Kim Q. Do

Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate

Supernatants (SN) of brain macrophages in culture induce death of cerebellar granule cells in vitro, while those of astrocytes and endothelial cells do not. This toxicity can be prevented by N-methyl-D-aspartate (NMDA) receptor antagonists. Macrophage SN contain high concentrations of glutamate. Reducing the glutamate level of macrophage SN, either by exposure to astrocytes or by enzymatic degradation abolished the toxic effect. Thus, macrophage neurotoxicity is mediated by glutamate acting on NMDA receptors, and might play a role in vivo in traumatic and cerebrovascular brain lesions.

Schizophrenia: glutathione deficit in cerebrospinal fluid and prefrontal cortex in vivo

Schizophrenia is a major psychiatric disease, which affects the centre of the personality, with severe problems of perception, cognition as well as affective and social behaviour. In cerebrospinal fluid of drug‐free schizophrenic patients, a significant decrease in the level of total glutathione (GSH) by 27% (P < 0.05) was observed as compared to controls, in keeping with the reported reduced level of its metabolite γ‐glutamylglutamine. With a new non‐invasive proton magnetic resonance spectroscopy methodology, GSH level in medial prefrontal cortex of schizophrenic patients was found to be 52% (P = 0.0012) lower than in controls. GSH plays a fundamental role in protecting cells from damage by reactive oxygen species generated among others by the metabolism of dopamine. A deficit in GSH would lead to degenerative processes in the surrounding of dopaminergic terminals resulting in loss of connectivity. GSH also potentiates the N‐methyl‐d‐aspartate (NMDA) receptor response to glutamate, an effect presumably reduced by a GSH deficit, leading to a situation similar to the application of phencyclidine (PCP). Thus, a GSH hypothesis might integrate many established biological aspects of schizophrenia.

N-acetyl cysteine as a glutathione precursor for schizophrenia--a double-blind, randomized, placebo-controlled trial.

BACKGROUND Brain glutathione levels are decreased in schizophrenia, a disorder that often is chronic and refractory to treatment. N-acetyl cysteine (NAC) increases brain glutathione in rodents. This study was conducted to evaluate the safety and effectiveness of oral NAC (1 g orally twice daily [b.i.d.]) as an add-on to maintenance medication for the treatment of chronic schizophrenia over a 24-week period. METHODS A randomized, multicenter, double-blind, placebo-controlled study. The primary readout was change from baseline on the Positive and Negative Symptoms Scale (PANSS) and its components. Secondary readouts included the Clinical Global Impression (CGI) Severity and Improvement scales, as well as general functioning and extrapyramidal rating scales. Changes following a 4-week treatment discontinuation were evaluated. One hundred forty people with chronic schizophrenia on maintenance antipsychotic medication were randomized; 84 completed treatment. RESULTS Intent-to-treat analysis revealed that subjects treated with NAC improved more than placebo-treated subjects over the study period in PANSS total [-5.97 (-10.44, -1.51), p = .009], PANSS negative [mean difference -1.83 (95% confidence interval: -3.33, -.32), p = .018], and PANSS general [-2.79 (-5.38, -.20), p = .035], CGI-Severity (CGI-S) [-.26 (-.44, -.08), p = .004], and CGI-Improvement (CGI-I) [-.22 (-.41, -.03), p = .025] scores. No significant change on the PANSS positive subscale was seen. N-acetyl cysteine treatment also was associated with an improvement in akathisia (p = .022). Effect sizes at end point were consistent with moderate benefits. CONCLUSIONS These data suggest that adjunctive NAC has potential as a safe and moderately effective augmentation strategy for chronic schizophrenia.

Glutathione Precursor, N-Acetyl-Cysteine, Improves Mismatch Negativity in Schizophrenia Patients

In schizophrenia patients, glutathione dysregulation at the gene, protein and functional levels, leads to N-methyl-D-aspartate (NMDA) receptor hypofunction. These patients also exhibit deficits in auditory sensory processing that manifests as impaired mismatch negativity (MMN), which is an auditory evoked potential (AEP) component related to NMDA receptor function. N-acetyl-cysteine (NAC), a glutathione precursor, was administered to patients to determine whether increased levels of brain glutathione would improve MMN and by extension NMDA function. A randomized, double-blind, cross-over protocol was conducted, entailing the administration of NAC (2g/day) for 60 days and then placebo for another 60 days (or vice versa). 128-channel AEPs were recorded during a frequency oddball discrimination task at protocol onset, at the point of cross-over, and at the end of the study. At the onset of the protocol, the MMN of patients was significantly impaired compared to sex- and age- matched healthy controls (p=0.003), without any evidence of concomitant P300 component deficits. Treatment with NAC significantly improved MMN generation compared with placebo (p=0.025) without any measurable effects on the P300 component. MMN improvement was observed in the absence of robust changes in assessments of clinical severity, though the latter was observed in a larger and more prolonged clinical study. This pattern suggests that MMN enhancement may precede changes to indices of clinical severity, highlighting the possible utility AEPs as a biomarker of treatment efficacy. The improvement of this functional marker may indicate an important pathway towards new therapeutic strategies that target glutathione dysregulation in schizophrenia.

