Author: asalamonczyk@ichf.edu.pl

Scientists have for the first time looked deep into the protein structure that may determine our vision – and discovered that it is much more dynamic than previously thought. RBP3 not only changes its shape depending on its load but may also play a role in protecting the retina from diseases ranging from diabetic retinopathy to retinitis pigmentosa.

Retinol-binding protein 3 (RBP3) is a glycoprotein of about 140 kDa, found in the intercellular space of the retina. It plays an important role in the transport of retinoids – molecules essential for properly functioning the vision process. Although the existence of RBP3 has been known for years, its structure and precise mechanisms of action have remained unknown until now. The lack of this information has been a significant gap in research on eye diseases, especially those leading to irreversible vision loss.

Retinitis pigmentosa (RP) is one such disease – insidious, progressive, and still incurable. It affects millions of people worldwide, leading to the gradual loss of photoreceptors and blindness. Previous studies suggested that abnormalities in RBP3 function may be one of the contributing factors to the development of the disease, but detailed knowledge of its structure and mechanism of action was lacking. The new findings shed light on this mystery, opening the way to potential therapies that could slow or even stop retinal degeneration.

An international team of scientists, including researchers from ICTER, used modern structural analysis methods to obtain for the first time an image of the native structure of RBP3 with such high accuracy. The results were published in the journal Open Biology in a paper titled “CryoEM structure and small-angle X-ray scattering analyses of porcine retinol-binding protein 3“.

• For the first time, we have managed to capture the full structure of native RBP3 in pigs with a resolution of 3.67 Å. This is an important step in understanding the function of this protein, especially in the context of its role in the transport of retinoids and fatty acids in the eye, says Dr Humberto Fernandes from ICTER.

Why is RBP3 so important?

Vision is a process that begins with the conversion of light into an electrical signal by photoreceptors in the retina. A key element of this transformation is the visual cycle – a complex chain of chemical reactions in which retinoids, or derivatives of vitamin A, play an essential role. However, for them to effectively perform their function, they must be transported between different cells of the retina. This transport is the responsibility of the retinol-binding protein 3 (RBP3), which acts as a “courier” delivering retinoids to where they are needed.

RBP3 is found in the interphotoreceptor extracellular matrix (IPM), the space between the retinal pigment epithelium and the photoreceptors. This is where the transport of molecules necessary for the proper functioning of the eye takes place, including oxygen, nutrients, and retinoids. RBP3 plays a key role in delivering all-trans-retinol (at-ROL) from photoreceptors to the retinal pigment epithelium, where it is converted into 11-cis-retinal (11c-RAL), a key molecule for vision. 11c-RAL then returns to the photoreceptors, where it binds to opsins to form light-sensitive pigments that enable vision. Without RBP3, this transport cycle would be much less efficient, which could lead to retinoid deficiencies in photoreceptors and, ultimately, retinal degeneration.

Structurally, RBP3 is a large glycoprotein consisting of four retinoid-binding modules. Each of these modules has a distinctive structure that allows it to interact with a variety of molecules, including retinoids and fatty acids such as docosahexaenoic acid (DHA). However, it has not been clear yet how exactly this protein interacts with its ligands and whether its structure changes depending on the type of molecule being transported.

However, it is known that RBP3 has additional protective functions. It protects retinoids from degradation under the influence of light by limiting their oxidation and disintegration. Its presence in the IPM stabilizes the biochemical environment of the retina, which is crucial for eye health. Moreover, mutations in the gene encoding RBP3 are associated with several eye diseases, including RP and some forms of myopia.

How was the study conducted?

To capture the three-dimensional structure of RBP3, the researchers used cryo-electron microscopy (cryoEM), a technique that allows for obtaining images of biomolecules in a nearly native state at cryogenic temperature. The study also used small-angle X-ray scattering (SAXS) analysis, which allowed for determining the conformational changes of the protein in solution.

The first step was to obtain the protein in its native form. To do this, the researchers isolated RBP3 from pig retinas obtained from local slaughterhouses. The tissues were stored in conditions that minimized protein degradation – on ice, in the dark. RBP3 was then purified using advanced chromatographic techniques, including affinity chromatography, ion exchange chromatography, and gel filtration. Each step of the procedure was aimed at obtaining a stable and functional form of the protein, which was crucial for further studies.

