1. Neuroscience

Dual leucine zipper kinase is required for mechanical allodynia and microgliosis after nerve injury

  1. Josette J Wlaschin
  2. Jacob M Gluski
  3. Eileen Nguyen
  4. Hanna Silberberg
  5. James H Thompson
  6. Alexander T Chesler  Is a corresponding author
  7. Claire E Le Pichon  Is a corresponding author
  1. National Institutes of Health, United States
https://doi.org/10.7554/eLife.33910
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Cite this article
  1. Josette J Wlaschin
  2. Jacob M Gluski
  3. Eileen Nguyen
  4. Hanna Silberberg
  5. James H Thompson
  6. Alexander T Chesler
  7. Claire E Le Pichon
(2018)
Dual leucine zipper kinase is required for mechanical allodynia and microgliosis after nerve injury
eLife 7:e33910.
https://doi.org/10.7554/eLife.33910
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6 figures, 2 videos, 2 tables and 1 additional file

Figures

Figure 1 with 3 supplements
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DLK is necessary for the development of mechanical allodynia and induction of injury markers after spared nerve injury (SNI).

(A) DLK deletion is protective in the von Frey behavior assay following SNI at 1, 2, 3 and 4 weeks post injury (wpi). Baseline (BL) mechanical sensitivity thresholds are comparable between DLK WT (n …

https://doi.org/10.7554/eLife.33910.002
Figure 1—source data 1

data plotted in Figure 1A; von Frey behavior testing in DLK WT and cKO mice.

https://doi.org/10.7554/eLife.33910.006
Figure 1—source data 2

data plotted in Figure 1E; p-cJun-positive nuclear counts in ipsilateral L4 DRG 7 days after SNI in DLK WT vs cKO mice.

https://doi.org/10.7554/eLife.33910.007
Figure 1—source data 3

data plotted in Figure 1H; ATF3-positive nuclear counts in ipsilateral L4 DRG 7 days after SNI in DLK WT vs cKO mice.

https://doi.org/10.7554/eLife.33910.008
Figure 1—source data 4

data plotted in Figure 1—figure supplement 1; dynamic brush scores 7 weeks after SNI in DLK WT vs cKO mice.

https://doi.org/10.7554/eLife.33910.009
Figure 1—source data 5

data plotted in Figure 1—figure supplement 2; von Frey testing in DLK WT vs cKO mice at 1, 3, 5 and 7 days post SNI.

https://doi.org/10.7554/eLife.33910.010
Figure 1—source data 6

data plotted in Figure 1E; p-cJun-positive nuclear counts in ipsilateral L4 DRG 48 hr after hind paw formalin injection in DLK WT vs cKO mice.

https://doi.org/10.7554/eLife.33910.011
Figure 1—source data 7

data plotted in Figure 1E; ATF3-positive nuclear counts in ipsilateral L4 DRG 48 hr after hind paw formalin injection in DLK WT vs cKO mice.

https://doi.org/10.7554/eLife.33910.012
Figure 1—figure supplement 1
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DLK deletion prevents allodynia to dynamic mechanical stimulus after spared nerve injury (SNI).

Dynamic brush testing at 7 weeks post injury showing that DLK cKO mice display less nocifensive and protective behaviors compared with DLK WT littermates (n = 6 per genotype group; 3 males and three …

https://doi.org/10.7554/eLife.33910.003
Figure 1—figure supplement 2
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DLK is necessary for the development of mechanical allodynia after spared nerve injury (SNI).

Von Frey testing at acute time points 1, 3, 5, and 7 days post injury (dpi) in DLK cKO (n = 11, 5 females and 6 males) and DLK WT littermates (n = 12, 6 per sex). DLK deletion is protective in the …

https://doi.org/10.7554/eLife.33910.004
Figure 1—figure supplement 3
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DLK deletion reduces injury markers in DRG in the formalin model of neuropathic pain.

(A–C) Example images of co-immunostaining for p-c-Jun (injury marker) and NeuN (all neuronal nuclei) in a wild type DRG to illustrate the % NeuN-positive neuron counts that were done for the data …

https://doi.org/10.7554/eLife.33910.005
Figure 2 with 1 supplement
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DLK is upstream of injury-associated genesimplicated in the establishment of neuropathic pain: Atf3, Gal, Npy, and Serpina3n.

