Direct Observation of Single Amyloid-β(1-40) Oligomers on Live Cells: Binding and Growth at Physiological Concentrations

Understanding how amyloid-β peptide interacts with living cells on a molecular level is critical to development of targeted treatments for Alzheimer's disease. Evidence that oligomeric Aβ interacts with neuronal cell membranes has been provided, but the mechanism by which membrane binding occurs and the exact stoichiometry of the neurotoxic aggregates remain elusive. Physiologically relevant experimentation is hindered by the high Aβ concentrations required for most biochemical analyses, the metastable nature of Aβ aggregates, and the complex variety of Aβ species present under physiological conditions. Here we use single molecule microscopy to overcome these challenges, presenting direct optical evidence that small Aβ(1-40) oligomers bind to living neuroblastoma cells at physiological Aβ concentrations. Single particle fluorescence intensity measurements indicate that cell-bound Aβ species range in size from monomers to hexamers and greater, with the majority of bound oligomers falling in the dimer-to-tetramer range. Furthermore, while low-molecular weight oligomeric species do form in solution, the membrane-bound oligomer size distribution is shifted towards larger aggregates, indicating either that bound Aβ oligomers can rapidly increase in size or that these oligomers cluster at specific sites on the membrane. Calcium indicator studies demonstrate that small oligomer binding at physiological concentrations induces only mild, sporadic calcium leakage. These findings support the hypothesis that small oligomers are the primary Aβ species that interact with neurons at physiological concentrations

Single particle tracking of alpha7 nicotinic AChR in hippocampal neurons reveals regulated confinement at glutamatergic and GABAergic perisynaptic sites

Alpha7 neuronal nicotinic acetylcholine receptors (alpha7-nAChR) form Ca(2+)-permeable homopentameric channels modulating cortical network activity and cognitive processing. They are located pre- and postsynaptically and are highly abundant in hippocampal GABAergic interneurons. It is unclear how alpha7-nAChRs are positioned in specific membrane microdomains, particularly in cultured neurons which are devoid of cholinergic synapses. To address this issue, we monitored by single particle tracking the lateral mobility of individual alpha7-nAChRs labeled with alpha-bungarotoxin linked to quantum dots in live rat cultured hippocampal interneurons. Quantitative analysis revealed different modes of lateral diffusion of alpha7-nAChR dependent on their subcellular localization. Confined receptors were found in the immediate vicinity of glutamatergic and GABAergic postsynaptic densities, as well as in extrasynaptic clusters of alpha-bungarotoxin labeling on dendrites. alpha7-nAChRs avoided entering postsynaptic densities, but exhibited reduced mobility and long dwell times at perisynaptic locations, indicative of regulated confinement. Their diffusion coefficient was lower, on average, at glutamatergic than at GABAergic perisynaptic sites, suggesting differential, synapse-specific tethering mechanisms. Disruption of the cytoskeleton affected alpha7-nAChR mobility and cell surface expression, but not their ability to form clusters. Finally, using tetrodotoxin to silence network activity, as well as exposure to a selective alpha7-nAChR agonist or antagonist, we observed that alpha7-nAChRs cell surface dynamics is modulated by chronic changes in neuronal activity. Altogether, given their high Ca(2+)-permeability, our results suggest a possible role of alpha7-nAChR on interneurons for activating Ca(2+)-dependent signaling in the vicinity of GABAergic and glutamatergic synapses

PICK1 interacts with alpha7 neuronal nicotinic acetylcholine receptors and controls their clustering

Central to synaptic function are protein scaffolds associated with neurotransmitter receptors. Alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) modulate network activity, neuronal survival and cognitive processes in the CNS, but protein scaffolds that interact with these receptors are unknown. Here we show that the PDZ-domain containing protein PICK1 binds to alpha7 nAChRs and plays a role in their clustering. PICK1 interacted with the alpha7 cytoplasmic loop in yeast in a PDZ-dependent way, and the interaction was confirmed in recombinant pull-down experiments and by co-precipitation of native proteins. Some alpha7 and PICK1 clusters were adjacent at the surface of SH-SY5Y cells and GABAergic interneurons in hippocampal cultures. Expression of PICK1 caused decreased alpha7 clustering on the surface of the interneurons in a PDZ-dependent way. These data show that PICK1 negatively regulates surface clustering of alpha7 nAChRs on hippocampal interneurons, which may be important in inhibitory functions of alpha7 in the hippocampus