A mental retardation gene, motopsin/neurotrypsin/prss12, modulates hippocampal function and social interaction

Abstract

Motopsin is a mosaic serine protease secreted from neuronal cells in various brain regions, including the hippocampus. The loss of motopsin function causes nonsyndromic mental retardation in humans and impairs long-term memory formation in Drosophila. To understand motopsin's function in the mammalian brain, motopsin knockout (KO) mice were generated. Motopsin KO mice did not have significant deficits in memory formation, as tested using the Morris water maze, passive avoidance and Y-maze tests. A social recognition test showed that the motopsin KO mice had the ability to recognize two stimulator mice, suggesting normal social memory. In a social novelty test, motopsin KO mice spent a longer time investigating a familiar mouse than wild-type (WT) mice did. In a resident-intruder test, motopsin KO mice showed prolonged social interaction as compared with WT mice. Consistent with the behavioral deficit, spine density was significantly decreased on apical dendrites, but not on basal dendrites, of hippocampal pyramidal neurons of motopsin KO mice. In contrast, pyramidal neurons at the cingulate cortex showed normal spine density. Spatial learning and social interaction induced the phosphorylation of cAMP-responsive element-binding protein (CREB) in hippocampal neurons of WT mice, whereas the phosphorylation of CREB was markedly decreased in mutant mouse brains. Our results indicate that an extracellular protease, motopsin, preferentially affects social behaviors, and modulates the functions of hippocampal neurons.

FIG.1

Motopsin knockout mice showed normal spatial memory. (A) Training session of Morris water maze test. Knockout (KO) mice showed spatial learning ability comparable to wild-type (WT) mice. Open circles, WT; Closed circles, KO. (B) Probe test. Both wild-type and motopsin KO mice spent a longer time swimming within the target quadrant compared to other quadrants 24 h after their final training session (* P < 0.05, post hoc analysis after a two-factor ANOVA). (C) Passive avoidance test. Motopsin KO mice showed similar latency to enter the dark box as WT mice 24 hr after the training. (D) Y-maze test. KO mice showed a proportion of alternation similar to that by WT mice in the Y-maze test. Values represent mean ± SEM.

FIG. 2

Abnormal social behavior in motopsin KO mice. Motopsin KO mice showed the ability to discriminate between familiar and unfamiliar mice; however, they investigated even a familiar mouse for longer than WT mice did. Values represent mean ± SEM. (A) Social recognition test. KO mice recognized the stranger mice, which is shown by the decline in sniffing time over the first four trials when they were presented with the same stranger mouse. When KO mice were exposed to an unfamiliar mouse at trial 5, the investigation time recovered to that at trial 2 which was significantly different from the investigation time of trial 4 (*P < 0.05, post hoc analysis after a two-factor ANOVA). (B) Sociability test. Both WT and KO mice sniffed the unfamiliar mouse significantly longer than the empty cage (***P < 0.0001), indicating that motopsin KO mice had normal sociability. (C) Social novelty test. Both WT and KO mice preferred the unfamiliar mouse to the familiar mouse (**P = 0.001). However, KO mice spent more time sniffing the familiar mouse than WT mice did (*P < 0.05). (D) Resident–intruder test. KO mice showed longer contact time than WT mice (*P < 0.05). However, the duration of active, passive, or aggressive behavior was not different between the genotypes.

FIG. 3

Normal olfactory behavior and exploratory activity in motopsin KO mice. (A) Olfactory test. Motopsin KO mice spent as much time finding buried food as WT mice (left). Motopsin KO mice ate more quickly than WT mice (right) (*P < 0.05). (B) Exploration test. Time spent investigating a novel object (left) or latency to the first investigation (right) was not different between the genotypes. Values represent mean ± SEM.

FIG. 4

Normal anxiety level of motopsin knockout mice. (A) Open field test. The mutant mice moved as much as WT mice (left). There is no difference between the genotypes in the proportion of distance passed in the center squares during 5 min sessions (right). (B) Elevated plus maze. There was no difference between genotypes in frequency of open arm entry (left) and time spent on open arms (right). (C) Light/dark box test. Time spent in the bright chamber (left) and latency to the first entry into the dark chamber (right) was not significantly different between WT and KO mice. Values represent mean ± SEM.

FIG. 5

Decreased spine density of apical dendrites in hippocampal neurons but not in cortical neurons. (A) The morphology of dendritic spines was not different between WT and KO mice, but the spine number appeared to be decreased in motopsin KO mice. Bar, 10 μm. (B) Spine density (per 20 μm) on apical dendrites of pyramidal neurons in the hippocampus was decreased in motopsin KO mice (*P < 0.05, n = 4 mice for each genotype), although the spine density on basal dendrites did not differ between the genotypes. The analyses were independently performed three times using N4, N6, and N10 in mouse brains. Declines were consistent across all generation groups tested. (C) There was no difference in the spine density of pyramidal neurons in the cingulate cortex between the genotypes. Values represent mean ± SEM.

FIG. 6

Decreased response in hippocampal neurons of motopsin knockout mouse to memory formation or social stimulation. (A) Immunohistochemistry using anti-CREB phosphorylated at Ser133 (pCREB) and anti-CREB. Hippocampal neurons were strongly stained by anti-pCREB antibody in WT mice, but barely in motopsin KO mice 90 min after the water maze training. Bar, 50 μm. (B) The ratio of fluorescence intensity of pCREB immunoreactivity (IR) to CREB IR after the water maze training. The ratio of pCREB/CREB IR in the hippocampus was significantly decreased in motopsin KO mice compared with WT mice (*P < 0.05). (C) After social stimulation, the pCREB/CREB IR ratio also significantly decreased in the motopsin KO mice (**P < 0.01).

FIG.7

Behavioral phenotype and CREB phosphorylation to PTZ administration. (A) Seizure score. The score was calculated in consideration of the severity and latency of seizure using an equation stated in Materials and Methods. The score of motopsin knockout mice was similar to that of wild-type mice. (B) The latency to tonic-clonic seizure. There is no difference between genotypes in the latency to tonic-clonic seizure after administration of PTZ. (C) High level of CREB phosphorylation was induced by PTZ administration at hippocampal neurons of both wild-type and mutant mice.