Serge Mazères

Thioflavin Derivatives as Markers for Amyloid‐β Fibrils: Insights into Structural Features Important for High‐Affinity Binding

Alzheimer’s disease (AD) is a common neurological disease associated with chronic dementia, memory loss, and cognitive impairment. Central to the neuropathology of AD are the senile plaques (SPs) and neurofibrillar tangles (NFTs), depositions composed of amyloid-b peptide (Ab) and tau protein, respectively. Aggregated Ab in senile plaques has a b-sheet secondary structure and is arranged as fibrils. 3] Soluble Ab prior to aggregation is predominantly unstructured. Formation and accumulation of aggregates of Ab peptides in the brain are critical factors in the development and progression of AD, in which oxidative stress represents a field of current intensive studies. There is a great interest in molecules able to bind specifically to Ab aggregates. Such markers of Ab fibrils would allow their early detection and specificity would permit identification of Ab fibrils from other amyloid deposits. This is of importance from basic research to clinical application. In particular, such molecules give access to molecular imaging (by Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET), or Magnetic Resonance Imaging (MRI)) therefore allowing a precise localisation and identification of the Ab aggregates in the brain. This is in contrast with the less specific information obtained either by cerebrospinal fluid analysis, or anatomic imaging by MRI. The progress of therapies that may affect Ab deposition in AD brain has added new significance to this pursuit. Three main categories of PET ligands of AD-associated aggregates are currently under investigation (Figure 1): Thioflavin T (ThT) derived compounds (for example, N-methyl-[(11)C]2-(4’-methylaminophenyl)-6-hydroxybenzothiazole (PIB)), including variations of the heteroaromatic core, styrylbenzene (SB), and compounds with an aminonaphthyl core (for example, 2-(1-{6-[(2-[F-18]fluoroethyl)ACHTUNGTRENNUNG(methyl)amino]-2-naphthyl}ethylidene)malononitrile (FDDNP)). ThT has been used for several decades to stain amyloids such as Ab. ThT is not specific for amyloids of Ab, as it reacts with many types of amyloids (for example, NFT, insulin, b2-microglobulin) but not all. Thus several derivatives of ThT have been generated with the aim of developing a biomarker of Ab fibrils with high affinity and high specificity, as exemplified by BTA-1, N-methyl-C-PIB, I-TZDM, etc. Yet not much is known about the structure–function relationships that explain the molecular nature of the interaction marker/Ab aggregate and most improvement of binding-affinity seemed to arise from empirical studies. The reported insights are scarce. It seems clear that the removal of the methyl group on the heterocyclic nitrogen of ThT and hence the removal of the positive charge increases the affinity to Ab fibrils by a factor of 40. Simultaneously, the removal of the charge increases the lipophilicity of the compounds and therefore eases crossing of the blood–brain barrier (BBB). Also removal of one of the two methyl groups on the amine nitrogen in the 4’-position did increase the affinity for a so far unknown reason. In contrast removal of the methyl group on the carbon 6 of the benzothiazole moiety did not significantly change the affinity. Better knowledge of the binding sites of the markers derived from ThT would allow a more rational ligand design. To address this question and to get a deeper insight into the nature of the interaction between uncharged ThT derivatives and in particular the role of the amine nitrogen in the 4’-position, we synthesized 18 ThT derivatives of the dimethylaminophenyl moiety and compared their binding affinity to Ab fibrils, which allowed us to propose some important features regarding the marker/Ab fibrils interaction. In the present work a highly convergent synthesis of ThT derivatives applied to 18 examples based on two similar reactions has been established (Scheme 1). Namely, the condensation of Figure 1. Structures of some known Ab aggregate markers.

Physical Association between Neuropeptide FF and μ-Opioid Receptors as a Possible Molecular Basis for Anti-opioid Activity

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF2 and μ-opioid (MOP) receptors tagged with variants of the green fluorescent protein. Importantly, cyan fluorescent protein-tagged NPFF2 receptors retained their capacity to antagonize opioid receptors. Fluorescence resonance energy transfer (FRET) and coimmunoprecipitation studies indicate that NPFF and MOP receptors are close enough to generate a basal FRET signal. The opioid agonist Tyr-d-Ala-Gly-NMe-Phe-Gly-ol disrupts by 20-30% this FRET signal, mainly because it concomitantly induces 40% internalization of receptors. In contrast, the NPFF analog 1DMe significantly increases by 10-15% the basal FRET signal, suggesting an association between both receptors. In addition, 1DMe reduces, by half, MOP receptor internalization, indicating that, besides a functional blockade of opioid receptors, the NPFF analog also inhibits their internalization. Finally, as a first report showing the modulation of the mobility of a G-protein-coupled receptor by another one, fluorescence recovery after photobleaching analysis reveals that 1DMe modifies the lateral diffusion of MOP receptors in the cell membrane, changing them from a confined to a freely diffusing state. By promoting NPFF-MOP receptor heteromerization, 1DMe could disrupt the domain organization of MOP receptors in the membrane, resulting in a reduction of opioid response.

Non invasive contact electrodes for in vivo localized cutaneous electropulsation and associated drug and nucleic acid delivery

For an effective tissue controlled electropermeabilization as requested for electrochemotherapy and electrogenotherapy, it is very important to have informations about the electric field distribution provided by a defined set of electrodes. Computer simulations using the finite element models approach predicted the associated field distributions and currents. Phantoms made of gels with well-defined electrical conductance were used to measure the current responses of a new electrode geometry (wires), A good agreement between the measured and predicted currents was observed supporting the validity of the prediction for the field distribution. Field distribution was observed to be very localized and highly homogeneous with the new concept of contact wire electrodes. They allowed to focus the field effect along the surface of the tissue to induce a controlled release of drugs or plasmids. Non invasive (contact) electrodes can be moved rapidly on the body and avoid puncturing the skin and the tissue. They can be used for large surface effects, to treat the skin and subcutaneous tumors. The use of contact electrodes after drug or DNA intradermal injection were validated by clinical treatment of large surface skin tumors and by in vivo imaging of permeabilization or of gene expression.

