- Top of page
- Material and methods
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 1D481N) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1G181R 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 1D481N mutation, mice with both the Dao1G181R and Grin 1D481N 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.
Schizophrenia is a chronic and severely debilitating mental illness that affects about 1% of the population worldwide. In addition to psychosis, schizophrenia is characterized by persistent negative symptoms of social withdrawal, flattened affect and decreased motivation, and by profound cognitive deficits in attention, working memory and executive functioning (Lewis & Gonzalez-Burgos 2006; Ross et al. 2006). Current antipsychotics have limited efficacy in reducing the negative and cognitive symptoms, and are often poorly tolerated (Lewis & Gonzalez-Burgos 2006; Ross et al. 2006). Consequently, there is a prevalent need for the development of improved therapeutic alternatives targeting the molecular alterations involved in schizophrenia.
Diminished glutamatergic neurotransmission mediated by the N-methyl-d-aspartate receptor (NMDAR) has been implicated in the biological mechanisms underlying schizophrenia (Coyle 2006). NMDAR inhibition with non-competitive antagonists exacerbates schizophrenic symptoms in patients and elicits psychotomimetic effects in healthy individuals (Javitt & Zukin 1991; Krystal et al. 1994). Furthermore, genetic studies have identified several risk genes for schizophrenia influencing NMDAR activity (Coyle 2006; Ross et al. 2006). These include a number of genes that modulate the selective endogenous NMDAR glycine site agonist, D-serine (Chumakov et al. 2002; Morita et al. 2006; Schumacher et al. 2004). D-serine is an important contributor to NMDAR activation, as reduction of D-serine concentrations have been shown to impede NMDAR-mediated signaling (Mothet et al. 2000; Panatier et al. 2006; Yang et al. 2003). Reduced levels of D-serine have been reported in the serum and cerebrospinal fluid (CSF) of patients with schizophrenia (Bendikov et al. 2007; Hashimoto et al. 2003), and postmortem studies indicate an abnormal increase in D-serine degradation (Madeira et al. 2008). Thus, diminished NMDAR function in schizophrenia may be related to lower D-serine availability, and by corollary augmented D-serine levels may be a beneficial form of treatment.
Endogenous D-serine availability in the brain is determined by its catabolic enzyme D-amino acid oxidase (DAO), its synthesis enzyme serine racemase and by various glial and neuronal transporters (Foltyn et al. 2005; Martineau et al. 2006). Compounds targeting these regulatory proteins may effectively enhance cerebral D-serine and occupancy of the NMDAR glycine site. Inhibition of DAO activity is of particular interest, as it would rectify the increase in DAO function reported in schizophrenia (Madeira et al. 2008) and avoid any nephrotoxic effects related to the metabolism of high levels of systemic D-serine (Maekawa et al. 2005a). D-amino acid oxidase is a peroxisomal flavoprotein that at physiological pH is highly selective for D-serine (Mothet et al. 2000), consistent with the inverse correlation between the distribution of DAO and D-serine in the central nervous system (CNS) (Moreno et al. 1999; Schell et al. 1995). In this study, we examined the efficacy of limited DAO activity in ameliorating behavioral phenotypes relevant to schizophrenia. We employed mice with a point mutation in the Dao1 gene (G181R) that produces a complete loss of DAO function and a subsequent elevation in brain D-serine (Labrie et al. 2008b; Sasaki et al. 1992). The Dao1G181R mutation is capable of augmenting NMDAR-mediated neurotransmission (Maekawa et al. 2005b; Wake et al. 2001), suggesting that diminished DAO function may benefit psychiatric conditions associated with reduced NMDAR activation. To evaluate the performance of Dao1G181R mice in a model pertinent to schizophrenia, the Dao1G181R mutation was combined with the Grin1D481N mutation. Grin1D481N mice have a fivefold decrease in NMDAR glycine affinity, and biochemical and electrophysiological studies have confirmed that reduced NMDAR activity in these mice can be reversed with exogenous D-serine or glycine application (Duffy et al. 2008; Kew et al. 2000). Moreover, these mice display behavioral abnormalities related to the negative and cognitive symptoms of schizophrenia, including deficient social approach, persistent latent inhibition and impaired spatial recognition and memory (Labrie et al. 2008c). Here, we report that DAO inactivation improves behavioral deficits associated with NMDAR hypofunction.
- Top of page
- Material and methods
Diminished DAO function enhanced D-serine levels in the brain and improved behavioral deficits associated with the negative and cognitive symptoms of schizophrenia. D-amino acid oxidase inactivation effectively reversed sociability deficits, persistent LI and spatial recognition impairments in mice with reduced NMDAR glycine occupancy. In addition, loss of DAO function partially normalized the elevated startle reactivity and long-term spatial memory disruption. These findings indicate that reduced DAO activity may be beneficial for the treatment of psychiatric symptoms associated with diminished NMDAR activation, thereby supporting the therapeutic potential of this novel approach in the clinic.
