Francesco Errico - Academia.edu (original) (raw)

Papers by Francesco Errico

High performance liquid chromatography determination of l-glutamate, l-glutamine and glycine content in brain, cerebrospinal fluid and blood serum of patients affected by Alzheimer’s disease

Amino Acids

Prenatal expression of d-aspartate oxidase causes early cerebral d-aspartate depletion and influences brain morphology and cognitive functions at adulthood

Amino Acids

Nature Precedings

In the present study, we demonstrated that D-aspartate acts as an in vitro and in vivo neuromodul... more In the present study, we demonstrated that D-aspartate acts as an in vitro and in vivo neuromodulatory molecule upon hippocampal NMDAR transmission. Accordingly, we showed that this D-amino acid, widely expressed during embryonic phase, was able to strongly influence hippocampus-related functions at adulthood. Thus, while up-regulated levels of D-aspartate increased LTP and spatial memory in four-month old adult mice, the prolonged deregulation of this molecule in thirteen-month old animals induced a substantial acceleration of age-dependent decay of synaptic plasticity and cognitive functions. Moreover, we highlighted a role for Daspartate in enhancing NMDAR-dependent synaptic plasticity through an inducible "turn-on/turnoff-like mechanism". Strikingly, we also showed that D-aspartate, when administered to aged mice, strongly rescued their physiological synaptic decay and attenuated their cognitive deterioration. In conclusion, our data suggest a tantalizing hypothesis for which this in-embryooccurring D-amino acid, might disclose plasticity windows in the aging brain.

Scientific Reports

Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor... more Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson's Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of Mptp-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in pD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy.

Experimental Neurology

In mammals, free D-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very... more In mammals, free D-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme D-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo −/−) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo −/− mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo −/− animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.

Frontiers in Psychiatry

Errico et al. Dysregulated D-Aspartate Metabolism in Schizophrenia suggest a possible involvement... more Errico et al. Dysregulated D-Aspartate Metabolism in Schizophrenia suggest a possible involvement of dysregulated embryonic D-aspartate metabolism in schizophrenia pathophysiology and, in turn, highlight the potential use of free D-aspartate supplementation as a new add-on therapy for treating the cognitive symptoms of this mental illness.

Scientific Reports

The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and... more The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene's regulatory regions. Furthermore, for DAO and DDO, our singlemolecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses. Free D-serine (D-Ser) and D-aspartate (D-Asp) act as a co-agonist and agonist at N-methyl-D-aspartate receptors (NMDARs), respectively, and influence numerous brain functions dependent by this subclass of glutamate receptors 1-4. These D-amino acids are present in the mammalian brain with an age-and region-specific distribution pattern 5-7. Based on the NMDAR hypofunction hypothesis of schizophrenia 8,9 , perturbation of D-Ser and D-Asp

PloS one, 2017

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-medi... more Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp,...

PloS one, 2017

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually a... more In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the put...

Scientific Reports, 2017

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). ... more D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the secondgeneration antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

npj Schizophrenia, 2017

It is long acknowledged that the N-methyl D-aspartate receptor co-agonist, D-serine, plays a cruc... more It is long acknowledged that the N-methyl D-aspartate receptor co-agonist, D-serine, plays a crucial role in several N-methyl Daspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides D-serine, another free Damino acid, D-aspartate, is involved in the activation of N-methyl D-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl D-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of D-aspartate and D-serine to stimulate N-methyl D-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two D-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of D-aspartate and D-serine. Consistent with previous work, we found that D-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for D-aspartate catabolism, D-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for D-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered D-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl D-aspartate receptor-mediated transmission in schizophrenia.

Epigenetics, 2016

We performed ultra-deep methylation analysis at single molecule level of the promoter region of d... more We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

Reviews in the Neurosciences, 2009

Within the pool of endogenous amino acids, serine and aspartate are the only two residues occurri... more Within the pool of endogenous amino acids, serine and aspartate are the only two residues occurring at significant concentrations in free Dform in mammalian tissues. D-Serine (D-Ser) is mainly localized in the forebrain structures of the CNS throughout embryonic development and postnatal phase. Compelling evidence demonstrates that D-Ser has a functional role as an endogenous co-agonist at JV-methyl-Daspartate receptors (NMDARs) and shows its beneficial involvement in psychiatric disorders including schizophrenia. On the other hand, knowledge concerning the role of free D-Asp in 'mammals has so far been less extensive. D-Asp occurs in the brain-as well as in peripheral tissues including the endocrine glands. In endocrine glands, D-Asp levels increase during the postnatal period in concomitance with their functional maturation. The involvement of D-Asp in the regulation of the synthesis and/or release of different hormones has been clearly demonstrated. However, its biological significance in the brain is still obscure. D-Asp appears with a peculiar temporal pattern of localization, being abundant during embryonic development and strongly decreasing after birth. This phenomenon is the result of the