Representative images and quantification of expression of 4 genes whose expression changes following sciatic nerve axotomy: Atf3 (A–C), Gal (D–F), Npy (G–I), and Serpina3n (J–L) by in situ …

https://doi.org/10.7554/eLife.33910.014
Figure 2—source data 1

data plotted in Figure 2; quantification of in situ hybridization for Atf3, Gal, Npy and Serpina3n in DLK WT vs cKO in ipsilateral L4 DRG 7 days post SNI.

https://doi.org/10.7554/eLife.33910.016
Figure 2—source data 2

data plotted in Figure 2—figure supplement 1; quantification of in situ hybridization for Cckbr, Ecel1, Gpr151, Nts, Sox11, and Sprr1a in DLK WT vs cKO in ipsilateral L4 DRG 7 days post SNI.

https://doi.org/10.7554/eLife.33910.017
Figure 2—figure supplement 1
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DLK is upstream of multiple genes implicated in the establishment of neuropathic pain.

(A–R) Representative images and quantification of expression of 6 genes whose expression changes following sciatic nerve axotomy: Cckbr (A–C), Ecel1 (D–F), Gpr151 (G–I), Nts (J–L), Sox11 (M–O), Sprr1…

https://doi.org/10.7554/eLife.33910.015
Figure 3
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DLK is necessary for Csf1 upregulation in DRG neurons and for spinal cord microgliosis elicited by SNI.

(A–C) Representative images and quantification of Csf1 gene expression changes following SNI by in situ hybridization in DLK WT vs DLK cKO L3 DRG 7 days post SNI. Scale bar 100 µm. **p<0.01, by …

https://doi.org/10.7554/eLife.33910.018
Figure 3—source data 1

data plotted in Figure 3C; quantification of in situ hybridization for Csf1 in DLK WT vs cKO in ipsilateral L4 DRG 7 days post SNI.

https://doi.org/10.7554/eLife.33910.019
Figure 3—source data 2

data plotted in Figure 3F; quantification of Iba1-positive signal in DLK WT vs cKO dorsal spinal cord ipsilateral to injury at 8 dpi.

https://doi.org/10.7554/eLife.33910.020
Figure 3—source data 3

data plotted in Figure 3I; quantification of Iba1-positive signal in DLK WT vs cKO dorsal spinal cord contralateral to injury at 8 dpi.

https://doi.org/10.7554/eLife.33910.021
Figure 4
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Time course of DLK-dependent gene expression in DRG neurons and corresponding spinal cord microgliosis.

(A–C) Representative images and quantification of gene expression changes in ipsilateral L4 DRGs of WT mice 6, 18, 24, 48 and 72 hr post-SNT (sciatic nerve transection). (A) and (D) Representative …

https://doi.org/10.7554/eLife.33910.023
Figure 4—source data 1

data plotted in Figure 4B,C and E; quantification of time course of expression of Atf3, Csf1 and Gal in ipsilateral L4 DRGs normalized by total neuronal volume sampled as labeled by Tubb3.

https://doi.org/10.7554/eLife.33910.024
Figure 4—source data 2

data plotted in Figure 4G; quantification of Iba1-positive volume in dorsal spinal cord ipsilateral to injury at the indicated time points.

DLK cKO samples are provided for comparison.

https://doi.org/10.7554/eLife.33910.025
Figure 5
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DLK inhibition prevents DLK-dependent gene upregulation, mechanical allodynia, and microgliosis after SNI.

(A–C) GNE-3511 treatment prevents upregulation of DLK-dependent genes in the DRG after SNI. (A) Representative images of Atf3 (green), Csf1 (orange), and Tubb3 (red) in situ hybridizations in …

https://doi.org/10.7554/eLife.33910.026
Figure 5—source data 1

data plotted in Figure 5B,C; quantification of in situ signal intensity at 5 dpi for Atf3 and Csf1 in ipsilateral and contralateral L4 DRGs normalized by total neuronal volume sampled as labeled by Tubb3.

https://doi.org/10.7554/eLife.33910.027
Figure 5—source data 2

data plotted in Figure 5E; von Frey thresholds (>5 responses out of 10 stimulations) in vehicle vs GNE-3511-treated at 7 dpi.

https://doi.org/10.7554/eLife.33910.028
Figure 5—source data 3

data plotted in Figure 5H; quantification of Iba1-positive signal in vehicle vs GNE-3511-treated dorsal spinal cord ipsilateral to injury at 8 dpi.

https://doi.org/10.7554/eLife.33910.029
Figure 6
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Schematic of DLK activation in DRG neurons following spared nerve injury.