After obtaining pure samples of the protein, the researchers began experiments using cryo-electron microscopy. RBP3 samples were cooled to extremely low temperatures and placed in an electron beam, which allowed them to obtain hundreds of thousands of images of single molecules. Assembling these images into a three-dimensional model allowed the reconstruction of the protein structure at an unprecedented resolution of 3.67 Å.

In parallel, SAXS analysis was used, which provided additional information about the conformational plasticity of RBP3 in solution. These experiments allowed them to observe how the protein changes its shape when bound to different molecules, including retinoids and fatty acids. Thanks to this, the researchers discovered that RBP3 adopts different conformations depending on the type of cargo it transports, which may be crucial for its function as a dynamic retinoid carrier.

• This is one of the most detailed structural studies of RBP3 ever performed. By combining cryoEM and SAXS, we have gained unique insight into how this protein works, explains Dr Humberto Fernandes from ICTER.

What was found?

One of the key findings of this study is the ability of RBP3 to change shape depending on the type of molecule being bound. Experiments have shown that after binding 11-cis-retinal (11c-RAL) and all-trans-retinol (at-ROL), the protein assumes different conformations – from compact to open. SAXS analyses have shown that at higher concentrations of these retinoids, RBP3 is elongated, which may suggest the mechanism of its action as a dynamic retinoid transporter.

• This finding is particularly interesting because it suggests that RBP3 may act as a flexible retinoid carrier, changing shape to optimize the transport of these molecules between the retinal pigment epithelium and photoreceptors – says Dr Vineeta Kaushik from ICTER.

Additional information on the plasticity of the protein was provided by molecular docking analyses, which indicated the presence of two main ligand binding sites. Interestingly, SAXS analysis showed that binding fatty acids such as DHA (docosahexaenoic acid) did not lead to significant changes in the structure of RBP3, indicating that its role in transporting these molecules may differ from that of retinoids.

This groundbreaking discovery changes our understanding of the role of RBP3 in the eye. Rather than being a passive transporter of retinoids, we are beginning to see it as an active, adaptive mechanism that can precisely regulate the delivery of key molecules to photoreceptors. Understanding these dynamics opens new avenues for research into the visual cycle and potential therapies for retinal degenerative diseases.

What’s next?

The discovery of the full structure of RBP3 and its conformational plasticity opens a new chapter in research on the functioning of the visual cycle and the mechanisms leading to retinal degeneration. The results may have key implications for the diagnosis and treatment of eye diseases, including diabetic retinopathy, retinitis pigmentosa, and high myopia.

Previous studies have suggested that reduced levels of RBP3 in the retina are associated with the progression of diabetic retinopathy, and its stabilization could have a protective effect. Now, thanks to precise structural data, scientists can focus on developing new therapeutic strategies that would modulate RBP3 activity and could slow down the progression of this disease. It is also possible to use RBP3 as a diagnostic biomarker, which could help identify patients at risk of vision loss at an early stage of the disease. The team plans to continue research on the dynamics of RBP3 function in both physiological and pathological conditions.

• This is just the beginning. Now that we have a 3D model of RBP3, we can study how exactly it interacts with other retinal proteins and how we can use this information to develop new treatments, concludes Dr Humberto Fernandes.

Authors of the paper “CryoEM structure and small-angle X-ray scattering analyses of porcine retinol-binding protein 3“: Vineeta Kaushik, Luca Gessa, Nelam Kumar, Matyáš Pinkas, Mariusz Czarnocki-Cieciura, Krzysztof Palczewski, Jiří Nováček and Humberto Fernandes.

Author: scientific editor Marcin Powęska

Eye diseases often develop asymptomatically for many years. ICTER scientists have developed the f-ORG technique, which analyzes the retina’s reaction to light, helping to detect danger before the first symptoms appear. New research proves that even the smallest changes in photoreceptors can be detected this way.

The retina is an extremely complex structure that acts as a biological “transducer” of light into neural signals. It is here, in the layer of photoreceptors—cones and rods—that the process of vision begins. Light hitting the outer segments of these cells initiates a series of biochemical reactions known as phototransduction. During this, the length of the photoreceptors changes, and these microscopic changes—invisible to the naked eye—carry information about the health of the retina.

Previous diagnostic methods, such as electroretinography (ERG), allowed for the assessment of photoreceptor function, but they had many limitations. They required contact with the eye surface, long-term adaptation to darkness, and complicated procedures. They were also uncomfortable for patients, especially children and the elderly.

