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Background. Stressful influences, depending on their severity and duration, can cause the development of pathological conditions. Repeated episodes of stress cause functional and structural changes in the central nervous system and can cause the development of depressive conditions. Depression is one of the leading mental illnesses. One of the most stress-sensitive brain structures is the hippocampus. Objective. To study is to evaluate structural changes in the hippocampus, which is considered as a heterogeneous structure with separate dorsal and ventral regions, to evaluate the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, serine racemase, synaptophysin in a mild stress model.Methods. A study of the effects of mild stress was carried out on 16 adult male Wistar rats (age 12 months, body weight 350–400 g). After acclimatization, the rats were divided into two equal groups (n = 8): intact (control) and stressed. When keeping animals, modeling and removing them from the experiment, we were guided by the Regulations for Carrying Out Work Using Laboratory Animals and the Declaration of Helsinki. Experimental modeling of depression in animals was induced by mild stress exposure for 7 days (30 minutes daily). Euthanasia was performed in a CO2 incubator. The brain was fixed in neutral buffered 10% formalin. Paraffin sections were made in the frontal plane, stained with hematoxylin and eosin, thionin using the Nissl method and examined at a level from –2.40 to –3.96 mm relative to bregma using an Axio Lab A1 microscope (Carl Zeiss Microscopy GmbH, Germany). Photo documentation was carried out with an AxioCam 105 color camera (Carl Zeiss Microscopy GmbH, Germany). Using the Image Analysis module of the ZEN 1.1.2.0 program (Carl Zeiss Microscopy GmbH, Germany) in the pyramidal layer of the hippocampus. Statistical analysis was performed with Microsoft Office Excel 2016 (Microsoft, USA) and Prism 6 (GraphPad Software Inc., USA). Comparisons of two conditions were made by nonparametric Mann-Whitney-U test to avoid a statistical bias of unequal data distribution. The level of significance was set at p < 0.05. The summarized data were presented as a as mean ± standard error of mean.Results. Functional research methods and assessment of pathological changes in hippocampal neurons are presented. An increase in the relative number of wrinkled hyperchromatic pyramidal neurons in the dorsal cornu ammonis field 3 in stressed rats was noted by 23.6% (p < 0.05) compared to the control. There was an increase in the relative number of inducible nitric oxide synthase-immunopositive neurons in the dorsal cornu ammonis field 3 by 40% ( p < 0.05) and the relative area of inducible nitric oxide synthase-immunoreactive material by 35% (p < 0.05) in the pyramidal layer of cornu ammonis field 3 in stressed rats. A decrease in the relative area of synaptophysin-immunopositive material in stressed rats was found in the ventral cornu ammonis field 3 compared to the control group by 16.8% (p < 0.05); decrease in the relative area of serine racemase-immunopositive material in dorsal cornu ammonis field 3 by 4.3% (p < 0.05) and ventral cornu ammonis field 3 by 7.8% (p < 0.05).Conclusion. The results of the study demonstrate that mild stress is an adequate model of depression in rats. In animals exposed to mild stress, pronounced morphological signs of damage to hippocampal neurons were revealed; motor and indicative exploratory activity decreases. Differences were found in morphofunctional changes in the dorsal and ventral parts of the hippocampus under the influence of mild stress. In cornu ammonis field 3 of the dorsal hippocampus, in contrast to the ventral section, more pronounced signs of damage to pyramidal layer neurons were observed. The increase in the relative number of inducible nitric oxide synthase-immunopositive neurons and the relative area of inducible nitric oxide synthase-immunoreactive material in the cornu ammonis field 3 pyramidal layer in stressed rats indicates an increase in nitric oxide production and the participation of nitrooxide-dependent free radical mechanisms of damage to hippocampal neurons. The decrease in the relative area of synaptophysin-immunoreactive material in stressed rats may contribute to changes in synaptic plasticity. A decrease in the relative area of serine racemase-immunoreactive material in the dorsal and ventral parts of cornu ammonis field 3 is considered to be a sign of a possible decrease in N-methyl-D-aspartate-dependent neurotransmission in the hippocampus under stress.

Background: D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas. Methods: Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line. Results and Discussion: D-cysteine is synthesized by serine racemase (SR) and SR−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR−/− mice. Conclusions: Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

It is crucial to regulate N-methyl-D-aspartate (NMDA) function bivalently depending on the central nervous system (CNS) conditions. CNS disorders with NMDA hyperfunction are involved in the pathogenesis of neurotoxic and/or neurodegenerative disorders with elevated D-serine, one of the NMDA receptor co-agonists. On the contrary, NMDA-enhancing agents have been demonstrated to improve psychotic symptoms and cognition in CNS disorders with NMDA hypofunction. Serine racemase (SR), the enzyme regulating both D- and L-serine levels through both racemization (catalysis from L-serine to D-serine) and β-elimination (degradation of both D- and L-serine), emerges as a promising target for bidirectional regulation of NMDA function. In this study, we explored using dimethyl malonate (DMM), a pro-drug of the SR inhibitor malonate, to modulate NMDA activity in C57BL/6J male mice via intravenous administration. Unexpectedly, 400 mg/kg DMM significantly elevated, rather than decreased (as a racemization inhibitor), D-serine levels in the cerebral cortex and plasma. This outcome prompted us to investigate the regulatory effects of dodecagalloyl-α-D-xylose (α12G), a synthesized tannic acid analog, on SR activity. Our findings showed that α12G enhanced the racemization activity of human SR by about 8-fold. The simulated and fluorescent assay of binding affinity suggested a noncooperative binding close to the catalytic residues, Lys56 and Ser84. Moreover, α12G treatment can improve behaviors associated with major CNS disorders with NMDA hypofunction including hyperactivity, prepulse inhibition deficit, and memory impairment in animal models of positive symptoms and cognitive impairment of psychosis. In sum, our findings suggested α12G is a potential therapeutic for treating CNS disorders with NMDA hypofunction. [ABSTRACT FROM AUTHOR]

Schizophrenia is a psychiatric disorder that affects over 20 million people globally. Notably, schizophrenia is associated with decreased density of dendritic spines and decreased levels of d-serine, a co-agonist required for opening of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that lowered d-serine levels associated with schizophrenia would enhance ion flux-independent signaling by the NMDAR, driving destabilization and loss of dendritic spines. We tested our hypothesis using the serine racemase knockout (SRKO) mouse model, which lacks the enzyme for d-serine production. We show that activity-dependent spine growth is impaired in SRKO mice, but can be acutely rescued by exogenous d-serine. Moreover, we find a significant bias of synaptic plasticity toward spine shrinkage in the SRKO mice as compared to wild-type littermates. Notably, we demonstrate that enhanced ion flux-independent signaling through the NMDAR contributes to this bias toward spine destabilization, which is exacerbated by an increase in synaptic NMDARs in hippocampal synapses of SRKO mice. Our results support a model in which lowered d-serine levels associated with schizophrenia enhance ion flux-independent NMDAR signaling and bias toward spine shrinkage and destabilization.

