Philippe Vallet

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia.

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.

A comparative study of histological and immunohistochemical methods for neurofibrillary tangles and senile plaques in Alzheimer's disease

SummarySeveral studies have demonstrated that the accurate visualization and quantification of pathological lesions in neurodegenerative disorders depend on the reliability of staining methods. In an attempt to gain a better assessment of the density and distribution of the neuropathological markers of Alzheimers disease, we compared the staining efficiency of a modified thioflavine S protocol for neurofibrillary tangles (NFT) and senile plaques (SP) to different argentic impregnation techniques (Bielchowsky, Gallyas, Globus, Campbell-Switzer-Martin) and to immunohistochemical stainings obtained with two different antibodies against the amyloid β protein A4 and the microtubule-associated tau protein. The modified thioflavine S technique (MTST) detects up to 60% more SP and up to 50% more NFT than the Bielschowsky and Globus methods, respectively. The results obtained with the specific antibodies are comparable to those obtained with the MTST, but these immunotechniques are more expensive and time consuming for routine neuropathological evaluation, and the appropriate antibodies are not always commercially available. Furthermore, the morphological appearance of NFT and SP with MTST is greatly improved when compared to the classical thioflavine S and the increased signal-to-noise ratio between specifically stained structures and background permits an accurate semi-automatic quantification.

An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer's disease

Large differences are usually observed when standard staining methods for a number of pathological lesions in neurodegenerative disorders are compared. With the modified thioflavine S method presented here (easy and cheap to perform), the morphological appearance of the stained neurofibrillary tangles (NFT) and senile plaques (SP) is greatly improved. Furthermore, the intense contrast between stained lesions and background obtained with this technique permits an accurate automatic quantification of NFT and SP using a computer-assisted image analysis system.

Resistance to cerebral ischemic injury in UCP2 knockout mice: evidence for a role of UCP2 as a regulator of mitochondrial glutathione levels

Uncoupling protein 2 (UCP2) is suggested to be a regulator of reactive oxygen species production in mitochondria. We performed a detailed study of brain injury, including regional and cellular distribution of UCP2 mRNA, as well as measures of oxidative stress markers following permanent middle cerebral artery occlusion in UCP2 knockout (KO) and wild‐type (WT) mice. Three days post ischemia, there was a massive induction of UCP2 mRNA confined to microglia in the peri‐infarct area of WT mice. KO mice were less sensitive to ischemia as assessed by reduced brain infarct size, decreased densities of deoxyuridine triphosphate nick end‐labelling (TUNEL)‐labelled cells in the peri‐infact area and lower levels of lipid peroxidation compared with WT mice. This resistance may be related to the substantial increase of basal manganese superoxide dismutase levels in neurons of KO mice. Importantly, we found a specific decrease of mitochondrial glutathione (GSH) levels in UCP2 expressing microglia of WT, but not in KO mice after ischemia. This specific association between UCP2 and mitochondrial GSH levels regulation was further confirmed using lipopolysaccharide models of peripheral inflammation, and in purified peritoneal macrophages. Moreover, our data imply that UCP2 is not directly involved in the regulation of ROS production but acts by regulating mitochondrial GSH levels in microglia.

Distinct patterns of neuronal loss and Alzheimer's disease lesion distribution in elderly individuals older than 90 years

To explore the characteristics of brain aging in very old individuals, we performed a quantitative analysis of neurofibrillary tangle (NFT) and senile plaque (SP) distribution and neuron densities in 13 nondemented patients, 15 patients with very mild cognitive impairment, and 22 patients with Alzheimers disease (AD), all older than 96 years of age. Non-demented cases displayed substantial NFT formation in the CA1 field and entorhinal cortex only. Very mild cognitive impairment cases were characterized by the presence of high NFT densities in layers V and VI of area 20, and AD cases had very high NFT densities in the CA1 field compared to nondemented cases. Moreover, high SP densities were found in areas 9 and 20 in AD, but not in cases with very mild cognitive impairment and nondemented cases. In contrast to previous reports concerning younger demented patients, neuron densities were preserved in the CA1 field, dentate hilus, and subiculum in centenarians with AD. In these cases, there was a marked neuronal loss in layers II and V of the entorhinal cortex, and in areas 9 and 20. In the present series, no correlation was found between neurofibrillary tangle and neuron densities in the areas studied, whereas there was a negative correlation between senile plaque and neuron densities in area 20. The comparison between the present data and those reported previously concerning younger cohorts suggests that there is a differential cortical vulnerability to the degenerative process near the upper age-limit of.

Excess of serotonin affects embryonic interneuron migration through activation of the serotonin receptor 6

The discovery that a common polymorphism (5-HTTLPR, short variant) in the human serotonin transporter gene (SLC6A4) can influence personality traits and increase the risk for depression in adulthood has led to the hypothesis that a relative increase in the extracellular levels of serotonin (5-HT) during development could be critical for the establishment of brain circuits. Consistent with this idea, a large body of data demonstrate that 5-HT is a strong neurodevelopmental signal that can modulate a wide variety of cellular processes. In humans, serotonergic fibers appear in the developing cortex as early as the 10th gestational week, a period of intense neuronal migration. In this study we hypothesized that an excess of 5-HT could affect embryonic cortical interneuron migration. Using time-lapse videometry to monitor the migration of interneurons in embryonic mouse cortical slices, we discovered that the application of 5-HT decreased interneuron migration in a reversible and dose-dependent manner. We next found that 5-HT6 receptors were expressed in cortical interneurons and that 5-HT6 receptor activation decreased interneuron migration, whereas 5-HT6 receptor blockade prevented the migratory effects induced by 5-HT. Finally, we observed that interneurons were abnormally distributed in the cerebral cortex of serotonin transporter gene (Slc6a4) knockout mice that have high levels of extracellular 5-HT. These results shed new light on the neurodevelopmental alterations caused by an excess of 5-HT during the embryonic period and contribute to a better understanding of the cellular processes that could be modulated by genetically controlled differences in human 5-HT homeostasis.