Persistent elevation of D-Aspartate enhances NMDA receptor-mediated responses in mouse substantia nigra pars compacta dopamine neurons

Neuropharmacology, 2016

Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive... more Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive functions. NMDA receptors play a crucial role in modulating the activity of these cells. Considering that the amino-acid D-Aspartate has been recently shown to be an endogenous NMDA receptor agonist, the aim of the present study was to examine the effects of D-Aspartate on the functional properties of nigral dopamine neurons. We compared the electrophysiological actions of D-Aspartate in control and D-aspartate oxidase gene (Ddo(-/-)) knock-out mice that show a concomitant increase in brain D-Aspartate levels, improved synaptic plasticity and cognition. Finally, we analyzed the effects of L-Aspartate, a known dopamine neuron endogenous agonist in control and Ddo(-/-) mice. We show that D- and L-Aspartate excite dopamine neurons by activating NMDA, AMPA and metabotropic glutamate receptors. Ddo deletion did not alter the intrinsic properties or dopamine sensitivity of dopamine neurons. However, NMDA-induced currents were enhanced and membrane levels of the NMDA receptor GluN1 and GluN2A subunits were increased. Inhibition of excitatory amino-acid transporters caused a marked potentiation of D-Aspartate, but not L-Aspartate currents, in Ddo(-/-) neurons. This is the first study to show the actions of D-Aspartate on midbrain dopamine neurons, activating not only NMDA but also non-NMDA receptors. Our data suggest that dopamine neurons, under conditions of high D-Aspartate levels, build a protective uptake mechanism to compensate for increased NMDA receptor numbers and cell hyper-excitation, which could prevent the consequent hyper-dopaminergia in target zones that can lead to neuronal degeneration, motor and cognitive alterations.

Neuropsychopharmacology, 2015

Rasd2 modulates prefronto-striatal phenotypes in humans and "schizophrenia-like behaviors" in mic... more Rasd2 modulates prefronto-striatal phenotypes in humans and "schizophrenia-like behaviors" in mice Supplementary Information Supplementary methods Human methods Prefrontal grey matter volume and prefrontal-striatal physiology in healthy humans Subjects. Healthy Caucasian subjects from the region of Puglia, Italy, were recruited for the study and were evaluated with the Structured Clinical Interview for DSM-IV (SCID) (First et al, 1996) to exclude any psychiatric disorder. Further exclusion criteria were history of drug or alcohol abuse, active drug use in the past year, head trauma with loss of consciousness, and any significant medical condition. Handedness (Edinburgh Inventory) (Oldfield, 1971) and total IQ (WAIS-R) were also measured. The study was approved by the local Institutional Review Board (Comitato Etico Locale Indipendente Azienda Ospedaliera "Ospedale Policlinico Consorziale" Bari). After complete description of the protocol and procedures, written informed consent was obtained by all participants, in accordance with the Helsinki Declaration. All subjects were genotyped for RASD2 rs6518956 and underwent the imaging protocols. Genotyping. Genotyping was performed by Real-Time PCR with TaqMan® SNP Genotyping Assay (Assay ID: C_29199375_10; Applied Biosystems). DNA was extracted from whole blood samples using standard procedures. All alleles displayed Hardy-Weinberg equilibrium in both the functional magnetic resonance imaging (fMRI) (χ2 = 0.50, p = 0.47) and voxel brain morphometry (VBM) (χ 2 = 0.36, p = 0.54) samples. RASD2 genotype distribution is reported respectively in Supplementary Table S1. sMRI acquisition parameters. Three-dimensional images were acquired with a 3.0 Tesla General Electric MR scanner using a T1-weighted SPGR sequence (TE = min full; flip angle, 6°; field of

