Autism-like behavior caused by deletion of vaccinia-related kinase 3 is improved by TrkB stimulation

Abstract

Vaccinia-related kinases (VRKs) are multifaceted serine/threonine kinases that play essential roles in various aspects of cell signaling, cell cycle progression, apoptosis, and neuronal development and differentiation. However, the neuronal function of VRK3 is still unknown despite its etiological potential in human autism spectrum disorder (ASD). Here, we report that VRK3-deficient mice exhibit typical symptoms of autism-like behavior, including hyperactivity, stereotyped behaviors, reduced social interaction, and impaired context-dependent spatial memory. A significant decrease in dendritic spine number and arborization were identified in the hippocampus CA1 of VRK3-deficient mice. These mice also exhibited a reduced rectification of AMPA receptor-mediated current and changes in expression of synaptic and signaling proteins, including tyrosine receptor kinase B (TrkB), Arc, and CaMKIIα. Notably, TrkB stimulation with 7,8-dihydroxyflavone reversed the altered synaptic structure and function and successfully restored autism-like behavior in VRK3-deficient mice. These results reveal that VRK3 plays a critical role in neurodevelopmental disorders and suggest a potential therapeutic strategy for ASD.

Figure 1.

VRK3-KO mice exhibit autism-like behavior. (A) Genotypes of VRK3-knockout (VRK3-KO) mice were determined by PCR of tail DNA, and verification of VRK3-KO mice, which contain a gene encoding β–galactosidase, was performed by Western blot analysis. (B) VRK3 mRNA level was quantified by real-time RT-PCR. VRK3 mRNA level was normalized to Rpl32 mRNA level (n = 7, 7 for WT and VRK3-KO mice, respectively; t₁₂ = 27.45, ***, P < 0.001, t test). (C) Western blot probed with an antibody directed against VRK3. Full-length VRK3 protein is absent in VRK3-KO mice. (D) The preference for either side of the three-chamber unit in the habituation period (n = 5, 5), and time spent (top) and number of entries (bottom) for the left (blank bar) and right chamber (filled bar) of the three-chamber unit. (E) Sociability (mouse 1 [blank bar] versus object [filled bar]) in a three-chamber assay, time spent investigating (left; n = 5, 5; interaction F_1,8 = 5.37, two-way ANOVA; **, P < 0.01, P > 0.05, Bonferroni’s post-test), total number of entries (middle; n = 11, 13; interaction F_1,44 = 10.81, P = 0.002, two-way ANOVA; *, P < 0.05, P > 0.05, Bonferroni’s post-test), and time spent in each chamber (right; n = 11, 13; WT vs. VRK3 KO F_1,44 = 18.36, P < 0.0001, interaction F_1,44 = 14.25, P = 0.0005, two-way ANOVA; ***, P < 0.001, P > 0.05, Bonferroni’s post-test). (F) Preference for social novelty (familiar mouse [blank bar] vs. novel mouse [filled bar] in a three-chamber assay, time spent investigating (left; n = 5, 5; WT vs. VRK3 KO F_1,8 = 7.68, *, P < 0.05, interaction F_1,8 = 5.82, *, P < 0.05, two-way ANOVA; **, P < 0.01, P > 0.05, Bonferroni’s post-test), total number of entries (middle; n = 11, 13; WT vs. VRK3 KO F_1,44 = 9.50; **, P < 0.01; two-way ANOVA; *, P < 0.05, P > 0.05, Bonferroni’s post-test), and time spent in each chamber (right; n = 11, 13; WT vs. VRK3 KO F_1,44 = 13.00, P = 0.0008, two-way ANOVA; **, P < 0.01, P > 0.05, Bonferroni’s post-test). (G) Duration of repetitive behavior in a grooming task (n = 11, 13; t₂₂ = 5.369, ***, P < 0.001, t test). (H) Latency to pup retrieval (n = 6, 7; WT vs. VRK3-KO female mice F_2,33 = 18.35, P < 0.0001, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). (I) Nest-building index over 3 d of a nesting behavior test (n = 6, 7; WT vs. VRK3 KO F_2,33 = 15.72, P < 0.0001, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). n.s., not significant. All values represent mean ± SEM.

Figure 2.

