Ran Inoue

Serine racemase is predominantly localized in neurons in mouse brain

D‐Serine is the endogenous ligand for the glycine binding site of the N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor (GluR) channel and is involved in the regulation of synaptic plasticity, neural network formation, and neurodegenerative disorders. D‐Serine is synthesized from L‐serine by serine racemase (SR), which was first reported to be localized in astrocytes. However, recently, SR mRNA and its protein have been detected in neurons. In this study, we examined the SR distribution in the brain during postnatal development and in cultured cells by using novel SR knockout mice as negative controls. We found that SR is predominantly localized in pyramidal neurons in the cerebral cortex and hippocampal CA1 region. Double immunofluorescence staining revealed that SR signals colocalized with those of the neuron‐specific nuclear protein, but not with the astrocytic markers glial fibrillary acid protein and 3‐phosphoglycerate dehydrogenase. In the striatum, we observed SR expression in γ‐aminobutyric acid (GABA)ergic medium‐spiny neurons. Furthermore, in the adult cerebellum, we detected weak but significant SR signals in GABAergic Purkinje cells. From these findings, we conclude that SR is expressed predominantly in many types of neuron in the brain and plays a key role in the regulation of brain functions under physiological and pathological conditions via the production of the neuromodulator D‐serine. J. Comp. Neurol. 510:641–654, 2008.

NMDA- and β-Amyloid1–42-Induced Neurotoxicity Is Attenuated in Serine Racemase Knock-Out Mice

d-Serine is detected in the brain and acts as a coagonist at the “glycine-site” of the NMDA-type glutamate receptor. Although d-serine can be directly produced from l-serine by serine racemase (SR), the relative contribution of SR in d-serine formation in vivo is not known. Pathological roles of brain d-serine mediating NMDA receptor overactivation are suggested in studies using in vitro culture systems. However, we have recently demonstrated the differential SR protein expression in vivo and in culture. Here, we reported an ∼90% decrease in forebrain d-serine content in SR knock-out (KO) mice. We also found a reduced neurotoxicity induced by NMDA- and Aβ1–42- peptide injections into the forebrain in SR KO mice. These results suggest that SR is the major enzyme for d-serine production in the brain, d-serine is the predominant endogenous coagonist of the NMDA receptor in the forebrain, and d-serine may be involved in controlling the extent of NMDA receptor-mediated neurotoxic insults observed in disorders including Alzheimers disease. The control of SR activity and d-serine level in the brain may lead to a novel strategy for neuroprotection against various neurodegenerative diseases.

Neuronal d-Serine and Glycine Release Via the Asc-1 Transporter Regulates NMDA Receptor-Dependent Synaptic Activity

d-Serine and glycine are coagonists of NMDA receptors (NMDARs), but their relative contributions for several NMDAR-dependent processes are unclear. We now report that the alanine–serine–cysteine transporter-1 (Asc-1) mediates release of both d-serine and glycine from neurons, and, in turn, this modulates NMDAR synaptic activity. Asc-1 antiporter activity is enhanced by d-isoleucine (d-Ile), which releases d-serine and glycine from Asc-1-transfected cells, primary neuronal cultures, and hippocampal slices. d-Ile has no effect on astrocytes, which do not express Asc-1. We show that d-Ile enhances the long-term potentiation (LTP) in rat and mouse hippocampal CA1 by stimulating Asc-1-mediated endogenous d-serine release. d-Ile effects on synaptic plasticity are abolished by enzymatically depleting d-serine or by using serine racemase knock-out (SR-KO) mice, confirming its specificity and supporting the notion that LTP depends mostly on d-serine release. Conversely, our data also disclose a role of glycine in activating synaptic NMDARs. Although acute enzymatic depletion of d-serine also drastically decreases the isolated NMDAR synaptic potentials, these responses are still enhanced by d-Ile. Furthermore, NMDAR synaptic potentials are preserved in SR-KO mice and are also enhanced by d-Ile, indicating that glycine overlaps with d-serine binding at synaptic NMDARs. Altogether, our results disclose a novel role of Asc-1 in regulating NMDAR-dependent synaptic activity by mediating concurrent non-vesicular release of d-serine and glycine. Our data also highlight an important role of neuron-derived d-serine and glycine, indicating that astrocytic d-serine is not solely responsible for activating synaptic NMDARs.

Localization of Serine Racemase and Its Role in the Skin

D-Serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)–type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.

Role of Serine Racemase in Behavioral Sensitization in Mice after Repeated Administration of Methamphetamine

Background The N-methyl-D-aspartate (NMDA) receptors play a role in behavioral abnormalities observed after administration of the psychostimulant, methamphetamine (METH). Serine racemase (SRR) is an enzyme which synthesizes D-serine, an endogenous co-agonist of NMDA receptors. Using Srr knock-out (KO) mice, we investigated the role of SRR on METH-induced behavioral abnormalities in mice. Methodology/Principal Findings Evaluations of behavior in acute hyperlocomotion, behavioral sensitization, and conditioned place preference (CPP) were performed. The role of SRR on the release of dopamine (DA) in the nucleus accumbens after administration of METH was examined using in vivo microdialysis technique. Additionally, phosphorylation levels of ERK1/2 proteins in the striatum, frontal cortex and hippocampus were examined using Western blot analysis. Acute hyperlocomotion after a single administration of METH (3 mg/kg) was comparable between wild-type (WT) and Srr-KO mice. However, repeated administration of METH (3 mg/kg/day, once daily for 5 days) resulted in behavioral sensitization in WT, but not Srr-KO mice. Pretreatment with D-serine (900 mg/kg, 30 min prior to each METH treatment) did not affect the development of behavioral sensitization after repeated METH administration. In the CPP paradigm, METH-induced rewarding effects were demonstrable in both WT and Srr-KO mice. In vivo microdialysis study showed that METH (1 mg/kg)-induced DA release in the nucleus accumbens of Srr-KO mice previously treated with METH was significantly lower than that of the WT mice previously treated with METH. Interestingly, a single administration of METH (3 mg/kg) significantly increased the phosphorylation status of ERK1/2 in the striatum of WT, but not Srr-KO mice. Conclusions/Significance These findings suggest first, that SRR plays a role in the development of behavioral sensitization in mice after repeated administration of METH, and second that phosphorylation of ERK1/2 by METH may contribute to the development of this sensitization as seen in WT but not Srr-KO mice.

