Glutathione deficit in schizophrenia: strategies to increase glutathione levels in " in vitro " and clinical studies

Summary: Decrease in glutathione (GSH) levels was observed in cerebrospinal fluid, prefrontal cortex and post-mortem striatum of schizophrenia patients. Evidences suggest a defect in GSH synthesis at the levels of the rate-limiting synthesizing enzyme, glutamate cysteine ligase (GCL). Indeed, polymorphisms in the gene of the modifier subunit of GCL (GCLM) was shown to be associated with the disease in three different populations, GCLM gene expression is decreaséd in fibroblasts from patients and the increase in GCL activity induced by an oxidative stress is lower in patients' fibroblasts compared to controls. GSH being a major antioxydant and redox regulator, its presence is of high importance for protecting cells against oxidative stress. The aim of the present work was to use various substances to increase GSH levels by diverse strategies. Since the synthesizing enzyme GCL is defective, bypassing this enzyme was the first strategy we used. GSH ethyl ester (GSHEE), a membrane permeable analog of GSH, succeeded in replenishing GSH levels in cultured neurons and astrocytes previously depleted in GSH by L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GCL. GSHEE also abolished dopamine-induced decrease of NMDA-mediated calcium response observed in BSO-treated neurons. y-Glutamylcysteine ethyl ester (GCSE), a membrane permeable analog of the product of GCL, increased GSH levels only in astrocytes. The second strategy was to boost the defective enzyme GCL. While quercetin (flavonoid) could increase GSH levels only in astrocytes, curcumin (polyphenol) and tertbutylhydroquinone (quinone) were successful in both neurons and astrocytes, via an increase in the gene expression of the two subunits of GCL and, consequently, an increase in the activity of the enzyme. However, FK506, an immunosupressant, was unefficient. Treating astrocytes from GCLM KO mice showed that the modulatory subunit is necessary for the action of the substances. Finally, since cysteine is the limiting precursor in the synthesis of GSH, we hypothesized that we could increase GSH levels by providing more of this precursor. N-acetyl-cysteine (NAC), a cysteine donor, was administered to schizophrenia patients, using adouble-blind and cross-over protocol. NAC significantly improved the mismatch negativity (MMN), a component of the auditory evoked potentials, thought to reflect selective current flowing through open, unblocked NMDA channels. Considering that NMDA function is reduced when GSH levels are low, increasing these levels with NAC could improve NMDA function as reflected by the improvement in the generation of the MMN. Résumé: Les taux de glutathion (GSH) dans le liquide céphalo-rachidien, le cortex préfrontal ainsi que le striatum post-mortem de patients schizophrènes, sont diminués. L'enzyme limitante dans la synthèse du GSH, la glutamyl-cysteine ligase (GCL), est défectueuse. En effet, des polymorphismes dans le gène de la sous-unité modulatrice de GCL (GCLM) sont associés à la maladie, l'expression du gène GCLM est diminuée dans les fibroblastes de patients et, lors d'un stress oxidative, l'augmentation de l'activité de GCL est plus faible chez les patients que chez les contrôles. Le GSH étant un important antioxydant et régulateur du status redox, sa présence est primordiale afin de protéger les cellules contre les stress oxydatifs. Au cours du présent travail, une variété de substances ont été utilisées dans le but d'augmenter les taux de GSH. Passer outre l'enzyme de synthèse GCL qui est défectueuse fut la première stratégie utilisée. L'éthylester de GSH (GSHEE), un analogue du GSH qui pénètre la membrane cellulaire, a augmenté les taux de GSH dans des neurones et des astrocytes déficitaires en GSH dû au L-buthionine-(S,R)-sulfoximine (BSO), un inhibiteur du GCL. Dans ces neurones, le GSHEE a aussi aboli la diminution de la réponse NMDA, induite parla dopamine. L'éthyl-ester de y-glutamylcysteine (GCEE), un analogue du produit de la GCL qui pénètre la membrane cellulaire, a augmenté les taux de GSH seulement dans les astrocytes. La seconde stratégie était d'augmenter l'activité de l'enzyme GCL. Tandis que la quercétine (flavonoïde) n'a pu augmenter les taux de GSH que dans les astrocytes, la curcumin (polyphénol) et le tert-butylhydroquinone (quinone) furent efficaces dans les deux types de cellules, via une augmentation de l'expression des gènes des deux sous-unités de GCL et de l'activité de l'enzyme. Le FK506 (immunosupresseur) n' a démontré aucune efficacité. Traiter des astrocytes provenant de souris GCLM KO a permis d'observer que la sous-unité modulatoire est nécessaire à l'action des substances. Enfin, puisque la cysteine est le substrat limitant dans la synthèse du GSH, fournir plus de ce présurseur pourrait augmenter les taux de GSH. Nacétyl-cystéine (NAC), un donneur de cystéine, a été administrée à des schizophrènes, lors d'une étude en double-aveugle et cross-over. NAC a amélioré le mismatch negativity (MMN), un composant des potentials évoqués auditifs, qui reflète le courant circulant via les canaux NMDA. Puisque la fonctionnalité des R-NMDA est diminuée lorsque les taux de GSH sont bas, augmenter ces taux avec NAC pourrait améliorer la fonction des R-NMDA, réflété par une augmentation de l'amplitude du MMN.

