Trodusquemine enhances Aß42 aggregation but suppresses its toxicity by displacing oligomers from cell membranes.

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-ß peptide (Aß42) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aß42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aß42-induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery.

BACKGROUND: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. NEW METHOD: We describe the development and application of an advanced machine vision system for characterising the behaviour of C. elegans, the Wide Field-of-View Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. RESULTS: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules for Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. COMPARISON WITH EXISTING METHODS: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. CONCLUSIONS: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. We anticipate that this approach will further extend the scope and utility of C. elegans as a model organism.

Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies.

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer's and Parkinson's diseases.

Selenium-enhanced electron microscopic imaging of different aggregate forms of a segment of the amyloid ß peptide in cells.

The aggregation of misfolded proteins is a common feature underlying a wide range of age-related degenerative disorders, including Alzheimer's and Parkinson's diseases. A key aspect of understanding the molecular origins of these conditions is to define the manner in which specific types of protein aggregates influence disease pathogenesis through their interactions with cells. We demonstrate how selenium-enhanced electron microscopy (SE-EM), combined with tomographic reconstruction methods, can be used to image, here at a resolution of 5-10 nm, the interaction with human macrophage cells of amyloid aggregates formed from Aß(25-36), a fragment of the Aß peptide whose self-assembly is associated with Alzheimer's disease. We find that prefibrillar aggregates and mature fibrils are distributed into distinct subcellular compartments and undergo varying degrees of morphological change over time, observations that shed new light on the origins of their differential toxicity and the mechanisms of their clearance. In addition, the results show that SE-EM provides a powerful and potentially widely applicable means to define the nature and location of protein assemblies in situ and to provide detailed and specific information about their partitioning and processing.

A highly amyloidogenic region of hen lysozyme.

Amyloid fibrils obtained after incubating hen egg-white lysozyme (HEWL) at pH 2.0 and 65 degrees C for extended periods of time have been found to consist predominantly of fragments of the protein corresponding to residues 49-100, 49-101, 53-100 and 53-101, derived largely from the partial acid hydrolysis of Asp-X peptide bonds. These internal fragments of HEWL encompass part of the beta-domain and all the residues forming the C-helix in the native protein, and contain two internal disulfide bridges Cys64-Cys80 and Cys76-Cys94. The complementary protein fragments, including helices A, B and D of the native protein, are not significantly incorporated into the network of fibrils, but remain largely soluble, in agreement with their predicted lower propensities to aggregate. Further analysis of the properties of different regions of HEWL to form amyloid fibrils was carried out by studying fragments produced by limited proteolysis of the protein by pepsin. Here, we show that only fragment 57-107, but not fragment 1-38/108-129, is able to generate well-defined amyloid fibrils under the conditions used. This finding is of particular importance, as the beta-domain and C-helix of the highly homologous human lysozyme have been shown to unfold locally in the amyloidogenic variant D67H, which is associated with the familial cases of systemic amyloidosis linked to lysozyme deposition. The identification of the highly amyloidogenic character of this region of the polypeptide chain provides strong support for the involvement of partially unfolded species in the initiation of the aggregation events that lead to amyloid deposition in clinical disease.