Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

Abstract

Reduced function of the N-methyl-d-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation (Grin 1(D481N)) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1(D481N) mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1(G181R) mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1(D481N) mutation, mice with both the Dao1(G181R) and Grin 1(D481N) mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.

Figure 1. Deficits in sociability are rescued by the Dao1^G181R mutation

Wild-type, +/Grin1, Dao/+, and double mutant mice were assessed in a social affiliations task. (a) Mean time (sec) spent in a chamber containing a stranger mouse, a central chamber, and a chamber with an empty cage in the sociability phase. (b) Mean time (sec) spent in a chamber containing a newly introduced mouse (stranger 2), a central chamber, and a chamber with a familiar mouse (stranger 1) in the social novelty phase. Number of chamber entries during the sociability (c) and social novelty (d) phase. n = 22 wild-type, 16 +/Grin1, 15 Dao1/+, and 15 double mutant mice; **P < 0.01, ***P < 0.001 compared to the chamber with an empty cage or a familiar mouse, within genotype; ~P < 0.05 compared to Dao1/+ mice in the same chamber.

Figure 2. Persistent latent inhibition is ameliorated by inactivation of DAO function

Mean suppression ratios of pre-exposed (PE) and non-preexposed (NPE) wild-type, +/Grin1, Dao/+, and double mutant mice given 2 CS-US (a) or 4 CS-US (b) pairings. n = 8–18 per group; *P < 0.05, **P < 0.01 compared to PE score, within genotype; #P < 0.05 compared to PE score of wild-type mice; ~P < 0.05 compared to NPE score of Dao1/+ mice; ^P < 0.05 compared to NPE score of +/Grin1 mice.

Figure 3. Prepulse inhibition and startle reactivity in mice with the Dao1^G181R and Grin1^D481N mutation

(a, left and right panel) Mean % inhibition of the acoustic startle response at prepulse intensities of 69, 73, and 81 dB in male and female wild-type, +/Grin1, Dao/+, and double mutant mice. (b, left and right panel) Mean startle amplitude in response to a stimulus (100 dB) given in absence of a prepulse. (c, left and right panel) Acoustic startle response to varying stimulus intensities (70 – 120 dB) administered without a prepulse. n = 7–11 per group; *P < 0.05, **P < 0.01, ***P < 0.001 compared to wild-type mice, within the same prepulse or startle stimulus intensity; ~P < 0.05, ~~P < 0.01, ~~~P < 0.001 double mutant mice compared to Dao1/+ mice, within the same prepulse or startle stimulus intensity; ^P < 0.05, ^^P < 0.01 +/Grin1 mice compared to double mutant mice, within the same startle stimulus intensity.

Figure 4. Impaired spatial recognition is improved by reduced DAO activity

Spatial and non-spatial object discrimination was examined in wild-type, +/Grin1, Dao/+, and double mutant mice. (a) Mean duration (sec) of locomotor activity in the sessions of habituation (S2–S5), spatial change (S6), and non-spatial change (S7). (b) Mean duration (sec) of object exploration in the habituation phase. (c) Mean time (sec) spent exploring the displaced and non-displaced objects during the last habituation phase (S5; left panel) and the spatial change phase (S6; right panel). (d) Mean time (sec) spent exploring the novel item and three familiar objects in the non-spatial change phase (S7). n = 19 wild-type, 19 +/Grin1, 19 Dao1/+, 17 double mutant mice; *P < 0.05, ***P < 0.001 compared to the time spent exploring the non-displaced or familiar objects, within genotype; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to wild-type mice exploring the same object category; ~~P < 0.01 compared to Dao1/+ exploring the non-displaced objects; ^P < 0.05, ^^^P < 0.001 compared to the +/Grin1 exploring the same object category.

Figure 5. Partial amelioration of long-term spatial memory in double mutant animals

Performance of wild-type, +/Grin1, Dao/+, and double mutant mice was evaluated in the Morris water maze (MWM) task. (a) Mean path length to reach a target platform in a visible platform session (day 1), and in a hidden-platform acquisition training phase (days 2–5). Spatial memory retention was assessed in a probe trial (b, c, and d) conducted 24 h after the last acquisition trial. During the probe trial, the time (sec) spent (a), frequency of platform crosses (b), and distance (cm) (c) from the correct target area and the averaged analogous non-target areas was determined. The dashed line represents chance level, corresponding to the ratio of the target area to the total pool area (2.8%). n = 14 wild-type, 9 +/Grin1, 14 Dao1/+, 8 double mutant mice. *P < 0.05 compared to wild-type mice, within the same platform location; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the target location, within genotype.