Myocilin-OLF oligomer reaction fractions ThT

Glaucoma is a group of neurodegenerative diseases that together are the leading cause of irreversible blindness worldwide. Myocilin-associated glaucoma is an inherited form of this disease caused by intracellular aggregation of misfolded mutant myocilin. <i>In vitro</i>, the myocilin C-terminal olfactomedin domain (OLF), the relevant domain for glaucoma pathogenesis, can be driven to form amyloid-like fibrils under mild conditions. Here we characterize a species present during <i>in vitro </i>fibrilization.<b> </b>Purified OLF was subjected to fibrillization at concentrations required for downstream electron microscopy imaging and NMR spectroscopy.ThT fluorescence measurements of OLF oligomer reactions fractions characterized throughout the work. The end-point fibril mixture contains the ThT-positive fibrils. The supernatant fractions contain largely non-ThT-positive species. The low fluorescence in the filtered supernatant sample confirms that the primary species is native OLF monomer.