In vivo over‐expression of interleukin‐10 increases resistance to focal brain ischemia in mice

Early studies showed that the administration of the anti‐inflammatory cytokine interleukin‐10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO‐related molecular mechanisms. IL10 was over‐expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase 3 compared with wild type mice. Under basal conditions, anti‐inflammatory factors such as nerve growth factor and GSH were up‐regulated and the pro‐inflammatory cytokine IL1β was down‐regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro‐inflammatory cytokines, including tumor necrosis factor‐α, interferon‐γ, and IL1β. Our data indicate that constitutive IL10 over‐expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.

Inhibition of post-ischemic brain injury by clusterin overexpression.

(Fig. 1e). In contrast, there was no prion transport in Caco-2 cultures without M cells (n = 9) (Fig. 1e), except for traces of infectivity (< 1 logLD50) in one case. Transport of prion infectivity by Raji B cells in M-cell–containing cocultures is extremely unlikely, since Raji B cells are restricted to the basolateral compartment (Fig. 1c) and are not capable of traversing the epithelial monolayer to the apical compartment. These findings indicate that M-cell differentiation is necessary and sufficient for active transepithelial prion transport in vitro. M-cell–dependent uptake of foreign antigens or particles is known to be followed by rapid transcytosis directly to the intraepithelial pocket, where key players of the immune system, such as macrophages, dendritic cells and lymphocytes, are located. Because at least some of these immune cells have been shown to be crucially involved in the process of neuroinvasion, prions might exploit the M-cell–dependent transcytosis to gain access to the immune system. These findings indicate that M cells are a plausible candidate for the mucosal portal of prion infection.

Postnatal distribution of cpp32/caspase 3 mRNA in the mouse central nervous system: An in situ hybridization study

Apoptotic cell death is a major feature of the developing nervous system and of certain neurodegenerative diseases. Various gene effectors and repressors of this type of cell death have been identified. Among them, bcl‐xl and bax, which encode for antiapoptotic and proapoptotic proteins, respectively, play major roles during development. The gene cpp32 encodes for the caspase 3 cysteine protease and is a critical mediator of cell death during embryonic development in the mammalian brain. To gain insight into the possible implications of these cell death genes during the postnatal development, we investigated the expression of bax, bcl‐xl, and cpp32 mRNAs by in situ hybridization in the mouse brain from birth to adulthood. Whereas bax and bcl‐xl mRNAs were expressed widely in neonates and adult mice, our results showed that cpp32 mRNA levels were decreased strongly from 12 postnatal days. From 1 postnatal day to 12 postnatal days, cpp32 mRNA was expressed ubiquitously in all brain nuclei, including areas where neurogenesis occurred. A positive correlation between areas displaying high levels of mRNA and apoptotic nuclei also was shown. In the adult, cpp32 mRNA was restricted to the piriform and entorhinal cortices, the neocortex, and to areas where neurogenesis is observed (e.g., olfactory bulb and dentate gyrus). The same pattern of expression was observed in adult mice over‐expressing the antiapoptotic protein Bcl‐2. These results demonstrate that the expression of cpp32 mRNA is highly regulated during the mouse postnatal period, leading to a specific distribution in the adult central nervous system. Moreover, the prevention of cell death by Bcl‐2 likely is not linked to the regulation of caspase mRNA levels. J. Comp. Neurol. 409:339–357, 1999.

Increased Infarct Size and Lack of Hyperphagic Response after Focal Cerebral Ischemia in Peroxisome Proliferator-Activated Receptor β-Deficient Mice:

Peroxisome proliferator-activated receptors (PPARs) are involved in energy expenditure, regulation of inflammatory processes, and cellular protection in peripheral tissues. Among the different types of PPARs, PPARβ is the only one to be widely expressed in cortical neurons. Using PPARβ knockout (KO) mice, we report here a detailed investigation of the role of PPARβ in cerebral ischemic damage, associated inflammatory and antioxidant processes as well as food intake regulation after middle cerebral artery occlusion (MCAO). The PPARβ KO mice had a two-fold increase in infarct size compared with wild-type (WT) mice. Brain oxidative stress was dramatically enhanced in these KO mice, as documented by an increased content of malondialdehyde, decreased levels of glutathione and manganese superoxide dismutase, and no induction of uncoupling protein 2 (UCP2) mRNA. Unlike WT mice, PPARβ KO mice showed a marked increase of prooxidant interferon-gamma but no induction of nerve growth factor and tumor necrosis factor alpha after MCAO. In WT mice, MCAO resulted in inflammation-specific transient hyperphagia from day 3 to day 5 after ischemia, which was associated with an increase in neuropeptide Y (NPY) mRNA. This hyperphagic phase and NPY mRNA induction were not observed in PPARβ KO mice. Furthermore, our study also suggests for the first time that UCP2 is involved in MCAO food intake response. These data indicate that PPARβ plays an important role in integrating and regulating central inflammation, antioxidant mechanisms, and food intake after MCAO, and suggest that the use of PPARβ agonists may be of interest for the prevention of central ischemic damage.