Molecular chaperones and associated cellular clearance mechanisms against toxic protein conformers in Parkinson's disease.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder marked by the loss of dopaminergic neurons (in particular in the substantia nigra) causing severe impairment of movement coordination and locomotion, associated with the accumulation of aggregated α-synuclein (α-Syn) into proteinaceous inclusions named Lewy bodies. Various early forms of misfolded α-Syn oligomers are cytotoxic. Their formation is favored by mutations and external factors, such as heavy metals, pesticides, trauma-related oxidative stress and heat shock. Here, we discuss the role of several complementing cellular defense mechanisms that may counteract PD pathogenesis, especially in youth, and whose effectiveness decreases with age. Particular emphasis is given to the 'holdase' and 'unfoldase' molecular chaperones that provide cells with potent means to neutralize and scavenge toxic protein conformers. Because chaperones can specifically recognize misfolded proteins, they are key specificity factors for other cellular defenses, such as proteolysis by the proteasome and autophagy. The efficiency of the cellular defenses decreases in stressed or aging neurons, leading to neuroinflammation, apoptosis and tissue loss. Thus, drugs that can upregulate the molecular chaperones, the ubiquitin-proteasome system and autophagy in brain tissues are promising avenues for therapies against PD and other mutation-, stress- or age-dependent protein-misfolding diseases.

Selective neurodegeneration induced in rotation-mediated aggregate cell cultures by a transient switch to stationary culture conditions: a potential model to study ischemia-related pathogenic mechanisms.

Aggregating brain cell cultures at an advanced maturational stage (20-21 days in vitro) were subjected for 1-3 h to anaerobic (hypoxic) and/or stationary (ischemic) conditions. After restoration of the normal culture conditions, cell loss was estimated by measuring the release of lactate dehydrogenase as well as the irreversible decrease of cell type-specific enzyme activities, total protein and DNA content. Ischemia for 2 h induced significant neuronal cell death. Hypoxia combined with ischemia affected both neuronal and glial cells to different degrees (GABAergic neurons>cholinergic neurons>astrocytes). Hypoxic and ischemic conditions greatly stimulated the uptake of 2-deoxy-D-glucose, indicating increased glucose consumption. Furthermore, glucose restriction (5.5 mM instead of 25 mM) dramatically increased the susceptibility of neuronal and glial cells to hypoxic and ischemic conditions. Glucose media concentrations below 2 mM caused selective neuronal cell death in otherwise normal culture conditions. GABAergic neurons showed a particularly high sensitivity to glucose restriction, hypoxia, and ischemia. The pattern of ischemia-induced changes in vitro showed many similarities to in vivo findings, suggesting that aggregating brain cell cultures provide a useful in vitro model to study pathogenic mechanisms related to brain ischemia.

PPARs in diseases: control mechanisms of inflammation.

The three isotypes of peroxisome proliferator-activated receptors (PPARs), PPARalpha, beta/delta and gamma, are ligand-inducible transcription factors that belong to the nuclear hormone receptor family. PPARs are implicated in the control of inflammatory responses and in energy homeostasis and thus, can be defined as metabolic and anti-inflammatory transcription factors. They exert their anti-inflammatory effects by inhibiting the induction of pro-inflammatory cytokines, adhesion molecules and extracellular matrix proteins or by stimulating the production of anti-inflammatory molecules. Furthermore, PPARs modulate the proliferation, differentiation and survival of immune cells including macrophages, B cells and T cells. This review discusses the molecular mechanisms by which PPARs and their ligands modulate the inflammatory response. In addition, it presents recent developments implicating PPAR specific ligands in potential treatments of inflammation-related diseases, such as atherosclerosis, inflammatory bowel diseases, Parkinson's and Alzheimer's diseases.