Differential regional distribution of phosphorylated tau and synapse loss in the nucleus accumbens in tauopathy model mice

Tauopathies differ in terms of the brain regions that are affected. In Alzheimers disease, basal forebrain and hippocampus are mainly involved, while frontotemporal lobar degeneration affects the frontal and temporal lobes and subcortical nuclei including striatum. Over 90% of human cases of tauopathies are sporadic, although the majority of established tau-transgenic mice have had mutations. This prompted us to establish transgenic mice expressing wild-type human tau (Tg601). Old (>14 months old) Tg601 mice displayed decreased anxiety in the elevated plus maze test and impaired place learning in the Morris water maze test. Immunoblotting of brain tissue identified that soluble tau multimer was increased with aging even though insoluble tau was not observed. In the striatum of old Tg601, the level of AT8- or AT180-positive tau was decreased compared with that of other regions, while PHF-1-positive tau levels remained equal. Phosphorylated tau-positive axonal dilations were present mainly in layers V and VI of the prefrontal cortex. Loss of synaptic dendritic spine and decreased immunohistochemical level of synaptic markers were observed in the nucleus accumbens. In vivo 2-[(18)F]fluoro-2-deoxy-d-glucose positron emission tomography analysis also showed decreased activity exclusively in the nucleus accumbens of living Tg601 mice. In Tg601 mice, the axonal transport defect in the prefrontal cortex-nucleus accumbens pathway may lead to decreased anxiety behavior. Differential distribution of hyperphosphorylated tau may cause region-specific neurodegeneration.

Neuromodulatory Effect of Gαs- or Gαq-Coupled G-Protein-Coupled Receptor on NMDA Receptor Selectively Activates the NMDA Receptor/Ca2+/Calcineurin/cAMP Response Element-Binding Protein-Regulated Transcriptional Coactivator 1 Pathway to Effectively Induce Brain-Derived Neurotrophic Factor Expression in Neurons

Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline β, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

Is d-aspartate produced by glutamic-oxaloacetic transaminase-1 like 1 (Got1l1): a putative aspartate racemase?

Abstractd-Aspartate is an endogenous free amino acid in the brain, endocrine tissues, and exocrine tissues in mammals, and it plays several physiological roles. In the testis, d-aspartate is detected in elongate spermatids, Leydig cells, and Sertoli cells, and implicated in the synthesis and release of testosterone. In the hippocampus, d-aspartate strongly enhances N-methyl-d-aspartate receptor-dependent long-term potentiation and is involved in learning and memory. The existence of aspartate racemase, a candidate enzyme for d-aspartate production, has been suggested. Recently, mouse glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) has been reported to synthesize substantially d-aspartate from l-aspartate and to be involved in adult neurogenesis. In this study, we investigated the function of Got1l1 in vivo by generating and analyzing Got1l1 knockout (KO) mice. We also examined the enzymatic activity of recombinant Got1l1 in vitro. We found that Got1l1 mRNA is highly expressed in the testis, but it is not detected in the brain and submandibular gland, where d-aspartate is abundant. The d-aspartate contents of wild-type and Got1l1 KO mice were not significantly different in the testis and hippocampus. The recombinant Got1l1 expressed in mammalian cells showed l-aspartate aminotransferase activity, but lacked aspartate racemase activity. These findings suggest that Got1l1 is not the major aspartate racemase and there might be an as yet unknown d-aspartate-synthesizing enzyme.

Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding.

Decreased susceptibility to seizures induced by pentylenetetrazole in serine racemase knockout mice.

The N-methyl-D-aspartate (NMDA)-type glutamate receptor plays a key role in excitatory synaptic transmission. The overactivation of the NMDA receptor has been implicated in the development of epileptic seizures. D-Serine is a coagonist of the NMDA receptor and its biosynthesis is catalyzed by serine racemase (SR). Here, we examined the effect of d-serine deficiency on the seizures induced by a single injection of pentylenetetrazole (PTZ) using SR knockout (KO) mice. We found that, compared with wild-type (WT) mice, SR-KO mice showed the attenuation of seizure expression in terms of a significantly shortened duration of generalized seizures and resistance to generalized clonic-tonic seizures. Consistently, immunohistochemical analysis of c-Fos demonstrated that the numbers of cells expressing c-Fos induced by high-dose PTZ in the cerebral cortex, hippocampal CA1, hippocampal CA3, and the basolateral nucleus of the amygdala in WT mice were significantly higher than those in SR-KO mice. Moreover, PTZ induced an increase in extracellular glutamate level in the dentate gyrus of WT mice at two different time phases. However, such a PTZ-induced increase in glutamate level was completely inhibited in SR-KO mice. The present findings suggest that SR may be a target for the development of new therapeutic strategies for epileptic seizures.