Kalirin regulates cortical spine morphogenesis and disease-related behavioral phenotypes

Abstract

Dendritic spine morphogenesis contributes to brain function, cognition, and behavior, and is altered in psychiatric disorders. Kalirin is a brain-specific guanine-nucleotide exchange factor (GEF) for Rac-like GTPases and is a key regulator of spine morphogenesis. Here, we show that KALRN-knockout mice have specific reductions in cortical, but not hippocampal, Rac1 signaling and spine density, and exhibit reduced cortical glutamatergic transmission. These mice exhibit robust deficits in working memory, sociability, and prepulse inhibition, paralleled by locomotor hyperactivity reversible by clozapine in a kalirin-dependent manner. Several of these deficits are delayed and age-dependent. Our study thus links spine morphogenic signaling with age-dependent, delayed, disease-related phenotypes, including cognitive dysfunction.

Fig. 1.

Absence of kalirin leads to a cortex-specific reduction in Rac1 activation. (A) Domain structure of major kalirin proteins and schematic structure of the KALRN gene with exons corresponding to key domains. Arrows indicate alternative translational start sites. (B) Gene targeting strategy. Exons 27–28, containing the active site of the GEF1 domain, have been replaced with the neo cassette. (C) PCR genotyping of KALRN-KO mice. (D) Kalirin isoforms are absent from the KALRN-KO mouse forebrain. *Indicate nonspecific bands (E) The expression of kalirin-7 and synaptic proteins in the hippocampus and frontal cortex of KO and WT mice. (F) KALRN-KO mice have reduced Rac1-GTP levels in the frontal cortex (P < 0.05), but no alteration in Rac1-GTP levels in the hippocampus or hypothalamus.

Fig. 2.

Brain region specific and age-dependent alterations in spine density in KALRN-KO mice. (A) Golgi-stained pyramidal neurons from frontal cortex layer V in KO and WT mice. (B) Quantification of (A) indicates that KO mice have a robust reduction in spine density in the frontal cortex relative to WT mice (***, P < 0.0001). (C) Golgi-stained hippocampal neurons from the CA1 field in KO and WT mice. (D) Quantification of (C) shows that spine density is unaffected in the CA1 field of KO mice relative to WT mice. (E) Two-photon imaging of layer V frontal cortical neurons in slices from 3- and 12-week-old mice. (F) Quantification of (E) shows that frontal cortex spine density was severely reduced in 12-week-old KALRN-KO mice (***, P < 0.0001). (G) Frontal cortical spine density was unaffected in 3-week-old KO mice. (H) KO mice show an age-mediated delayed reduction in spine density in the frontal cortex (*, P < 0.05). [Scale bars, 10 μm (A and C), 50 μm (E).] Data are mean ± SEM

Fig. 3.

Reduced AMPAR-mediated synaptic transmission in KALRN-KO mice. (A) Traces of AMPA mEPSC recordings from layer V pyramidal neurons in frontal cortical slices from WT and KO mice. (B) Quantification of (A) indicates that KO mice have a reduction in mEPSC frequency (*, P < 0.05), but no alteration in mEPSC amplitude. Data are median ± data distribution (box plots)

Fig. 4.

Behavioral phenotypes of KALRN-KO mice. (A) Two-trial matching-to-sample performance in the Morris water maze (MWM) shows that WT mice, but not KO mice, have a reduced path error on trial 2 relative to trail 1 (trial × genotype, P = 0.01). Data are means of each trial across 5 testing days. (B) MWM spatial reference memory (fixed platform test). Data are means of 6 trials for each day; main effects are observed for day and genotype (P < 0.001), but no effect of genotype by day (P > 0.05). (C) Additional analysis of MWM spatial reference memory. Means of trail 1 for each testing day; KO mice show an increased path error relative to WT mice on day 2 (*, P < 0.05), whereas performance between genotypes did not differ on any other days. (D) Water maze probe trail as a test of spatial reference memory shows the percentage of time animals search the target quadrant (Tar), the 2 adjacent quadrants (Adj1 and Adj2), and the quadrant opposite the target quadrant (Opp) for the removed platform. KO and WT mice spent a similar percentage of time searching in the target quadrant (P > 0.05). Line indicates chance level (25%). (E) Y-maze spontaneous alternation in 3- and 12-week-old KO and WT mice. Whereas the performance of 3-week-old KO and WT mice did not differ from each other, 12-week-old KO mice showed a deficit in spontaneous alternation relative to 12-week-old WT mice (***, P < 0.001). Line indicates chance performance (50%). Further analysis indicated a significant interaction of genotype by age (P < 0.001). (F) Prepulse inhibition: KO mice have a deficit in sensory motor gating across all prepulse intensities (P < 0.01). (G) Social behavior: In the presence of a stimulus mouse, KO mice spent less time in the social side and more time in the nonsocial side of the chamber relative to WT mice (**, P < 0.01); WT and KO mice show similar behavior in the absence of the stimulus mouse. (H) Additional testing for social behavior: social approach scores. Whereas WT mice show an increased social approach during the testing phase in which the stimulus mouse was present, relative to the habituation phase in which the stimulus mouse was absent, KO mice fail to show an increase in social approach between the 2 trials (P < 0.05). Data are mean ± SEM.

Fig. 5.

Locomotor hyperactivity in KALRN KO mice. (A) Twelve-week-old KO mice exhibit hyperactive behavior in an open-field environment (P = 0.001). Graphs show mean activity during 10-min bins for 1 h. Activity plots are shown below. (B) Choice-driven activity analysis in the Y-maze among 3- and 12-week-old WT and KO mice. Three-week-old WT and KO mice showed similar choice-driven activity levels. Twelve-week-old KO mice required less time to complete the Y-maze task than 12-week-old WT mice (**, P < 0.01), indicative of choice-driven hyperactivity in 12-week-old KO mice. (C) In the presence of intra-maze cues, Y-maze spontaneous alternation in 12-week-old WT and KO mice did not differ from each other (P > 0.05). (D) In the presence of intra-maze cues in the Y-maze, KO mice required less time to complete the task than WT mice, indicative of hyperactivity in KO mice (**, P < 0.01). (E) Heterozygous mice (HET) showed activity levels that were intermediate between KO and WT mice (P < 0.05). Clozapine (1 mg/kg) reversed the hyperactivity of HET mice (*, P < 0.05), but did not affect KO or WT mouse activity (P > 0.05). Data are mean ± SEM