Limiting DAO function was an effective means of augmenting D-serine concentrations in the brain, even in subjects with reduced NMDAR activity. Although the rise in D-serine was relatively modest, its chronicity may be an important factor in its capacity to modulate the effects of NMDAR hypofunction. Previous studies have shown that DAO inhibitors produce similar modest increases in brain D-serine levels in rodents, and chronic treatments have greater success in reversing behavioral deficits induced by NMDAR antagonism than acute DAO blockade (Adage et al. 2008; Ferraris et al. 2008). Moreover, inhibition of D-serine catabolism can allow for smaller elevations in D-serine levels to be efficacious, while larger doses of exogenous D-serine are necessary to surmount the effects of DAO activity. The Dao1G181R mutation in mice on a C57BL/6J genetic background has previously been shown to increase D-serine in the cortex, hippocampus and cerebellum (Labrie et al. 2008b). Changes in D-serine were most prevalent in caudal brain regions (Hashimoto et al. 1993; Labrie et al. 2008b), as DAO is highly abundant in glia of the hindbrain and cerebellum, while lower levels of DAO are present in neurons of the prefrontal cortex, hippocampus and substantia nigra (Moreno et al. 1999; Verrall et al. 2007). Importantly, diminished DAO activity and the related rise in D-serine significantly potentiate NMDAR function. The NMDAR glycine site is not saturated at the synapses of many brain regions (Fuchs et al. 2005), and Dao1G181R mice display an in vitro enhancement of NMDAR-mediated synaptic transmission in the hippocampus and spinal cord (Maekawa et al. 2005b; Wake et al. 2001) and an in vivo increase in cerebellar cGMP levels, indicative of greater NMDAR activation (Almond et al. 2006). Furthermore, Dao1G181R mice do not exhibit obvious compensatory changes affecting [3H]-D-serine reuptake or the expression of proteins relevant to the NMDAR signaling, including NR1, serine racemase, glycine transporter 1 and alanine-serine-cysteine transporter 1 (Almond et al. 2006). This suggests that behavioral improvements induced by the Dao1G181R mutation are specifically related to decreased DAO function.
The advantage of using the Grin1D481N mice to test the ameliorative effects of the Dao1G181R mutation is that in contrast to paradigms involving challenge with NMDAR antagonists, the Grin1D481N mice model the chronic and presumably developmental nature of NMDAR hypofunction proposed to occur in schizophrenia. In addition, decreased glycine site activation is relevant to the neural changes involved in schizophrenia, indicating that the Grin1D481N mice may be a more appropriate model for NMDAR hypofunction in schizophrenia than animals with limited expression of the NMDAR-NR1 subunit. The Grin1D481N mutation does not abrogate binding of agonists to the glycine site, but merely raises the K d. Consequently, the requirement for a ligand such as D-serine is simply shifted toward higher concentration ranges, predicting the reversal of phenotypes under conditions of decreased D-serine catabolism.
Poor social functioning, a common and enduring aspect of the negative symptoms of schizophrenia (Ellenbroek & Cools 2000), was improved in Grin1D481N mice by the Dao1G181R mutation. Social deficits and their amelioration were specific to the test of sociability, which examines social approach- and avoidance-related motivation (Sankoorikal et al. 2006). Reversal of the sociability impairment could not be attributed to the changes in olfactory acuity or to a non-specific increase in exploratory activity. Previous studies with Dao1G181R mice or exogenous D-serine treatments do not show anxiolytic-like effects (Labrie et al. 2008a; Schmitt et al. 1995), indicating that reduced anxiety responses are not likely to account for the improvement in sociability.
Deficits in selective attention and information processing are prevalent in schizophrenia and can be measured similarly in humans and rodents using an LI procedure (Moser et al. 2000). In accordance with previous findings, LI was abnormally persistent in Grin1D481N mice. Prolonged LI has similarly been observed in rodents treated with an inhibitor of the NMDAR glycine site (Labrie et al. 2008c) or the non-competitive NMDAR antagonist MK-801 (Gaisler-Salomon & Weiner 2003; Lipina et al. 2005). LI perseveration indicates an impaired switching ability, as subjects are unable to switch from ignoring an irrelevant stimulus to responding in a manner that shows a CS-US association. Behavioral inflexibility is characteristic of the negative symptoms in schizophrenia (Berman et al. 1997) and clinical studies have correlated LI persistence to the severity of negative symptoms in patients (Cohen et al. 2004; Rascle et al. 2001). We found that decreased DAO function was capable of reversing persistent LI, indicating that activation of the NMDAR glycine site facilitates appropriate switching responses. Persistent LI was normalized, in part, by a decrease in conditional learning (NPE score), as seen in other studies examining the reversal of LI persistence by proglycinergic treatments (Gaisler-Salomon et al. 2008; Labrie et al. 2008c; Lipina et al. 2005). Also, the Dao1G181R mutation and exogenous D-serine application have previously been shown to improve spatial reversal memory and accelerate extinction learning (Duffy et al. 2008; Labrie et al. 2008b). Thus, D-serine and decreased DAO activity improve adaptive learning and may be useful for the treatment of psychiatric disorders characterized by cognitive inflexibility.
