Gene deficiency and pharmacological inhibition of caspase-1 confers resilience to chronic social defeat stress via regulating the stability of surface AMPARs

Abstract

Both inflammatory processes and glutamatergic systems have been implicated in the pathophysiology of mood-related disorders. However, the role of caspase-1, a classic inflammatory caspase, in behavioral responses to chronic stress remains largely unknown. To address this issue, we examined the effects and underlying mechanisms of caspase-1 on preclinical murine models of depression. We found that loss of caspase-1 expression in Caspase-1^-/- knockout mice alleviated chronic stress-induced depression-like behaviors, whereas overexpression of caspase-1 in the hippocampus of wild-type (WT) mice was sufficient to induce depression- and anxiety-like behaviors. Furthermore, chronic stress reduced glutamatergic neurotransmission and decreased surface expression of glutamate receptors in hippocampal pyramidal neurons of WT mice, but not Caspase-1^-/- mice. Importantly, pharmacological inhibition of caspase-1-interleukin-1β (IL-1β) signaling pathway prevented the depression-like behaviors and the decrease in surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in stressed WT mice. Finally, the effects of chronic stress on both depression- and anxiety-like behaviors can be mimicked by exogenous intracerebroventricular (i.c.v.) administration of IL-1β in both WT and Caspase-1^-/- mice. Taken together, our findings demonstrate that an increase in the caspase-1/IL-1β axis facilitates AMPAR internalization in the hippocampus, which dysregulates glutamatergic synaptic transmission, eventually resulting in depression-like behaviors. These results may represent an endophenotype for chronic stress-induced depression.

Figure 1

Genetic ablation of caspase-1 in mice prevents chronic stress-induced depression-like behaviors. (a) The proposed schema depicting caspase-1-dependent IL-1β production and increased AMPA receptor internalization. (b) Horizontal scatter plot depicting the distribution of interaction ratios for control, susceptible, and resilient mice after CSDS. (c) Susceptible mice displayed anhedonia as measured by a reduction in 1% sucrose preference (n=15 mice/ group, mean±s.e.m., one-way ANOVA, Bonferroni’s test, ***P<0.001). (d) Caspase-1 mRNA level in the PBMC and hippocampus (Hip) after CSDS (n=5–8 mice/group, mean±s.e.m., one-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (e) Correlation of caspase-1 mRNA level in the hippocampus with social interaction ratio after CSDS (Pearson correlation, n=11–12 mice/group). (f) Representative immunoblots and quantification of caspase-1 protein expression in the hippocampus after CSDS (n=10 mice/group, mean±s.e.m., one-way ANOVA, Bonferroni’s test, **P<0.01, ***P<0.001). (g) CUMS increased the expression of caspase-1 protein in the hippocampus (n=10–11 mice/group, mean±s.e.m., Student’s t-test, ***P<0.001). (h) Social interaction times for WT, WT-CSDS, Caspase-1^−/− and Caspase-1^−/−-CSDS mice, and Caspase-1^−/− mice showed significant increase in social interaction after CSDS (n=10 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, ***P<0.001). (i) Caspase-1^−/− mice blocked anhedonia as measured by a reduction in 1% sucrose preference after CSDS (n=10 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, ***P<0.001). (j) Effects of CSDS on the object recognition test (n=8–10 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, **P<0.01). (k) Caspase-1^−/− mice reversed anhedonia as measured by a reduction in 1% sucrose preference after CRS (n=10–11 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01, ***P<0.001). (l and m) Caspase-1^−/− mice prevented CRS-induced increase in immobility time in FST (l) and TST (m) when compared to WT-CRS mice (n=10–11 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ANOVA, analysis of variance; CUMS, chronic unpredictable mild strss; CRS, chronic restraint stress; CSDS, chronic social defeat stress; FST, forced swim test; IL, interleukin; PBMC, periphery blood mononuclear cell; TST, tail suspension test; WT, wild-type.

