Metabolic role of aquaglyceroporin 9 in astrocytes

Aim: Aquaglyceroporin-9 (AQP9) is a member of the Aquaporin channel family involved in water flux through plasma membranes and exhibits the distinctive feature of also being permeable to glycerol and monocarboxylates. AQP9 is detected in astrocytes and catecholaminergic neurons.1 However, the presence of AQP9 in the brain is now debated after a recent publication claiming that AQP9 is not expressed in the brain.2 Based on our results,3 we have evidence of the presence of AQP9 in the brain and we further hypothesize that AQP9 plays a functional role in brain energy metabolism. Methods: The presence of AQP9 in brain of OF1 mice was studied by RT-PCR and immunohistochemistry. To address the role of AQP9 in brain, we used commercial siRNA against AQP9 to knockdown its expression in 2 cultures of astrocytes from two distinct sources (from differentiated stem cells4 and primary astrocyte cultures). After assessment of the decrease of AQP9, glycerol uptake was measured using [H3]-glycerol. Then, modifications of the astrocytic energy metabolism was evaluated by measurement of glucose consumption, lactate release5 and evaluation of the mitochondrial activity by MTT staining. Results: AQP9 is expressed in astrocytes of OF1 mouse brain (mRNA and protein levels). We also showed that AQP9 mRNA and protein are present in cultured astrocytes. Four days after AQP9 siRNA application, the level of expression is significantly decreased by 76% compared to control. Astrocytes with AQP9 knockdown exhibit a 23% decrease of glycerol uptake, showing that AQP9 is a glycerol channel in cultured astrocytes. In parallel, astrocytes with AQP9 knockdown have a 155% increase of their glucose consumption without modifications of lactate release. Moreover, considering the observed glucose consumption increase and the absence of proliferation induction, the significant MTT activity increase (113%) suggests an increase of oxidative metabolism in astrocytes with AQP9 knockdown. Discussion: The involvement of AQP9 in astrocyte energy metabolism adds a new function for this channel in the brain. The determination of the role of AQP9 in astrocytes provides a new perspective on the controversial expression of AQP9 in brain. We also suggest that AQP9 may have a complementary role to monocarboxylate transporters in the regulation of brain energy metabolism.

Nitric Oxide Induces the Expression of the Monocarboxylate Transporter MCT4 in Cultured Astrocytes by a cGMP-Independent Transcriptional Activation

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. (C) 2011 Wiley-Liss, Inc.

Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

New evidence of neuroprotection by lactate after transient focal cerebral ischaemia: extended benefit after intracerebroventricular injection and efficacy of intravenous administration.

BACKGROUND: Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously. METHODS: Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na L-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na L-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h. RESULTS: Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm(3), n = 7 vs. 12.1 ± 3.8 mm(3), n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm(3), n = 12 vs. 29.6 ± 25.4 mm(3), n = 12, p < 0.05). CONCLUSIONS: The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.

Mir-21-Sox2 Axis Delineates Glioblastoma Subtypes with Prognostic Impact.

UNLABELLED: Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21-Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21-Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21-Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT: Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21-Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

Imaging the time-integrated cerebral metabolic activity with subcellular resolution through nanometer-scale detection of biosynthetic products deriving from (13)C-glucose.

Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.

Redox dysregulation in schizophrenia : effect on myelination of cortical structures and connectivity