Bridging the gap between preclinical models of neurological and psychiatric disorders with their human manifestations is necessary to understand their underlying mechanisms, identify biomarkers, and develop novel therapeutics. Cognitive and social impairments underlie multiple neuropsychiatric and neurological disorders and are often comorbid with sleep disturbances, which can exacerbate poor outcomes. Importantly, many symptoms are conserved between vertebrates and invertebrates, although they may have subtle differences. Therefore, it is essential to determine the molecular mechanisms underlying these behaviors across different species and their translatability to humans. Genome-wide association studies have indicated an association between glutamatergic gene variants and both the risk and frequency of psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. For example, changes in glutamatergic neurotransmission, such as glutamate receptor subtype N-methyl-d-aspartate receptor (NMDAR) hypofunction, have been shown to contribute to the pathophysiology of schizophrenia. Furthermore, in neurological disorders, such as traumatic brain injury and Alzheimer's disease, hyperactivation of NMDARs leads to synaptic damage. In addition to glutamate binding, NMDARs require the binding of a co-agonist d-serine or glycine to the GluN1 subunit to open. d-serine, which is racemized from l-serine by the neuronal enzyme serine racemase (SRR), and both SRR and d-serine are enriched in cortico-limbic brain regions. d-serine is critical for complex behaviors, such as cognition and social behavior, where dysregulation of its synthesis and release has been implicated in many pathological conditions. In this review, we explore the role of d-serine in behaviors that are translationally relevant to multiple psychiatric and neurological disorders in different models across species. [ABSTRACT FROM AUTHOR]

Mammalian D‐Cysteine is racemized from L‐cysteine by serine racemase, a pyridoxal phosphate (PLP)‐dependent enzyme. Endogenous D‐Cysteine plays a role in neural development by inhibiting proliferation of neural progenitor cells (NPCs) via protein kinase B (AKT) signaling mediated by the FoxO family of transcription factors. D‐Cysteine binds to Myristoylated Alanine Rich C Kinase Substrate (MARCKS) and alters phosphorylation at Ser 159/163 and its translocation from the membrane. By racemizing serine and cysteine, mammalian serine racemase may play important roles in neural development highlighting its importance in psychiatric disorders. [ABSTRACT FROM AUTHOR]

Background: Neurodegenerative processes in Alzheimer’s disease (AD) are associated with excitotoxicity mediated by the N-methyl-D-aspartate receptor (NMDAR). D-Serine is an endogenous co-agonist necessary for NMDAR-mediated excitotoxicity. In the mammalian brain, it is produced by serine racemase (SRR) from L-serine, suggesting that dysregulation of L-serine, D-serine, or SRR may contribute to AD pathogenesis. Objective and methods: We examined the contributions of D-serine to AD pathology in the AppNL−G−F/NL−G−F gene knock-in (APPKI) mouse model of AD. We first examined brain SRR expression levels and neuropathology in APPKI mice and then assessed the effects of long-term D-serine supplementation in drinking water on neurodegeneration. To further confirm the involvement of endogenous D-serine in AD progression, we generated Srr gene-deleted APPKI (APPKI-SRRKO) mice. Finally, to examine the levels of brain amino acids, we conducted liquid chromatography–tandem mass spectrometry. Results: Expression of SRR was markedly reduced in the retrosplenial cortex (RSC) of APPKI mice at 12 months of age compared with age-matched wildtype mice. Neuronal density was decreased in the hippocampal CA1 region but not altered significantly in the RSC. D-Serine supplementation exacerbated neuronal loss in the hippocampal CA1 of APPKI mice, while APPKI-SRRKO mice exhibited attenuated astrogliosis and reduced neuronal death in the hippocampal CA1 compared with APPKI mice. Furthermore, APPKI mice demonstrated marked abnormalities in the cortical amino acid levels that were partially reversed in APPKI-SRRKO mice. Conclusion: These findings suggest that D-serine participates in the regional neurodegenerative process in the hippocampal CA1 during the amyloid pathology of AD and that reducing brain D-serine can partially attenuate neuronal loss and reactive astrogliosis. Therefore, regulating SRR could be an effective strategy to mitigate NMDAR-dependent neurodegeneration during AD progression. [ABSTRACT FROM AUTHOR]

Loss of dopaminergic midbrain neurons perturbs l-serine and d-serine homeostasis in the post-mortem caudate putamen (CPu) of Parkinson's disease (PD) patients. However, it is unclear whether the severity of dopaminergic nigrostriatal degeneration plays a role in deregulating serine enantiomers' metabolism. Here, through high-performance liquid chromatography (HPLC), we measured the levels of these amino acids in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and MPTP-plus-probenecid (MPTPp)-treated mice to determine whether and how dopaminergic midbrain degeneration affects the levels of serine enantiomers in various basal ganglia subregions. In addition, in the same brain regions, we measured the levels of key neuroactive amino acids modulating glutamatergic neurotransmission, including l-glutamate, glycine, l-aspartate, d-aspartate, and their precursors l-glutamine, l-asparagine. In monkeys, MPTP treatment produced severe denervation of nigrostriatal dopaminergic fibers (⁓75%) and increased the levels of serine enantiomers in the rostral putamen (rPut), but not in the subthalamic nucleus, and the lateral and medial portion of the globus pallidus. Moreover, this neurotoxin significantly reduced the protein expression of the astrocytic serine transporter ASCT1 and the glycolytic enzyme GAPDH in the rPut of monkeys. Conversely, concentrations of d-serine and l-serine, as well as ASCT1 and GAPDH expression were unaffected in the striatum of MPTPp-treated mice, which showed only mild dopaminergic degeneration (⁓30%). These findings unveil a link between the severity of dopaminergic nigrostriatal degeneration and striatal serine enantiomers concentration, ASCT1 and GAPDH expression. We hypothesize that the up-regulation of d-serine and l-serine levels occurs as a secondary response within a homeostatic loop to support the metabolic and neurotransmission demands imposed by the degeneration of dopaminergic neurons.

