KIBRA regulates activity-induced AMPA receptor expression and synaptic plasticity in an age-dependent manner

Abstract

A growing body of human literature implicates KIBRA in memory and neurodevelopmental disorders. Memory and the cellular substrates supporting adaptive cognition change across development. Using an inducible KIBRA knockout mouse, we demonstrate that adult-onset deletion of KIBRA in forebrain neurons impairs long-term spatial memory and long-term potentiation (LTP). These LTP deficits correlate with adult-selective decreases in extrasynaptic AMPA receptors under basal conditions, and we identify a role for KIBRA in LTP-induced AMPAR upregulation. In contrast, juvenile-onset deletion of KIBRA in forebrain neurons did not affect LTP and had minimal effects on basal AMPAR expression. LTP did not increase AMPAR protein expression in juvenile WT mice, providing a potential explanation for juvenile resilience to KIBRA deletion. These data suggest that KIBRA serves a unique role in adult hippocampal function through regulation of basal and activity-dependent AMPAR proteostasis that supports synaptic plasticity.

Keywords: Cellular neuroscience; Developmental neuroscience; Molecular neuroscience.

Graphical abstract

Figure 1

Tamoxifen treatment rapidly reduces KIBRA expression in the hippocampus of juvenile KIBRA cKO mice (A) Developmental expression profile of KIBRA in the hippocampus of WT mice. (B) Experiment timeline for juvenile Kibra^{floxed/floxed}:CaMK2α Cre^ERT2 mice. WT (Cre-negative, tamoxifen injected), WT’ (Cre-positive, vehicle injected), and cKO (Cre-positive, tamoxifen injected) mice received 1 injection (100mg/kg I.P.) per day for 3 days. Each mouse was given 5 days to recover from injections before experiments and tissue collection. Experiments were performed between P21-P25. (C) Hippocampal CA1 tissue was isolated following tamoxifen or vehicle injections. Endogenous KIBRA protein levels were assessed using an anti-KIBRA antibody. (D) Quantification normalized to VCP (one-way ANOVA, p = 0.010, post hoc comparisons shown in figure). WT, 100 ± 11%; WT′, 87 ± 17%; cKO, 40 ± 9%. (E and F) Juvenile-onset KIBRA deletion does not affect expression of the KIBRA homolog WWC2 (unpaired t-test (total and membrane) or Mann-Whitney test (synaptic) corrected for multiple comparisons). Total, WT = 100 ± 3%, cKO = 106 ± 5%; membrane, WT = 100 ± 7%, cKO = 104 ± 14%; synaptic, WT = 100 ± 17%, cKO = 93 ± 21%. ∗p < 0.05, 0.05 <#p < 0.1, n.s. p> 0.1. Data plotted as mean ± SEM, n = number of animals, indicated on each bar.

Figure 2

Juvenile-onset deletion of KIBRA does not affect basal synaptic transmission or long-term potentiation in the hippocampus (A−C) Representative traces from input-output curves, scale bars = 2mV/5ms, (B) Summary data from juvenile input-output analysis, (C) Slopes of individual input-output curves were quantified; no differences were observed across experimental conditions (one-way ANOVA, n.s.). Mean I-O slope: WT, 4.18 ± 0.45ms⁻¹; WT′, 3.92 ± 0.27 ms-1; cKO, 4.09 ± 0.49 ms⁻¹. (D) Paired pulse facilitation (fEPSP slope response 2/fEPSP slope response 1) is not altered in juvenile KIBRA cKO mice (RM two-way ANOVA, n.s. genotype X inter-stimulus interval interaction, main effect of genotype, and multiple comparisons at all inter-stimulus intervals). (E) Hippocampal LTP induced by four trains of theta-burst stimulation is unchanged by juvenile-onset deletion of KIBRA. Scale bars= 0.25mV/5ms. (F) STP magnitude is unaffected in juvenile KIBRA cKO mice (one-way ANOVA, n.s.). 5 min avg at gray bar, WT, 152 ± 7%; WT′, 148 ± 10%; cKO, 151 ± 7%. (G) LTP magnitude measured at 65–70 min post LTP induction is unaffected by KIBRA deletion in the juvenile hippocampus (one-way ANOVA, n.s.). 5 min. Avg at gray bar, WT, 120 ± 3%; WT′, 125 ± 4%; cKO, 123 ± 5%. All summary data presented as mean ± SEM. B-D: WT, n = 14 slices from 4 mice; WT′, n = 7 slices from 2 mice; cKO, n = 12 slices from 4 mice. E–G: WT, n = 9 slices from 4 mice; WT′, n = 8 slices from 2 mice; cKO, n = 9 slices from 4 mice. ‘n.s.’ = p > 0.05.

