Research ArticleNeurodegenerative Disease

Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease

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Science Translational Medicine  16 Aug 2017:
Vol. 9, Issue 403, eaag0394
DOI: 10.1126/scitranslmed.aag0394
  • Fig. 1. Aberrant JNK pathway activation in mouse models of ALS and in spinal cord tissue from patients with sporadic ALS.

    (A) Representative images of p-c-Jun immunostaining in nonTg and SOD1G93A mouse spinal cords at 9 and 14 weeks of age. Black arrowheads highlight p-c-Jun–positive nuclei. Scale bar, 40 μm. (B) Mean number of p-c-Jun–positive cells per section in spinal cords from SOD1G93A mice at 9 and 14 weeks of age. *P < 0.05 and ***P < 0.001. (C) Representative double staining for p-c-Jun (green), NeuN (red), and DAPI (4′,6-diamidino-2-phenylindole)–positive nuclei (blue) in 14-week-old SOD1G93A mouse spinal cord tissue. White arrowhead points to a NeuN-positive motor neuron. Scale bar, 10 μm. (D) Dlk (green) expression colocalizes with Tubb3 (red) by in situ hybridization in nonTg mouse spinal cord. Scale bar, 50 μm. (E to G) p-JNK and p-c-Jun in lumbar spinal cord lysates from patients with sporadic ALS (sALS). (E) Western blot of p-JNK and p-c-Jun in spinal cord lysates from patients with sALS. (F) p-JNK (54- and 46-kDa isoforms) and p-c-Jun are elevated in spinal cord tissue from sALS patients versus healthy controls. **P = 0.0095, two-tailed Mann-Whitney test. (G) Ratios of phosphoprotein to total protein for JNK 54 kDa (JNK 54), JNK 46 kDa (JNK 46), and c-Jun are elevated in spinal cord tissue from sALS patients versus healthy controls. **P = 0.0095 and *P = 0.0190, Mann-Whitney test. (H and I) Staining for p-c-Jun in brain cortical tissue from TDP-43A315T transgenic mice, a model of ALS. (H) Representative images of p-c-Jun staining in the cortex of nonTg (left) and TDP-43A351T transgenic littermates (right). Scale bar, 100 μm. (I) Mean number of cortical p-c-Jun–positive cells in TDP-43A351T transgenic and nonTg littermates. *P < 0.05, two-tailed Student’s t test.

  • Fig. 2. Aberrant JNK pathway activity in AD mouse models and in samples from patients with AD.

    (A) Representative images of p-c-Jun staining in the ventral hippocampus of 6-month-old nonTg and PS2APP transgenic mice. Boxed regions are high-magnification insets from the subiculum. Scale bars, 40 μm. (B) Quantification of p-c-Jun staining in hippocampal tissue from PS2APP versus nonTg littermates. *P < 0.05 and **P < 0.01, two-tailed Student’s t test; n = 3 to 6 per group and age. (C) Representative images of p-c-Jun staining in dorsal hippocampus of 6-month-old nonTg and TauP301L transgenic mice. Boxed regions are high-magnification insets from the subiculum/hippocampal CA1 region of the mouse brain. Scale bars, 40 μm. (D) Number of hippocampal p-c-Jun–positive cells per square millimeter of tissue from TauP301L transgenic versus nonTg littermates. *P < 0.05, two-tailed Student’s t test; n = 4 to 6 per group and age. (E) Representative images of p-c-Jun staining in dentate gyrus of AD patient and age-matched control autopsy brain tissue. p-c-Jun quantification scores noted as (0 to 5 of 5) (see Supplementary Materials and Methods). Scale bar, 40 μm. (F) p-c-Jun scores for human hippocampal sections from age-matched controls (CTL) and patients with early-stage AD (early AD) or confirmed AD (AD). n = 7 to 10 per group. **P < 0.01, Kruskal-Wallis analysis of variance (ANOVA), followed by Dunn’s multiple-comparison test. (G) Representative Western blots from the superior frontal gyrus of controls (C), early-stage AD patients (E), and confirmed AD patients (A) for p-c-Jun, total c-Jun, p-JNK, CP13, PHF1, Tau13, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CP13 and PHF1 antibodies recognize hyperphosphorylated Tau). Asterisk symbol (*) represents an AD patient that was excluded from analysis because of the absence of Tau pathology. (H) Quantification of p-c-Jun and p-JNK, each normalized to GAPDH. **P < 0.01, ANOVA, followed by Dunnett’s test. (I) Correlation between the amount of p-c-Jun and p-JNK with PHF1. *P < 0.05 and **P < 0.01, linear regression analysis. Open circles, controls; triangles, early-stage AD; filled circles, confirmed AD.