BackgroundNeurodegenerative processes in Alzheimer’s disease (AD) are associated with excitotoxicity mediated by the N-methyl-D-aspartate receptor (NMDAR). D-Serine is an endogenous co-agonist necessary for NMDAR-mediated excitotoxicity. In the mammalian brain, it is produced by serine racemase (SRR) from L-serine, suggesting that dysregulation of L-serine, D-serine, or SRR may contribute to AD pathogenesis.Objective and methodsWe examined the contributions of D-serine to AD pathology in the AppNL–G–F/NL–G–F gene knock-in (APPKI) mouse model of AD. We first examined brain SRR expression levels and neuropathology in APPKI mice and then assessed the effects of long-term D-serine supplementation in drinking water on neurodegeneration. To further confirm the involvement of endogenous D-serine in AD progression, we generated Srr gene-deleted APPKI (APPKI-SRRKO) mice. Finally, to examine the levels of brain amino acids, we conducted liquid chromatography–tandem mass spectrometry.ResultsExpression of SRR was markedly reduced in the retrosplenial cortex (RSC) of APPKI mice at 12 months of age compared with age-matched wild-type mice. Neuronal density was decreased in the hippocampal CA1 region but not altered significantly in the RSC. D-Serine supplementation exacerbated neuronal loss in the hippocampal CA1 of APPKI mice, while APPKI-SRRKO mice exhibited attenuated astrogliosis and reduced neuronal death in the hippocampal CA1 compared with APPKI mice. Furthermore, APPKI mice demonstrated marked abnormalities in the cortical amino acid levels that were partially reversed in APPKI-SRRKO mice.ConclusionThese findings suggest that D-serine participates in the regional neurodegenerative process in the hippocampal CA1 during the amyloid pathology of AD and that reducing brain D-serine can partially attenuate neuronal loss and reactive astrogliosis. Therefore, regulating SRR could be an effective strategy to mitigate NMDAR-dependent neurodegeneration during AD progression.

The NMDA receptor hypofunction has been implicated in schizophrenia, memory impairment, and Alzheimer's disease. Modulating the abundance of D-serine, a co-agonist of the NMDA receptor, is a strategy to treat symptoms of the NMDA receptor hypofunction. In contrast to D-amino acid oxidase (DAAO) inhibitors, which aim at decreasing the loss of D-serine, this study tried to identify serine racemase (SRR) agonists, which boost the conversion of L-serine to D-serine. We used holo and apo structures of human SRR for the molecular docking against the National Cancer Institute (NCI) and ZINC compound databases and validated their efficacy by in vitro SRR activity assay. We identified NSC294149 (2-amino-3-(3-nitroimidazo[1,2-a]pyridin-2-yl)sulfanylpropanoic acid) as a potential SRR agonist and confirmed its amelioration of the hazard ratio of survival of the AD model of fruit fly (Drosophila melanogaster). These results suggest that the SRR agonist could be a drug design target against the NMDA receptor hypofunction symptoms. [ABSTRACT FROM AUTHOR]

Activation of N -methyl- d -aspartate receptors (NMDARs) requires binding of a co-agonist in addition to l - glutamate. d - serine binds to the co-agonist site on GluN1 subunits of NMDARs and modulates glutamatergic neurotransmission. While loss of GluN1 subunits in mice results in neonatal death due to respiratory failure, animals that lack a d - serine synthetic enzyme, serine racemase (SR), show grossly normal growth. However, SR-independent origins of d - serine in the brain remain unclarified. In the present study, we investigated the origin of brain d - serine in mice. Loss of SR significantly reduced d - serine in the cerebral cortex, but a portion of d - serine remained in both neonates and adults. Although d - serine was also produced by intestinal bacteria, germ-free experiments did not influence d - serine levels in the cerebral cortex. In addition, treatment of SR-knockout mice with antibiotics showed a significant reduction of intestinal d - serine, but no reduction in the brain. On the other hand, restriction of dietary intake reduced systemic circulation of d - serine and resulted in a slight decrease of d - serine in the cerebral cortex, but did not account for brain d - serine found in the SR-knockout mice. Therefore, our findings show that endogenous d - serine of non-SR origin exists in the brain. Such previously unrecognized, SR-independent, endogenous d - serine may contribute baseline activity of NMDARs, especially in developing brain, which has minimal SR expression. • Mammalian brain contains d - serine unoriginated from serine racemase throughout life (83). • d - serine production by symbiotic microbes has no contribution to brain d - serine level (85). • Non-dietary d - serine derived not from serine racemase exists in the brain (73). [ABSTRACT FROM AUTHOR]

Impaired activation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) by D-serine is linked to cognitive aging. Whether this deregulation may be used to initiate pharmacological strategies has yet to be considered. To this end, we performed electrophysiological extracellular recordings at CA3/CA1 synapses in hippocampal slices from young and aged mice. We show that 0.1 nM of the soluble N-terminal recombinant fragment of the secreted amyloid-protein precursor-α (sAPPα) added in the bath significantly increased NMDAR activation in aged but not adult mice without impacting basal synaptic transmission. In addition, sAPPα rescued the age-related deficit of theta-burst-induced long-term potentiation. Significant NMDAR improvement occurred in adult mice when sAPPα was raised to 1 nM, and this effect was drastically reduced in transgenic mice deprived of D-serine through genetic deletion of the synthesizing enzyme serine racemase. Altogether, these results emphasize the interest to consider sAPPα treatment targeting D-serine-dependent NMDAR deregulation to alleviate cognitive aging. [ABSTRACT FROM AUTHOR]

