d-Aspartate oxidase influences glutamatergic system homeostasis in mammalian brain

Abstract

We have investigated the relevance of d-aspartate oxidase, the only enzyme known to selectively degrade d-aspartate (d-Asp), in modulating glutamatergic system homeostasis. Interestingly, the lack of the Ddo gene, by raising d-Asp content, induces a substantial increase in extracellular glutamate (Glu) levels in Ddo-mutant brains. Consistent with an exaggerated and persistent N-methyl-d-aspartate receptor (NMDAR) stimulation, we documented in Ddo knockouts severe age-dependent structural and functional alterations mirrored by expression of active caspases 3 and 7 along with appearance of dystrophic microglia and reactive astrocytes. In addition, prolonged elevation of d-Asp triggered in mutants alterations of NMDAR-dependent synaptic plasticity associated to reduction of hippocampal GluN1 and GluN2B subunits selectively located at synaptic sites and to increase in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-to-N-methyl-d-aspartate ratio. These effects, all of which converged on a progressive hyporesponsiveness at NMDAR sites, functionally resulted in a greater vulnerability to phencyclidine-induced prepulse inhibition deficits in mutants. In conclusion, our results indicate that d-aspartate oxidase, by strictly regulating d-Asp levels, impacts on the homeostasis of glutamatergic system, thus preventing accelerated neurodegenerative processes.

Introduction

Of the pool of endogenous amino acids, only serine and aspartate substantially occur in free d-form in mammalian tissues (Billard, 2012, Errico et al., 2012, Mothet and Snyder, 2012, Ota et al., 2012, Wolosker and Mori, 2012). d-Serine (d-Ser) is mainly localized in forebrain structures of the central nervous system throughout embryonic development and the postnatal phase. Compelling evidence demonstrates that d-Ser acts as an endogenous co-agonist at N-methyl-d-aspartate receptors (NMDARs) (Billard, 2012, Martineau et al., 2006, Snyder and Kim, 2000), and facilitation of d-Ser transmission could represent a therapeutic option in psychiatric disorders, including schizophrenia (Coyle et al., 2002). On the other hand, much less is known about the role of free d-aspartate (d-Asp) in the mammalian brain. d-Asp binds to and activates GluN2 subunits of NMDARs (Errico et al., 2011b). In line with its pharmacological features, nonphysiological elevated levels of d-Asp in knockout mice for the d-aspartate oxidase (Ddo) gene are associated with changes in NMDAR-dependent responses, including synaptic plasticity and spatial memory (Errico et al., 2011c, Errico et al., 2008a, Errico et al., 2008b). Furthermore, we have previously reported that 1-month oral administration of d-Asp in mice enhances NMDAR-mediated currents, basal cerebral blood volume in frontohippocampal areas, and increases dendritic length and spine density in the prefrontal cortex (PFC) and hippocampus (Errico et al., 2014). In agreement with mouse studies, we also found that genetic variation predicting reduced DDO expression in the postmortem human PFC mapped to greater prefrontal gray matter and activity during working memory (Errico et al., 2014). We also have recently documented a substantial reduction of d-Asp and NMDA in the postmortem PFC and striatum of schizophrenic patients compared with healthy controls, thus suggesting a potential involvement of this molecule in schizophrenia (Errico et al., 2013). Remarkably, d-Asp displays a peculiar temporal occurrence in the brain, that is, levels are high between embryonic and early postnatal stages and rapidly decrease to trace levels after birth (Dunlop et al., 1986, Hashimoto et al., 1993, Hashimoto et al., 1995, Lee et al., 1999, Neidle and Dunlop, 1990, Sakai et al., 1998, Wolosker et al., 2000) because of the concomitant expression of DDO, the only enzyme known to degrade bicarboxylic d-amino acids (D'Aniello et al., 1993, Still et al., 1949, Van Veldhoven et al., 1991). Given the pharmacological ability of d-Asp to act as a direct NMDAR agonist and in the light of the strict control exerted by DDO on postnatal brain levels of d-Asp, we investigated the biological significance of this enzyme in regulating glutamatergic system homeostasis in the hippocampus and PFC of mice with targeted deletion of Ddo gene.