Cette thèse traite du rôle qu'un facteur de risque génétique développé chez les patients souffrant de schizophrénie, à savoir un déficit de la synthèse du glutathion, peut jouer dans les anomalies de la connectivité cérébrale trouvées chez ces patients. L'essentiel du travail a été consacré à évaluer la structure de la substance blanche dans l'ensemble du cerveau chez un modèle animal par une méthode similaire à celle utilisée en recherche clinique avec l'imagerie par résonance magnétique (IRM). Cette approche de translation inverse chez la souris knock-out de glutamate-cystéine ligase modulateur sous-unité (Gclm KO), avait l'objectif d'étudier l'effet des défenses redox déficientes sur le développement des connexions cérébrales, tout en excluant celui des facteurs non liés au génotype. Après avoir établi le protocole de recherche, l'influence d'une manipulation environnementale a également été étudiée. Pour effectuer une analyse statistique fiable des données d'IRM obtenues, nous .avons d'abord créé un atlas du cerveau de la souris afin de l'utiliser comme modèle pour une segmentation précise des différentes régions du cerveau sur les images IRM obtenues in vivo. Les données provenant de chaque région d'intérêt ont ensuite été étudiées séparément. La qualité de cette méthode a été évaluée dans une expérience de simulation pour déduire la puissance statistique réalisable dans chaque région en fonction du nombre d'animaux utilisés. Ces outils d'analyse nous ont permis d'évaluer l'intégrité de la substance blanche dans le cerveau des souris durant le développement grâce à une expérience longitudinale, en utilisant l'imagerie du tenseur de diffusion (DTI). Nous avons ainsi observé des anomalies dans les paramètres dérivés du tenseur (diffusivité et anisotropie) dans la Commissure Antérieure et le Fimbria/Fornix des souris Gclm KO, par rapport aux animaux contrôles. Ces résultats suggèrent une substance blanche endommagée dans ces régions. Dans une expérience électrophysiologique, Pascal Steullet a montré que ces anomalies ont des conséquences fonctionnelles caractérisées par une réduction de la vitesse de conduction dans les fibres nerveuses. Ces données renforcent les conclusions des analyses d'imagerie. Le mécanisme par lequel une dérégulation redox affecte la structure de la substance blanche reste encore à définir, car une analyse immunohistochimique des protéines constituantes de la couche de myéline des fibres concernées n'a pas donné de résultats concluants. Nous avons également constaté un élargissement des ventricules dans les jeunes souris Gclm KO, mais pas chez les adultes et des anomalies neurochimiques déjà connues chez ces animaux (Duarte et al. 2011), à savoir une réduction du Glutathion et une augmentation de l'acide N-acétylaspartique, de l'Alanine et du ratio Glutamine/Glutamate. Nous avons ensuite testé l'effet d'un stress environnemental supplémentaire, l'élevage en isolement social, sur le phénotype. Ce stress n'a eu aucun effet sur la structure de la substance blanche évaluée par DTI, mais a réduit la concentration de myo-Inositol et augmenté le ratio de Glutamine/Glutamate dans le cortex frontal. Nous avons aussi reproduit dans ce groupe indépendant d'animaux les effets du génotype sur le profil neurochimique, sur la taille des ventricules et aussi sur les paramètres dérivés du tenseur de diffusion dans le Fimbria/Fornix, mais pas dans la Commissure Antérieure. Nos résultats montrent qu'une dérégulation redox d'origine génétique perturbe la structure et la fonction de la substance blanche dans des régions spécifiques, causant ainsi l'élargissement des ventricules. Ces phénotypes rassemblent certaines caractéristiques neuro-anatomiques de la schizophrénie, mais les mécanismes qui en sont responsables demeurent encore inconnus. L'isolement social n'a pas d'effet sur la structure de la substance blanche évaluée par DTI, alors qu'il est prouvé qu'il affecte la maturation des oligodendrocytes. La neurochimie corticale et en particulier le rapport Glutamine/Glutamate a été affecté par le dérèglement redox ainsi que par l'isolement social. En conséquence, ce ratio représente un indice prometteur dans la recherche sur l'interaction du stress environnemental avec le déséquilibre redox dans le domaine de la schizophrénie. -- The present doctoral thesis is concerned with the role that a genetic risk factor for the development of schizophrenia, namely a deficit in Glutathione synthesis, may play in the anomalies of brain connectivity found in patients. Most of the effort was devoted to perform a whole-brain assessment of white matter structure in the Glutamate-Cysteine ligase modulatory knockout mouse model (Gclm KO) using Magnetic Resonance Imaging (MRI) techniques similar to those used in state-of-the-art clinical research. Such reverse translational approach taking brain imaging from the bedside to the bench aimed to investigate the role that deficient redox defenses may play in the development of brain connections while excluding all influencing factors beside the genotype. After establishing the protocol, the influence of further environmental manipulations was also studied. Analysis of MRI images acquired in vivo was one of the main challenges of the project. Our strategy consisted in creating an atlas of the mouse brain to use as segmentation guide and then analyze the data from each region of interest separately. The quality of the method was assessed in a simulation experiment by calculating the statistical power achievable in each brain region at different sample sizes. This analysis tool enabled us to assess white matter integrity in the mouse brain along development in a longitudinal experiment using Diffusion Tensor Imaging (DTI). We discovered anomalies in diffusivity parameters derived from the tensor in the Anterior Commissure and Fimbria/Fornix of Gclm KO mice when compared to wild-type animals, which suggest that the structure of these tracts is compromised in the KO mice. In an elegant electrophysiological experiment, Pascal Steullet has provided evidence that these anomalies have functional consequences in form of reduced conduction velocity in the concerned tracts, thus supporting the DTI findings. The mechanism by which redox dysregulation affects WM structure remains unknown, for the immunohistochemical analysis of myelin constituent proteins in the concerned tracts produced inconclusive results. Our experiments also detected an enlargement of the lateral ventricles in young but not adult Gclm KO mice and confirmed neurochemical anomalies already known to affect this animals (Duarte et al. 2011), namely a reduction in Glutathione and an increase in Glutamine/Glutamate ratio, N-acetylaspartate and Alanine. Using the same methods, we tested the effect of an additional environmental stress on the observed phenotype: rearing in social isolation had no effect on white matter structure as assessed by DTI, but it reduced the concentration of myo-Inositol and increased the Glutamine/Glutamate ratio in the frontal cortex. We could also replicate in this separate group of animals the effects of genotype on the frontal neurochemical profile, ventricular size and diffusivity parameters in the Fimbria/Fornix but not in the Anterior Commissure. Our data show that a redox dysregulation of genetic origin may disrupt white matter structure and function in specific tracts and cause a ventricular enlargement, phenotypes that resemble some neuroanatomical features of schizophrenia. The mechanism responsible remains however unknown. We have also demonstrated that environmental stress in form of social isolation does not affect white matter structure as assessed by DTI even though it is known to affect oligodendrocyte maturation. Cortical neurochemistry, and specifically the Glutamine to Glutamate balance was affected both by redox dysregulation and social isolation, and is thus a good target for further research on the interaction of redox imbalance and environmental stress in schizophrenia.

