A marker chromosome in psychosis identifies glycine decarboxylase (GLDC) as a novel regulator of neuronal and synaptic function in the hippocampus.

The biological significance of a small supernumerary marker chromosome that results in dosage alterations to chromosome 9p24.1, including triplication of the GLDC gene encoding glycine decarboxylase, in two patients with psychosis is unclear. In an allelic series of copy number variant mouse models, we identify that triplication of Gldc reduces extracellular glycine levels as determined by optical fluorescence resonance energy transfer (FRET) in dentate gyrus (DG) but not in CA1, suppresses long-term potentiation (LTP) in mPP-DG synapses but not in CA3-CA1 synapses, reduces the activity of biochemical pathways implicated in schizophrenia and mitochondrial bioenergetics, and displays deficits in prepulse inhibition, startle habituation, latent inhibition, working memory, sociability and social preference. Our results thus provide a link between a genomic copy number variation, biochemical, cellular and behavioral phenotypes, and further demonstrate that GLDC negatively regulates long-term synaptic plasticity at specific hippocampal synapses, possibly contributing to the development of neuropsychiatric disorders.

Impact of schizophrenia GWAS loci converge onto distinct pathways in cortical interneurons vs glutamatergic neurons during development.

Remarkable advances have been made in schizophrenia (SCZ) GWAS, but gleaning biological insight from these loci is challenging. Genetic influences on gene expression (e.g., eQTLs) are cell type-specific, but most studies that attempt to clarify GWAS loci's influence on gene expression have employed tissues with mixed cell compositions that can obscure cell-specific effects. Furthermore, enriched SCZ heritability in the fetal brain underscores the need to study the impact of SCZ risk loci in specific developing neurons. MGE-derived cortical interneurons (cINs) are consistently affected in SCZ brains and show enriched SCZ heritability in human fetal brains. We identified SCZ GWAS risk genes that are dysregulated in iPSC-derived homogeneous populations of developing SCZ cINs. These SCZ GWAS loci differential expression (DE) genes converge on the PKC pathway. Their disruption results in PKC hyperactivity in developing cINs, leading to arborization deficits. We show that the fine-mapped GWAS locus in the ATP2A2 gene of the PKC pathway harbors enhancer marks by ATACseq and ChIPseq, and regulates ATP2A2 expression. We also generated developing glutamatergic neurons (GNs), another population with enriched SCZ heritability, and confirmed their functionality after transplantation into the mouse brain. Then, we identified SCZ GWAS risk genes that are dysregulated in developing SCZ GNs. GN-specific SCZ GWAS loci DE genes converge on the ion transporter pathway, distinct from those for cINs. Disruption of the pathway gene CACNA1D resulted in deficits of Ca2+ currents in developing GNs, suggesting compromised neuronal function by GWAS loci pathway deficits during development. This study allows us to identify cell type-specific and developmental stage-specific mechanisms of SCZ risk gene function, and may aid in identifying mechanism-based novel therapeutic targets.

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.

Clinical evidence that a dysregulated master neural network modulator may aid in diagnosing schizophrenia.

There are no validated biomarkers for schizophrenia (SCZ), a disorder linked to neural network dysfunction. We demonstrate that collapsin response mediator protein-2 (CRMP2), a master regulator of cytoskeleton and, hence, neural circuitry, may form the basis for a biomarker because its activity is uniquely imbalanced in SCZ patients. CRMP2's activity depends upon its phosphorylation state. While an equilibrium between inactive (phosphorylated) and active (nonphosphorylated) CRMP2 is present in unaffected individuals, we show that SCZ patients are characterized by excess active CRMP2. We examined CRMP2 levels first in postmortem brains (correlated with neuronal morphometrics) and then, because CRMP2 is expressed in lymphocytes as well, in the peripheral blood of SCZ patients versus age-matched unaffected controls. In the brains and, more starkly, in the lymphocytes of SCZ patients <40 y old, we observed that nonphosphorylated CRMP2 was higher than in controls, while phosphorylated CRMP2 remained unchanged from control. In the brain, these changes were associated with dendritic structural abnormalities. The abundance of active CRMP2 with insufficient opposing inactive p-CRMP2 yielded a unique lowering of the p-CRMP2:CRMP2 ratio in SCZ patients, implying a disruption in the normal equilibrium between active and inactive CRMP2. These clinical data suggest that measuring CRMP2 and p-CRMP2 in peripheral blood might reflect intracerebral processes and suggest a rapid, minimally invasive, sensitive, and specific adjunctive diagnostic aid for early SCZ: increased CRMP2 or a decreased p-CRMP2:CRMP2 ratio may help cinch the diagnosis in a newly presenting young patient suspected of SCZ (versus such mimics as mania in bipolar disorder, where the ratio is high).