For nearly fifty years, the dopamine hypothesis has dominated our understanding of the pathophysiology of schizophrenia and provided the lone target for drug development. However, with the exception of clozapine, the dopamine D2 receptor antagonizing anti-psychotic drugs have little impact on the negative symptoms and cognitive deficits, aspects of the disorder that robustly predict outcome. Pathologic studies reveal cortical atrophy and wide-spread loss of glutamatergic synaptic spines, unexplained by dopaminergic malfunction. Recent genome-wide association studies indicate that at least thirty risk genes for schizophrenia encode proteins localized to the glutamatergic synapse and inhibit glutamate neurotransmission, especially at the NMDA receptor. To function, the NMDA receptor requires the binding of glycine (primarily in the cerebellum and brainstem) or D-serine (in forebrain) to the NR1 channel subunit of the NMDA receptor. Genetically silencing the gene (srr) encoding serine racemase, the biosynthetic enzyme for D-serine, results in forebrain NMDA receptor hypofunction. The srr −/− mice have 90 % loss of endogenous D-serine and approximately 70 % decrease in NMDA receptor function. Several animal models of schizophrenia are based on behavioral and pharmacologic strategies, which have negligible validity with regard to the fundamental etiology of schizophrenia. We summarize here the results of a mouse model, in which srr , one of the two dozen or more risk gene for schizophrenia that affect NMDA receptor function, has been inactivated. The srr −/− mice exhibit striking similarities to schizophrenia including cortical atrophy, loss of cortico-limbic glutamatergic synapses, increased sub-cortical dopamine release, EEG abnormalities, and cognitive impairments. The limited efficacy of drugs targeting the glutamatergic synapse on DSM-5 diagnosed criteria for schizophrenia used in clinical trials may reflect the fact that only 30 % of the patients have impaired glutamatergic neurotransmission, resulting from the genetic heterogeneity of the disorder. [ABSTRACT FROM AUTHOR]

Serine racemase (SR) catalyzes L-serine racemization to activate the N-methyl-D-aspartate receptor (NMDAR). NMDAR activation is associated with the progression of acute-to-chronic neuropathic pain. This study aimed to investigate NMDAR antagonist interactions with SR to obtain potential chronic pain target therapy. Several NMDAR antagonist drugs were obtained from the drug bank, and malonate was used as a control inhibitor. Ligands were prepared using the open babel feature on PyRx. The SR structure was obtained from Protein data bank (PDB) (3l6B) and then docked with ligands using the AutoDock Vina. Haloperidol had a lower binding affinity than malonate and other ligands. Ethanol had the highest binding affinity than other drugs but could bind to the Adenosine triphosphate (ATP)-binding domain. Haloperidol is bound to reface that function for reprotonation in racemization reaction to produce D-serine. Halothane bond with Arg135 residues aligned negatively charged substrates to be reprotonated properly by reface. Tramadol is bound to amino acid residues in the triple serine loop, which determines the direction of the SR reaction. Several NMDAR antagonists such as haloperidol, halothane, ethanol, and tramadol bind to SR in the specific binding site. It reveals that SR potentially becomes an alternative target for chronic pain treatment.

Dysregulation of insulin signaling in the Alzheimer's disease (AD) brain has been extensively reported. Serine racemase (SR) modulates insulin secretion in pancreatic islets. This study aimed to examine whether SR regulates insulin synthesis and secretion in neurons, thereby modulating insulin signaling in the AD brain. Srr‐knockout (Srr−/−) mice generated with the CRISPR/Cas9 technique were used. Using immunofluorescence and fluorescence in situ hybridization, levels of insulin protein and insulin(ins2) mRNA were significantly increased in the hippocampal but not in hypothalamic sections of Srr−/− mice compared with WT mice. Real‐time quantitative PCR revealed that ins2 mRNA from primary hippocampal neuronal cultures of Srr−/− mice was significantly increased compared with that from cultured neurons of WT mice. Notably, the secretion of proinsulin C‐peptide was increased in Srr−/− neurons relative to WT neurons. By examining membrane fractional proteins with immunoblotting, Srr−/− neurons retained ATP‐dependent potassium channels on plasmalemma and correspondingly contained higher levels of p‐AMPK. After treatment with Aβ42, the phosphorylation levels of insulin receptor substrate at serine 616 636 (p‐IRS1ser616,636) were significantly lower, whereas p‐AKT308 and p‐AKT473 were higher in Srr−/− neurons than in WT neurons, respectively. The phosphorylated form of c‐Jun N‐terminal kinase decreased in the cultured Srr−/− neurons relative to the WT neurons upon Aβ42 treatment. In contrast, phosphorylated protein kinase R remained at the same levels. Further, reactive oxygen species were reduced in cultured Srr−/− neurons under Aβ42 treatment relative to the WT neurons. Collectively, our study indicated that Srr deletion promoted insulin synthesis and secretion of proinsulin C‐peptide, thereby reversing insulin resistance by Aβ42. This study suggests that targeting the neuronal SR may be utilized to enhance insulin signaling which is inhibited at the early stage of the AD brain. [ABSTRACT FROM AUTHOR]