  • Fig. 3. Dlk deletion reduces p-c-Jun in AD and ALS mouse models.

    (A) Representative staining for p-c-Jun in spinal cord ventral horn ofSOD1G93A;DLKcKO mice and nonTg controls. Scale bar, 100 μm. (B) p-c-Jun–positive cells per section in mouse L3 to L5 lumbar spinal cord by genotype. Each point represents the average value for one animal: 17 nonTg;DLKWT, 9 nonTg;DLKcKO, 16 SOD1G93A;DLKWT, and 22 SOD1G93A;DLKcKO. P values were determined by Tukey’s post hoc pairwise comparisons after ANOVA with significance for SOD1G93A genotype (F1,60 = 110.5, P < 0.0001), DLK genotype (F1,60 = 72.3, P < 0.0001), and an interaction (F1,60 = 8.3, P = 0.0055). (C) Western blot for DLK, p-MKK4, p-c-Jun, total c-Jun, and p-JNK in cortical lysates of 9-month-old PS2APP;DLKcKO and PS2APP;DLKWT mice. (D) Quantification of all markers in (C) from PS2APP;DLKcKO (n = 10) versus PS2APP;DLKWT (n = 12) mice. *P < 0.05, **P < 0.01, and ****P < 0.0001, two-tailed Student’s t test. (E) Representative p-c-Jun immunostaining in ventral hippocampus of PS2APP;DLKcKO and PS2APP;DLKWT mice. Scale bars, 40 μm. (F) Quantification of p-c-Jun–positive cells per square millimeter tissue in PS2APP;DLKcKO (n = 9) compared to PS2APP;DLKWT (n = 12) mice. (G) Western blot of DLK, p-MKK4, p-c-Jun, total c-Jun, and p-JNK in cortical lysates of 9-month-old TauP301L;DLKcKO and TauP301L;DLKWT mice. (H) Quantification of all markers in (G) from TauP301L;DLKcKO (n = 24) compared to TauP301L;DLKWT mice (n = 25). **P < 0.01 and ****P < 0.0001, two-tailed Student’s t test. (I) Representative p-c-Jun immunostaining in dorsal hippocampus of TauP301L;DLKcKO and TauP301L;DLKWT mice. Scale bars, 40 μm. (J) Mean number of p-c-Jun–positive cells per square millimeter in TauP301L;DLKcKO (n = 17) compared to TauP301L;DLKWT mice (n = 14). *P < 0.05, **P < 0.01, and ****P < 0.0001, two-tailed Student’s t test (F and I).

  • Fig. 4. Loss of DLK expression is neuroprotective in the SOD1G93A mouse model of ALS.

