation is an important and established event downstream of DLK, precisely how DLK mediates such diverse effects in neurons remains an open question.
Because one way to unravel the mode of action of DLK is to identify genes critical for its function in neurons, we characterized by next-generation sequencing (RNA-seq) the transcriptome of differentiated Neuro-2a neuroblastoma cells in which DLK has been depleted by RNA interference. Our results led to the identification of many genes whose expression was significantly altered upon DLK knockdown. Notably, among the identified genes, we focused on those encoding axon guidance molecules due to their crucial roles in many aspects of neuronal development, including axon pathfinding, axon growth, neuronal polarization, neuronal migration and dendrite formation, as well as in axon regeneration in the adult nervous system [22–25].
The polyclonal antiserum used for detection of DLK was described previously . The polyclonal or monoclonal antibodies against phospho-JNK (Thr183/Tyr185, #4671), JNK (#9252), phospho-c-Jun (Ser63, #9261), Nrp1 (#3725) and Plxna4 (#3816) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). The polyclonal antibody against γ-actin (#NB600-533) was from Novus Biologicals (Oakville, Ontario).
Mouse Neuro-2a neuroblastoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. When indicated, cells were differentiated by incubation in DMEM containing 0.1 % bovine serum albumin for 24 h.
Lentivirus production and infection of Neuro-2a cells
HEK 293T cells grown in DMEM supplemented with 10 % (v/v) FBS and antibiotics were cotransfected with the envelope protein expressing vector pMD2.G and the packaging protein expressing vector psPAX2, (kindly provided by Dr. Didier Trono University of Geneva Medical School, Geneva, Switzerland) and with either the transfer pLKO.1 empty lentiviral vector  (Addgene, Cambridge, MA, USA, plasmid 8453) or the pLKO.1-based lentiviral mouse DLK shRNA vector (clone TRCN0000022573 [sh73] or clone TRCN0000022569 [sh69], Open Biosystems, Huntsville, AL, USA) using Polyethylenimine Max (#24765, Polysciences Warrington, PA). At 72 h post-transfection, the culture medium containing lentiviruses was harvested, filtered through 0.45-μm filter, and used for infection. Neuro-2a cells, seeded at a density of 0.3 × 106 cells per well in six-well dishes, 0.5 × 106 cells in 60-mm dishes or 2.0 × 106 cells in 100-mm dishes 24 h before, were infected with viral supernatants supplemented with 8 μg/ml polybrene. Two days later, infected cells were treated with puromycin (2.5 μg/ml) and selected for 2 days, after which they were induced to differentiate as mentioned above.
Total RNA was extracted with the Direct-zol RNA MiniPrep kit (#R2050, Zymo Research) in combination with TRIzol (#15596-026, Life Technologies), following the manufacturer’s protocol. A 30 min on-column DNase treatment was performed before elution according to manufacturer’s instructions. RNA was quantified on a NanoDrop (Thermo Scientific) spectrophotometer. Total RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Reverse transcription was performed on 2.2 μg total RNA with Transcriptor reverse transcriptase, random hexamers, dNTPs (Roche Diagnostics), and 10 units of RNAseOUT (Invitrogen) following the manufacturer’s protocol in a total volume of 20 μl. All forward and reverse primers were individually resuspended to 20–100 μM stock solution in Tris-EDTA buffer (IDT) and diluted as a primer pair to 1 μM in RNase DNase-free water (IDT). Quantitative PCR (qPCR) reactions were performed in 10 μl in 96 well plates on a CFX-96 thermocycler (BioRad) with 5 μL of 2X iTaq Universal SYBR Green Supermix (BioRad), 10 ng (3 μl) cDNA, and 200 nM final (2 μl) primer pair solutions. The following cycling conditions were used: 3 min at 95 °C; 50 cycles: 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C. Relative expression levels were calculated using the qBASE framework  and the housekeeping genes Sdha, Txnl4b and Pum1 for mouse cDNA. Primer design and validation was evaluated as described elsewhere . In every qPCR run, a no-template control was performed for each primer pair and these were consistently negative. All primer sequences are available in Additional file 1: Table S1.
