Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.

Role of glutathione deficit in the disconnectivity syndrome in schizophrenia: from pathogenetic mechanisms to therapeutic interventions

In the cerebrospinal fluid of 26 drug-naive schizophrenics (DSM-III- R), we observed that the level of glutathione ([GSH]) and of its metabolite γ-Glu-Gln was decreased by 27% and 16% respectively. Using a new in-vivo method based on magnetic resonance spec- troscopy, [GSH] was measured in the medial prefrontal cortex of 18 schizophrenics and found to be 52 % lower than in controls (n = 20). This is consistent with the recently observed decreased mRNA levels in fibroblasts of patients (n=32) of the two GSH synthesizing en- zymes (glutathione synthetase (GSS), and glutamate-cysteine ligase M (GCLM) the modulatory subunit of glutamate-cysteine ligase). Moreover, the level of GCLM expression in fibroblasts correlates neg- atively with the psychopathology (positive, general and some nega- tive symptoms). Thus, the observed difference in gene expression is not only the cause of low brain [GSH], but is also related to the sever- ity of symptoms, suggesting that fibroblasts are adequate surrogate for brain tissue. A hypothesis was proposed, based on a central role of GSH in the pathophysiology of schizophrenia. GSH is an important endogenous redox regulator and neuroactive substance. GSH is pro- tecting cells from damage by reactive oxygen species generated, among others, by the metabolism of dopamine. A GSH deficit-in- duced oxidative stress would lead to lipid peroxidation and micro-le- sions in the surrounding of catecholamine terminals, affecting the synaptic contacts on dendritic spines of cortical neurones, where ex- citatory glutamatergic terminals converge with dopaminergic ones. This would lead to spines degeneration and abnormal nervous con- nections or structural disconnectivity, possibly responsible for posi- tive, perceptive and cognitive symptoms of schizophrenia. In addi- tion, a GSH deficit could also lead to a functional disconnectivity by depressing NMDA neurotransmission, in analogy to phencyclidine effects. Present experimental biochemical, cell biological and behav- ioral data are consistent with the proposed mechanism: decreasing pharmacologically [GSH] in experimental models, with or without blocking DA uptake (GBR12909), induces morphological and behav- ioral changes similar to those observed in patients. Dendritic spines: (a) In neuronal cultures, low [GSH] and DA induce decreased density of neural processes; (b) In developing rats (p5-p16), [GSH] deficit and GBR induce a decrease in normal spines in prefrontal pyramids and in GABA-parvalbumine but not of -calretinine immunoreactivity in anterior cingulate. NMDA-dependant synaptic plasticity: GSH deple- I/13 tion in hippocampal slices impairs long-term potentiation. Develop- ing rats with low [GSH] and GBR have deficit in olfactory integration and in object recognition which appears earlier in males than fe- males, in analogy to the delay of the psychosis onset between man and woman. In summary, a deficit of GSH and/or GSH-related enzymes during early development could constitute a major vulnerability fac- tor in schizophrenia.

Neuroscience: Astrocytes as aide-mémoires.

Memory formation is known to occur at the level of synaptic contacts between neurons. It therefore comes as a surprise that another type of brain cell, the astrocyte, is also involved in establishing memory.

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Adenosine A1 receptor activation is arrhythmogenic in the developing heart through NADPH oxidase/ERK- and PLC/PKC-dependent mechanisms.

Whether adenosine, a crucial regulator of the developing cardiovascular system, can provoke arrhythmias in the embryonic/fetal heart remains controversial. Here, we aimed to establish a mechanistic basis of how an adenosinergic stimulation alters function of the developing heart. Spontaneously beating hearts or dissected atria and ventricle obtained from 4-day-old chick embryos were exposed to adenosine or specific agonists of the receptors A(1)AR (CCPA), A(2A)AR (CGS-21680) and A(3)AR (IB-MECA). Expression of the receptors was determined by quantitative PCR. The functional consequences of blockade of NADPH oxidase, extracellular signal-regulated kinase (ERK), phospholipase C (PLC), protein kinase C (PKC) and L-type calcium channel (LCC) in combination with adenosine or CCPA, were investigated in vitro by electrocardiography. Furthermore, the time-course of ERK phosphorylation was determined by western blotting. Expression of A(1)AR, A(2A)AR and A(2B)AR was higher in atria than in ventricle while A(3)AR was equally expressed. Adenosine (100μM) triggered transient atrial ectopy and second degree atrio-ventricular blocks (AVB) whereas CCPA induced mainly Mobitz type I AVB. Atrial rhythm and atrio-ventricular propagation fully recovered after 60min. These arrhythmias were prevented by the specific A(1)AR antagonist DPCPX. Adenosine and CCPA transiently increased ERK phosphorylation and induced arrhythmias in isolated atria but not in ventricle. By contrast, A(2A)AR and A(3)AR agonists had no effect. Interestingly, the proarrhythmic effect of A(1)AR stimulation was markedly reduced by inhibition of NADPH oxidase, ERK, PLC, PKC or LCC. Moreover, NADPH oxidase inhibition or antioxidant MPG prevented both A(1)AR-mediated arrhythmias and ERK phosphorylation. These results suggest that pacemaking and conduction disturbances are induced via A(1)AR through concomitant stimulation of NADPH oxidase and PLC, followed by downstream activation of ERK and PKC with LCC as possible target.

