Rogerio Panizzutti

A CSF and postmortem brain study of D-serine metabolic parameters in schizophrenia

Clinical trials demonstrated that D-serine administration improves schizophrenia symptoms, raising the possibility that altered levels of endogenous D-serine may contribute to the N-methyl D-aspartate receptor hypofunction thought to play a role in the disease. We hypothesized that cerebro-spinal fluid (CSF) D-serine levels are decreased in the patients due to reduced synthesis and/or increased degradation in brain. We now monitored amino acid levels in CSF from 12 schizophrenia patients vs. 12 controls and in postmortem parietal-cortex from 15 control subjects and 15 each of schizophrenia, major-depression and bipolar patients. In addition, we monitored postmortem brain serine racemase and D-amino acid oxidase protein levels by Western-blot analysis. We found a 25% decrease in D-serine levels and D/L-serine ratio in CSF of schizophrenia patients, while parietal-cortex D-serine was unaltered. Levels of L-serine, L-glutamine and L-glutamate were unaffected. Frontal-cortex (39%) and hippocampal (21%) serine racemase protein levels and hippocampal serine racemase/D-amino acid oxidase ratio (34%) were reduced. Hippocampal D-amino-acid-oxidase protein levels significantly correlated with duration of illness (r=0.6, p=0.019) but not age. D-amino acid oxidase levels in patients with DOI>20 years were 77% significantly higher than in the other patients and controls. Our results suggest that reduced brain serine racemase and elevated D-amino acid oxidase protein levels may contribute to the lower CSF D-serine levels in schizophrenia.

Cofactors of serine racemase that physiologically stimulate the synthesis of the N-methyl-d-aspartate (NMDA) receptor coagonist d-serine

High levels of d-serine occur in the brain, challenging the notion that d-amino acids would not be present or play a role in mammals. d-serine levels in the brain are even higher than many l-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others. d-serine is synthesized by a serine racemase (SR) enzyme, which directly converts l- to d-serine. We now report that SR is a bifunctional enzyme, producing both d-serine and pyruvate in cultured cells and in vitro. Transfection of SR into HEK 293 cells elicits synthesis of d-serine and augmented release of pyruvate to culture media. We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate d-serine production by SR and elicit production of pyruvate. Experiments with recombinant enzyme reveal that Mg2+ and ATP present in the cell extracts are physiological cofactors and increase 5- to 10-fold the rates of racemization and production of pyruvate. As much as three molecules of pyruvate are synthesized for each molecule of d-serine produced by SR. This finding constitutes a previously undescribed mechanism underlying d-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria. Our data link the production of d-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.

Increased brain D-amino acid oxidase (DAAO) activity in schizophrenia.

D-serine has been shown to be a major endogenous coagonist of the N-methyl D-aspartate (NMDA) type of glutamate receptors. Accumulating evidence suggests that NMDA receptor hypofunction contributes to the symptomatic features of schizophrenia. d-serine degradation can be mediated by the enzyme d-amino acid oxidase (DAAO). An involvement of d-serine in the etiology of schizophrenia is suggested by the association of the disease with single nucleotide polymorphisms in the DAAO and its regulator (G72). The present study aims to further elucidate whether the DAAO activity is altered in schizophrenia. Specific DAAO activity was measured in postmortem cortex samples of bipolar disorder, major depression and schizophrenia patients, and normal controls (n=15 per group). The mean DAAO activity was two-fold higher in the schizophrenia patients group compared with the control group. There was no correlation between DAAO activity and age, age of onset, duration of disease, pH of the tissue and tissue storage time and no effect of gender, cause of death and history of alcohol and substance abuse. The group of neuroleptics users (including bipolar disorder patients) showed significantly higher D-amino acid oxidase activity. However, there was no correlation between the cumulative life-time antipsychotic usage and D-amino acid oxidase levels. In mice, either chronic exposure to antipsychotics or acute administration of the NMDA receptor blocker MK-801, did not change d-amino acid oxidase activity. These findings provide indications that D-serine availability in the nervous system may be altered in schizophrenia because of increased D-amino acid degradation by DAAO.

INHIBITION OF CREATINE KINASE BY S-NITROSOGLUTATHIONE

The sarcoplasmic reticulum‐bound creatine kinase from rabbit skeletal muscle was inhibited by the nitric oxide donor S‐nitrosoglutathione (GSNO). This led to a decrease in Ca2+ uptake in sarcoplasmic reticulum vesicles when the transport was driven by ATP generated from phosphocreatine and ADP. In contrast, the Ca2+ transport measured using 2 mM ATP as substrate was unaffected by GSNO up to 200 μM. GSNO (5–20 μM) inhibited the activity of both soluble and membrane‐bound creatine kinase. Oxyhemoglobin (15–40 μM) protected creatine kinase against inactivation by GSNO. The inhibition by 10 μM GSNO was reversed by the addition of dithiothreitol (2 mM). The results indicate that nitric oxide (NO, including NO+, NO and NO−) inactivates creatine kinase in vitro by promoting nitrosylation of critical sulphydryl groups of the enzyme.

