Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

Making sense of AMPA receptor trafficking by modeling molecular mechanisms of synaptic plasticity.

Synaptic plasticity involves a complex molecular machinery with various protein interactions but it is not yet clear how its components give rise to the different aspects of synaptic plasticity. Here we ask whether it is possible to mathematically model synaptic plasticity by making use of known substances only. We present a model of a multistable biochemical reaction system and use it to simulate the plasticity of synaptic transmission in long-term potentiation (LTP) or long-term depression (LTD) after repeated excitation of the synapse. According to our model, we can distinguish between two phases: first, a "viscosity" phase after the first excitation, the effects of which like the activation of NMDA receptors and CaMKII fade out in the absence of further excitations. Second, a "plasticity" phase actuated by an identical subsequent excitation that follows after a short time interval and causes the temporarily altered concentrations of AMPA subunits in the postsynaptic membrane to be stabilized. We show that positive feedback is the crucial element in the core chemical reaction, i.e. the activation of the short-tail AMPA subunit by NEM-sensitive factor, which allows generating multiple stable equilibria. Three stable equilibria are related to LTP, LTD and a third unfixed state called ACTIVE. Our mathematical approach shows that modeling synaptic multistability is possible by making use of known substances like NMDA and AMPA receptors, NEM-sensitive factor, glutamate, CaMKII and brain-derived neurotrophic factor. Furthermore, we could show that the heteromeric combination of short- and long-tail AMPA receptor subunits fulfills the function of a memory tag.

Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. GLIA 2014;62:1125-1141.

Hypoxie-ischémie cérébrale néonatale : rôle de la voie de JNK et implication de l'autophagie dans la mort neuronale