Imaging and quantification
Images of control and DLK-depleted Neuro-2a cells were acquired in bright-field using a Zeiss AxioObserver.Z1 inverted microscope equipped with a 20x/0.30 NA objective and a Zeiss Axiocam 506 camera. The length of approximately 200 neurites was measured from five random microscope fields per sample using the open source software Fiji  and the Simple Neurite Tracer plugin . GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA) was used to calculate and plot mean and standard error of the mean (SEM) of the measured neurite lengths. Neurites were also classified into five groups based on their length, and their frequency within each group was calculated for both the control and DLK-depleted cells. The mean ± SEM of the frequency of distribution within each group of neurites was determined using GraphPad Prism and presented in percentage. Significance of the results was assessed by Student’s t test.
RNA-seq sample preparation and analysis
Total RNA was isolated as described above from control, sh73/DLK- and sh69/DLK-depleted cells. The quality of total RNA was evaluated using the Agilent 2100 BioAnalyzer and the Agilent RNA 6000 Nano Kit (#5067-1511) according to the manufacturer’s instructions. For all samples, RNA integrity numbers were sufficiently high (>9.5) to perform mRNA sequencing. mRNA was purified from 5 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (#E7490, New England Biolabs, Whitby, Ontario) following manufacturer’s instructions. cDNA libraries were then prepared with 25 ng mRNA of each sample using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (#E7420, New England Biolabs) and barcoded by PCR for subsequent multiplexed sequencing. The quality and quantity of the librairies were assessed using the Agilent High Sensitivity DNA Kit (#5067-4626) on Agilent 2100 BioAnalyzer. Sequencing of the multiplexed cDNA libraries was performed on an Illumina HiSeq 2500 system (Illumina, San Diego, CA, USA) in two lanes at the Institut de Recherches Cliniques de Montréal (IRCM). The obtained raw reads were processed using the McGill University and Génome Québec Innovation Centre (MUGQIC) RNA-seq pipeline version 1.4 (https://bitbucket.org/mugqic/mugqic_pipelines). Briefly, the reads were trimmed using Trimmomatic version 0.32  to remove adapter sequences and low quality reads (Phred quality < 30 and minimum lenght of 32), and aligned onto the mouse reference genome (mm10 assembly) using TopHat version 2.0.11 and Bowtie version 2.2.2 [33, 34]. Read counts for each gene from the GRCm38.73 mouse assembly from the Ensembl database  were obtained using HTSeq version 0.6.1 . Differential gene expression analyses between control and DLK-depleted cells were performed using DESeq version 1.16  and edgeR version 3.6.8  from the Bioconductor package version 2.14 [39, 40] of R version 3.1.1. The identified genes were further filtered against the following criteria: (i) a minimum of 50 reads per gene, (ii) a fold-change threshold ≥ 2 between control and DLK-depleted cells, and (iii) transcripts altered in the same direction with both shDLK constructs and common to at least three of the four samples of DLK-depleted cells. Functional category and pathway analyses of the differentially expressed genes were performed using DAVID .
qRT-PCR and immunoblot data represent the mean ± SEM of at least three independently performed experiments. The statistical significance between mean values was determined by unpaired t test with Welch’s correction (two tails) using GraphPad Prism software. p-values of < 0.05 were considered to be statistically signifiant.
Depletion of DLK by RNA interference in Neuro-2a cells
Since DLK is an upstream activator of the JNK signaling pathway , we tested in parallel whether its knockdown by RNA interference in Neuro-2a cells would perturb the activity of JNK and c-Jun, a downstream target. The impact of DLK depletion on JNK signaling was assayed by Western blotting with antibodies specific to the phosphorylated, activated forms of JNK and c-Jun. Interestingly, loss of DLK attenuated by 70–95 % basal JNK or c-Jun activity (Fig. 1b and c), a result reminiscent of the effect of DLK gene disruption in mouse brain . Thus, these results indicate that DLK is required for basal activity of JNK and c-Jun in differentiated Neuro-2a cells.