Although the role of serine racemase (SR) in neuropsychiatric disorders has been extensively studied, its role in cell proliferation and differentiation remains unclear. Deletion of Srr, the encoding gene for SR, has been shown to reduce dendritic arborization and dendritic spine density in the brains of adult mice, whereas increased SR levels have been associated with differentiation in cell cultures. Previously, we demonstrated that valproic acid induces differentiation in the N2A neuroblastoma cell line, and that this differentiation is associated with increased SR expression. These observations suggest that SR may have a role in cell proliferation and differentiation. We herein found that both valproic acid and all‐trans retinoic acid induced N2A differentiation. In contrast, knockdown of SR reduced levels of differentiation, increased N2A proliferation, promoted cell cycle entry, and modulated expression of cell cycle‐related proteins. To further evaluate the effects of SR expression on cell proliferation and differentiation, we used an in vivo model of neuroblastoma in nude mice. N2A cells stably expressing scramble shRNA (Srrwt‐N2A) or specific Srr shRNA (Srrkd‐N2A) were subcutaneously injected into nude mice. The weights and volumes of Srrwt‐N2A‐derived tumors were lower than Srrkd‐N2A‐derived tumors. Furthermore, Srrwt‐N2A‐derived tumors were significantly mitigated by intraperitoneal injection of valproic acid, whereas Srrkd‐N2A‐derived tumors were unaffected. Taken together, our findings demonstrate for the first time that alterations in SR expression determine the transition between proliferation and differentiation in neural progenitor cells. Thus, in addition to its well‐established roles in neuropsychiatric disorders, our study has highlighted a novel role for SR in cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Schizophrenia (SCZ) is a multifactorial psychotic disorder characterized by positive and negative symptoms as well as cognitive impairments. To advance the current treatments, it is important to improve animal models by considering the multifactorial etiology, thus by combining different risk factors. The objective of our study was to explore in a new mouse model, the impact of genetic deletion of serine racemase (genetic vulnerability) combined with an early stress factor induced by maternal separation (early environmental exposure) in the context of SCZ development. The face validity of the model was assessed through a wide range of behavioral experiments. The 2-hit mice displayed an increased locomotor activity mimicking positive symptoms, working memory impairment, cognitive deficits and recognition memory alterations, which could reflect neophobia. This new multifactorial model therefore presents an interesting phenotype for modelling animal model with partial behavioral and cognitive deficits associated with SCZ., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, (Copyright © 2024. Published by Elsevier B.V.)

Background: D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas., Methods: Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line., Results and Discussion: D-cysteine is synthesized by serine racemase (SR) and SR -/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR -/- mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR -/- mice., Conclusions: Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Endometrial cancer (EC) kills about 76,000 women worldwide, with the highest incidence in industrialized countries. Because of the rise in disease mortality and new diagnoses, EC is now a top priority for women’s health. Serine racemase (SRR) is thought to play a role in the central nervous system, but its role in cancers, particularly in EC, is largely unknown. The current study starts with a pan-cancer examination of SRR’s expression and prognostic value before delving into SRR’s potential cancer-suppressing effect in patients with EC. SRR may affect the endometrial tumor immune microenvironment, according to subsequent immune-related analysis. SRR expression is also linked to several genes involved in specific pathways such as ferroptosis, N6-methyladenosine methylation, and DNA damage repair. Finally, we used the expression, correlation, and survival analyses to investigate the upstream potential regulatory non-coding RNAs of SRR. Overall, our findings highlight the prognostic significance of SRR in patients with EC, and we can formulate a reasonable hypothesis that SRR influences metabolism and obstructs key carcinogenic processes in EC. [ABSTRACT FROM AUTHOR]

D‐amino acids are being recognized as functionally important molecules in mammals. We recently identified endogenous D‐cysteine in mammalian brain. D‐cysteine is present in neonatal brain in substantial amounts (mM) and decreases with postnatal development. D‐cysteine binds to MARCKS and a host of proteins implicated in cell division and neurodevelopmental disorders. D‐cysteine decreases phosphorylation of MARCKS in neural progenitor cells (NPCs) affecting its translocation. D‐cysteine controls NPC proliferation by inhibiting AKT signaling. Exogenous D‐cysteine inhibits AKT phosphorylation at Thr 308 and Ser 473 in NPCs. D‐cysteine treatment of NPCs led to 50% reduction in phosphorylation of Foxo1 at Ser 256 and Foxo3a at Ser 253. We hypothesize that in the developing brain endogenous D‐cysteine is as a physiologic regulator of NPC proliferation by inhibiting AKT signaling mediated by Foxo1 and Foxo3a. Endogenous D‐cysteine may regulate mammalian neurodevelopment with roles in schizophrenia and Alzheimer's disease (AD). [ABSTRACT FROM AUTHOR]

The pupae of lepidopterans contain high concentrations of endogenous d -serine. In the silkworm Bombyx mori , d -serine is negligible during the larval stage but increases markedly during the pupal stage, reaching 50% of the total free serine. However, the physiological function of d -serine and the enzyme responsible for its production is unknown. Herein, we identified a new type of pyridoxal 5′-phosphate (PLP)-dependent serine racemase (SR) that catalyses the racemization of l -serine to d -serine in B. mori. This silkworm SR (BmSR) has an N-terminal PLP-binding domain that is homologous to mammalian SR and a C-terminal putative ligand-binding regulatory-like domain (ACT-like domain) that is absent in mammalian SR. Similar to mammalian SRs, BmSR catalyses the racemization and dehydration of both serine isomers. However, BmSR is different from mammalian SRs as evidenced by its insensitivity to Mg2+/Ca2+ and Mg-ATP—which are required for activation of mammalian SRs—and high d -serine dehydration activity. At the pupal stage, the SR activity was predominantly detected in the fat body, which was consistent with the timing and localization of BmSR expression. The results are an important first step in elucidating the physiological significance of d -serine in lepidopterans. [ABSTRACT FROM AUTHOR]

Serine racemase (SR) catalyzes L-serine racemization to activate the N-methyl-D-aspartate receptor (NMDAR). NMDAR activation is associated with the progression of acute-to-chronic neuropathic pain. This study aimed to investigate NMDAR antagonist interactions with SR to obtain potential chronic pain target therapy. Several NMDAR antagonist drugs were obtained from the drug bank, and malonate was used as a control inhibitor. Ligands were prepared using the open babel feature on PyRx. The SR structure was obtained from Protein data bank (PDB) (3l6B) and then docked with ligands using the AutoDock Vina. Haloperidol had a lower binding affinity than malonate and other ligands. Ethanol had the highest binding affinity than other drugs but could bind to the Adenosine triphosphate (ATP)-binding domain. Haloperidol is bound to reface that function for reprotonation in racemization reaction to produce D-serine. Halothane bond with Arg135 residues aligned negatively charged substrates to be reprotonated properly by reface. Tramadol is bound to amino acid residues in the triple serine loop, which determines the direction of the SR reaction. Several NMDAR antagonists such as haloperidol, halothane, ethanol, and tramadol bind to SR in the specific binding site. It reveals that SR potentially becomes an alternative target for chronic pain treatment. [ABSTRACT FROM AUTHOR]