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.

Altered neural oscillations and behavior in a genetic mouse model of NMDA receptor hypofunction.

Abnormalities in electroencephalographic (EEG) biomarkers occur in patients with schizophrenia and those clinically at high risk for transition to psychosis and are associated with cognitive impairment. Converging evidence suggests N-methyl-D-aspartate receptor (NMDAR) hypofunction plays a central role in the pathophysiology of schizophrenia and likely contributes to biomarker impairments. Thus, characterizing these biomarkers is of significant interest for early diagnosis of schizophrenia and development of novel treatments. We utilized in vivo EEG recordings and behavioral analyses to perform a battery of electrophysiological biomarkers in an established model of chronic NMDAR hypofunction, serine racemase knockout (SRKO) mice, and their wild-type littermates. SRKO mice displayed impairments in investigation-elicited gamma power that corresponded with reduced short-term social recognition and enhanced background (pre-investigation) gamma activity. Additionally, SRKO mice exhibited sensory gating impairments in both evoked-gamma power and event-related potential amplitude. However, other biomarkers including the auditory steady-state response, sleep spindles, and state-specific power spectral density were generally neurotypical. In conclusion, SRKO mice demonstrate how chronic NMDAR hypofunction contributes to deficits in certain translationally-relevant EEG biomarkers altered in schizophrenia. Importantly, our gamma band findings suggest an aberrant signal-to-noise ratio impairing cognition that occurs with NMDAR hypofunction, potentially tied to impaired task-dependent alteration in functional connectivity.

Factors regulating serine racemase and d-amino acid oxidase expression in the mouse striatum.

d-Serine plays an important role in modulating N-methyl-d-aspartate receptor (NMDAR) neurotransmission in the mammalian brain by binding to the receptor's glycine modulatory site (GMS). The cytosolic enzyme serine racemase (SR) converts L-serine to d-serine, while the peroxisomal enzyme d-amino acid oxidase (DAAO) catalyzes the breakdown of d-serine. Although it is important to understand how the activities of SR and DAAO regulate d-serine levels, very little is known about the mechanisms that regulate the expression of SR and DAAO. In this study, we investigated whether the different centrally active drugs affect the expression of SR and DAAO in adult mouse brain. We found that the NMDAR antagonist, MK801, and cocaine, psychotropic drugs that both augment glutamate release, reduce the expression of SR and DAAO. This regulation is brain region selective, and in the case of cocaine, is reversed in part byα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). However, d-serine and antipsychotics do not regulate SR and DAAO protein levels. In a genetic model of SR disruption, we found that DAAO expression was unaltered in SR conditional knockout mice, in which tissue d-serine content remains fairly stable despite marked reduction in SR expression. This study reveals a new mechanism by which AMPAR activity could regulate NMDAR function via d-serine availability.

Postsynaptic Serine Racemase Regulates NMDA Receptor Function.

d-serine is the primary NMDAR coagonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic d-serine remains limited. Though early studies suggested d-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and d-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional KO mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at 1 month of age, which could be rescued by exogenous d-serine. Interestingly, there was a restoration of LTP by 2 months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode of d-serine action.SIGNIFICANCE STATEMENT NMDARs are key regulators of neurodevelopment and synaptic plasticity and are unique in their requirement for binding of a coagonist, which is d-serine at most forebrain synapses. However, our understanding of the mechanisms regulating synaptic d-serine availability remains limited. d-serine is synthesized in the brain by the neuronal enzyme serine racemase (SR). Here, we show dendritic and postsynaptic localization of SR and d-serine in CA1 pyramidal neurons. In addition, using single-neuron genetic deletion of SR, we establish a role of postsynaptic SR in regulating NMDAR function. These results support an autocrine mode of d-serine action at synapses.

The Discovery and Characterization of Targeted Perikaryal-Specific Brain Lesions With Excitotoxins.

