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Introduction: Schizophrenia (SCZ) and autism spectrum disorder (ASD) are neurodevelopmental diseases characterized by different psychopathological manifestations and divergent clinical trajectories. Various alterations at glutamatergic synapses have been reported in both disorders, including abnormal NMDA and metabotropic receptor signaling., Methods: We conducted a bicentric study to assess the blood serum levels of NMDA receptors-related glutamatergic amino acids and their precursors, including L-glutamate, L-glutamine, D-aspartate, L-aspartate, L-asparagine, D-serine, L-serine and glycine, in ASD, SCZ patients and their respective control subjects. Specifically, the SCZ patients were subdivided into treatment-resistant and non-treatment-resistant SCZ patients, based on their responsivity to conventional antipsychotics., Results: D-serine and D-aspartate serum reductions were found in SCZ patients compared to controls. Conversely, no significant differences between cases and controls were found in amino acid concentrations in the two ASD cohorts analyzed., Discussion: This result further encourages future research to evaluate the predictive role of selected D-amino acids as peripheral markers for SCZ pathophysiology and diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Garofalo, De Simone, Motta, Nuzzo, De Grandis, Bruno, Boeri, Riccio, Pastore, Bravaccio, Iasevoli, Salvatore, Pollegioni, Errico, de Bartolomeis and Usiello.)

Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DβH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA., (© 2023. The Author(s).)

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1β, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

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., Competing Interests: Declaration of Competing Interest All authors declare no competing non-financial or financial interests to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

L-serine generated in astrocytes plays a pivotal role in modulating essential neurometabolic processes, while its enantiomer, D-serine, specifically regulates NMDA receptor (NMDAR) signalling. Despite their physiological relevance in modulating cerebral activity, serine enantiomers metabolism in Parkinson's disease (PD) remains elusive. Using High-Performance Liquid Chromatography (HPLC), we measured D- and L-serine levels along with other amino acids known to modulate NMDAR function, such as L-glutamate, L-aspartate, D-aspartate, and glycine, in the post-mortem caudate putamen (CPu) and superior frontal gyrus (SFG) of PD patients. Moreover, we examined these amino acids in the cerebrospinal fluid (CSF) of de novo living PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) patients versus subjects with other neurological disorders (OND), used as control. We found higher D-serine and L-serine levels in the CPu of PD patients but not in the SFG, a cerebral region that, in contrast to the CPu, is not innervated by nigral dopaminergic terminals. We also highlighted a significant elevation of both serine enantiomers in the CSF samples from PD but not in those of AD and ALS patients, compared with control subjects. By contrast, none or only minor changes were found in the amount of other NMDAR modulating amino acids. Our findings identify D-serine and L-serine level upregulation as a biochemical signature associated with nigrostriatal dopaminergic degeneration in PD., Competing Interests: Declaration of Competing Interest F.B. acted as Advisory Board members of Teva and Roche and received honoraria for speaking or consultation fees from Merck Serono, Teva, Bio-gen Idec, Sanofi, and Novartis and non-financial support from Merck Serono, Teva, Biogen Idec, and Sanofi. DC is an Advisory Board member of Almirall, Bayer Schering, Biogen, GW Pharmaceuticals, Merck Serono, Novar-tis, Roche, Sanofi19 Genzyme, and Teva and received honoraria for speaking or consultation fees from Almirall, Bayer Schering, Biogen, GW Pharmaceuticals, Merck Serono, Novartis, Roche, Sanofi-Genzyme, and Teva. He is also the principal investigator in clinical trials for Bayer Schering, Biogen, Merck Serono, Mitsubishi, Novartis, Roche, Sanofi-Genzyme, and Teva. His preclinical and clinical research was supported by grants from Bayer Schering, Biogen Idec, Celgene, Merck Serono, Novartis, Roche, Sanofi-Genzyme, and Teva. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. All the other authors declare no competing non-financial or financial interests to disclose., (Copyright © 2023. Published by Elsevier Inc.)

