Detection of proteins in the pathological deposits in Pseudoexfoliation syndrome using Atomic Force Microscopy

R. Creasey,¹ C. Gibson,¹ S. Sharma,¹ J.E. Craig,¹ T. Becker,² P. Hinterdorfer³ and N.H. Voelcker,¹ ¹Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia, ²Nanochemistry Research Institute, Curtin University, GPO Box U1987, Perth, WA 6845, Australia and ³Institute for Biophysics, Johannes Kepler University Linz, Altenberger St. 69, Linz 4040, Austria.

Protein aggregation is of significant interest to various disciplines; it can be the cause of debilitating diseases, or the foundation of advanced nanomaterials. One ocular disease hallmarked by protein aggregation is known as Pseudoexfoliation Syndrome (PEX). This condition is caused by the formation of insoluble aggregates, and is characterised by deposition of fibrillar proteinaceous material on the anterior lens capsule. PEX deposits in the eye block the aqueous outflow mechanisms, which can lead to an elevation in intraocular pressure and subsequent glaucoma. Glaucoma is the second leading cause of irreversible blindness worldwide, and PEX is the most common known risk factor for glaucoma.

Proteomic analyses have revealed an association of various genetic markers and protein expression with PEX; however a complete explanation for disease susceptibility is not yet available. As the aggregates are a complex arrangement of proteins, the ultrastructure is poorly characterised and many protein constituents of the aggregates remain unknown. This study addresses the critical issue of determining the molecular nature of PEX on lens capsules in their native state by atomic force microscopy (AFM) based antibody recognition imaging. The particular focus of this study is on a type of AFM methodology referred to as topography and recognition imaging (TREC). Proteins identified by proteomic data as being implicated in the PEX pathophysiology, such as clusterin and LOXL1, are detected by an AFM probe modified with the appropriate antibody. Topographical AFM images and antibody recognition images are obtained simultaneously to determine the specific location of proteins in and around PEX aggregates on the lens capsule anterior surface. These data, combined with data from alternative antibody-recognition imaging techniques, proteomic and genetic analyses, are leading to an improved understanding of the pathophysiological basis of PEX. A more complete understanding of the pathophysiological basis for the disease will lead to the development of earlier detection methods and treatments that target the disease instead of the subsequent glaucoma.