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior.

New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.

Genotoxic, neurotoxic and neuroprotective activities of apomorphine and its oxidized derivative 8-oxo-apomorphine

Apomorphine is a dopamine receptor agonist proposed to be a neuroprotective agent in the treatment of patients with Parkinson's disease. Both in vivo and in vitro studies have shown that apomorphine displays both antioxidant and pro-oxidant actions, and might have either neuroprotective or neurotoxic effects on the central nervous system. Some of the neurotoxic effects of apomorphine are mediated by its oxidation derivatives. In the present review, we discuss recent studies from our laboratory in which the molecular, cellular and neurobehavioral effects of apomorphine and its oxidized derivative, 8-oxo-apomorphine-semiquinone (8-OASQ), were evaluated in different experimental models, i.e., in vitro genotoxicity in Salmonella/microsome assay and WP2 Mutoxitest, sensitivity assay in Saccharomyces cerevisiae, neurobehavioral procedures (inhibition avoidance task, open field behavior, and habituation) in rats, stereotyped behavior in mice, and Comet assay and oxidative stress analyses in mouse brain. Our results show that apomorphine and 8-OASQ induce differential mutagenic, neurochemical and neurobehavioral effects. 8-OASQ displays cytotoxic effects and oxidative and frameshift mutagenic activities, while apomorphine shows antimutagenic and antioxidant effects in vitro. 8-OASQ induces a significant increase of DNA damage in mouse brain tissue. Both apomorphine and 8-OASQ impair memory for aversive training in rats, although the two drugs showed a different dose-response pattern. 8-OASQ fails to induce stereotyped behaviors in mice. The implications of these findings are discussed in the light of evidence from studies by other groups. We propose that the neuroprotective and neurotoxic effects of dopamine agonists might be mediated, in part, by their oxidized metabolites.

