Table 1.
qPCR primers used in this study.
Fig 1.
Treatment with low dose nocodazole activates DLK dependent signaling, resulting in transcriptional changes.
A) Immunoblot for phospho-MKK4, MKK4 and HSP90. Cultured DRGs were pretreated with DMSO or 500nM DLKi (GNE-3511) and subsequently treated with 200nM nocodazole or DMSO. Samples were collected at 4 and 16 Hours. Non-specific band in p-MKK4 blot is routinely noted in preparations of DRGs. B) Quantification of immunoblot. A two-way ANOVA revealed there was a statistically significant interaction on the ratio of pMKK4/MKK4 between the effect of DLKi and low dose nocodazole treatment (Df = 16, F = 9.329, p = 0.008). C) Immunoblot for phospho-Jun, cJun and HSP90. D) Quantification of immunoblot. A two-way ANOVA revealed there was a statistically significant interaction expression of cJun between the effect of DLKi and low dose nocodazole treatment (Df = 14, F = 17.04, p = 0.001). E) Representative images of cultured neuron axons after 16 hours of treatment. Lamellipodia structures are noted with an arrow; short branching structures are noted with a triple arrow and axonal swellings are noted with diamonds. F) Axons stained for Tuj1 after 16 hours of treatment. G) Normalized microtubule fragmentation after 16 hours of treatment. A two-way ANOVA revealed a statistically significant interaction between the effect of DLKi and low dose nocodazole treatment (Df = 153, F = 24.16, p = 2.26 x 10–6).
Fig 2.
DLK inhibition on low dose nocodazole treatment impacts gene expression.
A) Experimental design for RNA sequencing with two factors, pretreatment (DMSO, DLKi) and treatment (DMSO, Noc). B) Principal Component Analysis (PCA) of RNA sequencing experience for four conditions (DMSO, DMSO; DMSO, Noc; DLKi, DMSO; DLKi, Noc) with four replicates. C) Differential gene expression in cultured DRGs of DLKi in low dose nocodazole. Log2FoldChange was determined in a two factor with interaction design in DESeqEQ2. Log2FoldChange was plotted with regard to base mean gene expression. Large changes in transcription factors (Egr1, Jun and Atf3), DUSP and MAPK cascade genes are noted in red, blue and yellow. MAPK cascade (GO:0000165) is significantly enriched in genes with a greater than 1 log2FoldChange (p = 1.6 x 10–9). Genes with the greatest log2FoldChange (n = 500) were selected for further analysis using WebGestalt for Gene Set Enrichment. D) Biological Process enrichment in response to ER stress (value) and negative regulation of phosphorylation (value). Nocodazole treatment increases gene expression related to Stress Response and Signaling Regulation (ER stress (GO:0034976, NES = -1.9892, p = 0.00147, FDR = 0.09715) and Negative Regulation of Phosphorylation (GO:0042326, NES = -1.9969, p = 0.0037406, FDR = 0.12066)). Conversely, nocodazole treatment decreases expression of biological processes specific to mature neuronal behaviors including Pain Perception, Behavior Regulation and Feeding Behavior (Sensory Perception of Pain (GO:0019233, NES = 2.3965, p = <2.2e-16, FDR = 0.008895), Regulation of Behavior (GO:0050795, NES = 2.29, p = <2.2e-16, FDR = 0.01129), Neurotransmitter Transport (GO:0006836, NES = 2.3623, p = <2.2e-16, FDR = 0.00975), G-protein Coupled Receptor Signaling (GO:0007187, NES = 2.6953, p = <2.2e-16, FDR = <2.2e-16) and Feeding Behavior (GO:0007631, NES = 2.772, p<2.2e-16, FDR<2.2e-16)). E) Cell compartment enrichment in cell cortex (GO:0005938, FDR 0.28243, p = 0.0057389), neuron projection terminus (GO:0044306, FDR 0.46809, p = 0.006) F) Molecular function enrichment in DNA-binding Transcription Activator Activity increased after nocodazole treatment in a DLK-dependent manner (NES: -2.0360, FDR: 0.043388, GO:0001228, p = 0.003). Conversely, Peptide Receptor, Neurotransmitter, and G-protein Coupled Receptor Activity (peptide receptor activity (NES: 2.5456, FDR: 0.00084809, GO:0001653, p < 2.2e-16), and G protein-Coupled Receptor Binding (NES: 2.0976, FDR: 0.024595, GO:0001664, p = 0.0056338) had positive enrichment scores.