Schizophrenia is a psychiatric disorder that affects over 20 million people globally. Notably, schizophrenia is associated with decreased density of dendritic spines and decreased levels of d-serine, a co-agonist required for opening of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that lowered d-serine levels associated with schizophrenia would enhance ion flux-independent signaling by the NMDAR, driving destabilization and loss of dendritic spines. We tested our hypothesis using the serine racemase knockout (SRKO) mouse model, which lacks the enzyme for d-serine production. We show that activity-dependent spine growth is impaired in SRKO mice, but can be acutely rescued by exogenous d-serine. Moreover, we find a significant bias of synaptic plasticity toward spine shrinkage in the SRKO mice as compared to wild-type littermates. Notably, we demonstrate that enhanced ion flux-independent signaling through the NMDAR contributes to this bias toward spine destabilization, which is exacerbated by an increase in synaptic NMDARs in hippocampal synapses of SRKO mice. Our results support a model in which lowered d-serine levels associated with schizophrenia enhance ion flux-independent NMDAR signaling and bias toward spine shrinkage and destabilization.

Human serine racemase (hSR) is a pyridoxal-5′-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg2+, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction. [ABSTRACT FROM AUTHOR]

Simple Summary: Parotid, submandibular, and sublingual glands in rat were found to contain high concentrations of d-aspartic acid and low ones of d-serine and d-alanine. In addition to d-amino acid oxidase and d-aspartate oxidase, serine racemase was also detected in all three of these major salivary glands, as were N-methyl-d-aspartic acid receptor subunits NR1 and NR2D, but not NR2A, NR2B, or NR2C. Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands. [ABSTRACT FROM AUTHOR]

d-serine is synthesized by serine racemase (SR) and is a co-agonist at forebrain N-methyl-d-aspartate receptors (NMDARs). d-serine and SR are expressed primarily in neurons, but not in quiescent astrocytes. In this study, we examined the localization of d-serine and SR in the mouse striatum and the effects of genetically silencing SR expression in GABAergic interneurons (iSR−/−). iSR−/− mice had substantially reduced SR expression almost exclusively in striatum, but only exhibited marginal d-serine reduction. SR positive cells in the striatum showed strong co-localization with dopamine- and cyclic AMP-regulated neuronal phosphoprotein (DARPP32) in wild type mice. Transgenic fluorescent reporter mice for either the D1 or D2 dopamine receptors exhibited a 65:35 ratio for co-localization with D1and D2 receptor positive cells, respectively. These results indicate that GABAergic medium spiny neurons receiving dopaminergic inputs in striatum robustly and uniformly express SR. In behavioral tests, iSR−/− mice showed a blunted response to the hedonic and stimulant effects of cocaine, without affecting anxiety-related behaviors. Because the cocaine effects have been shown in the constitutive SR−/− mice, the restriction of the blunted response to cocaine to iSR−/− mice reinforces the conclusion that d-serine in striatal GABAergic neurons plays an important role in mediating dopaminergic stimulant effects. Results in this study suggest that SR in striatal GABAergic neurons is synthesizing d-serine, not as a glutamatergic co-transmitter, but rather as an autocrine whereby the GABAergic neurons control the excitability of their NMDARs by determining the availability of the co-agonist, d-serine. [ABSTRACT FROM AUTHOR]

The NMDA receptor hypofunction has been implicated in schizophrenia, memory impairment, and Alzheimer’s disease. Modulating the abundance of D-serine, a co-agonist of the NMDA receptor, is a strategy to treat symptoms of the NMDA receptor hypofunction. In contrast to D-amino acid oxidase (DAAO) inhibitors, which aim at decreasing the loss of D-serine, this study tried to identify serine racemase (SRR) agonists, which boost the conversion of L-serine to D-serine. We used holo and apo structures of human SRR for the molecular docking against the National Cancer Institute (NCI) and ZINC compound databases and validated their efficacy by in vitro SRR activity assay. We identified NSC294149 (2-amino-3-(3-nitroimidazo[1,2-a]pyridin-2-yl)sulfanylpropanoic acid) as a potential SRR agonist and confirmed its amelioration of the hazard ratio of survival of the AD model of fruit fly (Drosophila melanogaster). These results suggest that the SRR agonist could be a drug design target against the NMDA receptor hypofunction symptoms.

