Apolipoprotein E impairs amyloid-ß fibril elongation and maturation.

Alzheimer's disease (AD) is strongly linked to amyloid depositions of the Aß peptide (Aß). The lipid-binding protein apolipoprotein E (ApoE) has been found to interfere with Aß amyloid formation and to exert a strong clinical impact to the pathology of AD. The APOE gene exists in three allelic isoforms represented by APOE ε2, APOE ε3, and APOE ε4. Carriers of the APOE ε4 variant display a gene dose-dependent increased risk of developing the disease. Aß amyloids are formed via a nucleation-dependent mechanism where free monomers are added onto a nucleus in a template-dependent manner. Using a combination of surface plasmon resonance and thioflavin-T assays, we here show that ApoE can target the process of fibril elongation and that its interference effectively prevents amyloid maturation. We expose a complex equilibrium where the concentration of ApoE, Aß monomers, and the amount of already formed Aß fibrils will affect the relative proportion and formation rate of mature amyloids versus alternative assemblies. The result illustrates a mechanism which may affect both the clearance rate of Aß assemblies in vivo and the population of cytotoxic Aß assemblies.

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3.

Due to the essential roles of glutamate, detection and response to a large range of extracellular concentrations of this excitatory amino acid are necessary for the fine-tuning of brain functions. Metabotropic glutamate receptors (mGluRs) are implicated in shaping the activity of many synapses in the central nervous system. Among the eight mGluR subtypes, there is increasing interest in studying the mGlu3 receptor which has recently been linked to various diseases, including psychiatric disorders. This receptor displays striking functional properties, with a high and, often, full basal activity, making its study elusive in heterologous systems. Here, we demonstrate that Cl- ions exert strong positive allosteric modulation of glutamate on the mGlu3 receptor. We have also identified the molecular and structural determinants lying behind this allostery: a unique interactive "chloride-lock" network. Indeed, Cl- ions dramatically stabilize the glutamate-induced active state of the extracellular domain of the mGlu3 receptor. Thus, the mGlu3 receptors' large basal activity does not correspond to a constitutive activity in absence of agonist. Instead, it results mostly from a Cl-mediated amplified response to low ambient glutamate concentrations, such as those measured in cell media. This strong interaction between glutamate and Cl- ions allows the mGlu3 receptor to sense and efficiently react to sub-micromolar concentrations of glutamate, making it the most sensitive member of mGluR family.

Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy.

Efficient cell-to-cell communication relies on the accurate signalling of cell surface receptors. Understanding the molecular bases of their activation requires the characterization of the dynamic equilibrium between active and resting states. Here, we monitor, using single-molecule Förster resonance energy transfer, the kinetics of the reorientation of the extracellular ligand-binding domain of the metabotropic glutamate receptor (mGluR), a class C G-protein-coupled receptor. We demonstrate that most receptors oscillate between a resting- and an active-conformation on a sub-millisecond timescale. Interestingly, we demonstrate that differences in agonist efficacies stem from differing abilities to shift the conformational equilibrium towards the fully active state, rather than from the stabilization of alternative static conformations, which further highlights the dynamic nature of mGluRs and revises our understanding of receptor activation and allosteric modulation.

Pulsed interleaved excitation fluorescence spectroscopy with a supercontinuum source.

Pulsed Interleaved Excitation (PIE) improves fluorescence cross-correlation spectroscopy (FCCS) and single pair Förster Resonance Energy Transfer (spFRET) measurements, by correlating each detected photon to the excitation source that generated it. It relies on the interleaving of two picosecond laser sources and time correlated single photon counting (TCSPC) detection. Here, we present an optical configuration based on a commercial supercontinuum laser, which generates multicoulour interleaved picosecond pulses with arbitrary spacing and wavelengths within the visible spectrum. This simple, yet robust configuration can be used as a versatile source for PIE experiments, as an alternative to an array of picosecond lasers and drivers.