Fig 3.
Treatment with low dose nocodazole increases expression of cytoskeleton organization genes.
A) KEGG diagram for focal adhesion NES = -2.4 (mmu04510, FDR = 0.0026919, p = <2.2e-16). Genes with a log2FoldChange > -0.5 are noted in red. B) Differential gene expression of DLK inhibitor in low dose nocodazole in cultured DRGs, actin cytoskeleton organization (GO:0030036, padj = 1.979×10−7) noted in red, and cytoskeleton organization (GO:0007010, padj = 5.452×10−5) noted in blue. Cytoskeletal organization genes with a log2FoldChange greater than 2 noted. C) Differential expression of select genes (Stmn4, Zyx, Itga9) was confirmed by qPCR. Expression was normalized to Reference Genes (Ubc, Rpl27). There was a statistically significant interaction between low dose nocodazole treatment and DLK inhibitor on gene expression for Stmn4 (F = 19.45, p = 0.000851), Zyx (F = 4.580, p = 0.05190) and itga9 (F = 9.053, p = 0.0101).
Fig 4.
Low dose nocodazole increased filopodia along the axon in a DLK-dependent manner.
A) Cultured DRG neurons axons expressing GFP were stained for phalloidin after 16 hours of treatment with either low dose nocodazole or DMSO (control). Neurons were pretreated with either DLKi or DMSO. B) Differences in growth cone morphology were observed at 16 hours after treatment C) We further analyzed the number of filopodia along branches and found a significant increase in short branches (less than 10 μm) in neurons treated with DMSO+Noc (27.5 +/-10.1) compared to DMSO+DMSO treated neurons (4.9 +/- 2.1), DLKi+DMSO (1.4 +/- 1.35) DLKi+Noc (3.2 +/- 2.0). A Two-way ANOVA confirmed that the presence of actin rich branches in low dose nocodazole treated neurons was DLK dependent (Df = 35, F = 15.35, p = 0.0004). D) Normalized Growth Cone area was significantly increased after low dose nocodazole treatment. A two-way ANOVA demonstrated increased growth cone area in Noc (26.5% +/- 8.6%) compared to DMSO treated neurons (14.8% +/- 5.4%), DLKi (5.7% +/- 1.5%) DLKiNoc (5.6% +/- 1.5%). (Df = 76, F = 8.0, p = 0.006).
Fig 5.
Conservation of DLK-dependent injury gene expression after low dose nocodazole treatment and NGF deprivation.
Linear models were used to analyze the similarities between the different treatments. Correlation between log2FoldChange of differential gene expression of DRGs with respect to the effect of DLKi in low dose nocodazole and (A) log2FoldChange of DLKi vs NGF deprivation (correlation p-value < 1.2 e-12). (B,C) Transcriptional comparison of differential expression in Low Dose Nocodazole and NGF deprivation paradigms, genes differentially expressed in both data sets marked in blue. Volcano plot of -log10(p-adjusted) v log2Fold Change for (C) Low Dose Nocodazole and (D) NGF Deprivation. Genes differentially expressed in both data sets with greater than 2 log2FoldChange are noted. (D,E) Actin Filament based processes (GO:0030029) are enriched in differentially expressed genes sets for Low Dose Noc (p = 1.626×10−8) and NGF Deprivation (p = 8.656×10−11). Genes belonging to GO: Actin filament-based process (GO:0030029) displayed in red. Actin Cytoskeleton Organization genes (GO:0030029) with absolute log2Fold Change > 1 present in both datasets are noted (Zyx, Gab1, Fmnl1, Tacr1, Rhpn2).