Cataract and Presbyopia Research
||BSc PhD, Unit Head, NHMRC Senior Research Fellow
||BSc PhD, Postdoctoral Fellow
||PhD, Postdoctoral Fellow
||Postgraduate Student (Uni Wollongong)|
||Postgraduate Student (Uni Wollongong)
||Postgraduate Student (Uni Wollongong)
Research is focused on two common ocular conditions associated with aging: presbyopia (the inability to focus up close after middle age) and cataract (the major cause of world blindness).
On the basis of our research results, we have developed theories that can explain the origin of both of these debilitating visual conditions. It is proposed that nuclear cataract arises because of the age-related onset of a diffusion barrier within the lens. This ʻbarrier hypothesisʼ is now supported by a significant body of data. The end result of the barrier is protein oxidation in the lens nucleus. This arises because of a lack of sufficient antioxidants from the outer region reaching the centre of the lens. In addition, due to an increased residence time inside the barrier, there is an increase in the concentration of reactive species, derived from compounds such as UV filters. Together these factors lead to increased protein modification and nuclear opacification.
Our data indicate that presbyopia is the result of a massive stiffening of the human lens nucleus. The centre of our lenses becomes harder by a factor of approximately 1000 fold over our lifetime. As a result, we can no longer change the shape of our lenses to focus on nearby objects after age 45-50. We are actively trying to understand the biochemistry responsible for this huge change in physical properties.
UV filter Binding to Lens Proteins with Age
Lyons, Korlimbinis, Truscott
In 2006 we developed a novel method for measuring the amount of UV filters bound to proteins in human tissues. This involved treating the modified proteins with high levels of glutathione at pH 9.5. UV filters are released under these mild basic conditions and are trapped as GSH adducts that can then be quantified by HPLC. Using this methodology we showed that all normal lenses above the age of 50 have significant levels of UV filters bound to their proteins. Indeed the levels of bound are roughly equivalent to the free UV filter levels. This has major consequences. In collaborative work (with M. Davies, Heart Research Institute and J. Jamie, Macquarie University) we have also shown that when such modified proteins are illuminated by the wavelengths of light that pass the cornea, the proteins become oxidized. This may have relevance to the etiology of nuclear cataract. Thus the lenses of old people may be much more sensitive to the effects of light exposure than those of youngsters. We are collaborating with researchers in the USA (Prof Frank Giblin) to determine the structures of novel UV filters that are found in the lenses of squirrels. Squirrels and humans have very similar UV filters in their lenses and they therefore may be a useful model for investigating the effect of oxidation and UV light on cataract formation.
Posttranslational modification (PTM) in age-related cataract
Korlimbinis, Truscott, Hains
The reason for the development of age-related nuclear cataract is still unclear. This blinding affliction is associated with major oxidation and colouration of the lens proteins. By working out the nature of these modifications we hope to identify the major PTMs that have brought about this change in the properties of the lens. In this way we may be able to understand what causes cataract. We employ mass spectrometry as one of a number of techniques to enable these alterations in protein structure to be elucidated. Using this technique we have mapped all lens crystallins from older normal and cataract human lenses for the site and amount of deamidation. This PTM is one of the most abundant changes in old proteins and may lead to significant unfolding of the lens proteins.
Ageing of human lens (Presbyopia)
Human lens crystallins are present for the duration of a person’s lifetime. In this period they become modified and as a consequence alter their properties. We have been monitoring some of these changes and relating them to alterations in the physical and optical properties of the lens. In this way we hope to understand one aspect of the biochemistry of ageing. Lenses were examined for
stiffness and then were dissected into various regions and each extracted sequentially with buffer, 4M and 7Murea. Each fraction, including the membrane fraction, was analysed by SDS PAGE, bands quantified by scanning, and then each protein band identified by ingel tryptic digestion followed by mass spectrometry of the peptides.
Investigations into the lens barrier and its role in nuclear cataract
At middle age the lens nucleus becomes functionally uncoupled from the metabolically-active lens cortex. The resulting lack of adequate antioxidant defence renders the nucleus susceptible to oxidation. The existence of the lens barrier has been confirmed by NMR imaging. The consequences of the onset of the lens barrier are profound. It is not only the impediment to entry into the nucleus that is a problem; restricted exit from the lens centre also has deleterious consequences. The barrier hypothesis is increasingly recognized as the basis for understanding nuclear cataract. Ongoing studies into human lenses from the Lions Eye Bank are aimed at more precisely identifying the onset and the molecular basis of the barrier. This work involved the detailed proteomic analysis of the structure, function and interactions of molecules that play key roles in cell-cell communication.
A collaboration with Professor Kevin Schey (Medical University of South Carolina) is underway using new iTRAQ methodology to quantify the binding of proteins to the membranes of lenses at the barrier region.
Analysis of lens membrane components as a function of age
Deeley (Uni Wollongong), Truscott, Nealon (Uni Wollongong), Mitchell (Uni Wollongong), Stephen Blanksby (Uni Wollongong)
Over the life span of an individual, the lipid composition of the cell membranes in the lens changes substantially. The consequences of this are unknown, but such changes may contribute to the development of presbyopia – the inability to focus on nearby objects after age 50. In continuing studies, we are characterizing cortical and nuclear changes in lens lipid composition and relating these to separate measures of lens stiffness obtained by Dynamic Mechanical Analysis. We have shown that the membrane lipids of animals are quite different from those of humans. Human lens membranes also contain novel lipids that we have identified. In the next phase of research we are investigating how membrane changes influence the binding of denatured proteins – an important step in barrier formation.
Hooi, Friedrich, Lyons, Truscott
Old proteins are present at many sites in the body. We are investigating the changes that occur to these using a combination of accelerator mass spectrometry (at ANSTO) with sophisticated mass spectrometry approaches and HPLC to monitor the extent of amino acid racemisation.