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Indre Bielskus, Nicholas Pfahler, Thomas Cronin, James Haney, Paul A Knepper; Stop and stabilize β-amyloid: a novel bimodal approach for prevention of β-amyloid aggregation and toxicity. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3719. doi: https://doi.org/.
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Amyloid deposition is a feature in numerous human diseases, including primary open-angle glaucoma (POAG), Alzheimer’s disease (AD), and age-related macular degeneration (AMD). In POAG, amyloid deposits occur in the trabecular meshwork, ciliary body, retina and optic nerve. Amyloid formation occurs in a multistage process—monomeric nucleation, elongation, beta sheet formation, aggregation, and ultimately, a beta amyloid that is a cell toxic fibril causing devastating diseases. Our laboratory tested two potential anti-amyloidogenic compounds -the flavonoid curcumin (C) and disaccharide trehalose (T) - alone and in combination to test their effects on amyloid beta (1-42) (Aβ42) aggregation.
Monomeric Aβ42 was purchased from AnaSpec Inc, Fremont, CA and the assay followed the manufacturer’s protocol. Replicate samples (10μg Aβ42;n=3) were incubated in a non-binding 96-well black plate at 37°C for 2h,1,3,5 and 8 days with and without treatment with curcumin (Sigma-Aldrich;0.3, 3 and 30μM), trehalose (Sigma;20μM), and in combination. Aliquots were taken at each time point and stained with Thioflavin T, a benzothiazole dye that increases in fluorescence upon binding to beta sheet amyloid aggregates. Fluorescence readings were taken at Ex/Em=440/480nm at 5 min intervals over a 1h period at 37°C. Background fluorescence was subtracted, and results were compared to a Phenol Red positive control. All readings were expressed in relative fluorescence units (RFU). Statistical significance was determined by a student T-test.
Our results showed that curcumin and trehalose significantly decreased Aβ42 aggregate formation. After an 8 day incubation, treatment with C (3µM) significantly inhibited Aβ42 aggregate formation by 63% (p<0.0001), treatment with T (20µM) significantly inhibited Aβ42 aggregate formation by 35% (p=0.0028), and C (3µM) plus T (20µM) significantly inhibited Aβ42 aggregate formation by 94% (p<0.0001).
Our results indicate that in vitro, curcumin and trehalose are extremely effective in preventing Aβ42 aggregate formation. Curcumin mechanism of action is likely to be competitive binding to amyloid beta-sheet whereas trehalose is a solvent preventing tertiary structure formation. Our results indicate that curcumin and trehalose have potential as a novel bimodal approach in the prevention and treatment of amyloid diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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