Résumé : Malgré les immenses progrès réalisés depuis plusieurs années en médecine obstétricale ainsi qu'en réanimation néonatale et en recherche expérimentale, l'asphyxie périnatale, une situation de manque d'oxygène autour du moment de la naissance, reste une cause majeure de mortalité et de morbidité neurologique à long terme chez l'enfant (retard mental, paralysie cérébrale, épilepsie, problèmes d'apprentissages) sans toutefois de traitement pharmacologique réel. La nécessité de développer de nouvelles stratégies thérapeutiques pour les complications de l'asphyxie périnatale est donc aujourd'hui encore essentielle. Le but général de ce travail est l'identification de nouvelles cibles thérapeutiques impliquées dans des mécanismes moléculaires pathologiques induits par l'hypoxie-ischémie (HI) dans le cerveau immature. Pour cela, le modèle d'asphyxie périnatale (proche du terme) le plus reconnu chez le rongeur a été développé (modèle de Rice et Vannucci). Il consiste en la ligature permanente d'une artère carotide commune (ischémie) chez le raton de 7 jours combinée à une période d'hypoxie à 8% d'oxygène. Il permet ainsi d'étudier les lésions de type hypoxique-ischémique dans différentes régions cérébrales dont le cortex, l'hippocampe, le striatum et le thalamus. La première partie de ce travail a abordé le rôle de deux voies de MAPK, JNK et p38, après HI néonatale chez le raton à l'aide de peptides inhibiteurs. Tout d'abord, nous avons démontré que D-JNKI1, un peptide inhibiteur de la voie de JNK présentant de fortes propriétés neuroprotectrices dans des modèles d'ischémie cérébrale adulte ainsi que chez le jeune raton, peut intervenir sur différentes voies de mort dont l'activation des calpaïnes (marqueur de la nécrose précoce), l'activation de la caspase-3 (marqueur de l'apoptose) et l'expression de LC3-II (marqueur de macroautophagie). Malgré ces effets positifs le traitement au D-JNKI1 ne modifie pas l'étendue de la lésion cérébrale. L'action limitée de D-JNKI1 peut s'expliquer par une implication modérée des JNKs (faiblement activées et principalement l'isotype JNK3) après HI néonatale sévère. Au contraire, l'inhibition de la voie de nNOS/p38 par le peptide DTAT-GESV permet une augmentation de 20% du volume du tissu sain à court et long terme. Le second projet a étudié les effets de l'HI néonatale sur l'autophagie neuronale. En effet, l'autophagie est un processus catabolique essentiel au bien-être de la cellule. Le type principal d'autophagie (« macroautophagie » , que nous appellerons par la suite « autophagie ») consiste en la séquestration d'éléments à dégrader (protéines ou organelles déficients) dans un compartiment spécialisé, l'autophagosome, qui fusionne avec un lysosome pour former un autolysosome où le contenu est dégradé par les hydrolases lysosomales. Depuis peu, l'excès ou la dérégulation de l'autoptiagie a pu être impliqué dans la mort cellulaire en certaines conditions de stress. Ce travail démontre que l'HI néonatale chez le raton active fortement le flux autophagique, c'est-à-dire augmente la formation des autophagosomes et des autolysosomes, dans les neurones en souffrance. De plus, la relation entre l'autophagie et l'apoptose varie selon la région cérébrale. En effet, alors que dans le cortex les neurones en voie de mort présentent des caractéristiques mixtes apoptotiques et autophagiques, ceux du CA3 sont essentiellement autophagiques et ceux du CA1 sont principalement apoptotiques. L'induction de l'autophagie après HI néonatale semble donc participer à la mort neuronale soit par l'enclenchement de l'apoptose soit comme mécanisme de mort en soi. Afin de comprendre la relation pouvant exister entre autophagie et apoptase un troisième projet a été réalisé sur des cultures primaires de neurones corticaux exposés à un stimulus apoptotique classique, la staurosporine (STS). Nous avons démontré que l'apoptose induite par la STS était précédée et accompagnée par une forte activation du flux autophagique neuronal. L'inhibition de l'autophagie de manière pharmacologique (3-MA) ou plus spécifiquement par ARNs d'interférence dirigés contre deux protéines autophagiques importantes (Atg7 et Atg5) a permis de mettre en évidence des rôles multiples de l'autophagie dans la mort neuronale. En effet, l'autophagie prend non seulement part à une voie de mort parallèle à l'apoptose pouvant être impliquée dans l'activation des calpaïnes, mais est également partiellement responsable de l'induction des voies apoptotiques (activation de la caspase-3 et translocation nucléaire d'AIF). En conclusion, ce travail a montré que l'inhibition de JNK par D-JNKI1 n'est pas un outil neuroprotecteur efficace pour diminuer la mort neuronale provoquée par l'asphyxie périnatalé sévère, et met en lumière deux autres voies thérapeutiques beaucoup plus prometteuses, l'inhibition de nNOS/p38 ou de l'autophagie. ABSTRACT : Despite enormous progress over the last«decades in obstetrical and neonatal medicine and experimental research, perinatal asphyxia, a situation of lack of oxygen around the time of the birth, remains a major cause of mortality and long term neurological morbidity in children (mental retardation, cerebral palsy, epilepsy, learning difficulties) without any effective treatment. It is therefore essential to develop new therapeutic strategies for the complications of perinatal asphyxia. The overall aim of this work was to identify new therapeutic targets involved in pathological molecular mechanisms induced by hypoxia-ischemia (HI) in the immature brain. For this purpose, the most relevant model of perinatal asphyxia (near term) in rodents has been developed (model of Rice and Vannucci). It consists in the permanent ligation of one common carotid artery (ischemia) in the 7-day-old rat combined with a period of hypoxia at 8% oxygen. This model allows the study of the hypoxic-ischemic lesion in different brain regions including the cortex, hippocampus, striatum and thalamus. The first part of this work addressed the role of two MAPK pathways (JNK and p38) after rat neonatal HI using inhibitory peptides. First, we demonstrated that D-JNKI1, a JNK peptide inhibitor presenting strong neuroprotective properties in models of cerebral ischemia in adult and young rats, could affect different cell death mechanisms including the activation of calpain (a marker of necrosis) and caspase-3 (a marker of apoptosis), and the expression of LC3-II (a marker of macroautophagy). Despite these positive effects, D-JNKI1 did not modify the extent of brain damage. The limited action of D-JNKI1 can be explained by the fact that JNKs were only moderately involved (weakly activated and principally the JNK3 isotype) after severe neonatal HI. In contrast, inhibition of nNOS/p38 by the peptide D-TAT-GESV increased the surviving tissue volume by around 20% at short and long term. The second project investigated the effects of neonatal HI on neuronal autophagy. Indeed, autophagy is a catabolic process essential to the well-being of the cell. The principal type of autophagy ("macroautophagy", that we shall henceforth call "autophagy") involves the sequestration of elements to be degraded (deficient proteins or organelles) in a specialized compartment, the autophagosome, which fuses with a lysosome to form an autolysosome where the content is degraded by lysosomal hydrolases. Recently, an excess or deregulation of autophagy has been implicated in cell death in some stress conditions. The present study demonstrated that rat neonatal HI highly enhanced autophagic flux, i.e. increased autophagosome and autolysosome formation, in stressed neurons. Moreover, the relationship between autophagy and apoptosis varies according to the brain region. Indeed, whereas dying neurons in the cortex exhibited mixed features of apoptosis and autophagy, those in CA3 were primarily autophagíc and those in CA1 were mainly apoptotic. The induction of autophagy after neonatal HI seems to participate in neuronal death either by triggering apoptosis or as a death mechanism per se. To understand the relationships that may exist between autophagy and apoptosis, a third project has been conducted using primary cortical neuronal cultures exposed to a classical apoptotic stimulus, staurosporine (STS). We demonstrated that STS-induced apoptosis was preceded and accompanied by a strong activation of neuronal autophagic flux. Inhibition of autophagy pharmacologically (3-MA) or more specifically by RNA interference directed against two important autophagic proteins (Atg7 and AtgS) showed multiple roles of autophagy in neuronal death. Indeed, autophagy was not only involved in a death pathway parallel to apoptosis possibly involved in the activation of calpains, but was also partially responsible for the induction of apoptotic pathways (caspase-3 activation and AIF nuclear translocation). In conclusion, this study showed that JNK inhibition by D-JNKI1 is not an effective neuroprotective tool for decreasing neuronal death following severe perinatal asphyxia, but highlighted two more promising therapeutic approaches, inhibition of the nNOSlp38 pathway or of autophagy.

