Regulation of AMPA receptor function by the human memory-associated gene KIBRA

Abstract

KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.

Fig. 1

KIBRA directly binds PICK1 and forms a complex with GluA1/2 AMPAR subunits in vivo. (A) Schematic diagrams of yeast two-hybrid bait, prey and full-length proteins. (B) Representative images of HEK293T cells transfected with either HA-PICK1 or GFP-KIBRA alone or in combination. (C) HEK293T cells were transfected with GST-PICK1 and myc-KIBRA, lysed and immunoprecipitated with anti-myc antibodies in the presence or absence of antigenic blocking peptide. Proteins were resolved by Western blot and probed with anti-myc or anti-GST antibodies. (D) Mouse P2 brain fractions were immunoprecipitated with either anti-KIBRA antibodies or normal rabbit IgG. Samples were subjected to Western blot analyses using specific antibodies as indicated.

Fig. 2

KIBRA KD accelerates the rate of GluA2 recycling following NMDAR-induced internalization. Cultured hippocampal neurons were transfected with empty vectors (control), pSuper-KIBRA-shRNA-7, or pSuper-KIBRA-shRNA-7 and pRK5-shRNA resistant KIBRA rescue constructs together with the pH-GluA2 reporter and mCherry at DIV15. At DIV17, neurons were stimulated with 20 μM NMDA for 5 min and changes in pH-GluA2 fluorescence intensity were monitored by live-cell confocal microscopy. (A) Representative time-series images from control, KIBRA KD and KIBRA rescued neurons (scale bar: 50μm). Average time course of normalized pH-GluA2 fluorescence change (AF/F_o) in the soma (B) and (C) dendrite. Histograms of changes in pH-GluA2 fluorescence amplitude in response to NMDA (D) and recycling rate t_1/2 after NMDA washout (E). *P < 0.05, **P < 0.01, ANOVA (n = 8 to 14 cells) in the soma and dendrite.

Fig. 3

Impaired synaptic plasticity in adult KIBRA KO mice. (A) The slope of the input output curve is not altered in adult KIBRA KO mice. P > 0.05, two-tailed student's t-test (WT, n=29, average I-O slope = 3.4± 0.2 ms^-1; KO, n=28, 3.3 ± 0.2 ms^-1). (B) Paired pulse facilitation is not altered in adult KIBRA KO mice. A two-way repeated measures ANOVA revealed no significant genotype × inter-stimulus interval interaction or main effect of genotype. (WT n = 20, KO n = 17; p > 0.05 at all inter-stimulus intervals) (C) LTP induced with theta-burst stimulation is reduced in adult KIBRA KO mice. **P < 0.01, two-tailed student's t-test (wild-type, n = 13, 158±4%; KO, n = 10, 136±4%). (D) LTD induced with 900 stimuli at 1Hz is reduced in adult KIBRA KO mice. **P < 0.01, two-tailed student's t-test (wild-type, n = 11, 82±2%; KO, n = 12, 91±2%). Scale bars for all trace insets: 0.5mv, 5msec.

Fig. 4

Impaired learning and memory in adult KIBRA KO mice. (A) Schematic of fear conditioning protocol. (B) KIBRA KO mice exhibit delayed freezing to a 6 trial training protocol, but show levels of freezing comparable to wild-type by the end of the training session. A two-way repeated measures ANOVA revealed a significant genotype × interval interaction, P < 0.01, F_21,336 = 3.59 (wild-type, n = 8, KO, n = 10). *P < 0.05, **P < 0.01, Bonferroni post-hoc test, wild-type vs. KO. ITI, inter-train interval. (C) Freezing during the first minute of testing in the training context (**P < 0.01, two-tailed student's t-test) but not in a novel context (**P > 0.1, two-tailed student's t-test) is significantly impaired in KIBRA KO mice (wild-type, n = 8, KO, n = 10). (D) Trace fear conditioning during the first test block is impaired in KIBRA KO mice. A two-way repeated measures ANOVA revealed a significant genotype × interval interaction, P < 0.01, F_6,72 = 5.81 (wild-type, n = 5, KO, n = 9). *P < 0.05, **P < 0.01, Bonferroni post-hoc test, wild-type vs. KO.