VRK3-KO mice exhibit impaired learning and memory and abnormal behavior. (A) Passive avoidance. Step-through latencies into the dark compartment before (untrained) and 2 d after foot shock (test; n = 14, 15 for WT and VRK3-KO mice, respectively; WT vs. VRK3 KO F_1,54 = 115.63, P < 0.0001, untrained vs. test F_1,54 = 88.63, P < 0.0001; interaction F_1,54 = 82.15, P < 0.0001, two-way ANOVA; P > 0.05, ***, P < 0.001, Bonferroni’s post-test). (B) The novel object recognition test with discrimination index for a familiar objects (left; F1, familiar 1; F2, familiar 2), which was presented 24 h before the test (n = 5, 5; P > 0.05, t test), and discrimination index for novel object recognition ability (right; n = 5, 5; *, P < 0.05, t test). (C) Barnes circular maze with number of errors across each day of training (left; n = 15, 15; trial sessions F_1,110 = 4.35, P < 0.05; genotype F_3,110 = 32.30, P < 0.0001, two-way ANOVA; P > 0.05, Bonferroni’s post-test). Probe test on the fifth and 12th day of training with correct pokes (middle left; n = 14, 14 for WT and VRK3-KO mice; day 5 vs. day 12, F_1,45 = 7.90, P < 0.01; WT vs. VRK3 KO, F_1,45 = 13.44, P < 0.001, two-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001, Bonferroni’s post-test), and time in target (middle right; n = 14, 14; day 5 vs. day 12, F_1,45 = 11.45, P < 0.01; WT vs VRK3 KO, F_1,45 = 26.46, P < 0.0001, two-way ANOVA; **, P < 0.01, Bonferroni’s post-test), and distance moved (right; n = 14, 14; P > 0.05, t test). (D) Total locomotor activity (left) and time spent in the center region (right) of the open field (n = 11, 14; **, P < 0.01, t test). (E) Time spent in open arms (left) and total locomotor activity (right) in the elevated plus maze (n = 12, 12; **, P < 0.01, P > 0.05, t test). (F) Duration of time spent in the light compartment in the light-dark exploration test (n = 12, 14; ***, P < 0.001, t test). n.s., not significant. All values represent mean ± SEM.

Figure 3.

Alteration of synaptic structures in CA1 and DG regions of VRK3-KO mice. (A) Representative images of dendrites in CA1 pyramidal neurons of WT and VRK3-KO mice. Bars, 10 µm. (B) Quantification of spine density (spines per 10 µm) on apical and/or basal dendrites in the CA1 region (n = 44, 114 dendrites from 6, 8 mice of WT and VRK3-KO mice, respectively; **, P < 0.01, t test). (C) Quantification of spine length on apical and/or basal dendrites in the CA1 region (n = 665, 1,621 spines from 6, 8 mice; ***, P < 0.001; **, P < 0.01, t test). (D) Dendritic spines in the CA1 region were classified as thin, stubby, mushroom, or branched. Spine length for specific spine subtypes in the CA1 region (n = 88–574 spines from 6, 8 mice; ***, P < 0.001; *, P < 0.05, t test). (E) Comparable proportions of each type of spine on apical and basal dendrites in the CA1 region of WT and VRK3-KO mice (n = 6, 8 mice; P > 0.05, χ² test). (F) Representative images of dendrites in the DG region of WT and VRK3-KO mice. Bars, 10 µm. (G) Quantification of spine density (spines per 10 µm) in the DG region (n = 37, 40 dendrites from four mice per genotype; t₇₅ = 2.326, *, P < 0.05, t test). (H) Quantification of spine length in the DG region (P > 0.05, t test). (I) Spine length for specific spine subtypes in the DG region (P > 0.05, t test). (J) Comparable proportions of each type of spine in the DG region of WT and VRK3-KO mice (n = 4, 4 mice; P > 0.05, χ² test). (K) Representative electron micrographs of hippocampal CA1 synapses showing the presence (magnification 120,000×) of PSD (arrows), synaptic vesicles (arrowheads), and dendritic spines (asterisks). Bars, 200 nm. (L) PSD length (n = 214, 321 synapses from 3, 3 mice; ***, P < 0.001, t test). (M) PSD thickness (n = 155, 236 synapses from 3, 3 mice; ***, P < 0.001, t test). (N) Reconstructions of representative neurons of Golgi-stained pyramidal neurons. Bar, 50 µm. (O) Number of intersections of the dendrite at different distances (radius) from the soma (center of analysis) from Sholl analysis (n = 5 neurons). A repeated-measures ANOVA indicates a statistical significance for genotype (F_1,56 = 5.63, P = 0.02; *, P < 0.05), but not radius (distance) genotype, interaction (F_1,56 = 0.61, P = 0.84). n.s., not significant. All values represent mean ± SEM.