Cellular and molecular mechanisms underlying the effects of sleep loss on hippocampal synaptic plasticity

SUMMARY : The function of sleep for the organism is one of the most persistent and perplexing questions in biology. Current findings lead to the conclusion that sleep is primarily for the brain. In particular, a role for sleep in cognitive aspects of brain function is supported by behavioral evidence both in humans and animals. However, in spite of remarkable advancement in the understanding of the mechanisms underlying sleep generation and regulation, it has been proven difficult to determine the neurobiological mechanisms underlying the beneficial effect of sleep, and the detrimental impact of sleep loss, on learning and memory processes. In my thesis, I present results that lead to several critical steps forward in the link between sleep and cognitive function. My major result is the molecular identification and physiological analysis of a protein, the NR2A subunit of NMDA receptor (NMDAR), that confers sensitivity to sleep loss to the hippocampus, a brain structure classically involved in mnemonic processes. Specifically, I used a novel behavioral approach to achieve sleep deprivation in adult C57BL6/J mice, yet minimizing the impact of secondary factors associated with the procedure,.such as stress. By using in vitro electrophysiological analysis, I show, for the first time, that sleep loss dramatically affects bidirectional plasticity at CA3 to CA1 synapses in the hippocampus, a well established cellular model of learning and memory. 4-6 hours of sleep loss elevate the modification threshold for bidirectional synaptic plasticity (MT), thereby promoting long-term depression of CA3 to CA 1 synaptic strength after stimulation in the theta frequency range (5 Hz), and rendering long-term potentiation induction.more difficult. Remarkably, 3 hours of recovery sleep, after the deprivation, reset the MT at control values, thus re-establishing the normal proneness of synapses to undergo long-term plastic changes. At the molecular level, these functional changes are paralleled by a change in the NMDAR subunit composition. In particular, the expression of the NR2A subunit protein of NMDAR at CA3 to CA1 synapses is selectively and rapidly increased by sleep deprivation, whereas recovery sleep reset NR2A synaptic content to control levels. By using an array of genetic, pharmacological and computational approaches, I demonstrate here an obligatory role for NR2A-containing NMDARs in conveying the effect of sleep loss on CA3 to CAl MT. Moreover, I show that a genetic deletion of the NR2A subunit fully preserves hippocampal plasticity from the impact of sleep loss, whereas it does not alter sleepwake behavior and homeostatic response to sleep deprivation. As to the mechanism underlying the effects of the NR2A subunit on hippocampal synaptic plasticity, I show that the increased NR2A expression after sleep loss distinctly affects the contribution of synaptic and more slowly recruited NMDAR pools activated during plasticity-induction protocols. This study represents a major step forward in understanding the mechanistic basis underlying sleep's role for the brain. By showing that sleep and sleep loss affect neuronal plasticity by regulating the expression and function of a synaptic neurotransmitter receptor, I propose that an important aspect of sleep function could consist in maintaining and regulating protein redistribution and ion channel trafficking at central synapses. These findings provide a novel starting point for investigations into the connections between sleep and learning, and they may open novel ways for pharmacological control over hippocampal .function during periods of sleep restriction. RÉSUMÉ DU PROJET La fonction du sommeil pour l'organisme est une des questions les plus persistantes et difficiles dans la biologie. Les découvertes actuelles mènent à la conclusion que le sommeil est essentiel pour le cerveau. En particulier, le rôle du sommeil dans les aspects cognitifs est soutenu par des études comportementales tant chez les humains que chez les animaux. Cependant, malgré l'avancement remarquable dans la compréhension des mécanismes sous-tendant la génération et la régulation du sommeil, les mécanismes neurobiologiques qui pourraient expliquer l'effet favorable du sommeil sur l'apprentissage et la mémoire ne sont pas encore clairs. Dans ma thèse, je présente des résultats qui aident à clarifier le lien entre le sommeil et la fonction cognitive. Mon résultat le plus significatif est l'identification moléculaire et l'analyse physiologique d'une protéine, la sous-unité NR2A du récepteur NMDA, qui rend l'hippocampe sensible à la perte de sommeil. Dans cette étude, nous avons utilisé une nouvelle approche expérimentale qui nous a permis d'induire une privation de sommeil chez les souris C57BL6/J adultes, en minimisant l'impact de facteurs confondants comme, par exemple, le stress. En utilisant les techniques de l'électrophysiologie in vitro, j'ai démontré, pour la première fois, que la perte de sommeil est responsable d'affecter radicalement la plasticité bidirectionnelle au niveau des synapses CA3-CA1 de l'hippocampe. Cela correspond à un mécanisme cellulaire de l'apprentissage et de la mémoire bien établi. En particulier, 4-6 heures de privation de sommeil élèvent le seuil de modification pour la plasticité synaptique bidirectionnelle (SM). Comme conséquence, la dépression à long terme de la transmission synaptique est induite par la stimulation des fibres afférentes dans la bande de fréquences thêta (5 Hz), alors que la potentialisation à long terme devient plus difficile. D'autre part, 3 heures de sommeil de récupération sont suffisant pour rétablir le SM aux valeurs contrôles. Au niveau moléculaire, les changements de la plasticité synaptiques sont associés à une altération de la composition du récepteur NMDA. En particulier, l'expression synaptique de la protéine NR2A du récepteur NMDA est rapidement augmentée de manière sélective par la privation de sommeil, alors que le sommeil de récupération rétablit l'expression de la protéine au niveau contrôle. En utilisant des approches génétiques, pharmacologiques et computationnelles, j'ai démontré que les récepteurs NMDA qui expriment la sous-unité NR2A sont responsables de l'effet de la privation de sommeil sur le SM. De plus, nous avons prouvé qu'une délétion génétique de la sous-unité NR2A préserve complètement la plasticité synaptique hippocampale de l'impact de la perte de sommeil, alors que cette manipulation ne change pas les mécanismes de régulation homéostatique du sommeil. En ce qui concerne les mécanismes, j'ai .découvert que l'augmentation de l'expression de la sous-unité NR2A au niveau synaptique modifie les propriétés de la réponse du récepteur NMDA aux protocoles de stimulations utilisés pour induire la plasticité. Cette étude représente un pas en avant important dans la compréhension de la base mécaniste sous-tendant le rôle du sommeil pour le cerveau. En montrant que le sommeil et la perte de sommeil affectent la plasticité neuronale en régulant l'expression et la fonction d'un récepteur de la neurotransmission, je propose qu'un aspect important de la fonction du sommeil puisse être finalisé au règlement de la redistribution des protéines et du tracking des récepteurs aux synapses centraux. Ces découvertes fournissent un point de départ pour mieux comprendre les liens entre le sommeil et l'apprentissage, et d'ailleurs, ils peuvent ouvrir des voies pour des traitements pharmacologiques dans le .but de préserver la fonction hippocampale pendant les périodes de restriction de sommeil.