Effects of DLK depletion on neurite outgrowth in Neuro-2a cells
RNA-seq analysis of differentially expressed genes in DLK-depleted Neuro-2a cells
The RNA-seq data were subsequently analyzed using the HTSeq , DESeq  and edgeR  softwares to identify differentially expressed genes (DEGs) between control and DLK-depleted cells. As an additional filter on these data, we excluded from the analyses all genes with less than 50 reads on average per condition and with less than two-fold changes in expression as compared to control. To further narrow our candidate gene list, we then focused on up-regulated and down-regulated genes common to at least three of the four samples of sh73/DLK- and sh69/DLK-depleted cells (Fig. 3b). According to these parameters, 104 genes were found to be up-regulated after DLK depletion compared to control cells (Additional file 3: Table S3), whereas 63 were down-regulated (Additional file 4: Table S4).
SP-PIR keyword terms associated with dysregulated genes in DLK-depleted cells
Gene ontology terms associated with dysregulated genes in DLK-depleted cells
Protein amino acid phosphorylation
Enzyme linked receptor protein signaling pathway
Anatomical structure development
Extracellular structure organization
Phosphorus metabolic process
Phosphate metabolic process
KEGG pathway terms associated with dysregulated genes in DLK-depleted cells
Epha7, Rnd1, Nrp1, Plxna4, Sema6b, Unc5a
Itgb3, Itgb4, Lama2, Tnr, Thbs3
Hgf, Itgb3, Itgb4, Lama2, Pdgfra, Tnr, Thbs3
Arrhythmogenic right ventricular cardiomyopathy (ARVC)
Des, Itgb3, Itgb4, Lama2
Hypertrophic cardiomyopathy (HCM)
Des, Itgb3, Itgb4, Lama2
Fold change of the axon guidance genes dysregulated in DLK-depleted cells relative to control cells
Ensembl gene id
sh73 no. 1
sh73 no. 2
sh69 no. 1
sh69 no. 2
Eph receptor A7
Rho family GTPase 1
Unc-5 homolog A
Validation of RNA-seq data by qRT-PCR analysis and immunoblotting
As a complementary approach, we also analyzed whether the differences in transcript abundance between control and DLK-depleted cells translated into protein level changes for two of the identified axon guidance genes, namely Plxna4 and Nrp1. These two genes were of particular interest because they encode cell-surface receptors that act in complex to mediate repulsive responses of axons to class-3 semaphorins in most neurons . Moreover, they have been recognized as important regulators of neuronal migration , a process impaired in DLK knockout mouse brain . In accordance with RNA-seq and qRT-PCR data, immunoblot analysis showed that protein levels of Plxna4 and Nrp1 were significantly increased by 1.5- to 2-fold in Neuro-2a cells after DLK depletion compared with control (Fig. 5b and c). Taken together, these experiments confirm that DLK depletion has an effect on Plxna4 and Nrp1 expression in neuronal cells at both the transcriptional and translational levels, and shed light for the first time on the mechanisms by which DLK signalling contributes to nervous system development and function.
Recent exciting findings based on experiments carried out in vivo and in vitro have highlighted the potential role of DLK in nervous system assembly, maintenance and repair. These studies have indeed demonstrated the requirement for DLK in various aspects of neuronal cell physiology, ranging from migration, axon growth and apoptosis during development to nerve regeneration and degeneration in the adult (for a review, see ). One of the fundamental issues remaining to be solved in this context is elucidation of the mechanisms by which DLK regulates such a vast range of biological responses. In an attempt to gain insight into DLK’s mode of action, we have examined the global changes that occur in the transcriptome of differentiated Neuro-2a cells after knockdown of DLK by RNA interference. Our results demonstrate that DLK depletion leads to a decrease or an increase in the levels of numerous mRNAs, indicating that DLK contributes to both positive and negative regulation of gene expression even under basal conditions. At this point, it is not known whether these changes in gene expression are a cause or a consequence of DLK depletion. However, we assume that such a response is probably JNK-dependent because knockdown of DLK in Neuro-2a cells impairs JNK basal activity and JNK-mediated phosphorylation of c-Jun, a component of the AP-1 transcription factor (Fig. 1). This assumption is also supported by the studies of Hirai et al. (2006) who showed that JNK activity and phosphorylation of JNK substrates, including c-Jun, are significantly reduced in DLK−/− mouse embryonic brain. Previous reports found that JNK both positively and negatively regulates gene expression by phosphorylating DNA-bound proteins, such as transcription factors and histones, as well as by binding directly to transcriptionally active gene promoters [48, 49].