Single-trial multisensory memories affect later auditory and visual object discrimination.

Multisensory memory traces established via single-trial exposures can impact subsequent visual object recognition. This impact appears to depend on the meaningfulness of the initial multisensory pairing, implying that multisensory exposures establish distinct object representations that are accessible during later unisensory processing. Multisensory contexts may be particularly effective in influencing auditory discrimination, given the purportedly inferior recognition memory in this sensory modality. The possibility of this generalization and the equivalence of effects when memory discrimination was being performed in the visual vs. auditory modality were at the focus of this study. First, we demonstrate that visual object discrimination is affected by the context of prior multisensory encounters, replicating and extending previous findings by controlling for the probability of multisensory contexts during initial as well as repeated object presentations. Second, we provide the first evidence that single-trial multisensory memories impact subsequent auditory object discrimination. Auditory object discrimination was enhanced when initial presentations entailed semantically congruent multisensory pairs and was impaired after semantically incongruent multisensory encounters, compared to sounds that had been encountered only in a unisensory manner. Third, the impact of single-trial multisensory memories upon unisensory object discrimination was greater when the task was performed in the auditory vs. visual modality. Fourth, there was no evidence for correlation between effects of past multisensory experiences on visual and auditory processing, suggestive of largely independent object processing mechanisms between modalities. We discuss these findings in terms of the conceptual short term memory (CSTM) model and predictive coding. Our results suggest differential recruitment and modulation of conceptual memory networks according to the sensory task at hand.

Dyrk1A is dynamically expressed on subsets of motor neurons and in the neuromuscular junction: possible role in Down syndrome

Individuals with Down syndrome (DS) present important motor deficits that derive from altered motor development of infants and young children. DYRK1A, a candidate gene for DS abnormalities has been implicated in motor function due to its expression in motor nuclei in the adult brain, and its overexpression in DS mouse models leads to hyperactivity and altered motor learning. However, its precise role in the adult motor system, or its possible involvement in postnatal locomotor development has not yet been clarified. During the postnatal period we observed time-specific expression of Dyrk1A in discrete subsets of brainstem nuclei and spinal cord motor neurons. Interestingly, we describe for the first time the presence of Dyrk1A in the presynaptic terminal of the neuromuscular junctions and its axonal transport from the facial nucleus, suggesting a function for Dyrk1A in these structures. Relevant to DS, Dyrk1A overexpression in transgenic mice (TgDyrk1A) produces motor developmental alterations possibly contributing to DS motor phenotypes and modifies the numbers of motor cholinergic neurons, suggesting that the kinase may have a role in the development of the brainstem and spinal cord motor system.