Figure 3

Tamoxifen treatment reduces hippocampal KIBRA expression in the adult brain (A) Tamoxifen injection schedule for adult Kibra^{floxed/floxed}:CaMK2α Cre^ERT2 mice. WT (Cre-negative, tamoxifen injected), WT’ (Cre-positive, vehicle injected), and cKO (Cre-positive, tamoxifen injected) mice received 2 injections (100mg/kg I.P.) per day for 5 days. Each mouse was given 14 days to recover from injections before experiments and tissue collection. Injections and experiments were performed after each mouse turned 2 months of age and before 4.5 months of age. (B) Hippocampal CA1 tissue was isolated from Kibra^{floxed/floxed}:CaMK2α Cre^ERT2 mice following tamoxifen or vehicle injections. Endogenous KIBRA protein levels were assessed using an anti-KIBRA antibody. (C) Quantification normalized to VCP: One-way ANOVA with Holm-Sidak’s multiple comparisons test. (WT, 100 ± 10.58%; WT′, 60.44± 3.71%; cKO, 30.88 ± 3.39%). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Data plotted as mean ± SEM, n = number of animals, indicated on each bar.

Figure 4

KIBRA deletion from the adult brain impairs hippocampal long-term potentiation without affecting basal synaptic transmission (A−C) Representative traces from input-output curves, scale bars = 2mV/5ms, (B) Summary data from adult input-output analysis, (C) Slopes of individual input-output curves: WT, average I-O slope 4.98 ± 0.41 ms⁻¹; WT′, average I-O slope 3.83 ± 0.36 ms⁻¹; cKO, I-O slope 4.34 ± 0.39 ms⁻¹. No difference observed across experimental conditions (one-way ANOVA, n.s.). (D) Paired pulse facilitation (fEPSP slope response 2/fEPSP slope response1) is not altered in adult KIBRA cKO mice (repeated measures (RM) two-way ANOVA, n.s. genotype X inter-stimulus interval interaction, main effect of genotype, and multiple comparisons at all inter-stimulus intervals). (E) Hippocampal LTP induced by four trains of theta-burst stimulation is impaired after adult-onset KIBRA deletion. Scale bars= 0.25mV/5ms. (F) Trend toward decreased STP in adult KIBRA cKO mice (one-way ANOVA, p = 0.063). 5 min average at gray bar in E, WT, 174 ± 12%; WT′, 170 ± 13%; cKO, 141 ± 7. (G) LTP magnitude measured at 75–80 min after LTP induction is decreased after deletion of KIBRA in the adult hippocampus (one-way ANOVA, p = 0.021). 5 min average at gray bar in E, WT, 130 ± 8%; WT′, 130 ± 7%; cKO, 110 ± 4%. (H) LTP induction is not impaired in adult KIBRA cKO mice (one-way ANOVA, n.s. effect of genotype, genotype × train interaction, and multiple comparisons at all inter-stimulus intervals). LTP induction measured as charge transfer (Area UnderCurve, AUC) during each TBS train (40 fEPSPs per train, normalized to baseline fEPSP). (I) Sample traces from LTP induction quantified in H. Shown is the first burst of the first train from one example recording (not averaged), Scale bar = 1.5mV/50 ms. All summary data presented as mean ± SEM. ∗p < 0.05, # 0.5 < p < 0.1, n.s. (not significant) = p > 0.1. B–D, WT, n = 14 slices from 6 mice; WT′, n = 10 slices from 3 mice; cKO, n = 16 slices from 6 mice. E–H, WT, n = 9 slices from 5 mice; WT′, n = 8 slices from 3 mice; cKO, n = 13 slices from 5 mice.

