Deletion of Schizophrenia Susceptibility Gene Ulk4 Leads to Abnormal Cognitive Behaviors via Akt-GSK-3 Signaling Pathway in Mice

Abstract

Objectives: Despite of strenuous research in the past decades, the etiology of schizophrenia (SCZ) still remains incredibly controversial. Previous genetic analysis has uncovered a close association of Unc-51 like kinase 4 (ULK4), a family member of Unc-51-like serine/threonine kinase, with SCZ. However, animal behavior data which may connect Ulk4 deficiency with psychiatric disorders, particularly SCZ are still missing.

Methods: We generated Emx1-Cre:Ulk4flox/flox conditional knockout (CKO) mice, in which Ulk4 was deleted in the excitatory neurons of cerebral cortex and hippocampus.

Results: The cerebral cellular architecture was maintained but the spine density of pyramidal neurons was reduced in Ulk4 CKO mice. CKO mice showed deficits in the spatial and working memories and sensorimotor gating. Levels of p-Akt and p-GSK-3α/β were markedly reduced in the CKO mice indicating an elevation of GSK-3 signaling. Mechanistically, Ulk4 may regulate the GSK-3 signaling via putative protein complex comprising of two phosphatases, protein phosphatase 2A (PP2A) and 1α (PP1α). Indeed, the reduction of p-Akt and p-GSK-3α/β was rescued by administration of inhibitor acting on PP2A and PP1α in CKO mice.

Conclusions: Our data identified potential downstream signaling pathway of Ulk4, which plays important roles in the cognitive functions and when defective, may promote SCZ-like pathogenesis and behavioral phenotypes in mice.

Keywords: Akt; GSK-3; Unc-51-like kinase 4; cerebral cortex; schizophrenia; sensorimotor gating.

Fig. 1.

Generation and validation of Ulk4 CKO mice. (A) The expression of Ulk4 throughout the cerebral cortex (layers II/VI) and hippocampus. Scale bar = 400 μm in the left panel and 100 μm in the middle and right panels. (B) Diagram showing the genetic makeup of Ulk4 flox mice. (C) Using the cDNA as template and primer pair indicated in B, two bands (752 bp and 677 bp) were detected in Ulk4 CKO mice in the cerebral cortex and hippocampus where the cre recombinase was expressed. In the cerebellum, only the 752 bp band was amplified in both control and Ulk4 CKO mice. (D) qRT-PCR also showed that Ulk4 transcript was significantly reduced in the cerebral cortex and hippocampus but not in cerebellum of Ulk4 CKO mice. (E) The expression profile of other Unc-51-like family members (Ulk1-3 and STK36) were not changed in CKO mice. mRNA levels in CKO mice are normalized to that of control mice. N = 4 for each group; ** P < .01; one-way ANOVA with Bonferroni post hoc test. (F) No significant difference in the body weight was observed between adult CKO and control mice (4–6 month old). N = 10 for each group. Student’s t test. Ctx, cortex; Hippo, hippocampus; Cb, cerebellum; I-VI, cortical layers I-VI; CA1 and CA3, CA1-CA3 regions of hippocampus; DG, dentate gyrus.

Fig. 2.

Ulk4 CKO mice exhibited impaired spatial and working memories and defective sensorimotor gating. (A–C) In learning phase of Morris water maze (MWM) test, the Ulk4 CKO mice showed significantly longer latency to find platform than the control mice (A, two-way repeated ANOVA) and the Ulk4 CKO mice presented less platform crossing number during the memory trial (B) and longer latency to find the platform in test phase (C). N = 10 for control and N = 8 for CKO mice, ** P < .01; * P < .05. (D) The Ulk4 CKO mice showed a similar swimming velocity with control mice. N = 10 for control and N = 8 for CKO mice, Student’s t test. (E) Representative traveling trajectory of the control (top) and CKO mice (bottom) in the MWM test. (F, G) The Ulk4 CKO mice showed a reduced number of correct arm entries (F) and an increased latency to find sweet jelly when the interval last for 3 min (G). No difference was found when interval last for 1 min. N = 6 for control and N = 9 for CKO mice. Two-way repeated ANOVA, * P < .05; ** P < .01. (H, I) The Ulk4 CKO mice had a significant PPI deficit compared with control mice at three different pre-pulse intensities (H) but the startle response was comparable (I). Two-way repeated ANOVA for PPI test and Student’s t test for startle response comparison. *P < .05, ** P < .01. N = 14 for control and N = 13 for Ulk4 CKO.