Scientific Reports, 2015

Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls m... more Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses. The Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein discovered by subtractive hybridization 1 and highly expressed throughout the dorsal striatum and nucleus accumbens of rodent brain 2-4. Rhes transcription is regulated by thyroid hormones during development 4-7 and by dopamine (DA) in the adult rat brain 8. Early studies in cell lines 3,9 indicated that Rhes, most likely by binding to G α i, reduces G-protein-coupled receptor (GPCR)-mediated accumulation of cAMP 10. Accordingly, Rhes knockout (KO) mice have

Rasd2 Modulates Psychotomimetic Drug Effects in Mice and Schizophrenia-related Phenotypes in Humans

European Psychiatry, 2015

D-aspartate dysregulation in Ddo−/− mice modulates phencyclidine-induced gene expression changes of postsynaptic density molecules in cortex and striatum

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2015

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been considered a key alteration in schizo... more N-methyl-D-aspartate receptor (NMDAR) hypofunction has been considered a key alteration in schizophrenia pathophysiology. Thus, several strategies aimed at enhancing glutamatergic transmission, included the introduction in therapy of D-amino acids, such as D-serine and D-cycloserine augmentation, have been proposed to counteract difficult-to-treat symptoms or treatment-resistant forms of schizophrenia. Another D-amino acid, D-aspartate, has recently gained increasing interest for its role in NMDAR activation and has been found reduced in post-mortem cortex of schizophrenia patients. NMDAR is the core of the postsynaptic density (PSD), a postsynaptic site involved in glutamate signaling and responsive to antipsychotic treatment. In this study, we investigated striatal and cortical gene expression of key PSD transcripts (i.e. Homer1a, Homer1b/c, and PSD-95) in mice with persistently elevated brain D-aspartate-levels, i.e. the D-aspartate-oxidase knockout mice (Ddo(-/-)). These animal models were analyzed both in naive condition and after phencyclidine (PCP) treatment. Naive Ddo(-/-) mice showed decreased Homer1a expression in the prefrontal cortex, increased Homer1b/c expression in the striatum, and decreased PSD-95 expression in the striatum and in the cortex. Acute PCP treatment restored, and even potentiated, Homer1a expression in the prefrontal cortex of mutant mice, while it had limited effects on the other genes. These results suggest that persistently elevated D-aspartate, by enhancing NMDA transmission, may cause complex adaptive mechanisms affecting Homer1a, which in turn may explain the recently demonstrated protective effects of this D-amino acid against PCP-induced behavioral alterations, such as ataxic behavior.

d-Aspartate: An endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia

Journal of pharmaceutical and biomedical analysis, Jan 31, 2015

Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a p... more Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a physiological endogenous co-agonist for synaptic N-Methyl d-Aspartate (NMDA) receptors (NMDARs), and is involved in the pathophysiology of schizophrenia. Much less is known about the biological meaning of d-aspartate. d-Aspartate is present at high levels in the embryo brain and strongly decreases at post-natal phases. Temporal reduction of d-aspartate levels depends on the post-natal onset of d-aspartate oxidase (DDO), an enzyme able to selectively catabolize this d-amino acid. Pharmacological evidence indicates that d-aspartate binds to and activates NMDARs. Characterization of genetic and pharmacological mouse models with abnormally higher levels of d-aspartate has evidenced that increased d-aspartate enhances hippocampal NMDAR-dependent synaptic plasticity, dendritic morphology and spatial memory. In line with the hypothesis of a hypofunction of NMDARs in the pathogenesis of schizophr...

Bimodal effect of D-aspartate on brain aging processes: insights from animal models

Journal of biological regulators and homeostatic agents

Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryo... more Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryotes, including mammals. In particular, free D-serine and D-aspartate are found in the brain of mammals. Notably, D-aspartate occurs at substantial levels in the embryo brain to then consistently decrease at post-natal phases. Temporal regulation of D-aspartate content depends on the post-natal onset of D-aspartate oxidase expression, the only known enzyme able to catabolize this D-amino acid. Pharmacological evidence indicates that D-aspartate binds and activates NMDA receptors (NMDARs). To decipher the physiological function of D-aspartate in mammals, in the last years, genetic and pharmacological mouse models with abnormally higher levels of this D-amino acid have been generated. Overall, these animal models have pointed out a significant neuromodulatory role for D-aspartate in the regulation of NMDAR-dependent functions. Indeed, increased content of D-aspartate are able to increase hi...