The neurotoxic action of glutamic acid was first described by Lucas and Newhouse, who demonstrated neural degeneration in the inner layers of the neonatal mouse retina after systemic treatment with L-glutamate. Olney extended these findings by showing that neuronal degeneration affected other brain structures including neurons within the arcuate nucleus of the hypothalamus and the area postrema, that the lesion spared axons passing through these areas, and that the neurotoxic potency of glutamate analogs correlated with their excitatory potency, resulting in the designation "excitotoxins." As this method affected only a small number of brain regions, it was not suitable for targeted brain lesions. The Coyle laboratory showed that direct injection of the potent glutamate receptor agonist, kainic acid, into the rat striatum caused a rapid degeneration of intrinsic neurons while sparing axons of passage or termination including the unmyelinated dopaminergic terminals. Kainic acid also exhibited this perikaryal-specific and axon-sparing profile when injected into the cerebellum, hippocampus and eye. However, neuronal vulnerability was highly variable, with hippocampal CA3, pyriform cortex and amygdala neurons exhibiting great sensitivity due to kainate's high convulsive activity. In a comparison study, ibotenic acid, a potent glutamatergic agonist isolated from the amanita muscaria mushroom, was found to have excitotoxic potency comparable to kainate but was far less epileptogenic. Ibotenate produced spherical, perikaryal-specific lesions regardless of the site of injection, and experiments with specific glutamate receptor antagonists showed that its effects were mediated by the N-methyl-D-aspartate receptor. Because of this uniform neurotoxicity and near ubiquitous efficacy, ibotenic acid became the excitotoxic lesioning agent of choice. The discovery of the excitotoxic properties of the tryptophan metabolite quinolinic acid and of the anti-excitotoxic, neuroprotective effects of the related metabolite kynurenic acid in the Schwarcz laboratory then gave rise to the concept that these endogenous compounds may play causative roles in the neuropathology of a wide range of neurological and psychiatric disorders.

Activated microglia cause metabolic disruptions in developmental cortical interneurons that persist in interneurons from individuals with schizophrenia.

The mechanisms by which prenatal immune activation increase the risk for neuropsychiatric disorders are unclear. Here, we generated developmental cortical interneurons (cINs)-which are known to be affected in schizophrenia (SCZ) when matured-from induced pluripotent stem cells (iPSCs) derived from healthy controls (HCs) and individuals with SCZ and co-cultured them with or without activated microglia. Co-culture with activated microglia disturbed metabolic pathways, as indicated by unbiased transcriptome analyses, and impaired mitochondrial function, arborization, synapse formation and synaptic GABA release. Deficits in mitochondrial function and arborization were reversed by alpha lipoic acid and acetyl-L-carnitine treatments, which boost mitochondrial function. Notably, activated-microglia-conditioned medium altered metabolism in cINs and iPSCs from HCs but not in iPSCs from individuals with SCZ or in glutamatergic neurons. After removal of activated-microglia-conditioned medium, SCZ cINs but not HC cINs showed prolonged metabolic deficits, which suggests that there is an interaction between SCZ genetic backgrounds and environmental risk factors.

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.

Electroretinographic Abnormalities and Sex Differences Detected with Mesopic Adaptation in a Mouse Model of Schizophrenia: A and B Wave Analysis.

Purpose: Mesopic flash electroretinography (fERG) as a tool to identify N-methyl-d-aspartate receptor (NMDAR) hypofunction in subjects with schizophrenia shows great potential. We report the first fERG study in a genetic mouse model of schizophrenia characterized by NMDAR hypofunction from gene silencing of serine racemase (SR) expression (SR-/-), an established risk gene for schizophrenia. We analyzed fERG parameters under various background light adaptations to determine the most significant variables to allow for early identification of people at risk for schizophrenia, prior to onset of psychosis. SR is a risk gene for schizophrenia, and negative and cognitive symptoms antedate the onset of psychosis that is required for diagnosis. Methods: The scotopic, photopic, and mesopic fERGs were analyzed in male and female mice in both SR-/- and wild-type (WT) mice and also analyzed for sex differences. Amplitude and implicit time of the a- and b-wave components, b-/a-wave ratio, and Fourier transform analysis were analyzed. Results: Mesopic a- and b-wave implicit times were significantly delayed, and b-wave amplitudes, b/a ratios, and Fourier transform were significantly decreased in the male SR-/- mice compared to WT, but not in female SR-/- mice. No significant differences were observed in photopic or scotopic fERGs between genotype. Conclusions: The fERG prognostic capability may be improved by examination of background light adaptation, a larger array of light intensities, considering sex as a variable, and performing Fourier transform analyses of all waveforms. This should improve the ability to differentiate between controls and subjects with schizophrenia characterized by NMDAR hypofunction.

iPSC-derived homogeneous populations of developing schizophrenia cortical interneurons have compromised mitochondrial function.

