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

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Black JR (2016) Age-related macular degeneration: genome-wide association studies to translation. in Genetics in medicine : official journal of the American College of Medical Genetics

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Clark SJ (2018) The eye as a complement dysregulation hotspot. in Seminars in immunopathology

 
Description Elected to Executive Committee for Complement UK
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description Member of Professional Advisory Panel for the Macular Society
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Fight for Sight Project Grant
Amount £397,340 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2014 
End 08/2017
 
Description Fight for Sight Small Grant Award
Amount £14,547 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2014 
End 09/2015
 
Description Fight for Sight research grant
Amount £110,257 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2016 
End 08/2018
 
Description Macular Society Research Grant
Amount £217,343 (GBP)
Organisation Macular Society 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 09/2014 
End 08/2017
 
Title Establishment of an Eye Tissue Repository 
Description The Manchester Eye Bank corneal transplantation service obtains around 2000 pairs of eyes each year. We have set up a work-flow whereby eye tissue consented for research, after the cornea is removed for transplantation, is taken to our BioBank, dissected into its constituent components and stored appropriately for future studies. These eye tissues are phenotyped for AMD and genotyped using a comprehensive SNP array for AMD. While this is primarily a resource for our own use, we hope that in time we will have enough material and funding to make it a National Repository for eye research throughout the UK. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Provided enriched Bruch's membrane from known AMD donors for use in validating our targeted proteomic strategy. 
 
Title Generation of monoclonal anti FHR-4 antibody 
Description A novel monoclonal antibody capable of specifically identifying FHR-4 from the other Factor H-related proteins. Commercially available antibodies have proven to not be specific. 
Type Of Material Antibody 
Provided To Others? No  
Impact Identification of FHR-4 in the hallmark lesions of age-related macular degeneration (AMD) - the leading cause of blindness in the western world. This is the first time anyone has identified FHR-4 in drusen and will form the basis of a future grant application and publication. 
 
Title In house expression of recombinant FHL-1 
Description In house expression of the complement regulator factor H-like protein 1 (FHL-1) which will be used in biological experiments. Two forms have been made, representing each variant associated with the Y402H polymorphism present in the FH gene - the 402H form is associated with a 3-fold increased risk of blindness through AMD. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Only just completed, so none yet. 
 
Title In-house expression of FHL-1 C-terminal tail mutants 
Description FHL-1 has a unique four amino acid C-terminal tail which we believe to be important in recognising and binding various ligands. We have expressed truncated recombinant proteins representing the final two domains of FHL-1 with and without the C-terminal four amino acid tail. These will be used in biochemical assays to assess the role played in interactions with different ligands. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We have already discovered that the C-terminal tail plays a vital role in binding sulphated sugars (i.e. heparan sulphate). This is important, because HS is used to anchor FHL-1 to membranes in the human body and thus confers protection against aberrant complement attack. We have found that removing the C-terminal tail has a greater affect on the 402H form of FHL-1 (associated with AMD) over the 402Y form. 
 
Title New HTA master file and eye collection 
Description I have set up a new Human Tissue Authority master file and the necessary accompanying infrastructure in my new faculty, which allows the collection of human eyes for research. As such, I have recently begun collecting eye material for tissue culture and histological studies. 
Type Of Material Biological samples 
Provided To Others? No  
Impact This new set-up is vital if I am to investigate the role of immune homeostasis in the development of the most common form of blindness in the western world - age-related macular degeneration. 
 
Title Specific antiFHL-1 antibody 
Description I successfully raised an antibody capable of distinguishing the unique 4 amino acid C-terminal of factor H-like protein 1, enabling it's specific detection from the closely related complement factor H protein or the five factor H-related proteins. 
Type Of Material Antibody 
Provided To Others? No  
Impact The use of this antibody has lead to the identification of FHL-1 in the human eye and to demonstrate that it is responsible for immune homeostasis in the extracellular matrix, and not complement factor H as is currently assumed. This will change the way people attempt to treat the condition age-related macular degeneration. I have not yet published this data but intend to early next year. 
 
Title Targeted proteomic method for identifying factor H, factor H-like protein 1 and the five factor H-related proteins in a biological sample 
Description So far, no method is currently capable of differentiating between all seven factor H type proteins. This new methodology, based around targeted proteomics, can do this quantifiably in a biological sample. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact First to demonstrate the presence of FHL-1 and FHR1-3 on Bruch's membrane within the human eye - site of AMD pathogenesis. 
 