Effects of salbutamol on the contractile properties of human skeletal muscle before and after fatigue.

AIM: The study examined the effects of an oral acute administration of the beta2-agonist salbutamol (Sal) (6 mg) vs. placebo on muscle strength and fatigability in 12 non-asthmatic recreational male athletes in a randomized double-blind protocol. METHODS: Contractile properties of the right quadriceps muscle were measured during electrical stimulations, i.e. twitch, 1-s pulse trains at 20 (P(20) ) and 80 Hz (P(80) ) and during maximal voluntary isometric contraction (MVIC) before (PRE) and after (POST) a fatigue-producing protocol set by an electromyostimulation (30 contractions, frequency: 75 Hz, on-off ratio: 6.25-20s). In addition, the level of muscle voluntary activation was measured. RESULTS: In PRE and POST conditions, the peak torque (PT) of twitch, P(80) and MVIC were not modified by the treatment. The PT in POST P(20) was slightly, although not significantly, less affected by fatigue in Sal compared with placebo condition. Moreover, twitch half-relaxation time at PRE was smaller under Sal than under placebo (P < 0.05). No significant changes in the degree of voluntary activation were observed with Sal treatment in PRE or POST condition. CONCLUSION: Although these findings did not exclude completely an effect of Sal on peripheral factors of human skeletal muscle, oral acute administration of the beta2-agonist Sal seems to be without any relevant ergogenic effect on muscle contractility and fatigability in non-asthmatic recreational male athletes.

Ectodysplasin A (EDA) - EDA receptor signalling and its pharmacological modulation.

The TNF family ligand ectodysplasin A (EDA) regulates the induction, morphogenesis and/or maintenance of skin-derived structures such as teeth, hair, sweat glands and several other glands. Deficiencies in the EDA - EDA receptor (EDAR) signalling pathway cause hypohidrotic ectodermal dysplasia (HED). This syndrome is characterized by the absence or malformation of several skin-derived appendages resulting in hypotrychosis, hypodontia, heat-intolerance, dry skin and dry eyes, susceptibility to airways infections and crusting of various secretions. The EDA-EDAR system is an important effector of canonical Wnt signalling in developing skin appendages. It functions by stimulating NF-κB-mediated transcription of effectors or inhibitors of the Wnt, Sonic hedgehog (SHH), fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) pathways that regulate interactions within or between epithelial and mesenchymal cells and tissues. In animal models of Eda-deficiency, soluble EDAR agonists can precisely correct clinically relevant symptoms with low side effects even at high agonist doses, indicating that efficient negative feedback signals occur in treated tissues. Hijacking of the placental antibody transport system can help deliver active molecules to developing foetuses in a timely manner. EDAR agonists may serve to treat certain forms of ectodermal dysplasia.

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

Toll-like receptor 3 expressed by melanoma cells as a target for therapy?

PURPOSE: The immunomodulatory properties of Toll-like receptors (TLR) agonists have inspired their use as experimental adjuvants for vaccination of cancer patients. However, it is now well recognized that TLR expression is not restricted to immune cells but can also be found in many cell types, including those giving rise to tumors. It is therefore mandatory to explore the potential effects of TLR triggering directly on tumor cells. EXPERIMENTAL DESIGN: In the present work, we have investigated TLR3 protein expression in melanoma cell lines derived from patients, and analyzed the effects of TLR3 agonists on tumor cell survival. Moreover, we used RNA interference to stably knock down TLR3 expression and study the involvement of this receptor in dsRNA-induced effects on melanoma cells viability. RESULTS: Human melanoma cells can express functional TLR3 protein. Interestingly, the engagement of the receptor by TLR3 agonists can directly inhibit cell proliferation and induce tumor cell death when combined to treatment with either type I IFN or protein synthesis inhibitors. These effects were shown by RNA interference to be largely dependent on TLR3. Moreover, TLR3-mediated cell death involves the activation of caspases and engages both extrinsic and intrinsic apoptotic pathways. CONCLUSION: TLR3 protein can be expressed in human melanoma cells, where it can deliver proapoptotic and antiproliferative signaling. Altogether, these results suggest that TLR3 agonists represent very promising adjuvants for cancer vaccines not only based on their well-described immunostimulatory properties, but also due to their newly identified cytostatic and cytotoxic effects directly on tumor cells.