Glial transport of the neuromodulator D-serine.

D-Serine is an endogenous agonist of NMDA receptors that occurs in astrocytes in gray matter areas of the brain. D-Serine is synthesized from L-serine by the activity of a glial enriched serine racemase, but little is known on the properties of D-serine transport and factors regulating its synaptic concentration. In the present report we characterize the transport of D-serine in astrocytes. In primary astrocyte cultures, D-serine uptake is dependent on sodium ions and exhibits both low affinity and low specificity for D-serine. The kinetics of D-serine transport resembles that of ASCT type transporters as several small neutral amino acids strongly inhibit the uptake of D-serine. D-Serine fluxes are coupled to counter-movement of L-serine and to a less extent to other small neutral amino acids. Thus, addition of D-serine to cell cultures elicits robust efflux of intracellular L-serine. Conversely, physiological concentrations of L-serine induce efflux of preloaded D-serine from astrocytes. L-Serine was more effective than kainate, which have been previously shown to induce D-serine release from astrocytes upon stimulation of non-NMDA type of glutamate receptors. The features of D-serine transport we describe reveal possible new mechanisms controlling the synaptic concentration of D-serine.

Astrocyte-induced Synaptogenesis Is Mediated by Transforming Growth Factor β Signaling through Modulation of d-Serine Levels in Cerebral Cortex Neurons

Background: Synapse formation and function is modulated by intrinsic and extrinsic non-autonomous factors. Results: Astrocytes induce synapse formation through TGF-β1 pathway. TGF-β1 synaptogenic property is dependent on d-serine signaling. Conclusion: TGF-β induces excitatory glutamatergic synapses in vertebrates. Significance: This is a novel molecular mechanism that might impact synaptic function and shed light on new potential therapeutic targets for synaptic deficit diseases. Assembly of synapses requires proper coordination between pre- and postsynaptic elements. Identification of cellular and molecular events in synapse formation and maintenance is a key step to understand human perception, learning, memory, and cognition. A key role for astrocytes in synapse formation and function has been proposed. Here, we show that transforming growth factor β (TGF-β) signaling is a novel synaptogenic pathway for cortical neurons induced by murine and human astrocytes. By combining gain and loss of function approaches, we show that TGF-β1 induces the formation of functional synapses in mice. Further, TGF-β1-induced synaptogenesis involves neuronal activity and secretion of the co-agonist of the NMDA receptor, d-serine. Manipulation of d-serine signaling, by either genetic or pharmacological inhibition, prevented the TGF-β1 synaptogenic effect. Our data show a novel molecular mechanism that might impact synaptic function and emphasize the evolutionary aspect of the synaptogenic property of astrocytes, thus shedding light on new potential therapeutic targets for synaptic deficit diseases.

A new strategy to decrease N-methyl-d-aspartate (NMDA) receptor coactivation: Inhibition of d-serine synthesis by converting serine racemase into an eliminase

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

Natural Restoration of Critical Period Plasticity in the Juvenile and Adult Primary Auditory Cortex

Since its first description >40 years ago, the neurological “critical period” has been predominantly described as an early, plastic postnatal brain development stage that rather abruptly advances to an aplastic or less plastic “adult” stage. Here, we show that chronic exposure of juvenile or adult rats to moderate-level acoustic noise results in a broad reversal of maturational changes that mark the infant-to-adult progression in the primary auditory cortex. In time, noise exposure reinstates critical period plasticity. Cortical changes resulting from noise exposure are again reversed to reestablish a physically and functionally normal adult cortex, by returning animals to natural acoustic environments. These studies show that at least some of neurological changes believed to mark the transition from the infantile to the mature (adult) stage are, by their nature, reversible.

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-beta peptide (Abeta) play a central role in the pathogenesis of Alzheimers disease (AD). Identification of proteins that physiologically bind Abeta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Abeta(1-42), we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and Abeta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Abeta. Significantly, Abeta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Abeta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Abeta aggregation and Abeta-induced neuronal damage and that the Abeta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Abeta toxicity.

Plasma levels of D-serine in Brazilian individuals with schizophrenia

Changes in D-serine availability in the brain may contribute to the hypofunction of NMDA-glutamate receptors in schizophrenia; however, measurements of blood levels of D-serine in individuals with schizophrenia have not been consistent amongst previous studies. Here we studied plasma levels of D-serine and L-serine in 84 Brazilian individuals with schizophrenia and 75 gender- and age-matched controls. Plasma levels of D-serine and the ratio of plasma D-serine to total serine were significantly lower in individuals with schizophrenia as compared to the control group. Levels of D-serine were significantly and negatively correlated to the severity of negative symptoms of schizophrenia. We also observed that plasma levels of D-serine significantly decreased with aging in healthy controls. Our results suggest that the possible role of D-serine in the pathophysiology of schizophrenia should be further investigated, with possible implications for the drug treatment of this disorder.