Lipid rafts act as specialised domains for tetanus toxin binding and internalisation in neurons

Tetanus (TeNT) is a zinc protease that blocks neurotransmission by cleaving the synaptic protein vesicle-associated membrane protein/synaptobrevin. Although its intracellular catalytic activity is well established, the mechanism by which this neurotoxin interacts with the neuronal surface is not known. In this study, we characterize p15s, the first plasma membrane TeNT binding proteins and we show that they are glycosylphosphatidylinositol-anchored glycoproteins in nerve growth factor (NGF)-differentiated PC12 cells, spinal cord cells, and purified motor neurons. We identify p15 as neuronal Thy-1 in NGF-differentiated PC12 cells. Fluorescence lifetime imaging microscopy measurements confirm the close association of the binding domain of TeNT and Thy-1 at the plasma membrane. We find that TeNT is recruited to detergent-insoluble lipid microdomains on the surface of neuronal cells. Finally, we show that cholesterol depletion affects a raft subpool and blocks the internalization and intracellular activity of the toxin. Our results indicate that TeNT interacts with target cells by binding to lipid rafts and that cholesterol is required for TeNT internalization and/or trafficking in neurons.

Decoding auditory EEG responses in healthy and clinical populations: A comparative study.

BACKGROUND: Analyses of brain responses to external stimuli are typically based on the means computed across conditions. However in many cognitive and clinical applications, taking into account their variability across trials has turned out to be statistically more sensitive than comparing their means. NEW METHOD: In this study we present a novel implementation of a single-trial topographic analysis (STTA) for discriminating auditory evoked potentials at predefined time-windows. This analysis has been previously introduced for extracting spatio-temporal features at the level of the whole neural response. Adapting the STTA on specific time windows is an essential step for comparing its performance to other time-window based algorithms. RESULTS: We analyzed responses to standard vs. deviant sounds and showed that the new implementation of the STTA gives above-chance decoding results in all subjects (in comparison to 7 out of 11 with the original method). In comatose patients, the improvement of the decoding performance was even more pronounced than in healthy controls and doubled the number of significant results. COMPARISON WITH EXISTING METHOD(S): We compared the results obtained with the new STTA to those based on a logistic regression in healthy controls and patients. We showed that the first of these two comparisons provided a better performance of the logistic regression; however only the new STTA provided significant results in comatose patients at group level. CONCLUSIONS: Our results provide quantitative evidence that a systematic investigation of the accuracy of established methods in normal and clinical population is an essential step for optimizing decoding performance.

Representation of Sound Objects within Early-Stage Auditory Areas: A Repetition Effect Study Using 7T fMRI.

Environmental sounds are highly complex stimuli whose recognition depends on the interaction of top-down and bottom-up processes in the brain. Their semantic representations were shown to yield repetition suppression effects, i. e. a decrease in activity during exposure to a sound that is perceived as belonging to the same source as a preceding sound. Making use of the high spatial resolution of 7T fMRI we have investigated the representations of sound objects within early-stage auditory areas on the supratemporal plane. The primary auditory cortex was identified by means of tonotopic mapping and the non-primary areas by comparison with previous histological studies. Repeated presentations of different exemplars of the same sound source, as compared to the presentation of different sound sources, yielded significant repetition suppression effects within a subset of early-stage areas. This effect was found within the right hemisphere in primary areas A1 and R as well as two non-primary areas on the antero-medial part of the planum temporale, and within the left hemisphere in A1 and a non-primary area on the medial part of Heschl's gyrus. Thus, several, but not all early-stage auditory areas encode the meaning of environmental sounds.

The existence of FGFR1-5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons

Nkx2.1-derived astrocytes and neurons together with Slit2 are indispensable for anterior commissure formation.

Guidepost cells present at and surrounding the midline provide guidance cues that orient the growing axons through commissures. Here we show that the transcription factor Nkx2.1 known to control the specification of GABAergic interneurons also regulates the differentiation of astroglia and polydendrocytes within the mouse anterior commissure (AC). Nkx2.1-positive glia were found to originate from three germinal regions of the ventral telencephalon. Nkx2.1-derived glia were observed in and around the AC region by E14.5. Thereafter, a selective cell ablation strategy showed a synergistic role of Nkx2.1-derived cells, both GABAergic interneurons and astroglia, towards the proper formation of the AC. Finally, our results reveal that the Nkx2.1-regulated cells mediate AC axon guidance through the expression of the repellent cue, Slit2. These results bring forth interesting insights about the spatial and temporal origin of midline telencephalic glia, and highlight the importance of neurons and astroglia towards the formation of midline commissures.

Effects of adult dysthyroidism on the morphology of hippocampal neurons.