Figure 5

KIBRA regulates the basal expression of extrasynaptic AMPA receptors in the adult hippocampus (A, C, E, and G) Representative western blot images from sub-region CA1 of the adult hippocampus. Samples are normalized to loading control and quantified as % WT in B, D, F, and H (see STAR methods for details). (B) Adult-onset KIBRA deletion from neurons decreases the total expression GluA2 and GluA1, but not the excitatory synaptic scaffold PSD-95 (unpaired t-tests, corrected for multiple comparisons). GluA2, WT = 100 ± 3%, cKO = 67 ±3; GluA1, WT = 100 ± 2%, cKO = 80 ± 4%; PSD95, WT = 100 ± 3%, cKO = 93 ± 3%. (D) Loss of KIBRA from the adult brain decreases expression of membrane-localized GluA2 (unpaired t-tests with Welch’s correction, corrected for multiple comparisons). GluA2, WT = 100 ± 2%, cKO = 83 ± 5%; GluA1, WT = 100 ± 4%, cKO = 102 ± 2%; PSD95, WT = 100 ± 6%, cKO = 105 ± 4%. (F) Adult-onset KIBRA deletion does not alter basal expression of synaptic AMPA receptors (unpaired Mann-Whitney tests, corrected for multiple comparisons). GluA2, WT = 100 ± 3%, cKO = 98 ± 6%; GluA1, WT = 100 ± 2%, cKO = 94 ± 8%; PSD95, WT = 100 ± 8%, cKO = 106 ± 5%. (H) Decrease in expression of the KIBRA homolog WWC2 following adult-onset deletion of KIBRA (unpaired t-tests, corrected for multiple comparisons). Total, WT = 100 ± 4%, cKO = 70 ± 2%; membrane, WT = 100 ± 5%, cKO = 92 ± 6%; synaptic, WT = 100 ± 5%, cKO = 87 ± 7%. (I) Representative western blot with equal protein loaded for total, cytosolic, membrane and synaptic fractions, demonstrating depletion of the postsynaptic scaffold PSD95 from the cytosolic fraction and enrichment in the membrane fraction with further enrichment in the synaptic fraction. Data shown as mean ± SEM, n on bar graphs = number of animals.

Figure 6

Juvenile-onset deletion of KIBRA has minimal effect on hippocampal AMPAR expression (B, D, and F) Representative western blot images from sub-region CA1 of the juvenile hippocampus. (A) Acute reduction of KIBRA in the juvenile hippocampus decreases total expression of AMPAR subunit GluA2 but not GluA1 or PSD95 (unpaired t-tests, corrected for multiple comparisons, Welch’s correction for GluA2). GluA2, WT = 100 ± 1%, cKO = 86 ±3; GluA1, WT = 100 ± 3%, cKO = 95 ± 4%; PSD95, WT = 100 ± 4%, cKO = 95 ± 3%. (C) Larger decrease in total AMPAR expression in adult compared to juvenile KIBRA cKO mice (unpaired t-tests, corrected for multiple comparisons). For each group, data is shown as % decrease from respective WT (GluA2, juvenile cKO = −14 ± 4%, adult cKO = −33 ± 4%; GluA1, juvenile cKO = −3 ± 5%, adult cKO = −19 ± 4%). (E) Juvenile-onset deletion of KIBRA does not affect expression of membrane-associated AMPARs or PSD95 in the juvenile hippocampus (unpaired t- (GluA1, GluA2) or Mann-Whitney (PSD95) tests, corrected for multiple comparisons). GluA2, WT = 100 ± 6%, cKO = 95 ± 4%; GluA1, WT = 100 ± 6%, cKO = 101 ± 11%; PSD95, WT = 100 ± 3%, cKO = 98 ± 17%. (G) Loss of KIBRA in the juvenile brain does not alter basal expression of synaptic AMPA receptors or PSD95 (unpaired t- (GluA1, PSD95) or Mann-Whitney (GluA2) tests, corrected for multiple comparisons). GluA2, WT = 100 ± 14%, cKO = 89 ± 12%; GluA1, WT = 100 ± 7%, cKO = 85 ± 6%; PSD95, WT = 100 ± 7%, cKO = 96 ± 11%. Data shown as mean ± SEM, n on bar graphs = number of animals. ∗p < 0.05, ∗∗p < 0.01.

