Collagen hydrogel confinement of Amyloid-β (Aβ) accelerates aggregation and reduces cytotoxic effects

Simpson, Laura W.; Szeto, Gregory L.; Boukari, Hacène; Good, Theresa A.; Leach, Jennie B.

doi:10.1016/j.actbio.2020.05.030

Cited by 16 publications

(15 citation statements)

References 105 publications

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“…This is consistent with the Aβ pretreatment process that was used to ensure a consistent population of Aβ monomers at the start of each experiment. 15 All conditions showed the presence of aggregated Aβ (ThT fluorescence) that increased over time according to one of four trends: (1) a pronounced lag phase and then rapid aggregation, (2) a two-phase increase in fluorescence depicting an overall relatively slow rate of aggregation, (3) a brief slow phase and then rapid aggregation, and (4) a two-phase increase in fluorescence suggesting an overall relatively fast rate of aggregation.…”

Section: Resultsmentioning

confidence: 95%

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer’s Disease

et al. 2020

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The physiochemical properties of hydrogels utilized in 3D culture can be used to modulate cell phenotype and morphology with a striking resemblance to cellular processes that occur in vivo . Indeed, research areas including regenerative medicine, tissue engineering, in vitro cancer models, and stem cell differentiation have readily utilized 3D biomaterials to investigate cell biological questions. However, cells are only one component of this biomimetic milieu. In many models of disease such as Alzheimer’s disease (AD) that could benefit from the in vivo -like cell morphology associated with 3D culture, other aspects of the disease such as protein aggregation have yet to be methodically considered in this 3D context. A hallmark of AD is the accumulation of the peptide amyloid-β (Aβ), whose aggregation is associated with neurotoxicity. We have previously demonstrated the attenuation of Aβ cytotoxicity when cells were cultured within type I collagen hydrogels versus on 2D substrates. In this work, we investigated the extent to which this phenomenon is conserved when Aβ is confined within hydrogels of varying physiochemical properties, notably mesh size and bioactivity. We investigated the Aβ structure and aggregation kinetics in solution and hydrogels composed of type I 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 enhanced Aβ aggregation and Aβ cytotoxicity attenuation. We suggest that confinement itself imparts a profound effect, possibly by stabilizing Aβ structures and shifting the aggregate equilibrium toward larger species. If this phenomenon of altered protein aggregation in 3D hydrogels can be generalized to other contexts including the in vivo environment, it may be necessary to reevaluate aspects of protein aggregation disease models used for drug discovery.

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Section: Resultsmentioning

confidence: 95%

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer’s Disease

et al. 2020

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“…This discovery might partially explain why the Aβ targeting drugs fail in clinical trials since the much higher physiological concentration of Aβ is used in vitro. Recently, Simpson et al (2020) identified differences of Aβ aggregation and cytotoxicity between 2D in vitro models (typically culture cells on flat plastic or glass substrates) and 3D environments, such as collagen hydrogels in tissue culture models. Combined with FCS, they found that the size of Aβ aggregates in 3D collagen hydrogel is 25-200 times larger in diameter than 2D culture, suggesting a vastly different aggregation process.…”

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confidence: 99%

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

Cao

Loch

et al. 2020

Quart. Rev. Biophys.

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In neurodegenerative diseases, a wide range of amyloid proteins or peptides such as amyloid-beta and α-synuclein fail to keep native functional conformations, followed by misfolding and self-assembling into a diverse array of aggregates. The aggregates further exert toxicity leading to the dysfunction, degeneration and loss of cells in the affected organs. Due to the disordered structure of the amyloid proteins, endogenous molecules, such as lipids, are prone to interact with amyloid proteins at a low concentration and influence amyloid cytotoxicity. The heterogeneity of amyloid proteinscomplicates the understanding of the amyloid cytotoxicity when relying only on conventional bulk and ensemble techniques. As complementary tools, single-molecule techniques (SMTs) provide novel insights into the different subpopulations of a heterogeneous amyloid mixture as well as the cytotoxicity, in particular as involved in lipid membranes. This review focuses on the recent advances of a series of SMTs, including single-molecule fluorescence imaging, single-molecule force spectroscopy and single-nanopore electrical recording, for the understanding of the amyloid molecular mechanism. The working principles, benefits and limitations of each technique are discussed and compared in amyloid protein related studies.. We also discuss why SMTs show great potential and are worthy of further investigation with feasibility studies as diagnostic tools of neurodegenerative diseases and which limitations are to be addressed.

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“…Dynamics of hydrogel microstructures can be characterized by a) analysis of probe diffusion via fluorescence recovery after photobleaching (FRAP) and dynamic light scattering (DLS), b) estimation of mesh size from swelling data and Flory-Rehner-Huggins theory, and c) examination of mechanical properties by rheology and dielectric relaxation (Aggeli et al, 1997(Aggeli et al, , 2001; Barretta et al, 2000;Bohidar, 2001;Burne and Sellen, 1994;Canal and Peppas, 1989;Johnson et al, 1995;Pluen et al, 1999;Pusey and Van Megen, 1989;Simpson et al, 2020). Unfortunately, these techniques are most often incapable of distinguishing between features of the hydrogel and features of the protein because signatures related to the hydrogel typically overshadows the small signal generated by features of individual proteins.…”

Section: Confinementmentioning

confidence: 99%

“…Fluorescence correlation spectroscopy (FCS) detects the individual photons of fluorescently-labeled molecules that pass through a very small confocal volume. Photon fluctuation data can be used to generate a correlation function, which can then be used to calculate molecular diffusion and size (Michelman-Ribeiro et al, 2007;Sengupta et al, 2003;Vagias et al, 2013;Vagias et al, 2017;Zustiak et al, 2010;Simpson et al, 2020). In cases where fluorophore photobleaching is a concern, raster image correlation spectroscopy (RICS) is similar to FCS but applies multiphoton excitation and rasterized scanning to limit fluorophore exposure to light (Fig.…”

Section: Current Challenges and Opportunitiesmentioning

confidence: 99%

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Protein folding and assembly in confined environments: Implications for protein aggregation in hydrogels and tissues

Simpson

Good

Leach

2020

Biotechnology Advances

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In the biological milieu of a cell, soluble crowding molecules and rigid confined environments strongly influence whether the protein is properly folded, intrinsically disordered proteins assemble into distinct phases, or a denatured or aggregated protein species is favored. Such crowding and confinement factors act to exclude solvent volume from the protein molecules, resulting in an increased local protein concentration and decreased protein entropy. A protein's structure is inherently tied to its function. Examples of processes where crowding and confinement may strongly influence protein function include transmembrane protein dimerization, enzymatic activity, assembly of supramolecular structures (e.g., microtubules), nuclear condensates containing transcriptional machinery, protein aggregation in the contexts of disease and protein therapeutics. Historically, most protein structures have been determined from pure, dilute protein solutions or pure crystals. However, these are not the environments in which these proteins function. Thus, there has been an increased emphasis on analyzing protein structure and dynamics in more "in vivo-like" environments. Complex in vitro models using hydrogel scaffolds to study proteins may better mimic features of the in vivo environment. Therefore, analytical techniques need to be optimized for real-time analysis of proteins within hydrogel scaffolds.

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Collagen hydrogel confinement of Amyloid-β (Aβ) accelerates aggregation and reduces cytotoxic effects

Cited by 16 publications

References 105 publications

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer’s Disease

Impact of Four Common Hydrogels on Amyloid-β (Aβ) Aggregation and Cytotoxicity: Implications for 3D Models of Alzheimer’s Disease

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

Protein folding and assembly in confined environments: Implications for protein aggregation in hydrogels and tissues

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