This study investigates the effect of thyroid hormones on the morphology of hippocampal neurons in adult rats. Hypo- and hyperthyroidism were induced by adding 0.02% methimazole and 1% l-thyroxine, in drinking water from 40 days of age, respectively. When the rats were 89 days old their brains were removed and stained by a modified Golgi method and blood samples were collected in order to measure T4 serum levels. Neurons were selected and drawn using a camera lucida. Our results show that methimazole administration reduces the dendritic branching of the apical shafts of CA3 and CA1 pyramidal neurons mainly by increasing the distance to the first branch point in both types of neurons, and reducing branch points in the radius of 50 μm from the soma in CA1 neurons. Nevertheless, it was observed an increase of apical spine density in CA3 neurons from this group. Thyroxine reduces apical and basal tree of CA3 pyramidal neurons increasing the distance to the first branch point, reducing branch points in the radius of 50 μm from the soma and increases their apical and basal spine density. In CA1 field, thyroxine reduces the number of basal branch points. Both treatments seems to provoke alterations in the same direction reducing the dendritic branching and increasing spine density, although no significances appeared in some of the parameters analyzed. The effects are more evident in thyroxine than methimazole group; and in CA3 neurons than in CA1 neurons. In discussion it is pointed that the increase of spine density could be a mechanism to compensate the functionality reduction that can be provoke by the treatment effect on dendritic branching.

Neurites regrowth of cortical neurons by GSK3b inhibition independently of Nogo Receptor 1

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3 and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3 inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic

Enjeu: Déterminer si la macroautophagie est activée de façon excessive dans les neurones en souffrance dans l'encéphalopathie anoxique-ischémique du nouveau-né à terme. Contexte de la recherche: L'encéphalopathie anoxique-ischémique suite à une asphyxie néonatale est associée à une morbidité neurologique à long terme. Une diminution de son incidence reste difficile, son primum movens étant soudain, imprévisible voire non identifiable. Le développement d'un traitement pharmacologique neuroprotecteur post-anoxie reste un défi car les mécanismes impliqués dans la dégénérescence neuronale sont multiples, interconnectés et encore insuffisamment compris. En effet, il ressort des études animales que la notion dichotomique de mort cellulaire apoptotique (type 1)/nécrotique (type 3) est insuffisante. Une même cellule peut présenter des caractéristiques morphologiques mixtes non seulement d'apoptose et de nécrose mais aussi parfois de mort autophagique (type 2) plus récemment décrite. L'autophagie est un processus physiologique normal et essentiel de dégradation de matériel intracellulaire par les enzymes lysosomales. La macroautophagie, nommée simplement autophagie par la suite, consiste en la séquestration de parties de cytosol à éliminer (protéines et organelles) dans des compartiments intermédiaires, les autophagosomes, puis en leur fusion avec des lysosomes pour former des autolysosomes. Dans certaines conditions de stress telles que l'hypoxie et l'excitoxicité, une activité autophagique anormalement élevée peut être impliquée dans la mort cellulaire soit comme un mécanisme de mort indépendant (autodigestion excessive correspondante à la mort cellulaire de type 2) soit en activant d'autres voies de mort comme celles de l'apoptose. Description de l'article: Ce travail examine la présence de l'autophagie et son lien avec la mort cellulaire dans les neurones d'une région cérébrale fréquemment atteinte chez le nouveau- né humain décédé après une asphyxie néonatale sévère, le thalamus ventro-latéral. Ces résultats ont été comparés à ceux obtenus dans un modèle d'hypoxie-ischémie cérébrale chez le raton de 7 jours (dont le cerveau serait comparable à celui d'un nouveau-né humain de 34-37 semaines de gestation). Au total 11 nouveau-nés à terme décédés peu après la naissance ont été rétrospectivement sélectionnés, dont 5 présentant une encéphalopathie hypoxique- ischémique sévère et 6 décédés d'une cause autre que l'asphyxie choisis comme cas contrôle. L'autophagie et l'apoptose neuronale ont été évaluées sur la base d'une étude immunohistochimique et d'imagerie confocale de coupes histologiques en utilisant des marqueurs tels que LC3 (protéine dont la forme LC3-II est liée à la membrane des autophagosomes), p62/SQSTM1 (protéine spécifiquement dégradée par autophagie), LAMP1 (protéine membranaire des lysosomes et des autolysosomes), Cathepsin D ou B (enzymes lysosomales), TUNEL (détection de la fragmentation de l'ADN se produisant lors de l'apoptose), CASPASE-3 activée (protéase effectrice de l'apoptose) et PGP9.5 (protéine spécifique aux neurones). Chez le raton l'étude a pu être étendue en utilisant d'autres méthodes complémentaires telles que la microscopie électronique et le Western-blot. Une quantification des différents marqueurs montre une augmentation statistiquement significative de l'autophagie neuronale dans les cas d'asphyxie par rapport aux cas contrôles chez l'humain comme chez le raton. En cas d'asphyxie, les mêmes neurones expriment une densité accrue d'autophagosomes et d'autolysosomes par rapport aux cas contrôles. De plus, les neurones hautement autophagiques présentent des caractéristiques de l'apoptose. Conclusion: Cette étude montre, pour la première fois, que les neurones thalamiques lésés en cas d'encéphalopathie hypoxique-ischémique sévère présentent un niveau anormalement élevé d'activité autophagique comme démontré chez le raton hypoxique-ischémique. Ce travail permet ainsi de mettre en avant l'importance de considérer l'autophagie comme acteur dans la mort neuronale survenant après asphyxie néonatale. Perspectives: Récemment un certain nombre d'études in vitro ou sur des modèles d'ischémie cérébrale chez les rongeurs suggèrent un rôle important de la macroautophagie dans la mort neuronale. Ainsi, l'inhibition spécifique de la macroautophagie devrait donc être envisagée dans le futur développement des stratégies neuroprotectrices visant à protéger le cerveau des nouveau-nés à terme suite à une asphyxie.