Interestingly, decrease of DLK expression in Neuro-2a cells resulted in transcriptional dysregulation of a subset of genes involved in neuronal function, such as migration, differentiation, axonogenesis, and axon guidance, an observation that supports its critical role in nervous system development (Additional file 3: Table S3, Additional file 4: Table S4). To our knowledge, none of these genes have been previously identified as potential targets of DLK. In a recent work using a microarray approach, Watkins et al. (2013) showed that DLK is required for expression of proapoptotic- and regeneration-related genes in retinal ganglion cells (RGCs) after optic nerve crush. Consistent with this finding, it has also been reported that RGCs isolated from mice containing a floxed allele of DLK displayed resistance to death induced by axonal injury, together with a concomitant decrease in JNK phosphorylation and c-Jun expression . The basis for the difference in target gene specificity between Neuro-2a cells and RGCs is unclear, but could reflect cell type or cell context variability. Another possible explanation might be that the DLK-dependent transcriptional program varies between basal and stress-stimulated conditions, which inevitably lead to different cellular responses.
As stated above, the current results highlight for the first time the involvement of DLK in expression of axon guidance genes. This family of genes encodes proteins that act as attractants or repellents for axons, thereby guiding them towards or away from a specific region [23, 50]. The growth cone, a sensory structure located at the tip of extending axons, expresses receptors that recognize these guidance cues and trigger intracellular signaling cascades, resulting in extensive cytoskeletal rearrangement and subsequent axon steering . Different families of axon guidance cues and receptors have been identified, including ephrins and Eph receptors, semaphorins and plexin and neuropilin receptors, Slits and Robo receptors, Netrins, DCC and Unc5 receptors as well as RGM and noegenin receptors . Axon guidance proteins have been shown to be involved in various aspects of neural circuit development (e.g., growth, guidance, bundling, fasciculation and pruning of axons, and synaptogenesis), and in the control of synaptic plasticity in adults . Emerging data from human and animal models also implicate axon guidance molecules in neurological disorders and axon regeneration after injury [24, 53–55]. In our RNA-seq data, four axon guidance genes, including Epha7, Nrp1, Plxna4 and Unc5a, showed an increase in expression level after DLK depletion, whereas two, Rnd1 and Sema6b, were down-regulated (Additional file 3: Table S3 and Additional file 4: Table S4). qRT-PCR confirmed that these mRNA changes were significant for Epha7, Nrp1, Plxna4, Rnd1 and Sema6b (Fig. 5). Because there was no correlation between the RNA-seq and qRT-PCR results for Unc5a, we currently cannot draw any conclusion for this gene. A part of our findings was further validated by Western blot analysis, which shows that Nrp1 and Plxna4 protein levels were significantly induced in DLK-depleted cells (Fig. 5), thus demonstrating the ability of DLK to exert a repressing effect on Nrp1 and Plxna4 expression. Because Nrp1 and Plxna4 cooperate to mediate signaling in response to semaphorin 3A (Sema3A), a repulsive guidance cue for axons in most neurons , it is tempting to speculate that an increase in their expression, such as the one described here, could contribute to the defects of axonal growth previously described in DLK null mouse embryos and DLK-deficient cultured cortical neurons [11, 44]. This hypothesis is fully in line with results demonstrating that ectopic expression of Nrp1 or Plxna4 in neuronal cell types that are insensitive to Sema3a caused axon repulsion and growth cone collapse in response to Sema3A [45, 56]. The identification of Epha7 and Rnd1 as up- and down-regulated genes, respectively, in DLK-depleted cells is also of great interest in regard to the contribution of DLK to neuron projection development. Indeed, a recent study revealed that Epha7, which generally transduces repulsive signals , impairs