Serine deamination by human serine racemase synergizes with antibiotics to curtail the replication of Chlamydia trachomatis.

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 ß-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.

Impact of Serine Racemase Deletion on Nicotine Discrimination.

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.

Mammalian D-cysteine: A novel regulator of neural progenitor cell proliferation Mammalian D-cysteine: A novel regulator of neural progenitor cell proliferation Endogenous D-cysteine, the stereoisomer with rapid spontaneous in vitro racemization rate, has major neural roles

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).

D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain.

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.

Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor coagonist d-serine.

Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D1 and D3 receptors. Using in vivo microdialysis, we show that D1 and D3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D3R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.

Perturbation of serine enantiomers homeostasis in the striatum of MPTP-lesioned monkeys and mice reflects the extent of dopaminergic midbrain degeneration.

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.

Endogenous d-serine exists in the mammalian brain independent of synthesis by serine racemase.

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.

Alterations of serine racemase expression determine proliferation and differentiation of neuroblastoma cells.

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.

Cross species review of the physiological role of D-serine in translationally relevant behaviors.

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.

Mammalian D-Cysteine: A new addition to the growing family of biologically relevant D-amino acids.

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.

Deletion of serine racemase reverses neuronal insulin signaling inhibition by amyloid-ß oligomers.

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 Aß42, 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 Aß42 treatment. In contrast, phosphorylated protein kinase R remained at the same levels. Further, reactive oxygen species were reduced in cultured Srr-/- neurons under Aß42 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 Aß42. 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.

Reduced d-serine levels drive enhanced non-ionotropic NMDA receptor signaling and destabilization of dendritic spines in a mouse model for studying schizophrenia.

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.

Serine Racemase Expression by Striatal Neurons.

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.

Improved NMDA Receptor Activation by the Secreted Amyloid-Protein Precursor-α in Healthy Aging: A Role for D-Serine?

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.

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95.

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.

d-Serine and d-Alanine Regulate Adaptive Foraging Behavior in Caenorhabditis elegans via the NMDA Receptor.

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.SIGNIFICANCE STATEMENT In mammals, d-serine (d-Ser) functions as an important neuromodulator of the NMDA-type glutamate receptor, which regulates higher brain functions. In Caenorhabditis elegans, previous studies failed to clearly define the physiological significance of d-Ser, d-alanine (d-Ala), and their metabolic enzymes. In this study, we found that these d-amino acids and their associated enzymes are active during food deprivation, leading to an adaptive foraging behavior. We also found that this behavior involved NMDA receptor desensitization.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

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. Graphical abstract.

D-Serine, the Shape-Shifting NMDA Receptor Co-agonist.

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.

N-Methyl-d-aspartate receptor co-agonist availability affects behavioral and neurochemical responses to cocaine: insights into comorbid schizophrenia and substance abuse.

Both schizophrenia (SZ) and substance abuse (SA) exhibit significant heritability. Moreover, N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathophysiology of both SZ and SA. We hypothesize that the high prevalence of comorbid SA in SZ is due to dysfunction of NMDARs caused by shared risk genes. We used transgenic mice with a null mutation of the gene encoding serine racemase (SR), the enzyme that synthesizes the NMDAR co-agonist d-serine and an established risk gene for SZ, to recreate the pathology of SZ. We determined the effect of NMDAR hypofunction resulting from the absence of d-serine on motivated behavior by using intracranial self-stimulation and neurotransmitter release in the nucleus accumbens by using in vivo microdialysis. Compared with wild-type mice, SR-/- mice exhibited similar baseline intracranial self-stimulation thresholds but were less sensitive to the threshold-lowering (rewarding) and the performance-elevating (stimulant) effects of cocaine. While basal dopamine (DA) and glutamate release were elevated in the nucleus accumbens of SR-/- mice, cocaine-induced increases in DA and glutamate release were blunted. γ-Amino-butyric acid efflux was unaffected in the SR-/- mice. Together, these findings suggest that the impaired NMDAR function and a consequent decrease in sensitivity to cocaine effects on behavior are mediated by blunted DA and glutamate responses normally triggered by the drug. Projected to humans, NMDAR hypofunction due to mutations in SR or other genes impacting glutamatergic function in SZ may render abused substances less potent and effective, thus requiring higher doses to achieve a hedonic response, resulting in elevated drug exposure and increased dependence/addiction.

Glutamate hypothesis in schizophrenia.

Schizophrenia is a chronic and severe psychiatric disorder that has profound impact on an individual's life and on society. Thus, developing more effective therapeutic interventions is essential. Over the past quarter-century, an abundance of evidence from pharmacologic challenges, post-mortem studies, brain imaging, and genetic studies supports the role of glutamatergic dysregulation in the pathophysiology of schizophrenia, and the results of recent randomized clinical trials based on this evidence have yielded promising results. In this article, we review the evidence that alterations in glutamatergic neurotransmission, especially focusing on the N-methyl-d-aspartate receptor (NMDAR) function, may be a critical causative feature of schizophrenia, how this contributes to pathologic circuit function in the brain, and how these insights are revealing whole new avenues for treatment development that could reduce treatment-resistant symptoms, which account for persistent disability.