Raymond Pontcharraud

Effects of 4-hydroxynonenal, a lipid peroxidation product, on dopamine transport and Na+/K+ ATPase in rat striatal synaptosomes

Incubation of rat striatal synaptosomes in ascorbic acid induced the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and 4-hydroxynonenal (4-HNE), a lipid peroxidation aldehydic product. Incubations with 4-HNE, used at a range of concentrations comparable to those obtained during peroxidation, induced a simultaneous, dose-dependent decrease of dopamine (DA) uptake and Na+/K+ ATPase activity and a loss of sulfhydryl (SH) groups. Similar results were observed in a previous study when lipid peroxidation was induced after incubation of synaptosomes in ascorbic acid. Taken together, these data suggest that 4-HNE is an important mediator of oxidative stress and may alter DA uptake after binding to SH groups of the DA transporter and to Na+/K+ ATPase. These toxic events may contribute to the onset and progression of Parkinsons disease.

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease.

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimers disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.

Interaction of double-stranded RNA-dependent protein kinase (PKR) with the death receptor signaling pathway in amyloid beta (Abeta)-treated cells and in APPSLPS1 knock-in mice.

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APPSLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APPSLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT451-PKR and pS194-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.

Evidence of molecular links between PKR and mTOR signalling pathways in Abeta neurotoxicity: role of p53, Redd1 and TSC2.

The control of translation is disturbed in Alzheimers disease (AD). This study analysed the crosslink between the up regulation of double-stranded RNA-dependent-protein kinase (PKR) and the down regulation of mammalian target of rapamycin (mTOR) signalling pathways via p53, the protein Regulated in the Development and DNA damage response 1 (Redd1) and the tuberous sclerosis complex (TSC2) factors in two beta-amyloid peptide (Abeta) neurotoxicity models. In SH-SY5Y cells, Abeta42 induced an increase of P(T451)-PKR and of the ratio p66/(p66+p53) in nuclei and a physical interaction between these proteins. Redd1 gene levels increased and P(T1462)-TSC2 decreased. These disturbances were earlier in rat primary neurons with nuclear co-localization of Redd1 and PKR. The PKR gene silencing in SH-SY5Y cells prevented these alterations. p53, Redd1 and TSC2 could represent the molecular links between PKR and mTOR in Abeta neurotoxicity. PKR could be a critical target in a therapeutic program of AD.

Lactic acid-induced increase of extracellular dopamine measured by microdialysis in rat striatum: evidence for glutamatergic and oxidative mechanisms

Striatal lactacidosis was induced by direct lactic acid perfusion to obtain a local pH as close as possible to that observed in ischemia. In a previous study we showed that such lactacidosis produces a diphasic increase in extracellular dopamine (DA). The present work investigated whether DA accumulation is related to a glutamatergic mechanism and/or production of reactive oxygen species (ROS) in the striatum. Concentrations of extracellular DA, glutamate and hydroxyl radicals ((.)OH) were measured in the presence or absence of an N-methyl-D-aspartate (NMDA) receptor blocker (dizocilpine, MK-801) or an antioxidant (Trolox). Measurements were performed using high-performance liquid chromatography (HPLC) with electrochemical and fluorimetric detection on samples obtained by an in vivo microdialysis perfusion technique and stored at -80 degrees C. The increase in lactic acid-induced DA was entirely suppressed by MK-801 and Trolox. Lactacidosis also induced an increase in extracellular glutamate and (.)OH concentrations at the same time as the first DA accumulation, as well as another (.)OH accumulation which preceded and accompanied the second DA concentration peak. Glutamate release was totally inhibited by MK-801 or Trolox. The first peak of (.)OH production was completely suppressed by MK-801 and Trolox, but the second one was only suppressed by Trolox. These data showed that the increase in DA induced by lactic acid was related to glutamatergic excitotoxicity and ROS production, suggested that the kinetic of events was different for the two DA accumulations.

Pharmacological inhibition of PKR in APPswePS1dE9 mice transiently prevents inflammation at 12 months of age but increases Aβ42 levels in the late stages of the Alzheimer's disease.

The double-stranded RNA-dependent protein kinase (PKR) is switched on by a wide range of stimuli, including the amyloid peptide. Then, PKR transmits signals to the translational machinery, apoptosis and inflammatory signaling pathways by interacting with some adapters. In virus-infected cells, PKR engages the nucleus factor κB (NF-κB) pathway. In many models of Alzheimers disease (AD) and patients with AD, PKR was activated. Furthermore, there is strong evidence implicating the inflammatory process in the AD brain. However, the PKR involvement in inflammatory responses in AD is not elucidated. Based on our previous in vitro results, the aim of this study was to evaluate the effects of a pharmacological inhibition of PKR in inflammation in APPswePS1dE9 transgenic mice. Our results showed that PKR inhibition prevented the NF-κB activation and production of tumor necrosis factor alpha (TNFα) and interleukin (IL)-1β at 12 months of age without decrease of Aβ42 levels and memory deficits. Surprisingly, PKR inhibition failed to prevent IL-1β- mediated inflammation and induced a great increase in β-amyloid peptide (Aβ42) levels at 18 months of age. In this model, our findings highlight the lack of relationship between inflammation and Aβ42 levels. Moreover, the age-dependent inflammatory response must be carefully taken into account in the establishment of an anti-inflammatory therapy in AD.