Background: Neuroinflammation contributes to the onset and progression of neurodegenerative diseases, but has not been specifically investigated in patients affected by severe and milder forms of spinal muscular atrophy (SMA)., Methods: In this two-center retrospective study, we investigated signatures of neuroinflammation in forty-eight pediatric male and female SMA1 (n = 18), male and female SMA2 (n = 19), and female SMA3 (n = 11) patients, as well as in a limited number of male and female non-neurological control subjects (n = 4). We employed a Bio-Plex multiplex system based on xMAP technology and performed targeted quantitative analysis of a wide range of pro- and anti-inflammatory cytokines (chemokines, interferons, interleukins, lymphokines and tumor necrosis factors) and neurotrophic factors in the cerebrospinal fluid (CSF) of the study cohort before and after Nusinersen treatment at loading and maintenance stages., Results: We find a significant increase in the levels of several pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-2, IL-8, IL-12, IL-17, MIP-1α, MCP-1, and Eotaxin) and neurotrophic factors (PDGF-BB and VEGF) in the CSF of SMA1 patients relative to SMA2 and SMA3 individuals, who display levels in the range of controls. We also find that treatment with Nusinersen significantly reduces the CSF levels of some but not all of these neuroinflammatory molecules in SMA1 patients. Conversely, Nusinersen increases the CSF levels of proinflammatory G-CSF, IL-8, MCP-1, MIP-1α, and MIP-1β in SMA2 patients and decreases those of anti-inflammatory IL-1ra in SMA3 patients., Conclusions: These findings highlight signatures of neuroinflammation that are specifically associated with severe SMA and the neuro-immunomodulatory effects of Nusinersen therapy., (© 2023. The Author(s).)

Intrathecal delivery of Nusinersen-an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction-is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene., (© 2022. The Author(s).)

Schizophrenia is a disorder of synaptic plasticity and aberrant connectivity in which a major dysfunction in glutamate synapse has been suggested. However, a multi-level approach tackling diverse clusters of interacting molecules of the glutamate signaling in schizophrenia is still lacking. We investigated in the post-mortem dorsolateral prefrontal cortex (DLPFC) and hippocampus of schizophrenia patients and non-psychiatric controls, the levels of neuroactive D- and L-amino acids (L-glutamate, D-serine, glycine, L-aspartate, D-aspartate) by HPLC. Moreover, by quantitative RT-PCR and western blotting we analyzed, respectively, the mRNA and protein levels of pre- and post-synaptic key molecules involved in the glutamatergic synapse functioning, including glutamate receptors (NMDA, AMPA, metabotropic), their interacting scaffolding proteins (PSD-95, Homer1b/c), plasma membrane and vesicular glutamate transporters (EAAT1, EAAT2, VGluT1, VGluT2), enzymes involved either in glutamate-dependent GABA neurotransmitter synthesis (GAD65 and 67), or in post-synaptic NMDA receptor-mediated signaling (CAMKIIα) and the pre-synaptic marker Synapsin-1. Univariable analyses revealed that none of the investigated molecules was differently represented in the post-mortem DLPFC and hippocampus of schizophrenia patients, compared with controls. Nonetheless, multivariable hypothesis-driven analyses revealed that the presence of schizophrenia was significantly affected by variations in neuroactive amino acid levels and glutamate-related synaptic elements. Furthermore, a Machine Learning hypothesis-free unveiled other discriminative clusters of molecules, one in the DLPFC and another in the hippocampus. Overall, while confirming a key role of glutamatergic synapse in the molecular pathophysiology of schizophrenia, we reported molecular signatures encompassing elements of the glutamate synapse able to discriminate patients with schizophrenia and normal individuals., (© 2022. The Author(s).)

Schizophrenia (SCZ) is a polygenic severe mental illness. Genome-wide association studies (GWAS) have detected genomic variants associated with this psychiatric disorder and pathway analyses have indicated immune system and dopamine signaling as core components of risk in dorsolateral-prefrontal cortex (DLPFC) and hippocampus, but the mechanistic links remain unknown. The RasGRP1 gene, encoding for a guanine nucleotide exchange factor, is implicated in dopamine signaling and immune response. RasGRP1 has been identified as a candidate risk gene for SCZ and autoimmune disease, therefore representing a possible point of convergence between mechanisms involving the nervous and the immune system. Here, we investigated RasGRP1 mRNA and protein expression in post-mortem DLPFC and hippocampus of SCZ patients and healthy controls, along with RasGRP1 protein content in the serum of an independent cohort of SCZ patients and control subjects. Differences in RasGRP1 expression between SCZ patients and controls were detected both in DLPFC and peripheral blood of samples analyzed. Our results indicate RasGRP1 may mediate risk for SCZ by involving DLPFC and peripheral blood, thus encouraging further studies to explore its possible role as a biomarker of the disease and/or a target for new medication.