Ammonia toxicity to the brain.

Hyperammonemia can be caused by various acquired or inherited disorders such as urea cycle defects. The brain is much more susceptible to the deleterious effects of ammonium in childhood than in adulthood. Hyperammonemia provokes irreversible damage to the developing central nervous system: cortical atrophy, ventricular enlargement and demyelination lead to cognitive impairment, seizures and cerebral palsy. The mechanisms leading to these severe brain lesions are still not well understood, but recent studies show that ammonium exposure alters several amino acid pathways and neurotransmitter systems, cerebral energy metabolism, nitric oxide synthesis, oxidative stress and signal transduction pathways. All in all, at the cellular level, these are associated with alterations in neuronal differentiation and patterns of cell death. Recent advances in imaging techniques are increasing our understanding of these processes through detailed in vivo longitudinal analysis of neurobiochemical changes associated with hyperammonemia. Further, several potential neuroprotective strategies have been put forward recently, including the use of NMDA receptor antagonists, nitric oxide inhibitors, creatine, acetyl-L-carnitine, CNTF or inhibitors of MAPKs and glutamine synthetase. Magnetic resonance imaging and spectroscopy will ultimately be a powerful tool to measure the effects of these neuroprotective approaches.

Multi-scale integration and predictability in resting state brain activity.

The human brain displays heterogeneous organization in both structure and function. Here we develop a method to characterize brain regions and networks in terms of information-theoretic measures. We look at how these measures scale when larger spatial regions as well as larger connectome sub-networks are considered. This framework is applied to human brain fMRI recordings of resting-state activity and DSI-inferred structural connectivity. We find that strong functional coupling across large spatial distances distinguishes functional hubs from unimodal low-level areas, and that this long-range functional coupling correlates with structural long-range efficiency on the connectome. We also find a set of connectome regions that are both internally integrated and coupled to the rest of the brain, and which resemble previously reported resting-state networks. Finally, we argue that information-theoretic measures are useful for characterizing the functional organization of the brain at multiple scales.

Temporal changes in mRNA expression of the brain nutrient transporters in the lithium-pilocarpine model of epilepsy in the immature and adult rat.

The lithium-pilocarpine model mimics most features of human temporal lobe epilepsy. Following our prior studies of cerebral metabolic changes, here we explored the expression of transporters for glucose (GLUT1 and GLUT3) and monocarboxylates (MCT1 and MCT2) during and after status epilepticus (SE) induced by lithium-pilocarpine in PN10, PN21, and adult rats. In situ hybridization was used to study the expression of transporter mRNAs during the acute phase (1, 4, 12 and 24h of SE), the latent phase, and the early and late chronic phases. During SE, GLUT1 expression was increased throughout the brain between 1 and 12h of SE, more strongly in adult rats; GLUT3 increased only transiently, at 1 and 4h of SE and mainly in PN10 rats; MCT1 was increased at all ages but 5-10-fold more in adult than in immature rats; MCT2 expression increased mainly in adult rats. At all ages, MCT1 and MCT2 up-regulation was limited to the circuit of seizures while GLUT1 and GLUT3 changes were more widespread. During the latent and chronic phases, the expression of nutrient transporters was normal in PN10 rats. In PN21 rats, GLUT1 was up-regulated in all brain regions. In contrast, in adult rats GLUT1 expression was down-regulated in the piriform cortex, hilus and CA1 as a result of extensive neuronal death. The changes in nutrient transporter expression reported here further support previous findings in other experimental models demonstrating rapid transcriptional responses to marked changes in cerebral energetic/glucose demand.

Behavioural alteration in chronic pain: are brain glia involved?

