Rescue of impaired sociability and anxiety-like behavior in adult cacna1c-deficient mice by pharmacologically targeting eIF2α

Abstract

CACNA1C, encoding the Ca_v1.2 subunit of L-type Ca²⁺ channels, has emerged as one of the most prominent and highly replicable susceptibility genes for several neuropsychiatric disorders. Ca_v1.2 channels play a crucial role in calcium-mediated processes involved in brain development and neuronal function. Within the CACNA1C gene, disease-associated single-nucleotide polymorphisms have been associated with impaired social and cognitive processing and altered prefrontal cortical (PFC) structure and activity. These findings suggest that aberrant Ca_v1.2 signaling may contribute to neuropsychiatric-related disease symptoms via impaired PFC function. Here, we show that mice harboring loss of cacna1c in excitatory glutamatergic neurons of the forebrain (fbKO) that we have previously reported to exhibit anxiety-like behavior, displayed a social behavioral deficit and impaired learning and memory. Furthermore, focal knockdown of cacna1c in the adult PFC recapitulated the social deficit and elevated anxiety-like behavior, but not the deficits in learning and memory. Electrophysiological and molecular studies in the PFC of cacna1c fbKO mice revealed higher E/I ratio in layer 5 pyramidal neurons and lower general protein synthesis. This was concurrent with reduced activity of mTORC1 and its downstream mRNA translation initiation factors eIF4B and 4EBP1, as well as elevated phosphorylation of eIF2α, an inhibitor of mRNA translation. Remarkably, systemic treatment with ISRIB, a small molecule inhibitor that suppresses the effects of phosphorylated eIF2α on mRNA translation, was sufficient to reverse the social deficit and elevated anxiety-like behavior in adult cacna1c fbKO mice. ISRIB additionally normalized the lower protein synthesis and higher E/I ratio in the PFC. Thus this study identifies a novel Ca_v1.2 mechanism in neuropsychiatric-related endophenotypes and a potential future therapeutic target to explore.

Figure 1

Cacna1c fbKO mice display a deficit in sociability and impaired learning and memory. (a) Schematic of the three-chamber social approach apparatus. (b) In the three-chamber social approach test, WT but not fbKO adult mice spent significantly more time in the chamber (left; two-way ANOVA, genotype × chamber, F_1,34 = 8.865, P = 0.0053) and contact zone (right; two-way ANOVA, genotype × zone, F_1,34 = 6.6367, P =0.0165) containing the stranger mouse compared to the novel object (Bonferroni post hoc test, ***P<0.001 vs object; WT n = 11, fbKO n =8). (c) Representative heat maps showing time spent in the chambers (represented by rectangular compartments) and contact zones (represented by area between outer circle and inner circle) containing the stranger mouse and novel object (represented by inner circle). (d) WT and fbKO mice traveled similar distances during the social approach test (WT n =11, fbKO n =8). (e) In cue fear conditioning, WT and fbKO mice displayed similar percent freezing to the tone during training (two-way ANOVA, main effect of tone, F_{4, 60} = 55.06, P <0.0001) but during the twenty-four hour cue fear test, fbKO mice exhibited significantly higher freezing compared to WT mice (Two-way ANOVA, main effect of genotype, F_{1, 60} = 34.27, P <0.0001; Bonferroni post hoc test, *P <0.05, **P <0.01 vs WT). Each data point represents freezing during a single tone presentation (WT n =8, fbKO n =6). (f) During contextual fear conditioning, WT and fbKO mice displayed similar percent freezing to the context during training and the twenty-four hour test (WT n =6, fbKO n =6). (g) Schematic of the water-based Y-maze apparatus. (h) In the Y-maze during training, fbKO mice displayed increased latency in locating the submerged platform compared to WT mice (Two-way ANOVA, main effect of genotype, F_{1, 75} = 24.1, P <0.0001; Bonferroni post hoc test, *P <0.05 vs WT; WT n = 9, fbKO n = 8). (i) In the Y-maze memory tests, fbKO mice displayed significantly increased latency in locating the submerged platform at 1 h (Student’s t-test with Welch’s correction, t₍₈₎ = 3.188, P <0.05), 24 h (Student’s t-test with Welch’s correction, t₍₇₎ = 3.121, P <0.05) and 7 days (Mann–Whitney U-test, U = 13.00, P <0.05) post-training (*P<0.05, **P<0.01 vs WT; WT n =9, fbKO n =8). (j) WT and fbKO mice exhibit similar percent of spontaneous alternations during the working memory Y-maze task (WT n =8, fbKO n =8). (k) Schematic of the Morris water maze (MWM) apparatus. (l) In the MWM during training, WT and fbKO mice displayed similar latency in locating the submerged platform (Two-way ANOVA, main effect of day, F_{5, 72} = 26.41, P <0.0001; n = 7/genotype). (m–o) In the MWM probe tests, WT and fbKO mice spent significantly more time in the goal quadrant relative to the other quadrants at 1 h (m; Two-way ANOVA, main effect of quadrant, F_3,48 = 70.41, P <0.0001), 24 h (n; Two-way ANOVA, main effect of quadrant, F_3,48 = 23.01, P <0.0001) and 7 days (o; Two-way ANOVA, main effect of quadrant, F_3,48 = 18.19, P <0.0001) post-training (Bonferroni post hoc test, *P<0.05, **P<0.01, ***P<0.001 vs Goal; n =7/genotype). Error bars represent mean ± s.e.m. ANOVA, analysis of variance; fbKO, forebrain knockout; WT, wild type.

