A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse

Abstract

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

Figure 1. Generation of Snap-25 “knock-in” mouse.

(A) The targeting vector (middle) was constructed by insertion of the DNA fragment (lower), consisting of part of a mutated SNAP-25 minigene, the PGK 3′ end poly-A signal, the PGK promoter and the neo gene, into the SmaI site at exon 7 of the region between exon 6 and exon 8 of the wild-type allele (upper). The black box in the lower panel indicates the mutation point of the codon for Ser¹⁸⁷ to Ala¹⁸⁷. Arrows indicate the primers used in ES cell screening. (B) The genotyping of the mice was conducted by PCR using primers overlapping the 3′ and 5′ extremities of exon 7 (arrowheads in upper panel in (A)). The DNA fragments from wild-type and mutant alleles are indicated with “W” and “M”, respectively. Wt, wild-type mouse; He, Snap25^+/S187A mouse; Hm, Snap25^S187A/S187A mouse. Mw, molecular weight marker. (C) Northern blotting of total forebrain RNAs from each mouse genotype (P25) was conducted using SNAP-25b cDNA as a probe. The signals of transcripts from each allele are indicated as in (B). 18S, 18S rRNA; 28S, 28S rRNA. (D) Immunoblot analysis of the phosphorylation level of SNAP-25 and the expression levels of major synaptic proteins in mouse brain.

Figure 2. General locomotor activity in open field.

(A) Typical traces of WT (Wt), Snap25^+/S187A (He), and Snap25^S187A/S187A mice (Hm-1 and Hm-2) during 30 min. (B) Initial delay time before moving. (C) Percentage of time spent near the wall of the open-field box. (D) Total moving time in 30 min of test period. (E) Average velocity. (F) Time-course of rearing frequency during the 30 min. Values represent mean ± SD. **, p<0.01 and *, p<0.05 by paired Student's t test between Wt mice and Snap25^S187A/S187A mice.

Figure 3

(A) Traces of thirteen Wt (W1 to W13) and thirteen Snap25^S187A/S187A mice (H1 to H13) in open field performed three times with one week intervals. (B) Time courses of locomotor activities in open field shown in A. Total moving distances are plotted against time. Red, purple and yellow lines represent 1st, 2nd and 3rd trial, respectively. Sometimes, they start to move suddenly after a long stationary period (arrowhead), but at other times, they suddenly stopped after active movement (arrow). (C) Number of mice showing stopping period longer than 200 s during 30 min test period either 0 time (0/3), once (1/3), twice (2/3), or three times (3/3) in each three trials. (D) The value of standard deviation of moving distance in each three trials divided by the average of moving distance in Wt and Snap25^S187A/S187A mice (Hm). Values represent mean ± SD. ***, p<0.001 by paired Student's t test between Wt mice and Snap25^S187A/S187A mice.

Figure 4. Orbital of locomotor activity in light and dark boxes.

(A) Typical traces of WT (left five, Wt) and Snap25^S187A/S187A mice (right five, Hm) during 30 min. (B) % time in dark room. (C) Average moving velocity. (D) Latency to enter dark box. (E) Total number of rearing behaviors. Values represent mean ± SD. ***, p<0.001, **, p<0.01 and *, p<0.05 by paired Student's t test between Wt mice and Snap25^S187A/S187A mice.

Figure 5. Monoamine metabolisms in brain.

(A) Contents of dopamine (DA), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) in various brain regions of WT (red columns) and Snap25^S187A/S187A mice (blue columns). Fcx, frontal cortex; CP, striatum; Amy, amygdala; Hip, hippocampus; Th, thalamus; Hypo, hypothalamus; BS, brain stem; Ce, Cerebellum. *, p<0.05. (B) Spontaneous release of DA and 5-HT in amygdala of WT (red column) and Snap25^S187A/S187A mouse (blue column). **, p<0.01. (C) Time-dependent changes of high-K⁺ (100 mM)-evoked DA and 5-HT release in amygdala of WT (red circles) and Snap25^S187A/S187A mouse (blue circles). KCl concentration was elevated at a time indicated by arrows. *, p<0.05 compared with WT and Snap25^S187A/S187A mouse in each period.