High performance liquid chromatography determination of l-glutamate, l-glutamine and glycine content in brain, cerebrospinal fluid and blood serum of patients affected by Alzheimer’s disease

Amino Acids

Prenatal expression of d-aspartate oxidase causes early cerebral d-aspartate depletion and influences brain morphology and cognitive functions at adulthood

Amino Acids

Nature Precedings

In the present study, we demonstrated that D-aspartate acts as an in vitro and in vivo neuromodul... more In the present study, we demonstrated that D-aspartate acts as an in vitro and in vivo neuromodulatory molecule upon hippocampal NMDAR transmission. Accordingly, we showed that this D-amino acid, widely expressed during embryonic phase, was able to strongly influence hippocampus-related functions at adulthood. Thus, while up-regulated levels of D-aspartate increased LTP and spatial memory in four-month old adult mice, the prolonged deregulation of this molecule in thirteen-month old animals induced a substantial acceleration of age-dependent decay of synaptic plasticity and cognitive functions. Moreover, we highlighted a role for Daspartate in enhancing NMDAR-dependent synaptic plasticity through an inducible "turn-on/turnoff-like mechanism". Strikingly, we also showed that D-aspartate, when administered to aged mice, strongly rescued their physiological synaptic decay and attenuated their cognitive deterioration. In conclusion, our data suggest a tantalizing hypothesis for which this in-embryooccurring D-amino acid, might disclose plasticity windows in the aging brain.

Scientific Reports

Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor... more Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson's Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of Mptp-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in pD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy.

Experimental Neurology

In mammals, free D-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very... more In mammals, free D-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme D-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo −/−) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo −/− mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo −/− animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.

Frontiers in Psychiatry

Errico et al. Dysregulated D-Aspartate Metabolism in Schizophrenia suggest a possible involvement... more Errico et al. Dysregulated D-Aspartate Metabolism in Schizophrenia suggest a possible involvement of dysregulated embryonic D-aspartate metabolism in schizophrenia pathophysiology and, in turn, highlight the potential use of free D-aspartate supplementation as a new add-on therapy for treating the cognitive symptoms of this mental illness.

Scientific Reports

The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and... more The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene's regulatory regions. Furthermore, for DAO and DDO, our singlemolecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses. Free D-serine (D-Ser) and D-aspartate (D-Asp) act as a co-agonist and agonist at N-methyl-D-aspartate receptors (NMDARs), respectively, and influence numerous brain functions dependent by this subclass of glutamate receptors 1-4. These D-amino acids are present in the mammalian brain with an age-and region-specific distribution pattern 5-7. Based on the NMDAR hypofunction hypothesis of schizophrenia 8,9 , perturbation of D-Ser and D-Asp

PloS one, 2017

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-medi... more Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp,...

PloS one, 2017

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually a... more In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the put...

Scientific Reports, 2017

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). ... more D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the secondgeneration antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

npj Schizophrenia, 2017

It is long acknowledged that the N-methyl D-aspartate receptor co-agonist, D-serine, plays a cruc... more It is long acknowledged that the N-methyl D-aspartate receptor co-agonist, D-serine, plays a crucial role in several N-methyl Daspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides D-serine, another free Damino acid, D-aspartate, is involved in the activation of N-methyl D-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl D-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of D-aspartate and D-serine to stimulate N-methyl D-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two D-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of D-aspartate and D-serine. Consistent with previous work, we found that D-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for D-aspartate catabolism, D-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for D-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered D-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl D-aspartate receptor-mediated transmission in schizophrenia.

Epigenetics, 2016

We performed ultra-deep methylation analysis at single molecule level of the promoter region of d... more We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

Reviews in the Neurosciences, 2009

Within the pool of endogenous amino acids, serine and aspartate are the only two residues occurri... more Within the pool of endogenous amino acids, serine and aspartate are the only two residues occurring at significant concentrations in free Dform in mammalian tissues. D-Serine (D-Ser) is mainly localized in the forebrain structures of the CNS throughout embryonic development and postnatal phase. Compelling evidence demonstrates that D-Ser has a functional role as an endogenous co-agonist at JV-methyl-Daspartate receptors (NMDARs) and shows its beneficial involvement in psychiatric disorders including schizophrenia. On the other hand, knowledge concerning the role of free D-Asp in 'mammals has so far been less extensive. D-Asp occurs in the brain-as well as in peripheral tissues including the endocrine glands. In endocrine glands, D-Asp levels increase during the postnatal period in concomitance with their functional maturation. The involvement of D-Asp in the regulation of the synthesis and/or release of different hormones has been clearly demonstrated. However, its biological significance in the brain is still obscure. D-Asp appears with a peculiar temporal pattern of localization, being abundant during embryonic development and strongly decreasing after birth. This phenomenon is the result of the