Renal sodium handling in acute and chronic salt loading/depletion protocols: the confounding influence of acute water loading.

OBJECTIVES: Renal tubular sodium handling was measured in healthy subjects submitted to acute and chronic salt-repletion/salt-depletion protocols. The goal was to compare the changes in proximal and distal sodium handling induced by the two procedures using the lithium clearance technique. METHODS: In nine subjects, acute salt loading was obtained with a 2 h infusion of isotonic saline, and salt depletion was induced with a low-salt diet and furosemide. In the chronic protocol, 15 subjects randomly received a low-, a regular- and a high-sodium diet for 1 week. In both protocols, renal and systemic haemodynamics and urinary electrolyte excretion were measured after an acute water load. In the chronic study, sodium handling was also determined, based on 12 h day- and night-time urine collections. RESULTS: The acute and chronic protocols induced comparable changes in sodium excretion, renal haemodynamics and hormonal responses. Yet, the relative contribution of the proximal and distal nephrons to sodium excretion in response to salt loading and depletion differed in the two protocols. Acutely, subjects appeared to regulate sodium balance mainly by the distal nephron, with little contribution of the proximal tubule. In contrast, in the chronic protocol, changes in sodium reabsorption could be measured both in the proximal and distal nephrons. Acute water loading was an important confounding factor which increased sodium excretion by reducing proximal sodium reabsorption. This interference of water was particularly marked in salt-depleted subjects. CONCLUSION: Acute and chronic salt loading/salt depletion protocols investigate different renal mechanisms of control of sodium balance. The endogenous lithium clearance technique is a reliable method to assess proximal sodium reabsorption in humans. However, to investigate sodium handling in diseases such as hypertension, lithium should be measured preferably on 24 h or overnight urine collections to avoid the confounding influence of water.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Background: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.Results: The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.Conclusion: Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.