Schizophrenia (SCZ) is a mental illness characterized by aberrant synaptic plasticity and connectivity. A large bulk of evidence suggests genetic and functional links between postsynaptic abnormalities and SCZ. Here, we performed quantitative PCR and Western blotting analysis in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of SCZ patients to investigate the mRNA and protein expression of three key spine shapers: the actin-binding protein cyclase-associated protein 2 ( CAP2 ), the sheddase a disintegrin and metalloproteinase 10 ( ADAM10 ), and the synapse-associated protein 97 ( SAP97 ). Our analysis of the SCZ post-mortem brain indicated increased DLG1 mRNA in DLPFC and decreased CAP2 mRNA in the hippocampus of SCZ patients, compared to non-psychiatric control subjects, while the ADAM10 transcript was unaffected. Conversely, no differences in CAP2 , SAP97 , and ADAM10 protein levels were detected between SCZ and control individuals in both brain regions. To assess whether DLG1 and CAP2 transcript alterations were selective for SCZ, we also measured their expression in the superior frontal gyrus of patients affected by neurodegenerative disorders, like Parkinson's and Alzheimer's disease. Interestingly, also in Parkinson's disease patients, we found a selective reduction of CAP2 mRNA levels relative to controls but unaltered protein levels. Taken together, we reported for the first time altered CAP2 expression in the brain of patients with psychiatric and neurological disorders, thus suggesting that aberrant expression of this gene may contribute to synaptic dysfunction in these neuropathologies.

Excessive extracellular concentrations of L-glutamate (L-Glu) can be neurotoxic and contribute to neurodegenerative processes in multiple sclerosis (MS). The association between cerebrospinal fluid (CSF) L-Glu levels, clinical features, and inflammatory biomarkers in patients with MS remains unclear. In 179 MS patients (relapsing remitting, RR, N = 157; secondary progressive/primary progressive, SP/PP, N = 22), CSF levels of L-Glu at diagnosis were determined and compared with those obtained in a group of 40 patients with non-inflammatory/non-degenerative disorders. Disability at the time of diagnosis, and after 1 year follow-up, was assessed using the Expanded Disability Status Scale (EDSS). CSF concentrations of lactate and of a large set of pro-inflammatory and anti-inflammatory molecules were explored. CSF levels of L-Glu were slightly reduced in MS patients compared to controls. In RR-MS patients, L-Glu levels correlated with EDSS after 1 year follow-up. Moreover, in MS patients, significant correlations were found between L-Glu and both CSF levels of lactate and the inflammatory molecules interleukin (IL)-2, IL-6, and IL-1 receptor antagonist. Altered expression of L-Glu is associated with disability progression, oxidative stress, and inflammation. These findings identify CSF L-Glu as a candidate neurochemical marker of inflammatory neurodegeneration in MS., (© 2021 International Society for Neurochemistry.)

Altered glutamatergic neurotransmission is thought to play a crucial role in the progression of Alzheimer's disease (AD). Accordingly, the identification of peculiar biochemical patterns reflecting AD-related synaptopathy in blood and cerebrospinal fluid (CSF) could have relevant diagnostic and prognostic implications. In this study, we measured by High-Performance Liquid Chromatography the amount of glutamate, glutamine and glycine in post-mortem brain samples of AD patients, as well as in CSF and blood serum of drug-free subjects encompassing the whole AD clinical spectrum (pre-clinical AD, n = 18, mild cognitive impairment-AD, n = 29, dementia AD, n = 30). Interestingly, we found that glutamate and glycine levels, as well as total tau protein content, were significantly reduced in the superior frontal gyrus of patients with AD, compared with non-demented controls. No significant change was also found in glutamate, glutamine and glycine CSF concentrations between AD patients and neurological controls. Remarkably, serum glutamate levels were significantly higher in patients affected by early AD phases compared to controls, and were negatively correlated with CSF total tau levels. Conversely, serum glutamine concentration was significantly increased in AD patients, with a negative correlation with MMSE performances. Finally, we reported a significant correlation between serum L-glutamate concentrations and CDR score in female but not in male cohort of AD subjects. Overall, our results suggest that serum glutamate and glutamine levels in AD patients could vary across disease stages, potentially reflecting the progressive alteration of glutamatergic signaling during neurodegenerative processes.