Pharmacology and toxicology of diphenyl diselenide in several biological models

The pharmacology of synthetic organoselenium compounds indicates that they can be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumor and anti-infectious agents, and immunomodulators. In this review, we focus on the effects of diphenyl diselenide (DPDS) in various biological model organisms. DPDS possesses antioxidant activity, confirmed in several in vitro and in vivo systems, and thus has a protective effect against hepatic, renal and gastric injuries, in addition to its neuroprotective activity. The activity of the compound on the central nervous system has been studied since DPDS has lipophilic characteristics, increasing adenylyl cyclase activity and inhibiting glutamate and MK-801 binding to rat synaptic membranes. Systemic administration facilitates the formation of long-term object recognition memory in mice and has a protective effect against brain ischemia and on reserpine-induced orofacial dyskinesia in rats. On the other hand, DPDS may be toxic, mainly because of its interaction with thiol groups. In the yeast Saccharomyces cerevisiae, the molecule acts as a pro-oxidant by depleting free glutathione. Administration to mice during cadmium intoxication has the opposite effect, reducing oxidative stress in various tissues. DPDS is a potent inhibitor of d-aminolevulinate dehydratase and chronic exposure to high doses of this compound has central effects on mouse brain, as well as liver and renal toxicity. Genotoxicity of this compound has been assessed in bacteria, haploid and diploid yeast and in a tumor cell line.

Performance assessment of the single photon emission microscope: high spatial resolution SPECT imaging of small animal organs

The single photon emission microscope (SPEM) is an instrument developed to obtain high spatial resolution single photon emission computed tomography (SPECT) images of small structures inside the mouse brain. SPEM consists of two independent imaging devices, which combine a multipinhole collimator, a high-resolution, thallium-doped cesium iodide [CsI(Tl)] columnar scintillator, a demagnifying/intensifier tube, and an electron-multiplying charge-coupling device (CCD). Collimators have 300- and 450-µm diameter pinholes on tungsten slabs, in hexagonal arrays of 19 and 7 holes. Projection data are acquired in a photon-counting strategy, where CCD frames are stored at 50 frames per second, with a radius of rotation of 35 mm and magnification factor of one. The image reconstruction software tool is based on the maximum likelihood algorithm. Our aim was to evaluate the spatial resolution and sensitivity attainable with the seven-pinhole imaging device, together with the linearity for quantification on the tomographic images, and to test the instrument in obtaining tomographic images of different mouse organs. A spatial resolution better than 500 µm and a sensitivity of 21.6 counts·s-1·MBq-1 were reached, as well as a correlation coefficient between activity and intensity better than 0.99, when imaging 99mTc sources. Images of the thyroid, heart, lungs, and bones of mice were registered using 99mTc-labeled radiopharmaceuticals in times appropriate for routine preclinical experimentation of <1 h per projection data set. Detailed experimental protocols and images of the aforementioned organs are shown. We plan to extend the instrument's field of view to fix larger animals and to combine data from both detectors to reduce the acquisition time or applied activity.

La sécrétion de la protéine Tau : nouveau mécanisme de propagation de la pathologie de Tau dans la maladie d'Alzheimer

Tau est une protéine associée aux microtubules enrichie dans l’axone. Dans la maladie d’Alzheimer, Tau devient anormalement hyperphosphorylée, s’accumule dans le compartiment somato-dendritique et s’agrège pour former des enchevêtrements neurofibrillaires (NFTs). Ces NFTs se propagent dans le cerveau dans un ordre bien précis. Ils apparaissent d’abord dans le cortex transenthorinal pour ensuite se propager là où ces neurones projettent, c’est-à-dire au cortex entorhinal. Les NFTs s’étendent ensuite à l’hippocampe puis à différentes régions du cortex et néocortex. De plus, des études récentes ont démontré que la protéine Tau peut être sécrétée par des lignées neuronales et que lorsqu’on injecte des agrégats de Tau dans un cerveau de souris, ceux-ci peuvent pénétrer dans les neurones et induire la pathologie de Tau dans le cerveau. Ces observations ont mené à l’hypothèse que la protéine Tau pathologique pourrait être sécrétée par les neurones, pour ensuite être endocytée par les cellules avoisinantes et ainsi propager la maladie. L’objectif de la présente étude était donc de prouver la sécrétion de la protéine Tau par les neurones et d’identifier par quelle voie elle est secrétée. Nos résultats ont permis de démontrer que la protéine Tau est sécrétée par des neurones corticaux de souris de type sauvage ainsi que dans un modèle de surexpression dans des cellules HeLa et PC12. Nos résultats indiquent que la sécrétion de Tau se ferait par les autophagosomes. Finalement, nous avons démontré que la protéine Tau sécrétée est déphosphorylée et clivée par rapport à la protéine Tau intracellulaire non sécrétée.