Finite-element modeling of needle electrodes in tissue from the perspective of frequent model computation

Information about electric field distribution in tissue is very important for effective electropermeabilization. In heterogeneous tissues with complex geometry, finite-element (FE) models provide an alternative source of such information. In the present study, modeling of needle electrode geometry in the FE model was investigated in order to determine the most appropriate geometry by considering the need for frequent FE model computation present in electroporation models. The 8-faceted needle electrode geometry proposed /sub e/termined on a model with a single needle electrode pair by means of criteria function - consisted of the weighted sum of the relative difference between measured and computed total current, the relative difference in CPU time spent on solving model, and the relative difference in cross section surface of electrodes. Such electrode geometry was further evaluated on physical models with needle arrays by comparison of computed total current and measured current. The agreement between modeled and measured current was good (within 9% of measurement), except in cases with very thin gel. For voltage above 50 V, a linear relationship between current and voltage was observed in measurements. But at lower voltages, a nonlinear behavior was detected resulting from side (electrochemical) effects at electrode-gel interface. This effect was incorporated in the model by introducing a 50 V shift which reduced the difference between the model and the measurement to less than 3%. As long as material properties and geometry are well described by FE model, current-based validation can be used for a rough model validation. That is a routine assay compared with imaging of electric field, which is otherwise employed for model validation. Additionally, current estimated by model, can be preset as maximum in the electroporator in order to protect tissue against damage.

Optical Properties of Hybrid Dendritic–Mesoporous Titania Nanocomposite Films

New hybrid optical sensors have been prepared by grafting specifically designed fluorescent, functionalised, phosphorus-containing dendrimers onto a nanocrystalline mesoporous titania thin film formed by evaporation-induced self-assembly. The structural characterisation and optical behaviour of these new fluorescent probes have been studied both in solution and after being grafted onto an inorganic network, which resulted in the discovery of improved probing selectivity in the solid state. This new hybrid sensor exhibits high sensitivity to phenolic OH moieties (especially those from resorcinol and 2-nitroresorcinol), which induce the quenching of fluorescence more efficiently in the solid state than in solution. This effect is a result of the increased spatial proximity of the fluorescent molecules, which is induced by pore confinement that makes the formation of hydrogen bonds between the hydroxyl moieties of the quenchers and the carbonyl groups of the dendrimer easier.

CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells A CONFOCAL FLUORESCENCE RESONANCE ENERGY TRANSFER-BASED APPROACH

Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.

CD4 Interacts Constitutively with Multiple CCR5 at the Plasma Membrane of Living Cells A FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING AT VARIABLE RADII APPROACH

The entry of human immunodeficiency virus into target cells requires successive interactions of the viral envelope glycoprotein gp120 with CD4 and the chemokine receptors CCR5 or CXCR4. We previously demonstrated, by Förster resonance energy transfer experiments, the constitutive association of CD4 and CCR5 at the surface of living cells. We therefore speculated that this interaction may correlate with compartmentalization of CD4 and CCR5 within the plasma membrane. Here, we characterize the lateral distribution, the dynamics, and the stoichiometry of these receptors in living cells stably expressing CD4 and/or CCR5 by means of fluorescence recovery after photobleaching at variable radii experiments. We found that (i) these receptors expressed alone are confined into 1-μm-sized domains, (ii) CD4-CCR5 associations occur outside and inside smaller domains, and (iii) these interactions involve multiple CCR5 molecules per CD4.

Agonist-selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

Changes of the Membrane Lipid Organization Characterized by Means of a New Cholesterol-Pyrene Probe

We synthesized 3β-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): KDOPC ≫ KPOPC ≫ KDMPC > KDMPC/15 mol % Chol > KDMPC/30 mol % Chol. This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I1–I5) and excimer (IE) emission bands. In model membranes, I1/I3 and IE/I3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse δ-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse δ-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.

Annexin-A6 presents two modes of association with phospholipid membranes. A combined QCM-D, AFM and cryo-TEM study

Annexins are soluble proteins that bind to biological membranes in a Ca(2+)-dependent manner. Annexin-A6 (AnxA6) is unique in the annexin family as it consists of the repeat of two annexin core modules, while all other annexins consist of a single module. AnxA6 has been proposed to participate in various membrane-related processes, including endocytosis and exocytosis, yet the molecular mechanism of association of AnxA6 with biological membranes, especially its ability to aggregate membranes, is still unclear. To address this question, we studied the association of AnxA6 with model phospholipid membranes by combining the techniques of quartz crystal microbalance with dissipation monitoring (QCM-D), (cryo-) transmission electron microscopy (TEM) and atomic force microscopy (AFM). The properties of membrane binding and membrane aggregation of AnxA6 were compared to two reference systems, annexin A5 (AnxA5), which is the annexin prototype, and a chimerical AnxA5-dimer molecule, which is able to aggregate two membranes in a symmetrical manner. We show that AnxA6 presents two modes of association with lipid membranes depending on Ca(2+)-concentration. At low Ca(2+)-concentration ( approximately 60-150microM), AnxA6 binds to membranes via its two coplanar annexin modules and is not able to associate two separate membranes. At high Ca(2+)-concentration ( approximately 2mM), AnxA6 molecules are able to bind two adjacent phospholipid membranes and present a conformation similar to the AnxA6 3D crystallographic structure. Possible biological implications of these novel membrane-binding properties of AnxA6 are discussed.