Figure 4.

Altered synaptic transmission in VRK3-KO mice. (A and B) Representative traces, mean amplitude, and frequency of mEPSCs (A; n = 17, 14 neurons from 3, 3 mice of WT and VRK3-KO mice, respectively; P > 0.05, t test), and mIPSCs (B; n = 13, 14 neurons from 2, 2 mice; P > 0.05, t test). Bars, 10 pA, 1 s. (C and D) Theta-burst stimulation (TBS)–induced LTP (C; n = 12, 13 slices from 4, 4 mice; P > 0.05, t test.), and single-pulse low-frequency stimulation (SP-LFS)–induced LTD (D; n = 10, 7 slices from 4, 4 mice; P > 0.05, t test) in hippocampal SC-CA1 synapses. Traces were taken 1 min before TBS or SP-LFS (gray, light red) and at the end of the recording period (black, red). Bars, 0.5 mV, 10 ms. (E) Intrinsic excitability measured as firing rates against injected current (n = 21, 22 neurons from 3, 3 mice; P > 0.05, t test) Bars, 20 mV, 0.2 s. (F) Mean paired-pulse ratio (50-ms interstimulus interval) at SC-CA1 synapses (n = 9, 21 neurons from 3, 4 mice; t₂₈ = 0.289, P > 0.05, t test). Bars, 200 pA, 50 ms. (G) AMPA/NMDA receptor activity ratio (n = 15, 18 neurons from 4, 4 mice; P > 0.05, t test). Bars, 100 pA, 10 ms. (H) 20-Hz stimulation-induced response of hippocampal SC-CA1 synapses (n = 19, 15 neurons from 2, 2 mice; P > 0.05, t test). Bars, 100 pA, 0.2 s. (I) Current-voltage relationship of AMPA receptor/EPSCs after paired conditioning at Vh = −70, −60, −40, −20, 0, 20, and 40 mV (n = 15, 18 neurons from 4, 4 mice; **, P < 0.01, t test). Bars, 100 pA, 0.2 s. (J) The ratio of AMPA receptor/EPSC amplitudes at +40 mV and −70 mV holding is depicted (**, P < 0.01, t test). n.s., not significant. All values represent mean ± SEM.

Figure 5.

Biochemical changes in the hippocampal PSD fraction and whole lysates of VRK3-KO mice. (A) CaMKIIα, ERK1/2 (p42/44), mTOR, and S6K proteins. The level of phosphoproteins was normalized by KO/WT ratios of total proteins (n = 5–7 for WT and VRK3-KO mice; **, P < 0.01; *, P < 0.05, t test). (B) Levels of syntaxin, SynGAP, Homer1, PAK3, Arc, SAP97, and PSD-95; all proteins were normalized by α-tubulin or/and 14-3-3ζ (n = 5–7 for WT and VRK3-KO mice; **, P < 0.01; *, P < 0.05, t test). (C) Levels of mGluR1, GluN1, GluN2A, GluN2B, GluA1, GluA2, GluA3, and TrkB; all proteins were normalized by α-tubulin or/and 14-3-3ζ (n = 5–7 for WT and VRK3-KO mice; *, P < 0.05, t test). The same 14-3-3ζ loading control was used in both B and C, being the same test group. (D) Immunoblot analyses of PSD fractions and whole lysates from 12–13-wk old WT and VRK3-KO mice for the indicated proteins (left). Protein levels of GluA1, GluN1, PSD-95, phosphorylated TrkB, phosphorylated CaMKIIα, and Arc in the hippocampal whole lysates (middle) and PSD fractions (right) from VRK3-KO mice (n = 6 mice per genotype; ***, P < 0.001; **, P < 0.01; *, P < 0.05, t test). β-Actin was used as a loading control, and all values were normalized to the mean level of the respective protein in the PSD fractions or whole lysates from WT mice. n.s., not significant. All values represent mean ± SEM.

Figure 6.