d-Amino acids were believed to occur only in bacteria and invertebrates. Today, it is well known that d-amino acids are also present in mammalian tissues in a considerable amount. In particular, high levels of free d-serine (d-Ser) and d-aspartate (d-Asp) are found in the brain. While the functions of d-Ser are well known, many questions remain unanswered regarding the role of d-Asp in the central nervous system. d-Asp is very abundant at the embryonic stage, while it strongly decreases after birth because of the expression of d-aspartate oxidase ( Ddo ) enzyme, which catalyzes the oxidation of this d-amino acid into oxaloacetate, ammonium, and hydrogen peroxide. Pharmacologically, d-Asp acts as an endogenous agonist of N -methyl d-aspartate and mGlu5 receptors, which are known to control fundamental brain processes, including brain development, synaptic plasticity, and cognition. In this work, we studied a recently generated knockin mouse model ( R26 d do / d do ), which was designed to express DDO beginning at the zygotic stage. This strategy enables d-Asp to be almost eliminated in both prenatal and postnatal lives. To understand which biochemical pathways are affected by depletion of d-Asp, in this study, we carried out a metabolomic and lipidomic study of d do knockin brains at different stages of embryonic and postnatal development, combining nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. Our study shows that d-Asp deficiency in the brain influences amino acid pathways such as threonine, glycine, alanine, valine, and glutamate. Interestingly, d-Asp is also correlated with metabolites involved in brain development and functions such as choline, creatine, phosphocholine (PCho), glycerophosphocholine (GPCho), sphingolipids, and glycerophospholipids, as well as metabolites involved in brain energy metabolism, such as GPCho, glucose, and lactate.

Background: Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d-aspartate and d-serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out., Methods: Through HPLC and qPCR analyses we investigated d-aspartate and d-serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2 -/- , Shank3 -/- , and 16p11.2 +/- mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations., Results: Biochemical and gene expression mapping in Cntnap2 -/- , Shank3 -/- , and 16p11.2 +/- failed to find gross cerebral and serum alterations in d-aspartate and d-serine metabolism. Conversely, we found a striking and stereoselective increased d-aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d-aspartate oxidase, encoding the enzyme responsible for d-aspartate catabolism., Conclusions: Our results demonstrated the presence of disrupted d-aspartate metabolism in a widely used animal model of idiopathic ASD., General Significance: Overall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The diagnosis of Alzheimer's disease (AD) relies on the presence of amyloidosis and tauopathy, as reflected in cerebrospinal fluid (CSF), independently from the clinical stage. Recently, CSF d-serine has been proposed as a possible new AD biomarker, reflecting dysfunctional activation of neuronal glutamatergic N-methyl-d-aspartate receptor (NMDAR). In this study, we measured blood serum and CSF concentration of two NMDAR modulators, such as d-serine and d-aspartate, in a cohort of drug-free subjects encompassing the whole AD clinical spectrum. In addition, we also analyzed d-serine levels in a cohort of post-mortem AD and control cortex samples. We reported unaltered serum and CSF concentrations of d-serine and d-aspartate in AD patients both during the AD progression and compared to non-demented controls. Accordingly, no correlation was detected between serum or CSF d-serine content and mini-mental state examination or Clinical Dementia Rating. Similarly, cortical d-serine levels were also unaltered in post-mortem samples of AD patients. Overall, our results failed to confirm previous findings indicating the CSF d-serine as a novel biomarker for AD., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The endogenous amino acids serine and aspartate occur at high concentrations in free D-form in mammalian organs, including the central nervous system and endocrine glands. D-serine (D-Ser) is largely localized in the forebrain structures throughout pre and postnatal life. Pharmacologically, D-Ser plays a functional role by acting as an endogenous coagonist at N-methyl-D-aspartate receptors (NMDARs). Less is known about the role of free D-aspartate (D-Asp) in mammals. Notably, D-Asp has a specific temporal pattern of occurrence. In fact, free D-Asp is abundant during prenatal life and decreases greatly after birth in concomitance with the postnatal onset of D-Asp oxidase expression, which is the only enzyme known to control endogenous levels of this molecule. Conversely, in the endocrine system, D-Asp concentrations enhance after birth during its functional development, thereby suggesting an involvement of the amino acid in the regulation of hormone biosynthesis. The substantial binding affinity for the NMDAR glutamate site has led us to investigate the in vivo implications of D-Asp on NMDAR-mediated responses. Herein we review the physiological function of free D-Asp and of its metabolizing enzyme in regulating the functions of the brain and of the neuroendocrine system based on recent genetic and pharmacological human and animal studies.