Figure 2

Focal knockdown of cacna1c in the adult PFC recapitulates the social deficit and elevated anxiety-like behavior. (a) Outline of experimental design. AAV-Cre or AAV-GFP was stereotaxically injected into the PFC of adult cacna1c floxed mice and 5 weeks later tested in a battery of behavioral tests. (b) Representative image of GFP-positive cells expressed by AAV-Cre-GFP in the PFC of cacna1c floxed mice. (c) In the three-chamber social approach test, GFP but not Cre mice spent significantly more time in the chamber (left; Two-way ANOVA, genotype × chamber, F_1,44 = 24.28, P <0.0001) and contact zone (right; two-way ANOVA, genotype × zone, F_1,44 = 11.71, P = 0.0014) containing the stranger mouse and novel object (Bonferroni post hoc test, ***P<0.001 vs WT; GFP n = 12, Cre n =12). (d) Representative heat maps showing time spent in the chambers and contact zones containing the stranger mouse and novel object during the social approach test. (e) GFP and Cre mice traveled similar distances during the social approach test (GFP n =12, Cre n =12). (f) Cre mice spent significantly less time in the open arm of the elevated plus maze compared to GFP mice (Student’s t-test, t₍₁₃₎ = 2.156, P = 0.05; *P = 0.05 vs GFP; GFP n = 8, Cre n = 7). (g) In cue fear conditioning, GFP and Cre mice displayed similar percent freezing to the tone during training (two-way ANOVA, main effect of tone, F_{4, 70} = 12.7, P <0.0001) and the test. Each data point represents freezing during a single tone presentation (GFP n = 8, Cre n = 8). (h) In contextual fear conditioning, GFP and Cre mice displayed similar percent freezing to the context during training and the test (GFP n =8, Cre n = 8). (i) In the Y-maze, during training, Cre mice displayed slightly lower latency (main effect of treatment, F_1,75 = 5.374, P = 0.0232) in locating the submerged platform compared to GFP mice (GFP n =9, Cre n =8). (j) In the Y-maze memory tests, GFP and Cre mice displayed similar latencies in locating the submerged platform at 1 h and 24 h post-training (GFP n =9, Cre n =8). (k) GFP and Cre mice exhibit similar percent of spontaneous alternations during the working memory Y-maze task (GFP n =8, Cre n =8). Error bars represent mean ± s.e.m. ANOVA, analysis of variance; GFP, green fluorescent protein; PFC, prefrontal cortex.