Impaired glutathione synthesis in schizophrenia: Convergent genetic and functional evidence

Schizophrenia is a complex multifactorial brain disorder with a genetic component. Convergent evidence has implicated oxidative stress and glutathione (GSH) deficits in the pathogenesis of this disease. The aim of the present study was to test whether schizophrenia is associated with a deficit of GSH synthesis. Cultured skin fibroblasts from schizophrenia patients and control subjects were challenged with oxidative stress, and parameters of the rate-limiting enzyme for the GSH synthesis, the glutamate cysteine ligase (GCL), were measured. Stressed cells of patients had a 26% (P = 0.002) decreased GCL activity as compared with controls. This reduction correlated with a 29% (P < 0.001) decreased protein expression of the catalytic GCL subunit (GCLC). Genetic analysis of a trinucleotide repeat (TNR) polymorphism in the GCLC gene showed a significant association with schizophrenia in two independent case-control studies. The most common TNR genotype 7/7 was more frequent in controls [odds ratio (OR) = 0.6, P = 0.003], whereas the rarest TNR genotype 8/8 was three times more frequent in patients (OR = 3.0, P = 0.007). Moreover, subjects with disease-associated genotypes had lower GCLC protein expression (P = 0.017), GCL activity (P = 0.037), and GSH contents (P = 0.004) than subjects with genotypes that were more frequent in controls. Taken together, the study provides genetic and functional evidence that an impaired capacity to synthesize GSH under conditions of oxidative stress is a vulnerability factor for schizophrenia.

S‐Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography‐Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S‐nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S‐nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [35S]cysteine‐prelabeled slices suggested that S‐nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography‐mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using 15N‐labeled GSNO ([15N]‐GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.

Perineuronal nets protect fast-spiking interneurons against oxidative stress

A hallmark of schizophrenia pathophysiology is the dysfunction of cortical inhibitory GABA neurons expressing parvalbumin, which are essential for coordinating neuronal synchrony during various sensory and cognitive tasks. The high metabolic requirements of these fast-spiking cells may render them susceptible to redox dysregulation and oxidative stress. Using mice carrying a genetic redox imbalance, we demonstrate that extracellular perineuronal nets, which constitute a specialized polyanionic matrix enwrapping most of these interneurons as they mature, play a critical role in the protection against oxidative stress. These nets limit the effect of genetically impaired antioxidant systems and/or excessive reactive oxygen species produced by severe environmental insults. We observe an inverse relationship between the robustness of the perineuronal nets around parvalbumin cells and the degree of intracellular oxidative stress they display. Enzymatic degradation of the perineuronal nets renders mature parvalbumin cells and fast rhythmic neuronal synchrony more susceptible to oxidative stress. In parallel, parvalbumin cells enwrapped with mature perineuronal nets are better protected than immature parvalbumin cells surrounded by less-condensed perineuronal nets. Although the perineuronal nets act as a protective shield, they are also themselves sensitive to excess oxidative stress. The protection might therefore reflect a balance between the oxidative burden on perineuronal net degradation and the capacity of the system to maintain the nets. Abnormal perineuronal nets, as observed in the postmortem patient brain, may thus underlie the vulnerability and functional impairment of pivotal inhibitory circuits in schizophrenia.

In Vitro Release of Endogenous Excitatory Sulfur-Containing Amino Acids from Various Rat Brain Regions

Abstract: Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons‐medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 μg/ml). The 4‐N, N‐dimethylamino‐azobenzene‐4′‐isothiocyanate (DABITC) derivatization method of Chang [Biochem. J.199, 537–545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur‐containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+‐dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur‐containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur‐containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.

TORC1 is a calcium- and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity

A key feature of memory processes is to link different input signals by association and to preserve this coupling at the level of synaptic connections. Late-phase long-term potentiation (L-LTP), a form of synaptic plasticity thought to encode long-term memory, requires gene transcription and protein synthesis. In this study, we report that a recently cloned coactivator of cAMP-response element-binding protein (CREB), called transducer of regulated CREB activity 1 (TORC1), contributes to this process by sensing the coincidence of calcium and cAMP signals in neurons and by converting it into a transcriptional response that leads to the synthesis of factors required for enhanced synaptic transmission. We provide evidence that TORC1 is involved in L-LTP maintenance at the Schaffer collateral–CA1 synapses in the hippocampus.

Redox Dysregulation Affects the Ventral But Not Dorsal Hippocampus: Impairment of Parvalbumin Neurons, Gamma Oscillations, and Related Behaviors

Elevated oxidative stress and alteration in antioxidant systems, including glutathione (GSH) decrease, are observed in schizophrenia. Genetic and functional data indicate that impaired GSH synthesis represents a susceptibility factor for the disorder. Here, we show that a genetically compromised GSH synthesis affects the morphological and functional integrity of hippocampal parvalbumin-immunoreactive (PV-IR) interneurons, known to be affected in schizophrenia. A GSH deficit causes a selective decrease of PV-IR interneurons in CA3 and dendate gyrus (DG) of the ventral but not dorsal hippocampus and a concomitant reduction of β/γ oscillations. Impairment of PV-IR interneurons emerges at the end of adolescence/early adulthood as oxidative stress increases or cumulates selectively in CA3 and DG of the ventral hippocampus. Such redox dysregulation alters stress and emotion-related behaviors but leaves spatial abilities intact, indicating functional disruption of the ventral but not dorsal hippocampus. Thus, a GSH deficit affects PV-IR interneurons integrity and neuronal synchrony in a region- and time-specific manner, leading to behavioral phenotypes related to psychiatric disorders.