    (A to F) Histopathological comparisons between nonTg;DLKWT, nonTg;DLKcKO, SOD1G93A;DLKcKO, and SOD1G93A;DLKWT littermates at 14 weeks of age. (A) Representative ChAT-positive staining in mouse spinal cord ventral horn. (B) Quantification of ChAT-positive neurons. ***P < 0.001, Tukey’s test between SOD1G93A;DLKcKO and SOD1G93A;DLKWT mice. (C) Representative image of myelin staining in cross sections of mouse sciatic nerve. (D) Quantification of axonal lumen area. ***P < 0.001. (E) Iba1 immunostaining in mouse spinal cord ventral horn. (F) Quantification of Iba1-positive microglia. ****P < 0.0001, Tukey’s test between SOD1G93A;DLKcKO and SOD1G93A;DLKWT. Each data point in (B), (D), and (F) represents the average value for one animal in a cohort of nonTg;DLKWT (n = 16), nonTg;DLKcKO (n = 9), SOD1G93A;DLKWT (n = 16), and SOD1G93A;DLKcKO (n = 22) mice. Scale bars, 100 μm (A and E) and 10 μm (C). (G) Kaplan-Meier curves showing survival of SOD1G93A;DLKcKO versus SOD1G93A;DLKWT littermates (median, 162 versus 154 days): SOD1G93A;DLKcKO (red), n = 23 (with three being censored because of non-ALS death and not represented in graph); SOD1G93A;DLKWT (black), n = 21 (with none being censored). Log-rank test (*P = 0.0085) for the whole model, with a significant DLK genotype effect (**P = 0.0028); sex effect (not significant). (H) Latency to fall on the wire hang test in SOD1G93A;DLKcKO mice compared to SOD1G93A;DLKWT littermates. Repeated-measures ANOVA from 6 to 14 weeks: Significant effects for DLK genotype (P = 0.0062), SOD1G93A genotype (P < 0.0001), and sex (P = 0.0045); interaction of SOD1G93A genotype × sex (not significant). *P < 0.05, Student’s t test for individual time points comparing SOD1G93A;DLKWT andSOD1G93A;DLKcKO.

  • Fig. 5. Loss of DLK expression is neuroprotective in the PS2APP mouse model of AD.

    (A) Ex vivo imaging of spine densities (per micrometer) proximal to plaques (<100 μm) in layer II/III neurons of the somatosensory cortex of PS2APP;DLKlox;Crepos (DLKcKO, n = 7) compared to PS2APP;DLKlox;Creneg (DLKWT, n = 5) mice. Two-way ANOVA: Significant effects for DLK genotype (F1,10 = 14.0, P < 0.01), plaque location (F1,10 = 311.5, P < 0.0001), and an interaction (F1,10 = 12.76, P < 0.01). ****P < 0.0001 versus distal to plaque and ***P < 0.001 PS2APP;DLKcKO versus PS2APP;DLKWT, Sidak’s post hoc test. (B) Representative images of dendritic spines distal (left) and proximal (right) to plaques (blue) stained with Methoxy-X04. (C) Deletion of DLK at 10 weeks of age induced by tamoxifen (Tam) improved active avoidance learning in PS2APP mice when trained at 7 to 8 months of age. Percentage of active avoidance responses during behavioral training revealed an overall DLK effect (F1,36 = 13.2, P < 0.001) and DLK × block interaction (F1,36 = 11.3, P < 0.01) over the 3 days of training by two-way repeated-measures ANOVA. PS2APP;DLKcKO mice performed significantly better than PS2APP;DLKWT mice (F1,16 = 20.2, P < 0.001, two-way repeated-measures ANOVA) for 9 nonTg;DLKWT, 13 nonTg;DLKcKO, 10 PS2APP;DLKWT, and 8 PS2APP;DLKcKO mice. (D) PS2APP;DLKcKO mice (n = 13) in which DLK was deleted at 6 months of age performed significantly better in an active avoidance task than did PS2APP;DLKWT mice (n = 17) when trained at 9 to 10 months of age. Two-way repeated-measures ANOVA found a significant effect of DLKcKO (F1,28 = 9.06, P < 0.01) and a DLKcKO × block interaction (F14,392 = 4.42, P < 0.0001). (E) Representative images of cresyl violet staining in the subiculum of the hippocampus in 15-month-old TauP301L transgenic mice. (F) Quantification of subicular cell loss in TauP301L;DLKWT mice (n = 12) compared to TauP301L;DLKcKO mice (n = 13). *P = 0.0163, two-tailed Student’s t test.