Figure 7

KIBRA is required for LTP-induced increases in AMPAR expression in the adult hippocampus (A) Experimental design. Transverse hippocampal slices were collected from adult mice following basal stimulation (0.033 Hz) or LTP (TBS). The stimulated region of CA1 was microdissected 30 or 120 min after LTP or basal stimulation. Data from both time points was combined as no difference in AMPAR induction was observed between 30 and 120min post LTP. (B–D) Representative GluA2 and GluA1 immunoblot images after baseline stimulation or LTP from adult WT (cre-negative tamoxifen treated) or KIBRA cKO (cre-positive tamoxifen treated) mice. LTP increases GluA2 (C) and GluA1 (E) expression in WT but not KIBRA cKO mice. Data plotted as % increase over baseline stimulation, mean ± SEM (GluA2, WT mean= 151 ± 15%, cKO = 100 ± 8%; GluA1, WT mean = 119 ± 8%, cKO = 101 ± 4%). LTP-stimulated slices were compared to baseline-stimulated slices from the same animal. Number of slices is indicated on each bar. One sample t-test, ∗p < 0.05, ∗∗p < 0.01.

Figure 8

LTP does not increase AMPAR expression in juvenile WT or KIBRA cKO mice Transverse hippocampal slices were collected from juvenile mice following basal stimulation (0.033 Hz) or LTP (TBS). The stimulated region of CA1 was microdissected 30 min after LTP or basal stimulation. (A and C) Representative GluA2 and GluA1 immunoblot images after baseline stimulation or LTP from juvenile WT (cre-negative tamoxifen treated) or KIBRA cKO (cre-positive tamoxifen treated) mice. LTP did not induce increases in total GluA2 (B) or GluA1 (D) expression in juvenile WT or KIBRA cKO mice. Data plotted as % increase over baseline stimulation, mean ± SEM (GluA2, WT mean = 108 ± 11%, cKO = 107 ± 7%; GluA1, WT mean = 106 ± 6%, cKO = 114 ± 7%). LTP-stimulated slices were compared to baseline-stimulated slices from the same animal. For each group, n = 18 slices from 10 mice. One sample Wilcoxon test, p > 0.05 for all comparisons shown.

Figure 9

Adult-onset KIBRA deletion from forebrain neurons impairs memory in adult mice (A) Schematic of modified Barnes Maze arena. The target location is a covered box hidden from view by one of the identical walls positioned around the exterior of the maze. Training consisted of 4 trials per day over four days. (B) KIBRA mice show delayed memory acquisition during training, assessed by comparing latency to reach the target box on the first trial of day 2,3, and 4 to the first trial on day 1 (Welch’s RM one-way ANOVA, cKO p = 0.0013, WT p = 0.0006, post-hoc comparisons shown in figure). Lines in violin plots = median. (C) KIBRA cKO mice exhibit grossly normal learning as assessed by latency to reach the target box averaged across all 4 trials for each training day (RM two-way ANOVA, n.s. effect of genotype, genotype × day interaction, or WT vs KO post hoc comparison for any day). n = number of mice: WT, 6M + 5F; KIBRA cKO 8M +6F. (D) Example maze occupancy (top) and trajectory (bottom) plots during memory retention probe test from two example mice of each genotype. ‘Max’ time for occupancy scale is indicated at the bottom left corner of each plot. Target location is as depicted in panel A. (E) Percent occupancy per zone during memory retention (probe) test 7 days after the final training session. KIBRA cKO mice fail to show preference for target quadrant (Welch’s RM one-way ANOVA, cKO p = 0.0913, WT p = 0.0137, post-hoc comparisons shown in figure). Lines in violin plots = median. (F) Decreased memory exhibited by KIBRA cKO mice shown by the cumulative number of entries behind the target wall over the first 5 entries during the probe trial (RM two-way ANOVA, p = 0.0484 effect of genotype). (G and H) KIBRA cKO does not affect overall movement as shown by equivalent total distance traveled (G) and moving velocity (H, velocity when mice are moving > 3 cm/s) during the probe trial (unpaired t-test, corrected for multiple comparisons). Total velocity including pauses was also not different between genotypes (WT, 18 ±1 cm/s; cKO 16 ± 1 cm/s, ns). (I) KIBRA cKO and WT mice spend the same amount of time in the center of the maze during the probe trial (unpaired Mann-Whitney test). (J) No differences were observed between genotypes in weight lost because of time-restricted feeding (weight measured the last day of training/free feeding weight measured the day before maze habituation) (unpaired Mann-Whitney test). ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, 0.05 <#p < 0.1, n.s. p> 0.1. Panels C,F,G,H,I,J, data plotted as mean ± SEM.