dendrite formation when overexpressed in cortical neurons , while knockdown of Rnd1 in hippocampal neurons suppressed axon extension . Finally, for Sema6b, whose expression decreased upon DLK knockdown in Neuro-2a cells, results obtained from loss-of-function experiments demonstrated its requirement for proper projection of hippocampal granule cell axons . Taken together, these data are supportive of a model whereby DLK promotes axonal growth during neural development by regulating, at least in part, the expression of axon guidance genes. It is presently not known how DLK would modulate axon guidance molecule abundance in neurons, but one possible mechanism is control of synthesis, phosphorylation and/or activity of the transcription factors that regulate them. Work carried out over the past several years has established that transcriptional control of axon guidance cues and receptors is crucial to allow precise pathfinding decisions of neuronal growth cones [61, 62].
Although further work will be required to investigate the biological consequences of dysregulated axon guidance gene expression in DLK-depleted Neuro-2a cells, we anticipate as a potential outcome an alteration of the cytoskeleton. This prediction is supported by the fact that axon guidance cues and their receptors modulate actin and microtubule dynamics in the growth cone via activation of downstream signaling molecules, such as protein kinases, small GTPases and cytoskeleton-associated proteins [50, 63, 64]. As illustrated by studies in different systems, remodeling of the cytoskeleton is critically important for attraction, repulsion, growth cone collapse or axon extension . Interestingly, a functional link between DLK and cytoskeletal regulation has been recently suggested. In this study, Hirai et al. (2011) demonstrated by RNA interference that knockdown of DLK in cultured mouse embryonic neurons caused defects in axon formation, whereas concomitant treatment of cells with the microtubule-stabilizing drug taxol antagonized this response. These results implicate DLK as a key regulator of microtubule stabilization, a required event for axon formation [65, 66]. Consistent with an active role for DLK in microtubule dynamics, DLK gene disruption in mice resulted in reduced phosphorylation of the microtubule-stabilizing proteins Doublecortin and MAP2c . In light of these data and the results from the present study, it is conceivable to suggest that DLK regulation of axon growth in neurons may depend, at least in part, on cytoskeletal reorganization mediated either directly via phosphorylation of microtubule-associated proteins or indirectly through modulation of axon guidance gene expression.
We found that DLK plays an important role in regulating expression of genes recognized for their contribution in nervous system development and function. Future studies will be dedicated to understand how DLK influences the expression of Nrp1, Plxna4, Epha7, Rnd1 and Sema6b as well as the consequences of their up- or down-regulation on axon growth. Further experiments using the DLK knockout mice will also be required to assess the in vivo relevance of our in vitro findings.
DLK, dual leucine zipper kinase; Epha7, eph receptor A7; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; Nrp1, neuropilin 1; Plxna4, plexin A4; Rnd1, Rho family GTPase 1; Sema6b, semaphorin 6B; Unc5a, Unc-5 homolog A
We thank Dr. Alain Lavigueur for critical reading of the manuscript, Daniel Garneau for his indispensable help in microscopy and image processing, and the RNomics platform of the Université de Sherbrooke for the qRT-PCR analyses.
This work was made possible by grants from the Natural Sciences and Engineering Research Council of Canada (138068–2012) and the Université de Sherbrooke, and in part by support from Calcul Québec and Compute Canada.
Availability of data and material
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
AB, RB, JFL, DM, SR and PEJ designed the study. AB, JFL, LD and RB performed the experiments and analyzed the data. AB and RB supervised the experiments and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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