Glycogen synthase kinase-3 inhibitors reverse deficits in long-term potentiation and cognition in fragile X mice

Abstract

Background: Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes.

Methods: We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor-dependent long-term potentiation at medial perforant path synapses onto dentate granule cells and dentate gyrus-dependent cognitive behavioral tasks.

Results: GSK3 inhibitors completely rescued deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent cognitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects.

Conclusions: These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS.

Keywords: Cognition; fragile X syndrome; glycogen synthase kinase-3; learning; long-term potentiation (LTP); synaptic plasticity.

Figure 1

Deficits in LTP at MPP-DGC synapses in FX mice are accompanied by decreased inhibitory serine-phosphorylation of GSK3β. (A) Summary plots of the magnitude of LTP induced by HFS at CA3-CA1 Schaffer collateral synapses in slices from WT (n=7) and FX (n=6) mice. (B) Summary plots of the magnitude of LTP induced by HFS at MPP-DGC synapses in slices from WT (n=6) and FX (n=9) mice. (C) Representative Western blots showing a reduction in phospho-serine9-GSK3β (p-S9 GSK3) protein in dentate gyrus (DG) but not in CA1 from FX versus WT mice. Total GSK3β protein levels are not different between groups. β-Actin and PSD-95 were used as cytosolic and synaptic protein loading controls. (D) Quantitation of the ratio of p-S9 GSK3β to total GSK3β normalized to values of WT mice (CA1, n=4, and DG, n=6, for each genotype). *p<0.05 (Student’s t test).

Figure 2

Pharmacological blockade of GSK3 reverses the deficit in LTP at MPP-DGC synapses. Summary plots of the magnitude of LTP induced by HFS at MPP-DGC synapses in slices from (A) WT mice with (n=6) and without (n=6) bath application of LiCl (20 mM), and (B) FX mice with (n=6) and without (n=9) bath application of LiCl (20 mM). (C) Data from WT mice (from A) and FX mice (bath application of LiCl from B) replotted to compare the magnitudes of LTP.(D) Summary of data A–C. (E) WT mice with (n=8) and without (n=12) bath application of CT99021 (2 uM). (F) FX mice with (n=7) and without (n=7) bath application of CT99021. (G) WT (from E) and FX (bath application of CT99021 from F) replotted to compare the magnitudes of LTP. *p<0.05 (Student’s t test). (H) Summary of data E–F

Figure 3

Pharmacological blockade of GSK3 does not alter steady-state depolarization. (A) Averaged traces during the 4th tetanus from experiments in Fig 2A–C show reduced steady-state depolarization during tetanus in FX slices. (B) Pooled data from all LTP experiments in Fig 2 show LiCl treatment reverses the deficit in steady state depolarization in FX slices. (WT: 110±12 vs FX: 72±8) (FX: 71±8 vs FX+Li: 123±23) (WT: 110±12 vs FX+Li: 123±23) *p<0.05 (Student’s t test). (C) Averaged traces during the 4th tetanus from experiments in Fig 2D–E show reduced steady-state depolarization during tetanus in FX slices. (D) Pooled data from experiments in Fig 2 show CT99021 treatment reverses the deficit in steady state depolarization in FX slices. (WT: 116±8 vs FX: 71±8) (FX: 88±13 vs FX+CT99021: 117±20)(WT: 116±8 vs FX+CT99021: 117±20) *p<0.05 (Student’s t test)

Figure 4. Inhibition of GSK3 ameliorates cognitive impairments in FX mice

FX and WT mice were treated with 5 mg/kg of TDZD-8 (TD) or VP0.7 (VP) 1 hr prior to cognitive assessments. (A,B) Performance in the novelty detection for visual objects task. (A) Times spent exploring the novel (N) and familiar (F) object. **p<0.01 compared to time spent with familiar object (Student’s t test). (B) Exploration ratio. (C) Exploration ratio in the coordinate spatial processing task. (D) Exploration ratio in the categorical spatial processing task. (E,F) Performance in the temporal order for visual objects task. (E) Times spent exploring Object 5 and Object 7 (most recently explored). **p<0.01, *p<0.05 compared to time spent with Object 7 (Student’s t test). (F) Exploration ratio. B, C, D and F: **p<0.05 compared to untreated wild-type mice; *p<0.05 compared to same genotype without treatment (Kruskall-Wallis [genotype x treatment] with Dunn’s multiple comparison test). n=10–20 mice per group.

Figure 5

Synaptic and cognitive deficits in FX mice are not altered by mGluR inhibition. Summary plots of the magnitude of LTP induced by HFS at MPP-DGC synapses in slices from (A) WT mice with (n=5) and without (n=5) bath application of MPEP (100 uM), and (B) FX mice with (n=7) and without (n=6) bath application of MPEP. (C) Data