Inhibition of double-stranded RNA-dependent protein kinase strongly decreases cytokine production and release in peripheral blood mononuclear cells from patients with Alzheimer's disease.

Alzheimers disease (AD), a neurodegenerative disorder, is the most common form of dementia in the elderly individuals. Among the pathogenic mechanisms in AD, chronic systemic inflammation is described and characterized by massive production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMCs), which may contribute to an altered immune response and exacerbation of neurodegeneration. Studies have also reported increased double-stranded RNA-dependent protein kinase (PKR) activation in the PBMCs of patients with AD. Interestingly, PKR could be involved in NF-κB activation, leading to production of a wide range of cytokines. We proposed to decrease proinflammatory cytokines production and release by treating the PBMCs in 25 patients with AD with a specific inhibitor of PKR. Our results showed that PKR inhibition greatly decreased tumor necrosis factor , interleukin (IL)-1α, IL-1β, and IL-6 production and release but did not affect the chemokine RANTES. Moreover, inhibition of the proinflammatory factors was correlated with prevention of caspase-3 activation. These results indicated that specific inhibition of PKR at the peripheral level might decrease the inflammatory response in AD.

Copper-induced lipid peroxidation and hemolysis in whole blood: evidence for a lack of correlation

In order to establish a possible relationship between hemolytic and peroxidant activities of copper ions, lipid peroxidation was studied in plasma and whole blood incubated for 24 h with different concentrations of copper. The lipid peroxidation was investigated by the determination of thiobarbituric acid-reactive species, conjugated dienes and fluorescent lipid chromophores. The copper-induced lipoperoxidation was clearly demonstrated in plasma incubated with high concentrations of copper (12.10(-4) and 20.10(-4) M); in whole blood, all the lipoperoxidation products were increased in the plasma, while the fluorescent lipid chromophores remained unchanged in red cells. With a copper concentration similar to that found in acute copper intoxication (4.10(-4) M) no lipoperoxidation was observed and yet hemolysis occurred, reduced glutathione (GSH) decreased dramatically and methemoglobin (MetHb) increased. From these results, we assume that, despite its prooxidant activity and its capacity to produce lipoperoxidation, it has not been proven that copper ions at pathophysiological concentrations induce hemolysis by an oxidative mechanism.

Evidence that acidosis alters the high-affinity dopamine uptake in rat striatal slices and synaptosomes by different mechanisms partially related to oxidative damage

Several experimental studies have shown that acidosis impairs neurotransmitter uptake processes. The purpose of this study was to determine the mechanism underlying acidosis-induced alterations of the high-affinity dopamine (DA) uptake in rat striatal synaptosomes and slices. Acidosis (pH 5.5) performed either by lactic acid or phosphoric acid induced a decrease in the high-affinity DA uptake in the two striatal models, slices being lesser affected than synaptosomes. Addition of the acid prior to uptake measurement led to a strong reduction of the DA uptake velocity. This early inhibitory effect was completely reversed when acid was removed from the medium by washings. Conversely, when slices and synaptosomes were pre-incubated for different times with each acid, DA uptake remained inhibited in spite of washings. This later inhibition was accompanied by the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and was partially prevented by the antioxidant Trolox. Taken together, these results suggest that acidosis, in a degree encountered during ischemia, alters the high-affinity DA uptake by at least two ways: an early and direct effect of H(+) ions on the DA transporters, and subsequently an inhibition partially mediated by free radical damage.

Evidence of lipoperoxidation induced by lactic acid on kidney homogenates

Sawas and Gilbert (Arch. Int. Pharmacodyn. Ther., 276 (1985) 301-312) reported that the commercial solution of haloperidol induces lipoperoxidation of kidney homogenates from Sprague-Dawley rats. However, it would appear that this effect is attributable to the excipient, lactic acid, rather than to haloperidol itself. Lactic acid enhances susceptibility to lipoperoxidation of kidney homogenates in a dose- and time-dependent manner by increasing production of thiobarbituric acid-reactive substances and slightly decreasing polyunsaturated fatty acids such as arachidonic acid and docosahexaenoic acid. This stimulation of lipoperoxidation may be attributed to a mechanism less dependent on enzymatic action than on Fe2+ and Fe3+. Lactic acid may facilitate iron release and formation of iron complexes, factors which increase susceptibility to oxidative stress.