Persistent elevation of D-Aspartate enhances NMDA receptor-mediated responses in mouse substantia nigra pars compacta dopamine neurons

Neuropharmacology, 2016

Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive... more Dopamine neurons in the substantia nigra pars compacta regulate not only motor but also cognitive functions. NMDA receptors play a crucial role in modulating the activity of these cells. Considering that the amino-acid D-Aspartate has been recently shown to be an endogenous NMDA receptor agonist, the aim of the present study was to examine the effects of D-Aspartate on the functional properties of nigral dopamine neurons. We compared the electrophysiological actions of D-Aspartate in control and D-aspartate oxidase gene (Ddo(-/-)) knock-out mice that show a concomitant increase in brain D-Aspartate levels, improved synaptic plasticity and cognition. Finally, we analyzed the effects of L-Aspartate, a known dopamine neuron endogenous agonist in control and Ddo(-/-) mice. We show that D- and L-Aspartate excite dopamine neurons by activating NMDA, AMPA and metabotropic glutamate receptors. Ddo deletion did not alter the intrinsic properties or dopamine sensitivity of dopamine neurons. However, NMDA-induced currents were enhanced and membrane levels of the NMDA receptor GluN1 and GluN2A subunits were increased. Inhibition of excitatory amino-acid transporters caused a marked potentiation of D-Aspartate, but not L-Aspartate currents, in Ddo(-/-) neurons. This is the first study to show the actions of D-Aspartate on midbrain dopamine neurons, activating not only NMDA but also non-NMDA receptors. Our data suggest that dopamine neurons, under conditions of high D-Aspartate levels, build a protective uptake mechanism to compensate for increased NMDA receptor numbers and cell hyper-excitation, which could prevent the consequent hyper-dopaminergia in target zones that can lead to neuronal degeneration, motor and cognitive alterations.

Neuropsychopharmacology, 2015

Rasd2 modulates prefronto-striatal phenotypes in humans and "schizophrenia-like behaviors" in mic... more Rasd2 modulates prefronto-striatal phenotypes in humans and "schizophrenia-like behaviors" in mice Supplementary Information Supplementary methods Human methods Prefrontal grey matter volume and prefrontal-striatal physiology in healthy humans Subjects. Healthy Caucasian subjects from the region of Puglia, Italy, were recruited for the study and were evaluated with the Structured Clinical Interview for DSM-IV (SCID) (First et al, 1996) to exclude any psychiatric disorder. Further exclusion criteria were history of drug or alcohol abuse, active drug use in the past year, head trauma with loss of consciousness, and any significant medical condition. Handedness (Edinburgh Inventory) (Oldfield, 1971) and total IQ (WAIS-R) were also measured. The study was approved by the local Institutional Review Board (Comitato Etico Locale Indipendente Azienda Ospedaliera "Ospedale Policlinico Consorziale" Bari). After complete description of the protocol and procedures, written informed consent was obtained by all participants, in accordance with the Helsinki Declaration. All subjects were genotyped for RASD2 rs6518956 and underwent the imaging protocols. Genotyping. Genotyping was performed by Real-Time PCR with TaqMan® SNP Genotyping Assay (Assay ID: C_29199375_10; Applied Biosystems). DNA was extracted from whole blood samples using standard procedures. All alleles displayed Hardy-Weinberg equilibrium in both the functional magnetic resonance imaging (fMRI) (χ2 = 0.50, p = 0.47) and voxel brain morphometry (VBM) (χ 2 = 0.36, p = 0.54) samples. RASD2 genotype distribution is reported respectively in Supplementary Table S1. sMRI acquisition parameters. Three-dimensional images were acquired with a 3.0 Tesla General Electric MR scanner using a T1-weighted SPGR sequence (TE = min full; flip angle, 6°; field of