Short term impact of Tribulus terrestris intake on doping control analysis of endogenous steroids.

Tribulus terrestris is a nutritional supplement highly debated regarding its physiological and actual effects on the organism. The main claimed effect is an increase of testosterone anabolic and androgenic action through the activation of endogenous testosterone production. Even if this biological pathway is not entirely proven, T. terrestris is regularly used by athletes. Recently, the analysis of two female urine samples by GC/C/IRMS (gas chromatography/combustion/isotope-ratio-mass-spectrometry) conclusively revealed the administration of exogenous testosterone or its precursors, even if the testosterone glucuronide/epitestosterone glucuronide (T/E) ratio and steroid marker concentrations were below the cut-off values defined by World Anti-Doping Agency (WADA). To argue against this adverse analytical finding, the athletes recognized having used T. terrestris in their diet. In order to test this hypothesis, two female volunteers ingested 500 mg of T. terrestris, three times a day and for two consecutive days. All spot urines were collected during 48 h after the first intake. The (13)C/(12)C ratio of ketosteroids was determined by GC/C/IRMS, the T/E ratio and DHEA concentrations were measured by GC/MS and LH concentrations by radioimmunoassay. None of these parameters revealed a significant variation or increased above the WADA cut-off limits. Hence, the short-term treatment with T. terrestris showed no impact on the endogenous testosterone metabolism of the two subjects.

Student modeling based on fuzzy inference mechanisms

The paper presents a competence-based instructional design system and a way to provide a personalization of navigation in the course content. The navigation aid tool builds on the competence graph and the student model, which includes the elements of uncertainty in the assessment of students. An individualized navigation graph is constructed for each student, suggesting the competences the student is more prepared to study. We use fuzzy set theory for dealing with uncertainty. The marks of the assessment tests are transformed into linguistic terms and used for assigning values to linguistic variables. For each competence, the level of difficulty and the level of knowing its prerequisites are calculated based on the assessment marks. Using these linguistic variables and approximate reasoning (fuzzy IF-THEN rules), a crisp category is assigned to each competence regarding its level of recommendation.

Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: A challenge for neuroprotection.

There is currently no approved neuroprotective pharmacotherapy for acute conditions such as stroke and cerebral asphyxia. One of the reasons for this may be the multiplicity of cell death mechanisms, because inhibition of a particular mechanism leaves the brain vulnerable to alternative ones. It is therefore essential to understand the different cell death mechanisms and their interactions. We here review the multiple signaling pathways underlying each of the three main morphological types of cell death - apoptosis, autophagic cell death and necrosis - emphasizing their importance in the neuronal death that occurs during cerebral ischemia and hypoxia-ischemia, and we analyze the interactions between the different mechanisms. Finally, we discuss the implications of the multiplicity of cell death mechanisms for the design of neuroprotective strategies.

Molecular mechanisms involved in the activation and regulation of the alpha 1-adrenergic receptor subtypes.