Scientists from the International Centre for Eye Research (ICTER) decided to find a way to overcome these limitations. They developed an innovative technique – flicker optoretinography (f-ORG), which allows for fast, non-invasive, and precise monitoring of the processes occurring in photoreceptors. The method could revolutionize the diagnosis of retinal diseases, such as macular degeneration, retinitis pigmentosa, and congenital retinal dystrophies. The results were published in the journal Proceedings of the National Academy of Sciences (PNAS) in a paper titled “Photopic flicker optoretinography captures the light-driven length modulation of photoreceptors during phototransduction“.

• Our method enables tracking of molecular mechanisms of phototransduction without the need for prolonged exposure to darkness and without contact with the surface of the eye. This is a significant step forward in the diagnosis of retinal diseases – explains Professor Maciej Wojtkowski, co-author of the study.

“Ultrasound” for photoreceptors

Flicker electroretinography (f-ERG) is a valuable and successfully used tool for studying the physiological functions of the retina. However, it is not an ideal method, so ICTER scientists decided to develop its optical equivalent. Flicker optoretinography (f-ORG) is a technology that allows for real-time observation of changes occurring in the outer segments of the eye’s photoreceptors. This process is the result of conformational changes in the phosphodiesterase 6 (PDE6) protein.

Photoreceptors – cones and rods – are extremely sensitive cells that respond to light by lengthening or shortening their outer segments. These changes are a signal of their activity and the health of the retina. The f-ORG technique records these phenomena thanks to spatial-temporal optical tomography OCT (STOC-T), which allows for imaging of the structures of the retina with precision in the nanometer range.

This is yet another research work of the ICTER team focusing on f-ORG. In 2022, Prof. Wojtkowski’s team showed that it is possible to perform f-ORG measurements in a wide frequency range (up to 50 Hz), and in 2024, scientists proposed a new approach to f-ORG measurements that allows for the rapid determination of the frequency characteristics of photoreceptors.

• STOC-T is a real breakthrough. Thanks to it, we can non-invasively track how individual photoreceptors react to light. It’s like having a microscope that works directly in the patient’s eye – emphasizes Andrea Curatolo PhD.

Why is PDE6 so important?

Phosphodiesterase 6 (PDE6) is a key enzyme in the process of phototransduction, or the conversion of light into electrical signals that the brain interprets as images. It is located in the outer segments of photoreceptors – cones and rods – and acts as a light signal regulator. Its task is to break down cGMP (cyclic guanosine monophosphate), which keeps ion channels open in the dark, allowing sodium and calcium ions to flow into the cell.

When light falls on the retina, the rhodopsin signaling pathway is activated in the photoreceptors, as a result of which PDE6 is stimulated. This enzyme rapidly breaks down cGMP, which causes the ion channels to close and the flow of ions to decrease. As a result, the electrical potential of the cell changes, which is the first step in transmitting visual information to the brain.

• PDE6 is a molecular switch that regulates the sensitivity of photoreceptors to light. Its activation is like pressing the brake pedal in a car – light is the stimulus that starts this process, and PDE6 decides how strong the response will be – explains Sławomir Tomczewski PhD Eng, the main author of the study.

Phototransduction is a process that lasts fractions of a second, but our ability to see depends on its proper course. Disturbances in the functioning of PDE6 are associated with many retinal diseases, including retinitis pigmentosa and retinal dystrophies. The new f-ORG technique allows direct observation of the effects of this enzyme’s action in real-time, which gives scientists and doctors a new tool for studying these diseases and assessing the effectiveness of gene and pharmacological therapies.

How were the studies conducted?

The f-ORG technique was studied on a group of healthy volunteers, and it aimed to confirm the effectiveness of the method in tracking dynamic changes in photoreceptors and to understand the role of the PDE6 protein in this process. The participants underwent a short, one-minute adaptation to light, which is a significant difference compared to traditional methods requiring a long stay in the dark. Then, their retinas were stimulated with light of a variable frequency – from 1.5 Hz to 45 Hz – and changes in the length of the photoreceptor outer segments (OS) were recorded.

The STOC-T technique, performing about 200 three-dimensional scans per second, allowed for the observation of subtle oscillatory extensions of these structures under the influence of light. The studies showed that the extension of photoreceptors is consistent with theoretical predictions regarding the activation of the phototransduction cascade. In the next stage of the experiments, the effect of sildenafil – a PDE6 inhibitor, known for its blocking effect on the phototransduction process – on the photoreceptor response was examined. After its administration, a significant weakening of the photoreceptor response was observed, which confirmed the key role of PDE6 in the mechanism of elongation of the outer segments of photosensitive cells.