Figure 3

Cacna1c fbKO mice exhibit lower protein synthesis and lower mRNA translation initiation factors in the PFC. (a–c) Immunoblot analysis (top) and representative blots (bottom) of puromycin-labeled protein. Compared to WT mice, fbKO mice displayed significantly lower levels of puromycin-labeled proteins in the PFC (a; Student’s t-test, t₍₁₀₎ = 2.330, P<0.05) but not the somatosensory cortex (b) or hippocampus (c; WT n =6, fbKO n = 6). (d) Schematic representation indicating the relevant phosphorylation sites (indicative of their activity) of mTORC1, its substrates, and eIF2α in their regulation of mRNA translation. (e) Immunoblot analysis (left) and representative blots of markers of mRNA translation in the PFC from total protein lysates. Compared to WT mice, fbKO mice displayed significantly lower levels of P-mTOR S2448 (Student’s t-test, t₍₁₂₎ = 4.410, P = 0.0009), and its downstream substrates 4EBP1 (Student’s t-test, t₍₁₂₎ = 4.739, P = 0.0005), P-4EBP1 T37/46 (Student’s t-test, t₍₁₁₎ = 8.627, P<0.0001), eIF4B (Student’s t-test, t₍₁₁₎ = 2.494, P<0.05), and P-eIF4B S422 (Student’s t-test, t₍₁₁₎ = 3.651, P =0.0038) as well as significantly higher levels of P-eIF2α S51 (Student’s t-test, t₍₁₂₎ = 3.972, P <0.01) in the PFC (n = 6–7/genotype). (f) WT and fbKO mice display similar levels of mature BDNF protein levels in the PFC (WT n =7, fbKO n =9). *P<0.05, **P<0.01, ***P<0.001 vs WT. Error bars represent mean ± s.e.m. fbKO, forebrain knockout; PFC, prefrontal cortex; WT, wild type.

Figure 4

ISRIB normalizes the lower protein synthesis in the PFC and rescues the social deficit and elevated anxiety in cacna1c fbKO mice. (a) Schematic depiction of ISRIB’s inhibitory effect on the effect of P-eIF2α S51 on general mRNA translation. (b) Immunoblot analysis (left) and representative blots (right) of puromycin-labeled protein in the PFC. Compared to WT mice, vehicle treated fbKO mice displayed significantly lower levels of puromycin-labeled proteins that was normalized to WT levels with ISRIB treatment (Two-way ANOVA, main effect of genotype, F_1,14 = 5.934, P = 0.0288, main effect of treatment, F_1,14 = 26.78, P = 0.0001; Bonferroni post hoc test, *P <0.05 vs WT/Veh, †††P <0.001 vs fbKO/Veh; WT/Veh n =3, fbKO/Veh n =3, WT/ISRIB n =5, fbKO/ISRIB n = 6) (c) Outline of experimental design. Adult WT, fbKO and PFC-Cre mice were given a single systemic injection of either ISRIB (2.5 mg/kg, i.p) or vehicle and ninety minutes later tested in social approach followed immediately by EPM. (d) In the three-chamber social approach test, vehicle and ISRIB treated WT mice (WT/Veh and WT/ISRIB, respectively) spent significantly more time in the chamber containing the stranger mouse compared to the novel object (Two-way ANOVA, main effect of chamber, F_{1, 28} = 40.78, P <0.0001). In contrast, vehicle treated fbKO mice (fbKO/Veh) mice spent similar amounts of time in the chambers containing the stranger mouse and object while fbKO mice with ISRIB treatment (fbKO/ISRIB) displayed a normalization of this social deficit spending significantly more time with the stranger mouse compared to the object (Two-way ANOVA, treatment × chamber, F_{1, 20} = 8.12, P = 0.0099), looking similar to WT/Veh mice. Similarly, vehicle treated PFC Cre mice (PFC Cre/Veh) spent similar amounts of time in the chamber containing the stranger mouse and object whereas PFC Cre mice following ISRIB treatment (PFC Cre/ISRIB) exhibited a normalization of this social deficit by spending significantly more time with the stranger mouse compared to the novel object (Two-way ANOVA, treatment x chamber, F_{1, 26} = 6.535, P = 0.0168) looking similar to WT mice (Bonferroni post hoc test, ***P <0.0001 vs object; WT: Veh n = 7, ISRIB n = 9; fbKO: Veh n =5, ISRIB n =7; PFC-Cre: Veh n =6, ISRIB n =9). (e) All mice traveled similar distances during the social approach test (WT: Veh n =7, ISRIB n =9; fbKO: Veh n =5, ISRIB n =7; PFC Cre: Veh n =6, ISRIB n =9). (f) Representative heat maps showing time spent in the chambers containing the stranger mouse and novel object during the social approach test. (g) In the elevated plus maze, WT/Veh and WT/ISRIB mice spent significantly more time in the open arms relative to the closed arms of the maze, although there was a minor effect of ISRIB on anxiety- like behavior (Two-way ANOVA, treatment X arm, F_{1, 26} = 7.349, P = 0.0117). fbKO/Veh mice spent similar amounts of time in the open and closed arms of the maze while fbKO/ISRIB mice displayed normalization of this behavior spending significantly more time in the open arm relative to the closed arm of the maze (Two-way ANOVA, main effect of arm, F_{1, 18} = 19.64, P = 0.0003), looking similar to WT/Veh mice. In contrast, both PFC Cre/Veh and PFC Cre/ISRIB mice spent significantly less time in the open arm of the maze compared to the closed arm (Two-way ANOVA, main effect of arm, F_{1, 26} = 47.01, P <0.0001; Bonferroni post hoc test, *P <0.05, **P <0.01, ***P <0.001 vs closed arm; WT: Veh n =6, ISRIB n =9; fbKO: Veh n =5, ISRIB n =6; PFC Cre: Veh n =6, ISRIB n =9). (h) All mice traveled similar distances in the elevated plus maze (WT: Veh n =6, ISRIB n =9; fbKO: Veh n =5, ISRIB n =6; PFC Cre: Veh n =6, ISRIB n =9). (i) Representative heat maps showing time spent in the open and closed arms of the elevated plus maze. Error bars represent mean ±s.e.m. ANOVA, analysis of variance; EPM, elevated plus maze; fbKO, forebrain knockout; PFC, prefrontal cortex; WT, wild type.