Scientific Reports, 2015

Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls m... more Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses. The Ras homolog enriched in striatum (Rhes) is a small GTP-binding protein discovered by subtractive hybridization 1 and highly expressed throughout the dorsal striatum and nucleus accumbens of rodent brain 2-4. Rhes transcription is regulated by thyroid hormones during development 4-7 and by dopamine (DA) in the adult rat brain 8. Early studies in cell lines 3,9 indicated that Rhes, most likely by binding to G α i, reduces G-protein-coupled receptor (GPCR)-mediated accumulation of cAMP 10. Accordingly, Rhes knockout (KO) mice have

Rasd2 Modulates Psychotomimetic Drug Effects in Mice and Schizophrenia-related Phenotypes in Humans

European Psychiatry, 2015

D-aspartate dysregulation in Ddo−/− mice modulates phencyclidine-induced gene expression changes of postsynaptic density molecules in cortex and striatum

Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2015

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been considered a key alteration in schizo... more N-methyl-D-aspartate receptor (NMDAR) hypofunction has been considered a key alteration in schizophrenia pathophysiology. Thus, several strategies aimed at enhancing glutamatergic transmission, included the introduction in therapy of D-amino acids, such as D-serine and D-cycloserine augmentation, have been proposed to counteract difficult-to-treat symptoms or treatment-resistant forms of schizophrenia. Another D-amino acid, D-aspartate, has recently gained increasing interest for its role in NMDAR activation and has been found reduced in post-mortem cortex of schizophrenia patients. NMDAR is the core of the postsynaptic density (PSD), a postsynaptic site involved in glutamate signaling and responsive to antipsychotic treatment. In this study, we investigated striatal and cortical gene expression of key PSD transcripts (i.e. Homer1a, Homer1b/c, and PSD-95) in mice with persistently elevated brain D-aspartate-levels, i.e. the D-aspartate-oxidase knockout mice (Ddo(-/-)). These animal models were analyzed both in naive condition and after phencyclidine (PCP) treatment. Naive Ddo(-/-) mice showed decreased Homer1a expression in the prefrontal cortex, increased Homer1b/c expression in the striatum, and decreased PSD-95 expression in the striatum and in the cortex. Acute PCP treatment restored, and even potentiated, Homer1a expression in the prefrontal cortex of mutant mice, while it had limited effects on the other genes. These results suggest that persistently elevated D-aspartate, by enhancing NMDA transmission, may cause complex adaptive mechanisms affecting Homer1a, which in turn may explain the recently demonstrated protective effects of this D-amino acid against PCP-induced behavioral alterations, such as ataxic behavior.

d-Aspartate: An endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia

Journal of pharmaceutical and biomedical analysis, Jan 31, 2015

Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a p... more Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a physiological endogenous co-agonist for synaptic N-Methyl d-Aspartate (NMDA) receptors (NMDARs), and is involved in the pathophysiology of schizophrenia. Much less is known about the biological meaning of d-aspartate. d-Aspartate is present at high levels in the embryo brain and strongly decreases at post-natal phases. Temporal reduction of d-aspartate levels depends on the post-natal onset of d-aspartate oxidase (DDO), an enzyme able to selectively catabolize this d-amino acid. Pharmacological evidence indicates that d-aspartate binds to and activates NMDARs. Characterization of genetic and pharmacological mouse models with abnormally higher levels of d-aspartate has evidenced that increased d-aspartate enhances hippocampal NMDAR-dependent synaptic plasticity, dendritic morphology and spatial memory. In line with the hypothesis of a hypofunction of NMDARs in the pathogenesis of schizophr...

Bimodal effect of D-aspartate on brain aging processes: insights from animal models

Journal of biological regulators and homeostatic agents

Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryo... more Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryotes, including mammals. In particular, free D-serine and D-aspartate are found in the brain of mammals. Notably, D-aspartate occurs at substantial levels in the embryo brain to then consistently decrease at post-natal phases. Temporal regulation of D-aspartate content depends on the post-natal onset of D-aspartate oxidase expression, the only known enzyme able to catabolize this D-amino acid. Pharmacological evidence indicates that D-aspartate binds and activates NMDA receptors (NMDARs). To decipher the physiological function of D-aspartate in mammals, in the last years, genetic and pharmacological mouse models with abnormally higher levels of this D-amino acid have been generated. Overall, these animal models have pointed out a significant neuromodulatory role for D-aspartate in the regulation of NMDAR-dependent functions. Indeed, increased content of D-aspartate are able to increase hi...