• This was a breakthrough moment. After the administration of sildenafil, the photoreceptor response decreased significantly. The obtained results seem to confirm that it is indeed the conformational changes in the PDE6 protein that are responsible for the elongation of the outer segments under the influence of light – says Sławomir Tomczewski PhD Eng.

What can f-ORG be useful for?

Retinal diseases, such as macular degeneration (AMD), retinitis pigmentosa, or congenital dystrophies, often develop unnoticed for many years. Their early diagnosis is extremely difficult because the first clinical symptoms appear when a significant part of the photoreceptors is already irreversibly damaged. Previous diagnostic methods focused on visual observation of structural changes and measurements of electrical activity of the retina, omitting subtle structural changes at the molecular level. F-ORG fills this gap, allowing for the recording of changes in the length of the outer segments of photoreceptors, which is a direct indicator of the processes occurring in the retina during light reception.

• Thanks to f-ORG, we can observe the reactions of the retina in real-time. It is like monitoring the operation of an engine without having to dismantle it – explains Prof. Maciej Wojtkowski.

The potential applications of the f-ORG technique are enormous. Thanks to the possibility of recording the reactions of photoreceptors on a nanoscale, doctors can detect pathological changes much earlier than in the case of traditional methods. The new technique can be used not only in ophthalmology but also in neurology and research on neurodegeneration. The retina is a natural “window” to the nervous system and can provide valuable information on the functioning of the brain.

• The f-ORG technique allows us to understand the mechanisms of photoreceptor function, and in the future, it may help find the sources of neurodegenerative diseases at a level not seen before in ophthalmological research – emphasizes Sławomir Tomczewski PhD Eng.

The Future of f-ORG in clinical practice

ICTER scientists plan to further develop the f-ORG technology and adapt it for clinical applications. Preparations are currently underway for studies on patients with early symptoms of macular degeneration and retinitis pigmentosa. There are also plans to develop a portable version of the device that could be used in ophthalmologists’ offices and even during screening tests among populations at increased risk of retinal diseases.

• We want f-ORG to become a standard in ophthalmology. This is a technology that can help millions of patients around the world by enabling early detection of diseases and more effective treatment – says Professor Maciej Wojtkowski.

Authors of the paper “Photopic flicker optoretinography captures the light-driven length modulation of photoreceptors during phototransduction“: Sławomir Tomczewski, Andrea Curatolo, Andrzej Foik, Piotr Węgrzyn, Bartłomiej Bałamut, Maciej Wielgo, Wiktor Kulesza, Anna Galińska, Katarzyna Kordecka, Sahil Gulatie, Humberto Fernandes, Krzysztof Palczewski, Maciej Wojtkowski.

Author: scientific editor Marcin Powęska

ICTER scientists are opening a new window into the interior of the eye – the place where vision is born, but also where diseases begin. Thanks to the STOC-T technique, it is possible to look deeper than ever before, examining individual nerve cells without complex optical systems.

The human eye has fascinated poets, philosophers, and scientists for centuries. It is in it that the world is reflected, it is it that transmits colors, light, and movement to our minds. But deep inside this delicate structure lies a fragile system of nerve cells, which can be damaged imperceptibly – until it is too late. Glaucoma, one of the most dangerous neurodegenerative diseases, is slowly and inexorably depriving millions of people of sight around the world. Its insidiousness lies in the fact that for a long time, it remains asymptomatic, developing in silence, until suddenly it takes away what is most precious from a person.

Is it possible to look deep into the retina, see individual nerve cells, and capture the first signs of disease before irreversible changes occur? Thanks to a groundbreaking technique developed by ICTER scientists, this question is no longer just the domain of the future. Spatio-Temporal Optical Coherence Tomography (STOC-T) allows for the first time to see the retina in unprecedented resolution – capturing not only the light-sensitive cones and rods but also the ganglion cells themselves, which are crucial for transmitting images to the brain. The results were published in the journal Biocybernetics and Biomedical Engineering in a paper entitled “Imaging of retinal ganglion cells and photoreceptors using Spatio-Temporal Optical Coherence Tomography (STOC-T) without hardware-based adaptive optics“.

The intricate network of the retina

The retina is a complex, multi-layered structure whose precise organization allows it to receive, process, and transmit light stimuli to the brain. It is made up of several types of specialized cells, each of which performs a specific function in the mechanism of vision. The outer layer contains photoreceptors – cones responsible for color vision and image sharpness – and rods, which allow vision in low light.

