Background: Amyloids are native proteins that are misfolded such that aggregation is initiated, yielding insoluble fibrous deposits found in various diseases, including Alzheimer’s (AD), Parkinson’s, and Huntington’s. In AD, unstable β amyloid (Aβ) oligomers have a higher diffusibility, hydrophobicity and ability to interact with lipid membranes, as compared to Aβ fibrils. When confined in a 3D environment such as tissue or a tissue-like hydrogel, Aβ transport is altered vs in solution due to covalent and noncovalent binding and hindered diffusion. We hypothesize that 3D environment factors may stabilize unfolded oligomers and alter aggregation kinetics resulting in fewer toxic Aβ species formed. Methods: Pretreated Aβ42 films were dissolved in NaOH then diluted in PBS before being incorporated in gels including collagen I, SeaPlaque agarose, 4-arm PEG maleimide crosslinked with PEG dithiol and maleimide modified hyaluronic acid crosslinked with PEG dithiol. Neuroblastoma derived SY5Y cells were cultured in phenol red free DMEM/F12 w/ 1% B27. Multiple protein detection methods were utilized to classify the aggregate species over time including: Thioflavin T (ThT), Fluorescence Correlation Spectroscopy (FCS), TEM, and Dot blot. Cell viability assays and calcium imaging were used to determine the toxicity of the aggregate species on SY5Y cells. Results: ThT assays using 20µM Aβ showed a lag phase (~9 hr) in medium before β-sheet structures initiated the rapid increase in fluorescence vs minimal lag phase seen in the 3D gels representing rapid fibrilization. FCS found Aβ species in medium remained as small aggregates with only 5x slower diffusivities than monomer Aβ during the lag phase vs immediate larger aggregate species in all gels. SY5Y cells cultured on collagen substrates consistently have a cell viability &gt;90% but in the presence of 20µM Aβ, the cell viability decreases &lt;60% after 3 days compared to in 3D gels, the cell viability is no different with or without Aβ. Discussion: Results identify a lack of a lag phase during aggregation in 3D gels suggesting stabilization of β-sheet structures vs Aβ aggregation kinetics in medium. FCS confirms low molecular weight Aβ during the lag phase in medium and high molecular weight Aβ in gels. SY5Y cells cultured in 2D are susceptible to Aβ toxicity over 3 days while in 3D gels there is minimal difference in cell viability with or without Aβ, suggesting the 3D environment promotes fewer toxic Aβ species.

Bioengineered hydrogel models of Alzheimer's disease: Studying β-amyloid protein aggregation within 3D scaffolds

255th National Meeting (2018)

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