Nerve injury activates DLK (Ji and Strichartz, 2004) which induces a transcriptional response in DRG neurons (Basbaum et al., 2009), including Csf1 upregulation (von Hehn et al., 2012) which leads …

https://doi.org/10.7554/eLife.33910.031

Videos

Video 1
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untreated spinal cord, 5 dpi
https://doi.org/10.7554/eLife.33910.022
Video 2
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GNE-3511-treated spinal cord, 5 dpi
https://doi.org/10.7554/eLife.33910.030

Tables

Table 1
Top 25 genes with altered expression 7 days following sciatic nerve transection from (Guan et al., 2016)

Genes are sorted by significance, then by fold change. The genes we examined for DLK-dependence by in situ hybridization are indicated by an asterisk *

https://doi.org/10.7554/eLife.33910.013
Genelog2 fold_change
1Sprr1a *6.96879
2Npy *5.89862
3Gpr151 *5.72999
4Cckbr *5.35581
5Ecel1 *4.97284
6Atf3 *4.95333
7Gal *4.77653
8Nts *4.60215
9Hrk4.55133
10Fst4.09785
11Lmo73.76662
12Car33.48156
13Sox11 *3.37483
14Sema6a3.32589
15Mmp163.31338
16Flrt33.28661
17Loxl23.25419
18Flnc3.21128
19Sez6l3.20287
20Adam82.98222
21Stmn42.89975
22Xdh2.77339
23Gadd45a2.66746
24Sdc12.59248
25Csf1 *2.51895
Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Commercial
assay/kit- RNAscope		RNAscope Fluorescent
Multiplex Detection
Reagents		Advanced
Cell Diagnostics		ACD:320851
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Atf3; Atf3		Advanced
Cell Diagnostics		ACD:426891
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Serpina3n;
Serpina3n		Advanced
Cell Diagnostics		ACD:430191
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Csf1; Csf1		Advanced
Cell Diagnostics		ACD:315621
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Tubb3;
Tubb3		Advanced
Cell Diagnostics		ACD:423391
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Gal;
Galanin; Gal		Advanced
Cell Diagnostics		ACD:400961
Commercial
Assay/kit- RNAscope		RNAscope
Probe-Mm-Npy; Npy		Advanced
Cell Diagnostics		ACD:313321
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Cckbr; Cckbr		Advanced
Cell Diagnostics		ACD:439121
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Ecel1; Ecel1		Advanced
Cell Diagnostics		ACD:475331
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Gpr151; Gpr151		Advanced
Cell Diagnostics		ACD:317321
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Nts; Nts		Advanced
Cell Diagnostics		ACD:420441
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Sox11; Sox11		Advanced
Cell Diagnostics		ACD:440811
Commercial
assay/kit- RNAscope		RNAscope
Probe-Mm-Sprr1a; Sprr1a		Advanced
Cell Diagnostics		ACD:426871
Antibodyanti-phospho-c-Jun
S63 (rabbit polyclonal)		Cell SignalingCell Signaling:9261;
RRID:AB_2130162		1:500
Antibodyanti-NeuN
(mouse monoclonal)		MilliporeMillipore:MAB377;
RRID:AB_2298772		1:300
Antibodyanti-ATF3
(rabbit polyclonal)		Santa CruzSanta Cruz:sc-188;
RRID:AB_2258513		1:3000
Antibodyanti-Iba1
(rabbit polyclonal)		WakoWako:019–19741;
RRID:AB_2665520		1:200; 1:600
AntibodyDonkey anti-rabbit
AlexaFluor 647		ThermoFisherThermoFisher:A31573;
RRID:AB_2536183		1:500 for immunostaining;
1:600 for iDisco
AntibodyDonkey anti-rabbit
AlexaFluor 594		ThermoFisherThermoFisher:A21207;
RRID:AB_141637		1:500 for immunostaining;
1:600 for iDisco
AntibodyDonkey anti-mouse
AlexaFluor 488		ThermoFisherThermoFisher:A21202;
RRID:AB_141607		1:500 for immunostaining;
1:600 for iDisco
Strain, strain
background
(mus musculus)		Inbred strain C57BL/6J
(mus musculus)		The Jackson LaboratoryJax:000664;
RRID:IMSR_JAX:000664
Strain, strain
background
(mus musculus)		STOCK Tg(CAG-cre/Esr1*)
5Amc/J, C57BL/6J
(mus musculus)		The Jackson LaboratoryJax:004453;
RRID:IMSR_JAX:004453
Strain, strain
background
(mus musculus)		Map3k12 flox mouse,
C57BL/6J (mus musculus)		Genentech, by MTA;
PMID 24166713
Chemical compound,
drug		small molecule
GNE-3511 (DLK inhibitor)		Genentech, by MTA;
PMID 25341110
Software, algorithmFIJINIHRRID:SCR_002285
Software, algorithmarivis Vision 4DArivis

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.33910.032

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