Loss of DAO function partly reversed the augmented startle reactivity in Grin1D481N mice, supporting its efficacy in ameliorating the effects of chronic NMDAR hypoactivity. Although Grin1D481N mice displayed normal PPI, chronic inhibition of DAO has been shown to improve PPI disruptions induced by phencyclidine, an NMDAR antagonist (Adage et al. 2008). Acute DAO blockade in combination with D-serine administration has also been shown to attenuate PPI deficits in mice treated with MK-801 (Hashimoto et al. 2009). The absence of PPI differences in the Grin1D481N mice may reflect the more subtle effects of this glycine site mutation compared to other genetic and pharmacological models of NMDAR deficiency (Fradley et al. 2005; Geyer et al. 2001; Moy et al. 2006). Indeed, MK-801 dosages used disrupt PPI in rodents are generally higher than those required to induce persistent LI and deficits in social interactions and spatial recognition (Gaisler-Salomon & Weiner 2003; Roullet et al. 1996; Rung et al. 2005). Consequently, like most animal models pertinent to schizophrenia, the Grin1D481N mice display several relevant endophenotypes, but do not recapitulate all aspects of the disease. It will therefore be necessary to examine the effects of DAO elimination in other schizophrenia models to fully explore the therapeutic utility of this approach.
Since cognitive deficits are a core feature of schizophrenia and a major contributor to functional disability (Lewis & Gonzalez-Burgos 2006), we evaluated the capacity of limited DAO function and elevated D-serine to ameliorate disturbances in a visuospatial object discrimination task and in the MWM. In the object recognition task, Grin1D481N mice displayed a selective inability to detect a spatial change that was rescued by DAO inactivation. The improvement in spatial recognition was not related to perturbations in locomotor activity or object exploration during the habituation phase. In addition, all mice showed a similar progressive acclimatization to the environment and object configuration in the habituation session. Loss of DAO function also partially restored spatial memory in Grin1D481N mice in the MWM. Double mutant animals favored the correct target location in the probe trial; however, this preference did not reach significance compared to mice with only the Grin1D481N mutation. Previously, exogenous D-serine treatments have been shown to fully reverse spatial deficits in the object discrimination and MWM task (Duffy et al. 2008; Labrie et al. 2008c). The partial ameliorative effects of the Dao1G181R mutation in the MWM may be related to the less severe increases in D-serine induced by DAO inhibition (Smith et al. 2009). Also, spatial memory demand in the MWM is likely greater than in the object recognition task, due to the 24-h interval prior to the probe session. Nonetheless, these findings indicate that decreased DAO function may provide some benefit for the disturbances in perception and representation of spatial relationships in schizophrenia (Barnett et al. 2007; O’Donnell et al. 1996).
Clinical studies indicate that agonists of the NMDAR glycine site, including D-serine, produce symptomatic improvements in patients with schizophrenia when administered in conjunction with conventional antipsychotics (Coyle 2006; Heresco-Levy et al. 2005; Tsai et al. 1998). However, there are concerns with the administration of D-serine and similar compounds, as large doses are required to penetrate the blood–brain barrier. In contrast, DAO inhibitors cross the blood–brain barrier readily (Adage et al. 2008; Hashimoto et al. 2009) and may be a well-tolerated alternative for the modulation of D-serine levels and NMDAR glycine site occupancy in the clinical setting. Animals that chronically lack DAO activity exhibit normal development, longevity and reproductive potential (Konno & Yasumura 1983). Furthermore, chronic administration of D-serine at therapeutic doses has not been found to produce adverse effects in humans (Tsai et al. 1998). However, under conditions promoting excitotoxicity and neuroinflammation, high levels of D-serine can potentially compromise neuronal survival through excessive NMDAR activation (Martineau et al. 2006; Wu et al. 2004), suggesting that modest rises in D-serine, as observed following DAO reduction, may be advantageous.
In conclusion, chronic DAO inactivation and elevated D-serine improved behavioral deficits caused by reduced NMDAR glycine site occupancy in mice. This suggests that inhibition of DAO function may effectively reverse NMDAR hypofunction implicated in schizophrenia pathogenesis and could serve as a valuable therapeutic approach for the treatment of negative and cognitive symptoms. Further research will be necessary to determine the extent to which DAO inhibition can ameliorate other neurochemical events and behavioral disturbances associated with schizophrenia.