Figure 5

Cacna1c fbKO mice exhibit higher excitatory/inhibitory ratio in layer 5 pyramidal neurons of the PFC that is normalized with ISRIB. (a) Representative traces of mEPSCs from layer 5 pyramidal neurons of the PFC. (b) fbKO mice displayed significantly higher amplitude of mEPSCs that remained unchanged with ISRIB (Kruskal–Wallis test, H =8.242, P =0.0162; Dunn’s uncorrected post hoc test, **P<0.01 vs WT; WT n =8, fbKO n =12, fbKO/ISRIB n =9 cells, from n =2–4 mice per group). (c) fbKO mice displayed significantly higher frequency of mEPSCs that was normalized to WT levels with ISRIB (Kruskal–Wallis test, H =16.74, P =0.0002; Dunn’s uncorrected post hoc test, *P<0.05 vs WT, ^†††P<0.0001 vs fbKO; WT n =8, fbKO n =12 cells, fbKO/ISRIB n =9 cells, from n =2–4 mice per group). (d) Representative traces of mIPSCs from layer 5 pyramidal neurons of the PFC. (e) fbKO mice displayed similar amplitude of mIPSCs compared to WT mice that was unchanged with ISRIB (WT n =12, fbKO n =13, fbKO/ISRIB n =9 cells, from n =2–4 mice per group). (f) fbKO mice displayed significantly higher frequency of mIPSCs that was normalized to WT levels with ISRIB (one-way ANOVA, F_{2, 33} = 4.909, P = 0.0141; Bonferroni post hoc test, *P <0.05 vs WT, ^†P<0.05 vs fbKO; WT n =12, fbKO n =13, fbKO/ISRIB n =9 cells, from n =4 mice per group). (g) fbKO mice displayed significantly higher total charge transfer for mEPSCs that was normalized with ISRIB (Kruskal–Wallis test, H =14.2, P =0.0008; Dunn’s uncorrected post hoc test, **P<0.01 vs WT, ^†††P<0.001 vs fbKO; WT n =8, fbKO n = 12, fbKO/ISRIB n =9 cells, from n =2–4 mice per group). (h) fbKO mice displayed similar total charge transfer for mIPSCs compared to WT mice that was unchanged with ISRIB (WT n =12, fbKO n =13, fbKO/ISRIB n =9 cells, from n = 2–4 mice per group). (i) Higher relative change in mEPSC than mIPSC total charge transfer for each fbKO neuron normalized to the mean WT value that was corrected by ISRIB. (j) Compared to WT/Veh mice, fbKO/Veh mice had significantly higher levels of VGLUT1/VGAT ratio in the synaptoneurosome from the PFC while fbKO/ISRIB mice showed normalization of this elevated ratio (two-way ANOVA, treatment × genotype, F_{1, 23} = 5.797, P = 0.0245; Bonferroni post hoc test, *P <0.05 vs WT; WT: Veh n = 6, ISRIB n = 7; fbKO: Veh n = 6, ISRIB n = 7). Error bars represent mean ±s.e.m. ANOVA, analysis of variance; E, excitatory; fbKO, forebrain knockout, I, inhibitory; mEPSCs, miniature excitatory post synaptic currents; mIPSCs, miniature inhibitory post synaptic currents; WT, wild type.