Investigating the role of complement factor H-like protein-1 and factor H related proteins in age-related macular degeneration

About 50 million people worldwide are affected by Age-related Macular Degeneration (AMD), a condition that leads to a loss of central vision and blindness. The inside of the back of the eye is lined by the retina, which converts light into signals that are sent to the brain, and this is the basis of vision. In AMD there is damage to the central part of the retina which is called the macula. A variety of risk factors are associated with developing AMD such as smoking and dietary factors, but increasingly it is becoming obvious that our genes have a large influence on our susceptibility to the disease. Over recent years a series of alterations in genes for part of the immune system (known as the complement cascade) have been shown to strongly increase an individuals risk of developing AMD. An alteration in the gene encoding complement factor H (or CFH for short) represent a particularly strong risk factor. CFH is responsible for making sure that the complement cascade does not accidently attack our own tissues and cells. CFH achieves this by sticking to our tissues and cells and thus prevents activation of our complement system that would otherwise damage our tissue. A common alteration (called a polymorphism) in CFH is referred to as the Y402H polymorphism and results in a change in the function of CFH. Around 35% of people of European descent have this polymorphism. We have shown previously that the two forms of CFH (known as 402Y and 402H) bind the macula of human eyes differently. The disease form of CFH (402H) binds less well to a specific region of the macula, called the Bruch's membrane, and therefore can't regulate the complement cascade well enough. This leaves the macula open to tissue damage and ultimately cell death, resulting in the loss of central vision.
My recent research has shown that, surprisingly, there is very little CFH in the macula but instead it mainly contains a smaller, closely related protein called factor H-like protein 1 (FHL-1). FHL-1 comes from the same gene as CFH but cuts off one third of the way through. That means FHL-1 is identical to the first third of CFH before abruptly ending. However, the Y402H polymorphism occurs early in the CFH gene and therefore FHL-1 is also subject to the same polymorphism. It appears that the scientific community may have incorrectly assumed that it is full-length CFH that is preventing immune activation and tissue damage in the macula. I believe that this FHL-1 protein, and not CFH, is protecting the Bruch's membrane. I also hypothesise that a group of closely related genes, the factor H-related genes (FHR), produce proteins that play a role in regulation of the immune system in the macula. I believe that the function of these proteins changes with the progression of disease, making things worse and amplifying the disease process. With access to one of the largest Eye Banks in Europe I will be able to test these hypotheses by studying all the proteins in question and comparing their localisation in the macula of human donor eyes (60+ years old) that have been segregated into healthy, early AMD and late AMD. Also, by using FHL-1 and FHR proteins made in the laboratory, I will compare their ability to regulate complement in human eye tissue from varying ages. This multidisciplinary project will be well supported by world-class research facilities at the University of Manchester and will be supported by a network of scientific collaborators (both in the UK and abroad) providing access to specialised biochemicals and cutting-edge technologies. This work will improve our understanding of how and why AMD starts and then progresses into such a devastating disease. Furthermore, I anticipate that this research will identify targets for therapeutic intervention potentially to both prevent AMD and slow down its progression.

Age-related macular degeneration (AMD) is the most common form of blindness in the Western world and is characterised by the presence of extracellular debris including drusen at the interface between the retinal pigment epithelium (RPE) and Bruch's membrane. Drusen contain complement proteins suggesting a central role for the complement cascade in their pathogenesis. The common Y402H polymorphism in the gene encoding complement factor H (CFH) has been associated with an increased risk of developing AMD. Recently, I have obtained preliminary evidence showing that a protein resulting from a splice variation of the CFH gene, factor H-like protein 1 (FHL-1) that is also subject to the Y402H polymorphism, is the main regulator on the Bruch's membrane. I hypothesise that FHL-1 confers protection to the Bruch's membrane and that interplay exists between this protein and proteins encoded by related, but separate complement genes, referred to as the factor H-related proteins. My aim is to test this hypothesis by examining the macula of genotyped human eye tissues: donor tissue genotype will be mapped for all the disease-associated genes. Donor eyes will be analysed histologically (including using Picro-Mallory staining) and grouped into unaffected, early or late AMD. Fluorescent immunohistochemistry will show the endogenous localisation of the FHL/FHR proteins in the various disease states. Immuno-EM will elucidate the macromolecular nature of protein/Bruch's interaction and how this changes during the disease process. Functional assays will determine the effect of the Y402H polymorphism on FHL-1 regulatory activity and how this alters with disease. This project is focused on understanding the biochemistry of the early stages of AMD progression, and as such may help guide improvements in treating this disease early in it pathogenesis, before the devastating, life-changing, symptoms occur.

The aim of this study is to determine in Age-related Macular Degeneration (AMD) how the factor H-like protein 1 (FHL-1) regulates the complement cascade, how its function is modified by the Y402H polymorphism and the role of the factor H-related proteins (FHR). In particular this project will elucidate the interplay between FHL-1 and the FHR proteins FHR, whose functions are currently completely unknown, but it is known that a deletion of the genes for FHR1 and FHR3 is protective against AMD. It is likely that this project will have major impacts on our understanding of the molecular pathology underpinning AMD and it may identify novel therapeutic targets for the treatment and prevention of this devastating disease. AMD is the leading cause of blindness in the western world and its incidence is only going to risk with the increasing elderly population. With ever greater numbers of AMD suffers, there will be an escalating socioeconomic burden on healthcare services (such as the NHS), carers and the patients themselves. AMD results in the loss of central vision and therefore the contribution of suffers to the nation's workforce will be diminished. The work proposed in this application focuses on understanding the mechanisms of disease and is aimed at developing novel therapeutic strategies to address the progression of early AMD to the late, and devastating, stages of AMD. This may have direct financial benefits for the economy through the generation of IP, the setting up of spinout companies and drug sales. Not only is the finding that FHL-1 plays a greater role in complement regulation on the Bruch's membrane novel, elucidating the interplay between FHL-1 and the FHR proteins may lead to a new disease mechanism. Subsequently, changes in the scientific and clinical communities, based on this new information, may well alter the way this disease is diagnosed and handled and as such may have an impact on reducing the socioeconomic burden of AMD. I am currently involved in various public engagement activities organised through the Faculties of Medicine and Human Sciences and Life Sciences, aimed at raising the awareness of ocular disease, such as AMD, which may also facilitate early diagnosis.