Figure 2

Overexpression of caspase-1 in the hippocampus increases depression- and anxiety-like behaviors. (a, top) Schematic representation of construct showing mouse caspase-1 subcloned into an AAV plasmid under transcriptional regulation of the CMV promoter (AAV-caspase-1-3FLAG-SV40-EYFP). AAV-3FLAG-SV40-EYFP plasmid without encoding caspase-1 served as the control. Bottom, the timeline of experimental procedure. (b) Representative photomicrographs of injection sites in the hippocampus. Scale bars, 200 μm. (c) Representative western blotting of hippocampal pro-Caspase-1, Caspase-1 p20, Caspase-1 p20/pro-Caspase-1, IL-1β after subthreshold social defeat stress (left). Mean (±s.e.m.) fold change in protein from hippocampus microdissections from AAV-GFP or AAV-caspase-1-injected mice (right) (n=6–8 mice/group, mean±s.e.m., Student’s t-test, *P<0.05, **P<0.01, ***P<0.001). (d and e) AAV-caspase-1-injected mice increased social avoidance (d) and decreased sucrose preference (e) after subthreshold social stress when compared with their respective AAV-GFP (n=10–11 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, **P<0.01). (f) Summary of the TST and FST in AAV-GFP and AAV-caspase-1-injected mice. Graphs showed an increase in time spent immobile in AAV-caspase-1-injected mice (n=10–11 mice/group, mean±s.e.m., Student’s t-test, *P<0.05). (g and h) AAV-caspase-1-injected mice displayed an anxiogenic phenotype in OF (g) and EPM (h) when compared with their respective AAV-GFP (n=10–11 mice/group, mean±s.e.m., Student’s t-test, *P<0.05, **P<0.01). AAV, adeno-associated virus; ANOVA, analysis of variance; CMV, cytomegalovirus; EPM, elevated plus maze; FST, forced swim test; GFP, green fluorescent protein; OF, open-field test; TST, tail suspension test.

Figure 3

Genetic ablation of caspase-1 blocks CSDS-induced synaptic plasticity impairment in the hippocampus. (a) Input–output curves illustrating the relationship between the magnitudes of stimulation and evoked response for fEPSPs recorded in hippocampal slices from WT, WT-CSDS, Caspase-1^−/− and Caspase-1^−/−-CSDS groups. Insets are typical superimposed fEPSPs recorded by increasing stimulation intensity (n=10 slices from four to seven mice per group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, *P<0.05). (b, left) time-course of LTP induced by HFS in hippocampal slices from different groups. Insets are superimposed fEPSPs recorded for each condition before (1) and 60 min after the application of HFS (2). (right) The histogram showing LTP magnitude averaged from the last 15 min of recordings from different groups (n=8–11 slices from four to seven mice per group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01, ***P<0.001). (c) Representative immunoblots and quantification of surface expression of GluA1 and GluA2 in the hippocampus from different groups (n=7 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (d) Representative immunoblots and quantification of total expression of hippocampal GluA1, GluA2 and PSD95 proteins from different groups (n=6–7 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (e, left) Representative whole-cell voltage-clamp traces of AMPAR-mediated mEPSC in the hippocampal pyramidal neurons. Scale is depicted on the middle right. (right) Mean±s.e.m. amplitude and frequency of AMPAR-mediated mEPSC from different groups (n=10–12 cells from five to six mice per group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (f) Immunofluorescence of PSD95 in CA1 pyramidal neurons. Scale bars, 100 μm. (g) The levels of IL-1β in the serum were determined by ELISA in the different groups (n=10–12 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, ***P<0.001). (h) Analysis of IL-1β mRNA expression in the hippocampus by qRT-PCR. (n=5 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (i) Representative immunoblots and quantification of hippocampal IL-1β, p-p38 MAPK and BDNF proteins (n=6–7 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; ANOVA, analysis of variance; BNDF, brain-derived neurotrophic factor; CSDS, chronic social defeat stress; fEPSP, field excitatory postsynaptic potential; HFS, high-frequency; stimulation; I, intracellular; IL, interleukin; LTP, long-term potentiation; mEPSC, miniature excitatory postsynaptic current; S, surface; qRT-PCR, quantitative PCR with reverse transcription; WT, wild-type.

