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Visualising age- and cataract-related changed within cell membranes of human eye lens using molecular rotors

Periodic Reporting for period 1 - Cata-rotors (Visualising age- and cataract-related changed within cell membranes of human eye lens using molecular rotors)

Reporting period: 2019-11-01 to 2021-10-31

The eye lens is considered as one of ideal models of human ageing due to no turnover of its major components, proteins and lipids. Frequently normal age-related changes are followed by the development of opaque and non-transparent regions within the lens, i.e. cataract, which is one of the main reason for the vision loss worldwide. Currently, the surgery is the only way of cataract treatment with significant costs and a heavy burden to National Health Services. Therefore, there is a demand for the understanding of cataract progression at the molecular level, which is currently still unknown.
The main goal of this project was to shed the light on age-related changes within plasma membranes of lens cells, which play a crucial role in the transport of nutrients within this avascular tissue. I suggested to use a novel method, Fluorescent Lifetime Imaging Microscopy (FLIM) in combination with fluorescent probes sensitive to polarity and viscosity. The current project was mainly focused on the study of age-related and photo-induced changes in viscosity within plasma membranes of human and porcine eye lenses.
Systematic studies of photo-induced damage to plasma membranes have revealed different changes in viscosity depending on the reaction mechanisms (electron transfer or singlet oxygen). The work with non-fixed human lens has shown that morphology of cells is significantly affected by the tissue fixation and demonstrated the presence of cells with three different structures and dynamic properties of plasma membranes within a non-fixed tissue. The results illustrate that FLIM method is highly promising to study ocular tissues, providing new data about fundamental processes about normal ageing and disease progression.
1. The search of most optimal fluorescent probes
The implementation of this project requires the use of fluorescent probes for viscosity and polarity for systematic work with animal (porcine) and human eye lenses. A set of available molecular rotors including newly sensitized fluorescent dyes (in total 18 molecules) was tested for their localisation within plasma membranes, the signal intensity, and the functioning as viscosity reporters. Based on this screening two molecules were chosen as the major molecular rotors for this project. Viscosity calibration curves were revised with the purpose to extend the existing calibration to high viscosity region, 2000 – 12 000 cP, a significant improvement on previous studies. One of the newly reported polarity probes (pyrene-based probe, PK) has revealed a bright signal and good localisation within plasma membranes for both young porcine and aged human lenses.

2. Photodamage to plasma membranes of eye lens
The Solar UV-A radiation (315-400 nm) on the Earth’s surface is considered as the most important external source of modifications of proteins and lipids within the eye lens. Proteins are currently considered to be the major targets for photodamage, and little is known about photodamage to plasma membranes.
In this work the impact of different photoreactions via electron / proton transfer (Type I) or singlet oxygen (Type II) on the plasma membranes of eye lens was studied with porcine eye lenses. We demonstrated that these two types of photodamage result in clearly distinct changes in viscosity – a decrease in the case of Type I damage and an increase in the case of Type II processes. At the final step of this study, we simulated age-related changes that occur in vivo via an exposure of an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts. These results were published in a full paper in J. Photochem. Photobiol. B: Biology.

3. Age-related changes in viscosity of plasma membranes of human eye lenses
Experiments with non-fixed human lenses revealed significant differences in the morphology of cells as compared with fixed tissues in previously published histology works. Three types of cell morphologies have been revealed by our studies, with clear distinction between different types of cells. Polarity and viscosity profiles were recorded across slices giving unexpected results in the observed viscosities. The correct interpretation of these results requires the accumulation of further statistics as well as the measurement of viscosities within cytosol. Unfortunately, the schedule of this work was affected by the COVID-19 pandemic and restrictions, primarily affecting the delivery of human samples.

Exploitation and dissemination
The microscopy work with lens tissue has not been attempted previously, yielding many new protocols in procedures for sample preparation, acquisition and analysis of data. The main outcomes of this project are (i) data used by other lab members working with biological tissues, (ii) new directions of further research involving ocular tissues, (iii) publications resulting from this work.
Due to COVID-19 induced restrictions to mobility, the results were mainly disseminated via group seminars. The Fellow participated in one on-line meeting organised by RSC Photophysics and Photochemistry Group with the delivery of results as a pre-recorded video. Another important channel of dissemination was organising new collaborations and research meetings with groups of Prof M. Wormstone (University of East Anglia, the UK) and Prof Y. Rotenstreich (Sheba Medical Centre, Israel), which expressed large interest in the application of FLIM method in their eye-related studies.

Personal development
At Imperial College I received training on microscopy, fluorescence imaging and work with live cells, which were used to highlight differences in structures between live cells and fibrous lens cells. From my side, I transferred my knowledge in the work with ocular tissues and their preparations for microscopy and different ways of photodamage to the whole or sliced tissues.
The project implementation brought following progress beyond the state-of-the-art:
1. Discovery of new fluorescent probes visualising the viscosity distributions within densely packed ocular tissues and at the interface between plasma membranes and cytosol.
2. Visualisation of viscosity within membranes of epithelial cells on the anterior surface of lens.
3. Direct measurements of viscosity within plasma membranes have shown a marked decrease in viscosity after UV-A impact that is opposite to an age-related increase of membrane stiffness assumed from previous indirect measurements.
4. Observation of cells with different morphologies within an aged eye lens and transition zones between different types of cells may be fingerprints of a diffusion barrier retarding the metabolite transport between central and peripheral areas of lens and previously suggested as a key factor for the cataract onset.

One of main project impacts is the demonstration that FLIM – fluorescent probe approach could be successfully applied to study non-fixed ex vivo tissues and to provide new knowledge about age- and disease-related changes in dynamic properties of various cellular components within a tissue.
Visualisation of viscosity changes within plasma membranes of eye lens in different photoreactions.