  • Fig. 6. Treatment with a DLK inhibitor is neuroprotective and reverses stress-induced gene expression changes.

    (A) The amount of p-c-Jun measured by enzyme-linked immunosorbent assay in retinal lysates after optic nerve crush in wild-type C57BL/6J mice dosed with vehicle or GNE-8505 at 3, 7, or 18 mg/kg (n = 8 per group). Data are normalized to vehicle-treated samples for each time point. Symbols denote significant differences compared with the vehicle group for each time point. *P < 0.05, ^P < 0.001, #P ≤ 0.0001, Dunnett’s test. (B) The pharmacokinetic/pharmacodynamic relationship between total drug exposure and reductions in p-c-Jun measured in (A) was used to calculate an in vivo IC50 of 0.983 ± 0.106 μM (mean ± SEM). (C) Heat map of injury-induced gene expression changes in mouse retinas 72 hours after optic nerve crush injury measured by RNA-seq for vehicle-treated or GNE-3511–treated mice (75 mg/kg; dosed 60 and 68 hours after crush). Comparison of injury-induced genes that are differentially regulated by GNE-3511 (1.2-fold, P < 0.05) identified 148 up-regulated and 105 down-regulated genes. (D) Representative staining of Brn3-positive (cyan) and activated caspase 3–positive (yellow) neurons in flat-mounted retinal samples (white arrowheads). (E and F) Quantification of Brn3-positive neurons [****P < 0.0001, two-tailed Student’s t test (E)] and active caspase 3–positive neurons [**P < 0.01, two-tailed Student’s t test (F)] in mouse retinas after optic nerve crush with or without GNE-3511 treatment.

  • Fig. 7. Treatment with DLK inhibitors reduces p-c-Jun and protects against neuronal and synaptic loss in vitro and in ALS mouse models.

    (A) Quantification of p-c-Jun–positive cells/spinal cord section in SOD1G93A mice after treatment with vehicle (0 mg/kg) or GNE-3511 (37.5 or 75 mg/kg) (n = 5 per group). ANOVA: Significant treatment effect (F2,12 = 11.3, P < 0.0017). *P < 0.05 and **P < 0.01,Tukey’s test, versus vehicle (0 mg/kg). (B) Quantification of p-c-Jun–positive cells per section after treatment with vehicle (0 mg/kg) or GNE-8505 (35 mg/kg) (n = 4 per group; ***P < 0.001). (C) Representative images of each dose group shown in (A). Scale bar, 100 μm. (D) Representative images of HB9-GFP–positive motor neurons in the presence (+TF) or absence (−TF) of trophic factors or without trophic factors plus 3.34 μM GNE-3511. (E) Quantification of GFP-positive neurite area for trophic factor–deprived motor neurons exposed to increasing concentrations of GNE-3511. ANOVA: Significant treatment effect (F3,12 = 9.529, P < 0.01). **P < 0.01, Dunnett’s test, versus vehicle (0 mg/kg) dose group. (F) Quantification of α-bungarotoxin–positive neuromuscular junctions (NMJs) that were either denervated or fully innervated [as measured by proximity to vesicular acetylcholine transporter (VAChT)–positive pixels] in GNE-3511–treated (n = 14) versus vehicle-treated (n = 17) SOD1G93Amice. *P = 0.0289 and **P = 0.0094 versus control littermates. (G) Ratio of denervated to innervated neuromuscular junctions is decreased in GNE-3511–treated SOD1G93Amice. **P < 0.01. (H) Representative images of neuromuscular junction synapses from vehicle- and GNE-3511–treated SOD1G93A mice. Red, presynaptic side (VAChT); green, postsynaptic side (α-bungarotoxin). White arrows indicate innervated synapses. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/403/eaag0394/DC1

    Materials and Methods

    Fig. S1. p-c-Jun is expressed in ChAT-positive motor neurons of SOD1 mice, and Dlk is coexpressed with neuronal markers.

