Feature

Cataracts In Focus

Our aging population is at risk from blindness from cataracts, but research is providing a clearer view of what causes eyes to cloud over.

By Paul Donaldson and Joerg Kistler

A major health problem worldwide is that of cataracts, where the lenses in the eyes become progressively opaque, ultimately leading to blindness.

The disease may be inherited or caused by environmental factors such as malnutrition, high exposure to ultraviolet or other forms of radiation, drug therapies or lens injury. By far the highest risk factor for lens opacification, however, is simply old age.

Almost a third of people over 70 years of age need cataract surgery. With populations aging worldwide, the size of the problem is enormous. In the US, for example, cataract extraction and insertion of a plastic lens is already the most common in-patient procedure. In India, which has a particularly high cataract incidence, 1.5 million cataract extractions are performed annually.

It has been estimated that more than five million extractions would have to be done to deal with the four million new cataract cases annually and to reduce the existing backlog. Waiting lists are growing at alarming rates as even the wealthy nations struggle with exploding health budgets.

Lens Physiology

Consequently, high priority has been given to research aimed at gaining a better understanding of how cellular and molecular mechanisms maintain lens transparency. A knowledge of lens physiology and of what goes wrong can form a basis for new therapies aimed at preventing or delaying lens opacification.

The lens is an avascular tissue, which means it has no blood vessels. Its nutrients are transported from the aqueous humour within the eyeball across the rear epithelial cell layer and into the lens fibre cells. These fibre cells make up the bulk of the lens, and they are highly ordered to minimise light scattering within the lens.

In cataractogenesis, this tissue order is disrupted by swelling of the fibre cells, rupture of the plasma membranes and aggregation of proteins. These all result in increased light scattering and opacification. This mechanism is thought to be common to both major cataract forms, old-age cataract and diabetic cataract. For the latter, drugs have been developed which successfully delay lens opacification in animal diabetic models. However, clinical trials in humans have proven less successful and the results obtained so far are at best controversial.

Channel Network

Our research group has been investigating the extensive network of cell-to-cell channels which allow the rapid diffusion of nutrient molecules, ions and metabolites across plasma membranes and between the cells of the lens. These channels play an important role for lens homeostasis and transparency.

The structure of the lens and these channels suggests that opacification would progress uniformly. However, this is not the case. Both diabetic and old-age cataracts start as discrete patches consisting of deteriorating cell groups in otherwise seemingly-normal tissue. The most obvious interpretation is that cell-to-cell channels close up in some regions in response to a cellular insult, effectively disconnecting groups of fibre cells from adjacent areas. As a consequence, cells in these uncoupled regions lose homeostatic control and deteriorate, creating an opaque area.

We are examining these channels, looking at their molecular composition and structure, their distribution in the lens, and their regulation. In the longer term this knowledge should provide us with a basis for the development of new anti-cataract drugs, capable of modulating cell-to-cell communication and thereby delaying the formation of lens opacities.

Our group was the first to identify a cell-to-cell channel-specific protein and to produce antibody markers against this polypeptide. These antibodies are useful tools for examining the lens channel network at the microscopic level. They have also been useful for the isolation and biochemical analysis of cell-to-cell channel complexes.

While much of this work was carried out in Auckland, we have made our reagents available to laboratories overseas, hence increasing the momentum of our research by collaboration.

We now know that lens cell-to-cell channels consist of two very similar proteins, connexin46 and connexin50. Not only have these polypeptides been purified, they have also been cloned and their DNAs sequenced. The proteins form the cell-to-cell channel structures which cross the cell membranes and connect the cytoplasms of adjacent cells.

Cell Regulation

Since it is our aim to develop new drugs with the potential to regulate these channels in the cataractogenic lens, we need to establish techniques capable of measuring the current flow through single channels. Biological channels can open and close spontaneously, often with opening times in the millisecond range. Cells control the amount of current flow by modulating the amount of time a channel is open.

To observe single channel events, sophisticated electronics and computing equipment are necessary, as single channel currents are in the pico-ampere (10^-12amps) range. This makes such currents barely distinguishable from background noise levels. Fortunately, state-of-the-art equipment is commercially available.

We use two systems in our laboratory. Firstly, there is a planar lipid bilayer system which we use to study the function of biochemically purified lens channel complexes.

We use sheep lenses, and purify the fibre cell plasma membranes, which are biochemically almost indistinguishable from human lens membranes. These membranes are dissociated using detergents to allow the channel complexes to be purified in soluble form and their purity assessed by electron microscopy.

Purified channel complexes are then introduced into a planar lipid bilayer apparatus, which consists of two small chambers connected via a 200-micrometre hole. An artificial membrane covers this pinhole and acts as an electrical seal between the two chambers.

When stirred, channel complexes hit this membrane and some will be incorporated, allowing minuscule currents to flow from one chamber to the other. These currents can be measured, and the activity related to lens cell-to-cell channels isolated.

The second system allows the measurement of currents through cell-to-cell channels in their natural environment. This is important, as other cell functions may regulate channel opening and closing, and these cannot be investigated using the cell-free planar lipid bilayer system.

Cell pairs are kept alive on a specially equipped light microscope stage. Glass pipettes pulled to a tip diameter of only a few micrometres are lowered onto the surface of these cells using micromanipulators. A gentle suction is then applied, resulting in the glass and the cell membrane forming a high resistance. After seal formation, the underlying membrane patch is ruptured by a strong current pulse.

This approach allows us to gain access to the interior of each cell of the pair. Using appropriate amplifiers, we are able to measure currents flowing through cell-to-cell channels. Generally, the total current flowing through the cell-to-cell channels is measured, but occasionally or with the help of channel blockers, single channel events can also be measured.

We can use this system to investigate reagents which can regulate intercellular communication between lens cells, and are in the process of doing this.

Taken together, structure and function studies on lens cell-to-cell channels in our laboratory and by our colleagues overseas will doubtless produce new insights into the mechanisms responsible for cataract formation. This knowledge will serve as a basis from which we can develop reagents with the potential to halt this.

We are fortunate that our colleagues in the Department of Optometry at the Auckland School of Medicine have already established an animal cataract model. Diabetic rabbits develop visible cataracts over a period of ten weeks, allowing potential drugs to be tested for their ability to delay the onset of lens opacification.

Cataract research is a multidisciplinary effort. It serves as an example where techniques as diverse as molecular biology, physiology and optometry have joined together to help solve one of the most common health problems in modern society.

Paul Donaldson and Joerg Kistler are in the Department of Cellular and Molecular Biology at Auckland University. Their work has been supported by the Health Research Council, the Lottery Grants Board, the Auckland Medical Research Foundation and the NZ Optometric Vision Research Foundation.
Paul Donaldson and Joerg Kistler are in the Department of Cellular and Molecular Biology at Auckland University. Their work has been supported by the Health Research Council, the Lottery Grants Board, the Auckland Medical Research Foundation and the NZ Optometric Vision Research Foundation.