Early glutathione deficit impairs parvalbumin expression in gaba interneurons and kainate-induced gamma oscillations

An increased oxidative stress and alteration of the antioxidant systems have been observed in schizophrenia. Glutathione (GSH), a major redox regulator, is decreased in patients' cerebrospinal fluid, prefrontal cortex in vivo and striatum post-mortem tissue. Most importantly, there is genetic and functional evidence for the implication of the gene of the glutamate cysteine ligase (GCL) catalytic subunit, the key GSH-synthesizing enzyme. We have developed animal models for a GSH deficit to study the consequences of such deficit on the brain development. A GSH deficit combined with elevated dopamine (DA) during development leads to reduced parvalbumin (PV) expression in a subclass of GABA interneurons in rat anterior cingulate cortex (ACC). Similar changes are observed in postmortem brain tissue of schizophrenic patients. GSH dysregulation increases vulnerability to oxidative stress, that in turn could lead to cortical circuit anomalies in the schizophrenic brain. In the present study, we use a GCL modulatory subunit (GCLM) knock-out (KO) mouse model that presents up to 80% decreased brain GSH levels. During postnatal development, a subgroup of animals from each genotype is exposed to elevated oxidative stress induced by treatment with the DA reuptake inhibitor GBR12909. Results reveal a significant genotype-specific delay International Congress on Schizophrenia Research 136 10. 10. Neuroanatomy, Animal Downloaded from http://schizophreniabulletin.oxfordjournals.org at Bibliotheque Cantonale et Universitaire on June 18, 2010 in cortical PV expression at postnatal day P10 in GCLM-KO mice, as compared to wild-type. This effect seems to be further exaggerated in animals treated with GBR12909 from P5 to P10. At P20, PV expression is no longer significantly reduced in GCLM-KO ACC without GBR but is reduced if GBR is applied from P10 to P20. However, our result show that GCLM-KO mice exhibit increased oxidative stress, cortical altered myelin development as shown by MBP marker, and more specifically impairment of the peri-neuronal net known to modulate PV connectivity. In addition, we also observe a reduced PV expression in the ventro-temporal hippocampus of adult GCLM-KO mice, suggesting that anomalies of the PV interneurons prevail at least in some brain regions throughout the adulthood. Interestingly, the power of kainate-induced gamma oscillations, known to be dependent on proper activation of PV interneuron's, is also lower in hippocampal slices of adult GCLM KO mice. These results suggest that the PV positive GABA interneurons is particularly vulnerable to increased oxidative stress

Thyroid function in severely traumatized patients with or without head injury.

The pattern of thyroid function changes following severe trauma was assessed prospectively in 35 patients during the first 5 days after injury. Patients were divided into 2 groups to evaluate the effect of head injury: group I, patients with severe head injury; group II, patients with multiple injuries without head injury. The results demonstrate a low T3 and low T4 syndrome throughout the study, with decreases in both total and free levels of T3 and T4, normal or increased rT3 levels, and normal TSH levels. The presence of severe head injury was associated with lower levels of TSH and free T3. Mortality was 37%. Survival was associated with higher TSH and T3 levels, but not with higher T4 levels. TSH levels exceeding 1 mU/l on the first day were only observed in survivors. These findings show that a typical low T3 and low T4 syndrome is present after severe trauma in patients with multiple injury as well as with head injury. Primary hypothyroidism can be excluded, pituitary or hypothalamic hypothyroidism is likely in these patients.

Optogenetic Interrogation of Dopaminergic Modulation of the Multiple Phases of Reward-Seeking Behavior.

Phasic activation of dopaminergic neurons is associated with reward-predicting cues and supports learning during behavioral adaptation. While noncontingent activation of dopaminergic neurons in the ventral tegmental are (VTA) is sufficient for passive behavioral conditioning, it remains unknown whether the phasic dopaminergic signal is truly reinforcing. In this study, we first targeted the expression of channelrhodopsin-2 to dopaminergic neurons of the VTA and optimized optogenetically evoked dopamine transients. Second, we showed that phasic activation of dopaminergic neurons in freely moving mice causally enhances positive reinforcing actions in a food-seeking operant task. Interestingly, such effect was not found in the absence of food reward. We further found that phasic activation of dopaminergic neurons is sufficient to reactivate previously extinguished food-seeking behavior in the absence of external cues. This was also confirmed using a single-session reversal paradigm. Collectively, these data suggest that activation of dopaminergic neurons facilitates the development of positive reinforcement during reward-seeking and behavioral flexibility.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.