Experience-based Auditory Predictions Modulate Brain Activity to Silence as Do Real Sounds.

Interactions between stimuli's acoustic features and experience-based internal models of the environment enable listeners to compensate for the disruptions in auditory streams that are regularly encountered in noisy environments. However, whether auditory gaps are filled in predictively or restored a posteriori remains unclear. The current lack of positive statistical evidence that internal models can actually shape brain activity as would real sounds precludes accepting predictive accounts of filling-in phenomenon. We investigated the neurophysiological effects of internal models by testing whether single-trial electrophysiological responses to omitted sounds in a rule-based sequence of tones with varying pitch could be decoded from the responses to real sounds and by analyzing the ERPs to the omissions with data-driven electrical neuroimaging methods. The decoding of the brain responses to different expected, but omitted, tones in both passive and active listening conditions was above chance based on the responses to the real sound in active listening conditions. Topographic ERP analyses and electrical source estimations revealed that, in the absence of any stimulation, experience-based internal models elicit an electrophysiological activity different from noise and that the temporal dynamics of this activity depend on attention. We further found that the expected change in pitch direction of omitted tones modulated the activity of left posterior temporal areas 140-200 msec after the onset of omissions. Collectively, our results indicate that, even in the absence of any stimulation, internal models modulate brain activity as do real sounds, indicating that auditory filling in can be accounted for by predictive activity.

Intrahemispheric cortico-cortical connections of the human auditory cortex.

The human auditory cortex comprises the supratemporal plane and large parts of the temporal and parietal convexities. We have investigated the relevant intrahemispheric cortico-cortical connections using in vivo DSI tractography combined with landmark-based registration, automatic cortical parcellation and whole-brain structural connection matrices in 20 right-handed male subjects. On the supratemporal plane, the pattern of connectivity was related to the architectonically defined early-stage auditory areas. It revealed a three-tier architecture characterized by a cascade of connections from the primary auditory cortex to six adjacent non-primary areas and from there to the superior temporal gyrus. Graph theory-driven analysis confirmed the cascade-like connectivity pattern and demonstrated a strong degree of segregation and hierarchy within early-stage auditory areas. Putative higher-order areas on the temporal and parietal convexities had more widely spread local connectivity and long-range connections with the prefrontal cortex; analysis of optimal community structure revealed five distinct modules in each hemisphere. The pattern of temporo-parieto-frontal connectivity was partially asymmetrical. In conclusion, the human early-stage auditory cortical connectivity, as revealed by in vivo DSI tractography, has strong similarities with that of non-human primates. The modular architecture and hemispheric asymmetry in higher-order regions is compatible with segregated processing streams and lateralization of cognitive functions.