Schizophrenia (SCZ) is a neurodevelopmental disorder. Thus, studying pathogenetic mechanisms underlying SCZ requires studying the development of brain cells. Cortical interneurons (cINs) are consistently observed to be abnormal in SCZ postmortem brains. These abnormalities may explain altered gamma oscillation and cognitive function in patients with SCZ. Of note, currently used antipsychotic drugs ameliorate psychosis, but they are not very effective in reversing cognitive deficits. Characterizing mechanisms of SCZ pathogenesis, especially related to cognitive deficits, may lead to improved treatments. We generated homogeneous populations of developing cINs from 15 healthy control (HC) iPSC lines and 15 SCZ iPSC lines. SCZ cINs, but not SCZ glutamatergic neurons, show dysregulated Oxidative Phosphorylation (OxPhos) related gene expression, accompanied by compromised mitochondrial function. The OxPhos deficit in cINs could be reversed by Alpha Lipoic Acid/Acetyl-L-Carnitine (ALA/ALC) but not by other chemicals previously identified as increasing mitochondrial function. The restoration of mitochondrial function by ALA/ALC was accompanied by a reversal of arborization deficits in SCZ cINs. OxPhos abnormality, even in the absence of any circuit environment with other neuronal subtypes, appears to be an intrinsic deficit in SCZ cINs.

Correction: iPSC-derived homogeneous populations of developing schizophrenia cortical interneurons have compromised mitochondrial function.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Targeted Treatment of Individuals With Psychosis Carrying a Copy Number Variant Containing a Genomic Triplication of the Glycine Decarboxylase Gene.

BACKGROUND: The increased mutational burden for rare structural genomic variants in schizophrenia and other neurodevelopmental disorders has so far not yielded therapies targeting the biological effects of specific mutations. We identified two carriers (mother and son) of a triplication of the gene encoding glycine decarboxylase, GLDC, presumably resulting in reduced availability of the N-methyl-D-aspartate receptor coagonists glycine and D-serine and N-methyl-D-aspartate receptor hypofunction. Both carriers had a diagnosis of a psychotic disorder. METHODS: We carried out two double-blind, placebo-controlled clinical trials of N-methyl-D-aspartate receptor augmentation of psychotropic drug treatment in these two individuals. Glycine was used in the first clinical trial, and D-cycloserine was used in the second one. RESULTS: Glycine or D-cycloserine augmentation of psychotropic drug treatment each improved psychotic and mood symptoms in placebo-controlled trials. CONCLUSIONS: These results provide two independent proof-of-principle demonstrations of symptom relief by targeting a specific genotype and explicitly link an individual mutation to the pathophysiology of psychosis and treatment response.

Neurotoxic astrocytes express the d-serine synthesizing enzyme, serine racemase, in Alzheimer's disease.

Although ß-amyloid plaques are a well-recognized hallmark of Alzheimer's disease (AD) neuropathology, no drugs reducing amyloid burden have shown efficacy in clinical trials, suggesting that once AD symptoms emerge, disease progression becomes independent of Aß production. Reactive astrocytes are another neuropathological feature of AD, where there is an emergence of neurotoxic (A1) reactive astrocytes. We find that serine racemase (SR), the neuronal enzyme that produces the N-methyl-d-aspartate receptor (NMDAR) co-agonist d-serine, is robustly expressed in A1-reactive neurotoxic astrocytes in the hippocampus and entorhinal cortex of AD subjects and an AD rat model. Furthermore, we observe intracellular signaling changes consistent with increased extra-synaptic NMDAR activation, excitotoxicity and decreased neuronal survival. Thus, reducing neurotoxic d-serine release from A1 inflammatory astrocytes could have therapeutic benefit for mild to advanced AD, when anti-amyloid strategies are ineffective.

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.