Introduction: The high comorbidity between schizophrenia and cigarette smoking points to a possible shared heritable factor predisposing individuals with schizophrenia to nicotine addiction. The N-methyl-D-aspartate (NMDA) receptor has been highly implicated in both schizophrenia and nicotine addiction., Methods: In the present study, we used mice with a null mutation on the serine racemase gene (srr), an established risk gene for schizophrenia, which encodes the enzyme to produce the NMDA receptor co-agonist D-serine, to model the pathology of schizophrenia and to determine whether NMDA receptor hypofunction reduced the ability of srr-/- mice to identify nicotine's subjective effects. Established nicotine discrimination procedures were used to train srr-/- and wild-type (WT) mice to discriminate 0.4 mg/kg nicotine under a 10-response fixed-ratio (FR10) schedule of food reinforcement., Results: Results show that WT mice reliably acquired 0.4 mg/kg nicotine discrimination in about 54 training session, whereas srr-/- mice failed to acquire robust 0.4 mg/kg nicotine discrimination even after extended (>70) training sessions. These results show that NDMA receptor hypofunction in srr-/- mice decreased sensitivity to the interoceptive effects of nicotine., Conclusions: Projected to humans, NMDA receptor hypofunction caused by mutations to the serine racemase gene in schizophrenia may reduce sensitivity to nicotine's subjective effects leading to increased nicotine consumption to produce the same effects as those unaffected by schizophrenia., Implications: There is high comorbidity between schizophrenia and nicotine dependence as well as possible shared genetic risk factors between the two. The serine racemase knockout mouse (srr-/-) with NMDA receptor hypofunction has been developed a model for schizophrenia. We found that srr-/- mice were unable to acquire 0.4 mg/kg nicotine discrimination, whilst wild-type mice readily discriminated nicotine. These results show that decreased NMDA receptor function present in srr-/- mice and patients with schizophrenia may result in reduced sensitivity to nicotine's interoceptive effects, leading to increased nicotine consumption to produce the same subjective effects as those unaffected by schizophrenia., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via β-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human serine racemase (hSR) catalyzes the biosynthesis of D‐serine, an obligatory co‐agonist of the NMDA receptors. It was previously found that the reversible S‐nitrosylation of Cys113 reduces hSR activity. Here, we show by site‐directed mutagenesis, fluorescence spectroscopy, mass spectrometry, and molecular dynamics that S‐nitrosylation stabilizes an open, less‐active conformation of the enzyme. The reaction of hSR with either NO or nitroso donors is conformation−dependent and occurs only in the conformation stabilized by the allosteric effector ATP, in which the ε‐amino group of Lys114 acts as a base toward the thiol group of Cys113. In the closed conformation stabilized by glycine—an active‐site ligand of hSR—the side chain of Lys114 moves away from that of Cys113, while the carboxyl side‐chain group of Asp318 moves significantly closer, increasing the thiol pKa and preventing the reaction. We conclude that ATP binding, glycine binding, and S‐nitrosylation constitute a three‐way regulation mechanism for the tight control of hSR activity. We also show that Cys113 undergoes H2O2‐mediated oxidation, with loss of enzyme activity, a reaction also dependent on hSR conformation. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. Methods: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood–retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid–Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. Results: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood–retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood–retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. Conclusions/interpretation: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Human serine racemase (hSR) is a pyridoxal-5′-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg2+, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction.

Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands.

Aging is associated with a decrease in N-methyl-D-aspartate (NMDA) receptor function, which is critical for maintaining synaptic plasticity, learning, and memory. Activation of the NMDA receptor requires binding of the neurotransmitter glutamate and also the presence of co-agonist D-serine at the glycine site. The enzymatic conversion of L-serine to D-serine is facilitated by the enzyme serine racemase (SR). Subsequently, SR plays a pivotal role in regulating NMDA receptor activity, thereby impacting synaptic plasticity and memory processes in the central nervous system. As such, age-related changes in the expression of SR could contribute to decreased NMDA receptor function. However, age-associated changes in SR expression levels in the medial and lateral prefrontal cortex (mPFC, lPFC), and in the dorsal hippocampal subfields, CA1, CA3, and dentate gyrus (DG), have not been thoroughly elucidated. Therefore, the current studies were designed to determine the SR expression profile, including protein levels and mRNA, for these regions in aged and young male and female Fischer-344 rats. Our results demonstrate a significant reduction in SR expression levels in the mPFC and all hippocampal subfields of aged rats compared to young rats. No sex differences were observed in the expression of SR. These findings suggest that the decrease in SR levels may play a role in the age-associated reduction of NMDA receptor function in brain regions crucial for cognitive function and synaptic plasticity.

Xerosis is a common sign of both type 1 and type 2 diabetes mellitus (DM), and patients with DM and mouse models for DM show a compromised epidermal permeability barrier. Barrier defects then allow the entry of foreign substances into the skin, triggering inflammation, infection, and worsening skin symptoms. Characterizing how barrier abnormalities develop in DM could suggest treatments for xerosis and other skin disease traits. Because the proper ratio, as well as proper bulk amounts, of heterogeneous ceramide species are keys to forming a competent barrier, we investigated how ceramide metabolism is affected in type 1 DM using a mouse model (induced by streptozotocin). Chronic inflammation, evident in the skin of mice with DM, leads to (i) decreased de novo ceramide production through serine racemase activation-mediated attenuation of serine palmitoyl transferase activity by D-serine; (ii) changes in ceramide synthase activities and expression that modify the ratio of ceramide molecular species; and (iii) increased ceramide-1-phosphate, a proinflammatory lipid mediator, that stimulates inflammatory cytokine expression (TNFα and IFN-γ). Together, chronic inflammation affects ceramide metabolism, which attenuates epidermal permeability barrier formation, and ceramide-1-phosphate could amplify this inflammation. Alleviation of chronic inflammation is a credible approach for normalizing barrier function and ameliorating diverse skin abnormalities in DM., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

D-Serine (D-Ser) is a coagonist for NMDA-type glutamate receptors and is thus important for higher brain function. D-Ser is synthesized by serine racemase and degraded by D-amino acid oxidase. However, the significance of these enzymes and the relevant functions of D-amino acids remain unclear. Here, we show that in the nematode Caenorhabditis elegans, the serine racemase homolog SERR-1 and D-amino acid oxidase DAAO-1 control an adaptive foraging behavior. Similar to many organisms, C. elegans immediately initiates local search for food when transferred to a new environment. With prolonged food deprivation, the worms exhibit a long-range dispersal behavior as the adaptive foraging strategy. We found that serr-1 deletion mutants did not display this behavior, whereas daao-1 deletion mutants immediately engaged in long-range dispersal after food removal. A quantitative analysis of D-amino acids indicated that D-Ser and D-alanine (D-Ala) are both synthesized and suppressed during food deprivation. A behavioral pharmacological analysis showed that the long-range dispersal behavior requires NMDA receptor desensitization. Long-term pretreatment with D-Ala, as well as with an NMDA receptor agonist, expanded the area searched by wild-type worms immediately after food removal, whereas pretreatment with D-Ser did not. We propose that D-Ser and D-Ala are endogenous regulators that cooperatively induce the long-range dispersal behavior in C. elegans through actions on the NMDA receptor. [ABSTRACT FROM AUTHOR]