Deeper lies a layer of bipolar cells, which collect information from photoreceptors and pass it on to retinal ganglion cells (GCC). They are crucial for transmitting visual signals to the brain, and processing them into an image. The axons of the ganglion cells connect to form the optic nerve – a kind of “cable” through which electrical impulses reach the visual cortex of the brain.

The delicate balance of this system can be disrupted by neurodegenerative diseases such as glaucoma. In its course, the retinal ganglion cells gradually die, and damage to the optic nerve leads to irreversible vision loss. This process can be asymptomatic for a long time, which is why early detection of changes in the structure of the retina is so important. Modern imaging methods, which allow us to see these subtle changes at the cellular level, are becoming a key tool in the fight against this insidious disease.

• Precise diagnostics of the retina at the level of single cells is of great importance for the early detection of pathological changes. One of the greatest challenges in this field is imaging nerve cells, which are almost transparent and have a very similar refractive index to the surrounding structures – says Marta Mikuła-Zdańkowska PhD Eng. from ICTER, the first author of the publication.

The previous technology allowed for high-resolution imaging of the retina, but required the use of adaptive optics (AO-OCT), i.e. systems that dynamically correct aberrations, which was associated with high costs and complicated calibration.

STOC-T reveals the “invisible”

Developed by ICTER scientists, spatial-temporal optical tomography OCT (STOC-T) allows for imaging of the retina at the cellular level without the need for adaptive optics. By using special image averaging and aberration correction techniques, it is possible to obtain images of nerve cell bodies in in vivo studies.

• In our method, we used a dynamic deformable mirror, which allows for active mixing of light modes and reduction of interference noise. Thanks to this, we can eliminate noise and obtain images of quality comparable to adaptive optics methods, but without the need to use complicated and expensive scanning systems – says Marta Mikuła-Zdańkowska PhD Eng.

Optimization of optical elements allowed to achieve a lateral resolution of approx. 3 micrometers, which allows for precise visualization of retinal ganglion cell bodies and photoreceptors, including the cone mosaic. This is particularly important in the context of diagnosing neurodegenerative diseases such as glaucoma, in which the first symptoms often appear at the level of changes in the retinal nerve cells. High imaging quality allows for more accurate monitoring of the process of neuronal loss, which may be crucial for implementing therapy in the early stages of the disease. STOC-T also enables imaging of deeper layers, including amacrine cell bodies, which play an important role in processing visual information and whose damage may be associated with various neurodegenerative diseases.

• Thanks to STOC-T, we can significantly increase patient comfort by shortening the examination time and eliminating the need for long, complicated measurement procedures. Our method may be a breakthrough in glaucoma diagnostics, but we also see its wide application in research on neurodegeneration and other retinal diseases – explains Marta Mikuła-Zdańkowska PhD Eng.

One of the greatest advantages of this technique is the significant reduction in examination time, which can be performed in less than one minute. Compared to OCT tomography methods using adaptive optics, which require at least 15 minutes of imaging and complex calibration procedures, STOC-T opens up new possibilities for clinical practice. Shorter examination time minimizes the impact of eye movements on image quality, increases patient comfort, and facilitates the implementation of this technology in standard ophthalmological offices, and not only in specialized research laboratories.

• We would like our method to become a standard in modern retinal diagnostics. Our research shows that high-quality retinal images can be obtained in real-time, which significantly increases the clinical potential of this technology – emphasizes Marta Mikuła-Zdańkowska PhD Eng.

Research conducted by ICTER scientists confirms the effectiveness of STOC-T in retinal imaging at the cellular level, which opens the way for further development of this technique and its integration with modern diagnostic tools. The ability to precisely track neurodegenerative changes in real-time means that this method can be widely used not only in ophthalmology but also in neurology and research on diseases such as Alzheimer’s disease or Parkinson’s disease, in which changes in the retina can be one of the early diagnostic criteria.

Authors of the paper “Imaging of retinal ganglion cells and photoreceptors using Spatio-Temporal Optical Coherence Tomography (STOC-T) without hardware-based adaptive optics“: Marta Mikuła-Zdańkowska, Dawid Borycki, Piotr Węgrzyn, Karolis Adomavicius, Egidijus Auksorius, Maciej Wojtkowski.

Author: scientific editor Marcin Powęska