TrkB stimulation restores dysregulated Arc-induced synaptic plasticity in VRK3-KO mice. (A–C) Representative Western blots (A) and quantitative plots of the time courses of 7,8-DHF–induced ERK (B) and Arc expression (C). Arc protein was normalized by 14-3-3ζ (B: n = 4/4/4/4/4 and n = 4/4/4/4/4 for vehicle/7,8-DHF(1hr)/7,8-DHF(2hr)/7,8-DHF(4hr)/7,8-DHF(8hr)-treated WT and VRK3-KO mice, respectively; *, P < 0.05, t test; C: n = 4/4/4/4/4 for WT and n = 4/4/4/4/4 for KO; **, P < 0.01; *, P < 0.05, t test; vehicle-treated VRK3 KO vs. 7,8-DHF [8 h]–treated VRK3 KO; **, P < 0.01, t test). (D–G) Representative Western blots and quantitative plots of the time course of 7,8-DHF–induced TrkB (D), PSD-95 (E), mTOR (F), and S6K (G) activation in VRK3-KO mice compared with vehicle-treated WT mice (D: n = 5/5/5/5/5 for WT and n = 5/5/5/5/5 for KO; WT vs. VRK3 KO; t₈ = 2.577; *, P < 0.05, t test; vehicle-treated VRK3 KO vs. 7,8-DHF [2 h]–treated VRK3 KO t₈ = 2.325; *, P < 0.05; E: n = 4/4/4/4/4 for WT and n = 4/4/4/4/4 for KO; P > 0.05, t test; vehicle-treated VRK3 KO vs. 7,8-DHF [2 h]–treated VRK3 KO; *, P < 0.05, t test; F: n = 4/4/4/4/4 for WT and n = 4/4/4/4/4 for KO; P = 0.11; **, P < 0.01; *, P < 0.05, t test; G: n = 4/4/4/4/4 for WT and n = 4/4/4/4/4 for KO; t₆ = 1.538, P = 0.17, t test; vehicle-treated VRK3 KO vs. 7,8-DHF [4 h]–treated VRK3 KO; *, P < 0.05, t test). Each densitometry analysis of Western blots in KO mice was normalized by calculation of relative protein levels compared with corresponding values of the vehicle-treated (1 h) and 7,8-DHF–treated (1, 2, 4, and 8 h) WT control group after normalization to 14-3-3ζ levels. The same 14-3-3ζ loading control was used in both D and E, being the same test group. All values represent mean ± SEM.

Figure 7.

Improvement of the synaptic changes in VRK3-KO mice by TrkB stimulation. (A) Immunoblot analyses of PSD fractions and whole lysates from mice at 12 wk after final chronic treatment with 7,8-DHF. Protein levels of GluA1, GluN1, PSD-95, phosphorylated TrkB, phosphorylated CaMKIIα, and Arc in the hippocampal whole lysates and PSD fractions (n = 6 mice per genotype; *, P < 0.05, t test). β-Actin was used as a loading control, and all values were normalized to the mean level of the respective protein in the PSD fractions or whole lysates from WT mice. (B) Current–voltage relationship of AMPA receptor/EPSCs after paired conditioning at V_h = −70, −60, −40, −20, 0, 20, and 40 mV (middle), and rectification index (right) with or without 7,8-DHF treatment. Bars, 100 pA, 0.2 s. The ratio of AMPA receptor/EPSC amplitudes at +40 mV and −70 mV holding is also depicted (n = 12 neurons from 3 mice for WT_vehicle; n = 13, 3 for VRK3 KO_vehicle; n = 15, 3 for WT_7,8-DHF; n = 14, 2 for VRK3 KO_7,8-DHF; **, P < 0.01 and *, P < 0.05 for WT_vehicle vs. VRK3 KO_vehicle; ^#, P < 0.05 for VRK3 KO_vehicle vs. VRK3 KO_7,8-DHF, two-way ANOVA with Horm–Sidak’s post-test). (C) Quantification of spine density (spines per 10 µm) on apical (AC) and/or basal (BS) dendrites in the CA1 region (n = 3 mice per genotype; ***, P < 0.001, t test). n.s., not significant. All values represent mean ± SEM.

Figure 8.