Behavioural symptoms such as abnormal emotionality (including anxious and depressive episodes) and cognition (for instance weakened decision-making) are highly frequent in both chronic pain patients and their animal models. The theory developed in the present article posits that alterations in glial cells (astrocytes and microglia) in cortical and limbic brain regions might be the origin of such emotional and cognitive chronic pain-associated impairments. Indeed, in mood disorders (unipolar depression, anxiety disorders, autism or schizophrenia) glial changes in brain regions involved in mood control (prefrontal and cingulate cortices, amygdala and the hippocampus) have been recurrently described. Besides, glial cells have been undoubtedly identified as key actors in the sensory component of chronic pain, owing to the profound phenotypical changes they undergo throughout the sensory pathway. Hence, the possibility arises that brain astrocytes and microglia react in upper brain structures as well, mediating the related mood and cognitive dysfunctions in chronic pain. So far, only very few studies have provided results in this prospect, mainly indirectly in pain-independent researches. Nevertheless, the first scant available data seem to merge in a unified description of a brain glial reaction occurring after chronic peripheral lesion. The present article uses this scarce literature to formulate the provocative theory of a glia-driven mood and cognitive dysfunction in chronic pain, expounding upon its validity and putative therapeutical impact as well as its current limitations and expected future developments.

Rôle de la MGMT et implications cliniques dans les tumeurs cérébrales [Role of MGMT and clinical applications in brain tumours]

Glioblastoma multiforme is the most common and most malignant primary brain tumour with a dismal prognosis. The advent of new chemotherapies with alkylating agents crossing the blood-brain barrier, like temozolomide, have permitted to notably ameliorate the survival of a subgroup of patients. Improved outcome was associated with epigenetic silencing of the MGMT (O6-methylguanin methyltransferase) gene by promotor methylation, thereby blocking its repair capability, thus rendering the alkylating agents more effective. This particularity can be tested by methylation specific PCR on resected tumour tissue, best on fresh frozen biopsies, and allows identification of patients more susceptible to respond favourably to the treatment.

Cell locations for AQP1, AQP4 and 9 in the non-human primate brain.

The presence of three water channels (aquaporins, AQP), AQP1, AQP4 and AQP9 were observed in normal brain and several rodent models of brain pathologies. Little is known about AQP distribution in the primate brain and its knowledge will be useful for future testing of drugs aimed at preventing brain edema formation. We studied the expression and cellular distribution of AQP1, 4 and 9 in the non-human primate brain. The distribution of AQP4 in the non-human primate brain was observed in perivascular astrocytes, comparable to the observation made in the rodent brain. In contrast with rodent, primate AQP1 is expressed in the processes and perivascular endfeet of a subtype of astrocytes mainly located in the white matter and the glia limitans, possibly involved in water homeostasis. AQP1 was also observed in neurons innervating the pial blood vessels, suggesting a possible role in cerebral blood flow regulation. As described in rodent, AQP9 mRNA and protein were detected in astrocytes and in catecholaminergic neurons. However additional locations were observed for AQP9 in populations of neurons located in several cortical areas of primate brains. This report describes a detailed study of AQP1, 4 and 9 distributions in the non-human primate brain, which adds to the data already published in rodent brains. This relevant species differences have to be considered carefully to assess potential drugs acting on AQPs non-human primate models before entering human clinical trials.

Differential phosphorylation of MAP1b during postnatal development of the cat brain.

Microtubule-associated protein 1b, previously also referred to as microtubule-associated protein 5 or microtubule-associated protein 1x, is a major component of the juvenile cytoskeleton, and is essential during the early differentiation of neurons. It is required for axonal growth and its function is influenced by phosphorylation. The distribution of microtubule-associated protein 1b in kitten cerebellum and cortex during postnatal development was studied with two monoclonal antibodies. Hybridoma clone AA6 detected a non-phosphorylated site, while clone 125 detected a site phosphorylated by casein-kinase II. On blots, both monoclonal antibodies stained the same two proteins of similar molecular weights, also referred to as microtubule-associated protein 5a and 5b. Antibody 125 detected a phosphorylated epitope on both microtubule-associated protein 1b forms; dephosphorylation by alkaline phosphatase abolished the immunological detection. During development of cat cortex and cerebellum, AA6 stained the perikarya and dendrites of neurons during their early differentiation, and especially labelled newly generated axons. The staining decreased during development, and axonal staining was reduced in adult tissue. In contrast to previous reports which demonstrated that antibodies against phosphorylated microtubule-associated protein 1b label exclusively axons, antibody 125 also localized microtubule-associated protein 1b in cell bodies and dendrites, even in adulthood. Some nuclear staining was observed, indicating that a phosphorylated form of microtubule-associated protein 1b may participate in nuclear function. These results demonstrate that microtubule-associated protein 1b is subject to CK2-type phosphorylation throughout neuronal maturation and suggest that phosphorylation of microtubule-associated protein 1b may participate in juvenile and mature-type microtubule functions throughout development.