Shape-shifting, a phenomenon wide-spread in folklore, refers to the ability to physically change from one identity to another, typically from an innocuous entity to a destructive one. The amino acid d-serine over the last 25 years has "shape-shifted" into several identities: a purported glial transmitter activating N-methyl-d-aspartate receptors (NMDARs), a co-transmitter concentrated in excitatory glutamatergic neurons, an autocrine that is released at dendritic spines to prime their post-synaptic NMDARs for an instantaneous response to glutamate and an excitotoxic moiety released from inflammatory (A1) astrocytes. This article will review evidence in support of these scenarios and the artifacts that misled investigators of the true identity of d-serine. [ABSTRACT FROM AUTHOR]

Serine racemase (SR) is a pyridoxal 5′‐phosphate (PLP)‐containing enzyme that converts l‐serine to d‐serine, an endogenous co‐agonist for the N‐methyl‐d‐aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates d‐serine levels by the reversible racemization of l‐serine to d‐serine, as well as the catabolism of serine by α,β‐elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78–155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four‐stranded β‐sheet of the small domain, including changes in the ϕ–ψ angles of all three residues in the C‐terminal β‐strand (residues 149–151). In the malonate‐bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six‐residue β‐strand (residues 78–83), but in the holoenzyme the β‐strand is only four residues (78–81) and His82 has ϕ–ψ values in the α‐helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure‐guided design of small‐molecule modulators for this important but to date undrugged target. [ABSTRACT FROM AUTHOR]

Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury. [ABSTRACT FROM AUTHOR]

Hamdy N El-Tallawy,1 Tahia H Saleem,2 Abdallah MAA El-Ebidi,3 Mohammed H Hassan,4 Romany H Gabra,1 Wafaa MA Farghaly,1 Nagwa Abo El-Maali,5 Hoda S Sherkawy3 1Department of Neuropsychiatry, 2Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Assiut University, Assiut, 3Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Aswan University, Aswan, 4Department of Medical Biochemistry and Molecular Biology, Qena Faculty of Medicine, South Valley University, Qena, 5Department of Chemistry, Faculty of Science, Assiut University, Assiut, Egypt Background: Schizophrenia is a typical N-methyl-d-aspartate receptor (NMDA-R) hypofunction disorder. Decreased d-serine (d-Ser) levels in the periphery occur in schizophrenia and may reflect decreased availability of d-Ser to activate NMDA-R in the brain.Objective: The objective of this study was to investigate the role of d-Ser metabolism in the pathogenesis of schizophrenia via biochemical assays and correlates, the serum level of d-Ser, d-serine racemase (SR) (responsible for its formation from l-serine [l-Ser]) and d-amino acid oxidase (DAAO) (responsible for its catabolism), among different clinical types of schizophrenia patients.Patients and methods: This cross-sectional case–control study was carried out on 100 patients and 50 controls. They were recruited from the outpatients’ psychiatric unit of the Neuropsychiatric Department of Assiut University Hospital, Upper Egypt. The type of schizophrenia was determined according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), while the severity of schizophrenia was determined according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5). Serum d-Ser levels were estimated using high-performance liquid chromatography (HPLC), while serum SR and DAAO were measured using commercially available enzyme-linked immunosorbent assay kits.Results: There were significantly lower mean serum levels of d-Ser and SR and significantly higher mean serum levels of DAAO (P-value 35.6 pg/mL for DAAO.Conclusion: The present study confirms that disturbed d-Ser metabolism could be implicated in the pathogenesis of schizophrenia. Keywords: d-serine, serine racemase, d-amino acid oxidase, schizophrenia, HPLC

A seizure is one of the leading neurological disorders. NMDA receptor-mediated neuronal excitation has been thought to be essential for epileptogenesis. As an endogenous co-agonist of the NMDA receptor, D-serine has been suggested to play a role in epileptogenesis. However, the underlying mechanisms remain unclear. In the current study, we investigated the effects of antagonizing two key enzymes in D-serine metabolism on the development of seizures and the downstream signaling. Our results showed that serine racemase (SR), a key enzyme in regulating the L-to-D-serine conversion, was significantly up-regulated in hippocampal astrocytes in rats and patients who experienced seizure, in comparison with control rats and patients. L-aspartic acid β-hydroxamate (LaaβH), an inhibitor of SR, significantly prolonged the latencies of seizures, shortened the durations of seizures, and decreased the total EEG power in rats. In contrast, D-amino acid oxidase inhibitor 5-chlorobenzo[d]isoxazol-3-ol (CBIO), which can increase D-serine levels, showed the opposite effects. Furthermore, our data showed that LaaβH and CBIO significantly affected the phosphorylation of Extracellular Signal-regulated Kinase (ERK). Antagonizing or activating ERK could significantly block the effects of LaaβH/CBIO on the occurrence of seizures. In summary, our study revealed that D-serine is involved in the development of epileptic seizures, partially through ERK signaling, indicating that the metabolism of D-serine may be targeted for the treatment of epilepsy.

Serine racemase (SR) is the first racemase enzyme to be identified in human biology and converts L-serine to D-serine, an important neuronal signaling molecule that serves as a co-agonist of the NMDA (N-methyl-D-aspartate) receptor. This overview describes key molecular features of the enzyme, focusing on the side chains and binding motifs that control PLP (pyridoxal phosphate) cofactor binding as well as activity modulation through the binding of both divalent cations and ATP, the latter showing allosteric modulation. Discussed are catalytically important residues in the active site including K56 and S84—the si- and re-face bases, respectively,—and R135, a residue that appears to play a critical role in the binding of both negatively charged alternative substrates and inhibitors. The interesting bifurcated mechanism followed by this enzyme whereby substrate L-serine can be channeled either into D-serine (racemization pathway) or into pyruvate (β-elimination pathway) is discussed extensively, as are studies that focus on a key loop region (the so-called “triple serine loop”), the modification of which can be used to invert the normal in vitro preference of this enzyme for the latter pathway over the former. The possible cross-talk between the PLP enzymes hSR and hCBS (human cystathionine β-synthase) is discussed, as the former produces D-serine and the latter produces H2S, both of which stimulate the NMDAR and both of which have been implicated in neuronal infarction pursuant to ischemic stroke. Efforts to gain a more complete mechanistic understanding of these PLP enzymes are expected to provide valuable insights for the development of specific small molecule modulators of these enzymes as tools to study their roles in neuronal signaling and in modulation of NMDAR function.