Recovery of social interaction deficits by acute treatment with 7,8-DHF in VRK3-KO mice. (A and B) Sociability and preference for social novelty at 1 d (test 1) after acute treatment with 7,8-DHF or vehicle; sociability with mouse 1 (blank bar) versus object (filled bar; A: n = 7, 8 and 8, 9 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,56 = 49.99, P < 0.0001, interaction F_3,56 = 3.61, P = 0.0186, two-way ANOVA; ***, P < 0.001; **, P < 0.01; Bonferroni’s post-test) and preference for social novelty with familiar mouse (blank bar) versus novel mouse (filled bar; B: WT vs. VRK3 KO F_1,56 = 22.17, P < 0.0001, interaction F_3,56 = 4.58, P = 0.0069, two-way ANOVA; ***, P < 0.001; **, P < 0.01; *, P < 0.05; Bonferroni’s post-test). (C and D) Sociability and preference for social novelty at 7 d (test 2) after acute treatment with 7,8-DHF or vehicle, sociability (C: n = 7 and 8, 9 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,42 = 10.01, P = 0.0029, interaction F_2,42 = 4.55, P = 0.0163, two-way ANOVA; ***, P < 0.001, Bonferroni’s post-test), and preference for social novelty (D: WT vs. VRK3 KO F_1,42= 4.13, P = 0.0487, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). n.s., not significant. All values represent mean ± SEM.

Figure 9.

Recovery of social interaction deficits and stereotyped behaviors by chronic treatment with 7,8-DHF in VRK3-KO mice. (A) Duration of grooming behavior 1 wk (test 1) after final chronic treatment with 7,8-DHF or vehicle in WT and VRK3-KO mice (n = 5–7 for WT and VRK3-KO mice; ***, P < 0.001; **, P < 0.01; t test). (B and C) Sociability and preference for social novelty at 1 wk (test 1) after final chronic treatment with 7,8-DHF or vehicle, sociability with mouse 1 (blank bar) versus object (filled bar; B: n = 6, 7 and 6, 9 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,48 = 31.50, P < 0.0001, interaction F_3,48 = 7.17, P = 0.0005, two-way ANOVA; ***, P < 0.001; **, P < 0.01, Bonferroni’s post-test), and preference for social novelty with familiar mouse (blank bar) versus novel mouse (filled bar; C: n = 6, 7 and 6, 9 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,48 = 25.13, P < 0.0001, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). (D and E) Sociability and preference for social novelty at 5 wk (test 2) after final chronic treatment with 7,8-DHF or vehicle, sociability (D: n = 5 and 6, 8 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,32= 16.84, P = 0.0003, two-way ANOVA; **, P < 0.01; Bonferroni’s post-test), and preference for social novelty (E: n = 5 and 6, 8 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,32= 8.89, P = 0.0055, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). (F and G) Sociability and preference for social novelty at 13 wk (Test 3) after final chronic treatment with 7,8-DHF or vehicle; sociability (F: n = 5 and 6, 8 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,32= 20.13, P < 0.0001, two-way ANOVA; **, P < 0.01; Bonferroni’s post-test), and preference for social novelty (G: n = 5 and 6, 8 for WT and VRK3-KO mice; WT vs. VRK3 KO F_1,32= 5.04, P = 0.0315, two-way ANOVA; *, P < 0.05, Bonferroni’s post-test). n.s., not significant. All values represent mean ± SEM.

Figure 10.

Acute and chronic TrkB stimulation have no effect on anxiety in VRK3-KO mice. (A) Duration of time spent (left) and total locomotor activity (right) in the open arms of the elevated plus maze 1 d (test) after acute 7,8-DHF or vehicle treatment in WT and VRK3-KO mice (n = 6, 6 and 7, 10 for WT and VRK3-KO mice). (B) Locomotor activity in the center region of the open field as shown by time spent in the center 1 d (test) after acute 7,8-DHF or vehicle treatment (n = 7, 8 and 8, 10 for WT and VRK3-KO mice). Chronic treatment with 7,8-DHF and test schemes. (C and D) Duration of time spent in the open arms of the elevated plus maze (C) and in the center region of the open field (D) at 1 wk (test 1) after final chronic treatment with 7,8-DHF or vehicle (n = 5–8 for WT and VRK3-KO mice). (E and F) Duration of time spent in the open arms of the elevated plus maze (E) and in the center region of the open field (F) at 13 wk (test 2) after chronic treatment with 7,8-DHF or vehicle (n = 6–10 for WT and VRK3-KO mice). n.s., not significant. *, P < 0.05, two-tailed t test. All values represent mean ± SEM.