Amyloid β (Aβ) oligomers may play a decisive role in Alzheimer’s disease related neurodegeneration, but their structural properties are poorly understood. In this report, sedimentation velocity centrifugation, small angle neutron scattering (SANS) and molecular modelling were used to identify the small oligomeric species formed by the 42 amino acid residue long isoform of Aβ (Aβ42) in solution, characterized by a sedimentation coefficient of 2.56 S, and a radius of gyration between 2 and 4 nm. The measured sedimentation coefficient is in close agreement with the sedimentation coefficient calculated for Aβ42 hexamers using MD simulations at µM concentration. To the best of our knowledge this is the first report detailing the Aβ42 oligomeric species by SANS measurements. Our results demonstrate that the smallest detectable species in solution are penta- to hexamers. No evidences for the presence of dimers, trimers or tetramers were found, although the existence of those Aβ42 oligomers at measurable quantities had been reported frequently.

Background: Protofibrils of the amyloid-␤ peptide (A␤) are neurotoxic oligomers implicated in development and progression of Alzheimer disease. Results: The dissociation of A␤ protofibrils into their monomeric subunits is a slow process, occurring on the time scale of hours. Conclusion: A␤ protofibrils possess a high kinetic stability toward dissociation into monomers. Significance: The longevity of A␤ protofibrils permits sustained toxic effects.

The amyloid-beta peptide (Aβ) plays a major role in the progression of Alzheimer’s disease. Due to its high toxicity, the 42 amino acid long isoform Aβ42 has become of considerable interest. The Aβ42 monomer is prone to aggregation down to the nanomolar range which makes conventional structural methods such as NMR or X-ray crystallography infeasible. Conformational information, however, will be helpful to understand the different aggregation pathways reported in the literature and will allow to identify potential conditions that favour aggregation-incompetent conformations. In this study, we applied fluorescence correlation spectroscopy (FCS) to investigate the unfolding of Alexa Fluor 488 labelled monomeric Aβ42 using guanidine hydrochloride as a denaturant. We show that our Aβ42 pre-treatment and the low-nanomolar concentrations, typically used for FCS measurements, strongly favour the presence of monomers. Our results reveal that there is an unfolding/folding behaviour of monomeric Aβ42. The existence of a cooperative unfolding curve suggests the presence of structural elements with a Gibbs free energy of unfolding of about 2.8 kcal/mol.

Ischaemia-reperfusion (IR) injury is a feature of multiple diseases including myocardial infarction, renal failure and stroke, occurring when tissue blood supply is restricted and subsequently restored. While reperfusion is essential for tissue survival, it is also associated with significant ROS-mediated damage, triggering inflammatory responses and ultimately cell death. The rational development of targeted therapeutic interventions require both a detailed understanding of the mechanisms underlying reperfusion injury and a convenient means of assessing the efficacy of putative therapeutics. This need has driven the development of a number of in vitro IR models including pharmacological ATP depletion, oil overlays to induce autohypoxia, glucose oxidase addition to cause sample deoxygenation, and N 2 purged hypoxia chambers to induce a slow reduction in dissolved oxygen. The utility of these models has however been limited as they do not facilitate the induction of the rapid, controlled, transient, ischemic shock and reperfusion necessary to replicate IR injury condition in vitro . Critically, neither do they facilitate real-time cellular oxygenation monitoring to allow accurate IR characterisation or parallel measurements of critical IR injury parameters such as ROS and mitochondrial membrane potential (MMP). Here we present a model which addresses these limitations through the combined use of iPS-derived cardiomyocytes (Cor4U), plate reader with integrated atmospheric control facilitating rapid [O 2 ] modulation (ClarioSTAR) and a novel intracellular probe capable of reporting cellular oxygenation in real time (MitoXpress-Intra). Oxygenation monitoring facilitates precise control of an ischemic insult whereby instrument [O 2 ] can be modulated (21% to 0.1% O 2 ) to provide the desired depth and duration of hypoxia. The chamber can then be vented to model rapid reperfusion (1 to 10% in less than 10min), with parallel monitoring of ROS and MMP, facilitating detailed metabolic characterisation of the short-term metabolic implications of reperfusion and the efficacy of model therapeutic intervention.