Quantitative proteomics and localisation of complement proteins in human macular and peripheral Bruch’s membrane/choroid

Abstract

Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly worldwide. Drusen, extracellular deposits of debris accumulating in Bruch's membrane, are considered hallmarks of early AMD. Dysregulation of the complement cascade is believed to be a driver in the pathogenesis of AMD. However, why the macular region is particularly susceptible to AMD remains unclear. In this thesis, I investigate differences in molecular composition of the macular and peripheral Bruch's membrane with associated choroidal extracellular matrix (enriched Bruch's membrane) from eyes donated by older individuals without AMD. Furthermore, I analyse and compare the localisation of complement proteins in eyes from older donors with and without early AMD. In this study, using isobaric tags for relative and absolute quantitation (iTRAQ) mass spectrometry, a total of 1,722 proteins were identified in enriched Bruch's membrane from donors who did not have AMD. Quantitative analysis revealed that 14 proteins were significantly more abundant in the macular region than in the peripheral region, including complement proteins (C3, C5, C6 and C7). Conversely, seven proteins were significantly more abundant in the peripheral region than in the macula. Subsequent immunohistochemistry studies demonstrated that C5b-9, FHR-4 and FB localised mainly in the intercapillary septa and around the capillaries of the choriocapillaris in AMD donors, but these were barely seen within the Bruch's membrane structure itself in non-AMD donors. However, in eyes with early AMD, these complement components were observed as an intense band throughout Bruch's membrane and within drusen. Overall, this study shows regional differences in the proteins of enriched Bruch's membrane, with an increased concentration of C3 and terminal pathway proteins in the macula compared to the periphery; this mirrors the macular predilection for AMD. Additionally, the translocation of complement proteins from the intercapillary septa to within Bruch's membrane itself between non-AMD and AMD donor eyes indicates that the complement pathway is initially activated in the intercapillary septa and immediately around the capillaries of the choriocapillaris. As the disease progresses Bruch's membrane becomes more permeable to complement proteins allowing them to permeate through its layers and enter drusen.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Paul Bishop (Supervisor) & Simon Clark (Supervisor)