Fig. 3.

Unchanged cortical layers but reduced spine density of pyramidal neurons in CKO mice. (A–E) Compared to the control mice, the CKO mice presented normal cortical lamination as shown by Nissl staining (A) and layer-specific markers, including Cux2 for layer II-IV (B), Rorβ for layer IV (C), CDP for layer II–IV (D), TLE4 for layer V–VI (D), PlxnD1 for layer V (E) in P7 mice. Scale bar = 50 μm. (F) Quantification of the thickness of the individual layers shown by Nissl staining. Student’s t test. N = 3 for each group. (G) Representative images of dendritic spines from the neurons in the layers II/III and layer V/VI in the two groups. Scale bar = 10 μm. (H) Decreased spine density was found in Ulk4 CKO mice in comparison with control mice. Student’s t test, ** P < .01, N = 34–40 in each group.

Fig. 4.

Ulk4 CKO mice presented significantly down-regulated expression of p-Akt and p-GSK-3 in the cerebral cortex and hippocampus. (A) Representative western blots for the proteins of interest in the cerebral cortex and hippocampus. (B) Quantitative data of the relative protein expression levels showed significantly decreased p-Akt, p-GSK-3α, and p-GSK-3β in the cerebral cortex (left) and hippocampus (right) of Ulk4 CKO mice. Student’s t test, *P < .05, N = 5 for each group. Ctx, cerebral cortex; Hippo, hippocampus. (C) Representative western blots for the proteins of interest in the 293T cells transfected with control plasmids and Flag-ULK4-overexpressing plasmid. (D) Quantitative data of the relative protein expression levels showed significantly increased p-Akt, p-GSK-3α and p-GSK-3β in ULK4-overexpressing group (Flag-ULK4) compared with control group. Student’s t test, *P < .05, N = 5 for each group. (E) SB-216763 restored the impaired PPI in CKO mice at pre-pulse intensity of 82dB. ** P < .01, One-way ANOVA with Bonferroni post hoc test. (F) The startle responses were not altered among the four groups. N = 6 in each group. One-way ANOVA with Bonferroni post hoc test.

Fig. 5.

Ulk4 regulated Akt-GSK-3 pathway via PP2A and PP1α. (A) The workflow of ULK4 interactor discovery. (B) The enrichment of PPP2R2A (PP2A B), PPP1CA (PP2A C) and PPP2R1A (PP1α) was confirmed by co-immunoprecipitation. (C) Representative blots for p-PP2A C subunit, PP2A C subunit, p-PP1α subunit, PP1α and PP2A B subunit (left). Quantitative analysis showed no difference between control and Ulk4 CKO mice (right). Student’s t test, N = 3 for each group. (D, E) Representative blots (left) of p-Akt, Akt, p-GSK-3α, GSK-3α, p-GSK-3β and GSK-3β for the cortices (D) and hippocampus (E) of control and Ulk4 CKO mice before and after the administration of PBS or OA. The right panel was quantitative analysis. N = 3 for each group; ** P < .01; One-way ANOVA with Bonferroni post hoc test. n.s, no significance. Ctx, cerbral cortex; Hippo, hippocampus; OA, okadaic acid. (F) A schematic diagram depicting Ulk4-involved pathways/interactions in regulating brain functions.