The adrenergic receptors (ARs) belong to the superfamily of membrane-bound G protein coupled receptors (GPCRs). Our investigation has focused on the structure-function relationship of the alpha 1b-AR subtype used as the model system for other GPCRs. Site-directed mutagenesis studies have elucidated the structural domains of the alpha 1b-AR involved in ligand binding, G protein coupling or desensitization. In addition, a combined approach using site-directed mutagenesis and molecular dynamics analysis of the alpha 1b-AR has provided information about the potential mechanisms underlying the activation process of the receptor, i.e. its transition from the 'inactive' to the 'active' conformation.

Analysis of LEDGF/p75 expression regulation

Background To replicate, retroviruses must insert DNA copies of their RNA genomes into the host genome. This integration process is catalyzed by the viral integrase protein. The site of viral integration has been shown to be non-random and retrovirus-specific. LEDGF/p75, a splice variant encoded by PSIP1 gene and described as a general transcription coactivator, was identified as a tethering factor binding both to chromatin and to lentiviral integrases, thereby affecting integration efficiency as well as integration site selection. LEDGF/p75 is still a poorly characterized protein, and its cellular endogenous function has yet to be fully determined. In order to start unveiling the roles of LEDGF/p75 in the cell, we started to investigate the mechanisms involved in the regulation of LEDGF/p75. Materials and methods To identify PSIP1 minimal promoter and associated regulatory elements, we cloned a region starting 5 kb upstream the transcription start site (TSS, +1 reference position) to the ATG start codon (+816), as well as systematic truncations, in a plasmid containing the firefly luciferase reporter gene. These constructs were co-transfected into HEK293 cells with a plasmid encoding the Renilla luciferase under the pTK promoter as an internal control for transfection efficiency. Both luciferase activities were assessed by luminescence as an indicator of promoter activity. Results Luciferase assays identified regions -76 to +1 and +1 to +94 as two independent minimal promoters showing respectively a 3.7x and 2.3x increase in luciferase activity. These two independent minimal promoters worked synergistically increasing luciferase activity up to 16.3x as compared to background. Moreover, we identified five regulatory blocks which modulated luciferase activity depending on the DNA region tested, three enhancers (- 2007 to -1159, -284 to -171 and +94 to +644) and two silencers (-171 to -76 and +796 to +816). However, the silencing effect of the region -171 to -76 is dependent on the presence of the +94 to +644 region, ruling out the enhancer activity of the latter. Computational analysis of PSIP1 promoter revealed the absence of TATA box and initiator (INR) sequences, classifying this promoter as nonconventional. TATA-less and INR-less promoters are characterized by multiple Sp1 binding sites, involved in the recruitment of the RNA pol II complex. Consistent with this, PSIP1 promoter contains multiple putative Sp1 binding sequences in regions -76 to +1 and +1 to +94.

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA.

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.

Effects of lactate on glucose metabolism in healthy subjects and in severely injured hyperglycemic patients.

Hepatic glucose production is autoregulated during infusion of gluconeogenic precursors. In hyperglycemic patients with multiple trauma, hepatic glucose production and gluconeogenesis are increased, suggesting that autoregulation of hepatic glucose production may be defective. To better understand the mechanisms of autoregulation and its possible alterations in metabolic stress, lactate was coinfused with glucose in healthy volunteers and in hyperglycemic patients with multiple trauma or critical illness. In healthy volunteers, infusion of glucose alone nearly abolished endogenous glucose production. Lactate increased gluconeogenesis (as indicated by a decrease in net carbohydrate oxidation with no change in total [13C]carbohydrate oxidation) but did not increase endogenous glucose production. In patients with metabolic stress, endogenous glucose production was not suppressed by exogenous glucose, but lactate did not further increase hepatic glucose production. It is concluded that 1) in healthy humans, autoregulation of hepatic glucose production during infusion of lactate is still present when glycogenolysis is suppressed by exogenous glucose and 2) autoregulation of hepatic glucose production is not abolished in hyperglycemic patients with metabolic stress.