Description Generation of novel monoclonal antibody against FHR-4 
Organisation Cardiff University
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution We provided Prof. Paul Morgan (Cardiff University) and his laboratory team recombinant FHR-4 protein, which they used to raise monoclonal antibodies in mice.
Collaborator Contribution Prof. Morgan produced several clones of monoclonal antibody hybridoma against the FHR-4 protein, at least one of which has been shown to specifically identify FHR-4 in human blood.
Impact Obtained three clones of anti-FHR-4 antobody
Start Year 2014
 
Description Richard Unwin for the development of target proteomic methodologies 
Organisation University of Manchester
Department Centre for Advanced Discovery and Experimental Therapeutics
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Hospitals 
PI Contribution Collaboration to develop targeted proteomic analysis of complement regulatory proteins. We provided specific protein sequences, and synthesised peptides for mass spectrometry calibration and validation. We also provided human eye tissue for the measurement of proteins in biological samples.
Collaborator Contribution Richard Uniwn developed a specific mass spectrometry methodology, based on our protein data, that is now capably of quantifiably identifying all seven complement regulatory proteins in human tissue (i.e. factor H, factor H-like protein 1, and the five factor H-related proteins). This is, to our knowledge, the first time any methodology can quantifiably identify all seven proteins in a biological sample. We are now trying to develop this for analysis of blood samples.
Impact A novel methodology for distinguishing between all seven factor H, factor H-like or factor H-related proteins in a biological sample.
Start Year 2013
 
Description age-related metal ion accumulation in human eyes 
Organisation University of Manchester
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Provided phenotyped and genotyped eye material and expertise in eye research.
Collaborator Contribution Performed metal ion analysis by ICMS mass spectrometry and performed RNAseq experiments
Impact one published paper: Langford-Smith, A., Tilakaratna, V., Lythgoe, P.R., Clark, S.J., Bishop, P.N. and Day, A.J. Age and smoking related changes in metal ion levels in human lens: Implications for cataract formation. PLoS One, accepted 6th Jan 2016. and one further paper in preparation, as well as a new grant application to fund further research in this area.
Start Year 2014
 
Title ELISA specific for FHR-4 protein in human blood samples 
Description Using our unique and specific anti-FHR-4 protein antibody, generated as part of a collaboration with Cardiff University and through work funded by the MRC (MR/K024418/1), we have developed an ELISA capable of selectively detecting FHR-4 in human blood samples. This is the first, truly specific, ELISA currently available. Although very helpful to researchers, our preliminary data suggests that systemic levels of FHR-4 may well change with disease, and therefore this ELISA may become useful in a diagnostic setting. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2015
Development Status Under active development/distribution
Impact This product is now widely used by the complement community and has begun to contribute to new disease mechanistic discoveries. Current work suggests that measuring FHR-4 levels in blood could be used as a selection criteria for new treatments of AMD coming through the clinical trial pipeline. 
 
Description 'Discovering Diabetes' A-Level study day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A total of 22 AS and A2 Level students from Cardinal Newman College in Preston and Salford City College attended the study day, which was fully subscribed within 2 hours of being advertised on the Museum's website.

Evaluations carried out at the end of the day (using the Turning point voting pads) showed that the over 90% of the students felt the day directly contributed to what they were learning in college and felt that they had a better understanding of diabetes research. In addition they were keen to study science at degree level and found the interactions with the scientists a useful and invaluable experience. Moreover the teaching staff who attended with the students were very positive and recommended that the day should be repeated again next year.
Year(s) Of Engagement Activity 2012,2013,2014
 
Description Invited speaker at Eyecare 2017, Glasgow, UK, 22nd Jan 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I gave a talk about new treatments being developed for Dry AMD to eye care health professionals and patients. Official feedback was collected and provided (please see URL provided below). Briefly, my talk was the third highest attended talk of the two day conference (80); where 67% of the audience ranked it as "Excellent", and a further 30% as "Good". I also took part in the most attended session of the conference - the 'Ask the Experts' Q & A panel.
Year(s) Of Engagement Activity 2017
URL https://gallery.mailchimp.com/75705ffde3df8421f476475ac/files/94884fc5-64b5-49cb-9a55-ad0630a6b7ca/E...
 
Description Invited speaker, Macular Society 'Top Doctor' event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact There was much discussion and interaction both during and after the talks. Had lunch with audience and interacted for a good 2 hours afterwards.

N/A
Year(s) Of Engagement Activity 2015
 
Description Invited talk to the Women's Institute Science Club, Grazely, Berkshire, 22nd Sept. 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I was invited to talk about AMD, its affects on people and the economy and new treatments being developed that will make a difference in the future. We also raised money for the charity Fight for Sight.
Year(s) Of Engagement Activity 2016