    Fig. S2. Elevations in p-c-Jun and c-Jun are detected in mouse models of AD.

    Fig. S3. Dlk deletion is neuroprotective by histological and behavioral measures in the SOD1 mouse model of ALS.

    Fig. S4. SOD1 transgene copy number data for the histological and survival studies shown in Fig. 4 and fig. S3.

    Fig. S5. PS2APP;DLKcKO behavior in the water maze and fear conditioning.

    Fig. S6. DLK deletion in PS2APP mice results in slight elevation of plaque-associated gliosis and hAPP expression without altering BACE1.

    Fig. S7. DLK deletion in TauP301L mice does not alter total Tau or p-Tau species.

    Fig. S8. Chemical structure, selectivity, and pharmacokinetics of GNE-8505.

    Fig. S9. qPCR of optic nerve crush mouse retinas for genes selected on the basis of the RNA-seq results.

    Fig. S10. Short-term treatment with DLK inhibitors reduces p-c-Jun and total c-Jun levels in PS2APP and TauP301L mice.

    Fig. S11. Classification of synaptic loss in the neuromuscular junction of SOD1 mice.

    Table S1. Distribution of SOD1 mice in survival study according to euthanasia criteria.

    Table S2. GNE-8505 inhibition constant in a DLK biochemical assay.

    Reference (50)

  • Supplementary Material for:

    Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease

    Claire E. Le Pichon, William J. Meilandt,* Sara Dominguez, Hilda Solanoy, Han Lin, Hai Ngu, Alvin Gogineni, Arundhati Sengupta Ghosh, Zhiyu Jiang, Seung-Hye Lee, Janice Maloney, Vineela D. Gandham, Christine D. Pozniak, Bei Wang, Sebum Lee, Michael Siu, Snahel Patel, Zora Modrusan, Xingrong Liu, York Rudhard, Miriam Baca, Amy Gustafson, Josh Kaminker, Richard A. D. Carano, Eric J. Huang, Oded Foreman, Robby Weimer, Kimberly Scearce-Levie, Joseph W. Lewcock*

    *Corresponding author. Email: lewcock{at}dnli.com (J.W.L.); meilandt.william{at}gene.com (W.J.M.)

    Published 16 August 2017, Sci. Transl. Med. 9, eaag0394 (2017)
    DOI: 10.1126/scitranslmed.aag0394

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. p-c-Jun is expressed in ChAT-positive motor neurons of SOD1 mice, and Dlk is coexpressed with neuronal markers.
    • Fig. S2. Elevations in p-c-Jun and c-Jun are detected in mouse models of AD.
    • Fig. S3. Dlk deletion is neuroprotective by histological and behavioral measures in the SOD1 mouse model of ALS.
    • Fig. S4. SOD1 transgene copy number data for the histological and survival studies shown in Fig. 4 and fig. S3.
    • Fig. S5. PS2APP;DLKcKO behavior in the water maze and fear conditioning.
    • Fig. S6. DLK deletion in PS2APP mice results in slight elevation of plaque-associated gliosis and hAPP expression without altering BACE1.
    • Fig. S7. DLK deletion in TauP301L mice does not alter total Tau or p-Tau species.
    • Fig. S8. Chemical structure, selectivity, and pharmacokinetics of GNE-8505.
    • Fig. S9. qPCR of optic nerve crush mouse retinas for genes selected on the basis of the RNA-seq results.
    • Fig. S10. Short-term treatment with DLK inhibitors reduces p-c-Jun and total c-Jun levels in PS2APP and TauP301L mice.
    • Fig. S11. Classification of synaptic loss in the neuromuscular junction of SOD1 mice.
    • Table S1. Distribution of SOD1 mice in survival study according to euthanasia criteria.
    • Table S2. GNE-8505 inhibition constant in a DLK biochemical assay.
    • Reference (50)

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