Figure 4

Pharmacological inhibition of caspase-1 signaling pathway prevents CSDS-induced depression-like behaviors and synaptic plasticity impairment. (a) I.c.v. infusion of AC-YVAD-CMK 30 min before daily social defeat for 10 days rendered the mice more resilient to CSDS in social interaction (left) and sucrose preference (right) test (n=9–11 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (b) Representative immunoblots and quantification of hippocampal caspase-1 and IL-1β proteins from DMSO, 10 μg kg⁻¹ AC-YVAD-CMK, CSDS-DMSO and CSDS-10 μg kg⁻¹ AC-YVAD-CMK mice (n=6 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (c) Representative immunoblots and quantification of surface expression of GluA1 and GluA2 in the hippocampus from different groups (n=6 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05). S, surface; I, intracellular. (d, left) Representative whole-cell voltage-clamp traces of AMPAR-mediated mEPSC in the hippocampal pyramidal neurons. Scale is depicted on the middle right. (right) Mean±s.e.m. amplitude of AMPAR-mediated mEPSC from different groups (n=10–11 cells from three to four mice per group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (e) Continuous administration of IL-1ra during CSDS improved social interaction (left) and sucrose preference (right) reduction in stressed mice (n=10–11 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). (f) Representative immunoblots and quantification of surface expression of GluA1 and GluA2 in the hippocampus (n=7 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05). (g) Representative immunoblots and quantification of hippocampal p-p38 MAPK, BDNF, PSD95, total GluA1 and GluA2 proteins from Vehicle, IL-1ra, CSDS-vehicle and CSDS-IL-1ra mice, and the reduction in the hippocampus of stressed mice were significantly prevented by IL-1ra administration (n=5 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (h, left) Representative whole-cell voltage-clamp traces of AMPAR-mediated mEPSC in the hippocampal pyramidal neurons. Scale is depicted on the middle right. (right) Mean±s.e.m. amplitude of AMPAR-mediated mEPSC from different groups (n=10–12 cells from four to six mice per group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, **P<0.01). AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CSDS, chronic social defeat stress; DMSO, dimethylsulphoxide; I, intracellular; IL, interleukin; S, surface.

Figure 5

Chronic IL-1β exposure induces depression and anxiety-like behaviors in Caspase-1^−/− mice. (a) Timeline of experimental procedure. D, day. (b) The body weight was measured during the experimental procedure, and there were no changes in WT-vehicle, WT-IL-1β, Caspase-1^−/−-vehicle and Caspase-1^−/−-IL-1β (n=10–12 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test). (c and d) WT and Caspase-1^−/− mice displayed decreased social interaction following IL-1β administration for 10 days when compared to their respective vehicle-injected WT and Caspase-1^−/− controls (n=10–12 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (e) Chronic i.c.v. infusion of IL-1β induced anhedonia as measured by 1% sucrose preference. Both WT and Caspase-1^−/− mice that received repeated IL-1β infusion showed significant reduction on days 10 and 12 (n=10 mice/group, mean±s.e.m., repeated-measures ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). (f and g) Chronic i.c.v. infusion of IL-1β in both WT and Caspase-1^−/− mice significantly increased anxiety in OF (f) and EPM (g) when compared to their controls (n=10–12 mice/group, mean±s.e.m., two-way ANOVA, Bonferroni’s test, *P<0.05, **P<0.01). ANOVA, analysis of variance; EPM, elevated plus maze; IL, interleukin; OF, open-field test; WT, wild-type.