Szeto, G. | bioRxiv Science

bioRxiv2019

Collagen hydrogel confinement of amyloid-β accelerates aggregation and reduces cytotoxic effects

Alzheimers disease (AD) is the most common form of dementia and is associated with the accumulation of amyloid-{beta} (A{beta}), a peptide whose aggregation has been associated with neurotoxicity. Drugs targeting A{beta} have shown great promise in 2D in vitro models and mouse models, yet preclinical and clinical trials for AD have been highly disappointing. We propose that current in vitro culture systems for discovering and developing AD drugs have significant limitations; specifically, that A{beta} aggregation is vastly different in these 2D cultures carried out on flat plastic or glass substrates vs. in a 3D environment, such as brain tissue, where A{beta} confinement very likely alters aggregation kinetics and thermodynamics. In this work, we identified attenuation of A{beta} cytotoxicity in 3D hydrogel culture compared to 2D cell culture. We investigated A{beta} structure and aggregation in solution vs. hydrogel using Transmission Electron Microscopy (TEM), Fluorescence Correlation Spectroscopy (FCS), and Thioflavin T (ThT) assays. Our results reveal that the equilibrium is shifted to stable {beta}-sheet aggregates in hydrogels and away from the relatively unstable/unstructured presumed toxic oligomeric A{beta} species in solution. Volume exclusion imparted by hydrogel confinement stabilizes unfolded, presumably toxic species, promoting stable extended {beta}-sheet fibrils. These results, taken together with the many recent reports that 3D hydrogel cell cultures enable cell morphologies and epigenetic changes that are more similar to cells in vivo compared to 2D cultures, strongly suggest that AD drugs should be tested in 3D culture systems as a step along the development pathway towards new, more effective therapeutics.

bioengineering↗

bioRxiv2019

Impact of four common hydrogels on amyloid-β (Aβ) aggregation and cytotoxicity: Implications for 3D models of Alzheimer’s disease

The properties of a hydrogel utilized in 3D culture can influence cell phenotype and morphology, yielding striking similarities to cellular processes that occur in vivo. Indeed, research areas including regenerative medicine, tissue engineering, cancer models, and stem cell cultures have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this milieu. Macromolecules play roles as bioactive factors and physical structures. Yet, investigations of macromolecular biophysics largely focus on pure molecules in dilute solution. Biophysical processes such as protein aggregation underlie diseases including Alzheimers disease, which is hallmarked by accumulated neurotoxic amyloid-{beta} (A{beta}) aggregates. Previously, we demonstrated that A{beta} cytotoxicity is attenuated when cells are cultured within type I collagen hydrogels vs. on 2D substrates. Here, we investigated whether this phenomenon is conserved when A{beta} is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated A{beta} structure and aggregation kinetics in solution and in hydrogels (collagen, agarose, hyaluronic acid and polyethylene glycol) using fluorescence correlation spectroscopy and thioflavin T assays. Our results reveal that all hydrogels tested were associated with A{beta} cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing A{beta} structures and shifting the aggregate equilibrium toward larger species. It is likely that the milieu that exist within cells and tissues also influences protein-protein interactions; thus, we suggest that it is critical to evaluate whether protein structure, function, and stability are altered in 3D systems vs. ideal solutions and 2D culture.

bioengineering↗