Diagnostic assays for molecular alterations highly correlated with prognosis, predictive efficacy or safety of therapeutics are valuable clinical tools and in some cases approved as companion diagnostics (CDx) by the Federal Food and Drug Administration. For example, assays that determine echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation status have been approved as CDx assay for therapies that target this molecular alteration. Characterizing the parameters that may compromise diagnostic accuracy for molecular biomarkers is critical for optimal patient care. To investigate the impact of pre-analytical handling and processing of tumor tissue on commonly used diagnostic immunohistochemistry-based assays for ALK and mesenchymal epithelial transition protein [c-mesenchymal epithelial transition (c-MET)], we investigated the effects of cold ischemia, fixative type, fixation time, and cut-slide age on staining consistency and intensity using human lung xenograft tumor tissue. Cold ischemia times for up to 5 to 6 hours for c-MET or ALK, respectively had minimal impact on staining. The optimal fixation conditions for both assays were found to be at least 6 hours and up to 48 hours for c-MET or 72 hours for ALK, in 10% neutral buffered formalin and Zinc formalin. The ALK antigen demonstrated marked staining intensity differences across non-neutral buffered formalin fixative types and times. Finally, cut-slide age influenced assay performance for both ALK and c-MET, with maximum stability observed when cut slides were stored at ambient temperatures (30°C) for no longer than 3, and 5 months, respectively. This study highlights the potential for pre-analytical factors to confound diagnostic test result interpretation.

The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson's disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA-induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice ( RasGRP1 -/- ) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA-induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice are viable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

Despite the great effort of the scientific community in the field, the pathogenesis of frontotemporal dementia (FTD) remains elusive. Recently, a role for autoimmunity and altered glutamatergic neurotransmission in triggering disease onset has been put forward. We reported the presence of autoantibodies recognizing the GluA3 subunit of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in about 25% of FTD cases. In this study, we evaluated the mechanisms involved in anti-GluA3 autoimmunity, through molecular/neurochemical analyses conducted on patients' brain specimens with frontotemporal lobar degeneration-tau neuropathology. We then corroborated these results in vivo in FTD patients with transcranial magnetic stimulation and glutamate, D-serine, and L-serine dosages in the cerebrospinal fluid and serum. We observed that GluA3 autoantibodies affect glutamatergic neurotransmission, decreasing glutamate release and altering GluA3-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor levels. These alterations were accompanied by changes of scaffolding proteins involved in receptor synaptic retention/internalization. The above results were confirmed by transcranial magnetic stimulation, suggesting a significant impairment of indirect measures of glutamatergic neurotransmission in FTD patients compared with controls, with further add-on harmful effect in those FTD patients with anti-GluA3 antibodies. Finally, FTD patients showed a significant increase of glutamate, D-serine, and L-serine levels in the cerebrospinal fluid., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as D-serine and D-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of D-amino acids are found. We recently demonstrated that Ddo, the D-aspartate degradation gene, is actively demethylated to ultimately reduce D-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate D-ser levels during brain development has been poorly investigated to date., Methods: We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based D-amino acids level analyses of genes controlling the mammalian brain levels of D-serine and D-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis., Results: We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar D-serine a few days after mouse birth. High amount of 5'-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process., Conclusion: The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of D-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

Previous evidence pointed out a role for the striatal-enriched protein Rhes in modulating dopaminergic transmission. Based on the knowledge that cocaine induces both addiction and motor stimulation, through its ability to enhance dopaminergic signaling in the corpus striatum, we have now explored the involvement of Rhes in the effects associated with this psychostimulant. Our behavioral data showed that a lack of Rhes in knockout animals caused profound alterations in motor stimulation following cocaine exposure, eliciting a significant leftward shift in the dose-response curve and triggering a dramatic hyperactivity. We also found that Rhes modulated either short- or long-term motor sensitization induced by cocaine, since lack of this protein prevents both of them in mutants. Consistent with this in vivo observation, we found that lack of Rhes in mice caused a greater increase in striatal cocaine-dependent D1R/cAMP/PKA signaling, along with considerable enhancement of Arc, zif268, and Homer1 mRNA expression. We also documented that lack of Rhes in mice produced cocaine-related striatal alterations in proteomic profiling, with a differential expression of proteins clustering in calcium homeostasis and cytoskeletal protein binding categories. Despite dramatic striatal alterations associated to cocaine exposure, our data did not reveal any significant changes in midbrain dopaminergic neurons as a lack of Rhes did not affect: (i) DAT activity; (ii) D2R-dependent regulation of GIRK; and (iii) D2R-dependent regulation of dopamine release. Collectively, our results strengthen the view that Rhes acts as a pivotal physiological "molecular brake" for striatal dopaminergic system overactivation induced by psychostimulants, thus making this protein of interest in regulating the molecular mechanism underpinning cocaine-dependent motor stimulatory effects.

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., (Copyright © 2019. Published by Elsevier Inc.)

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.