Final Report Summary - TAM IN AMD (Role of TAM signaling in Retinal Homeostasis)
Photoreceptors are the light-sensitive neurons that sense the light signal and are the first step in a chain of events which eventually enables vision. Without healthy, functional photorecptors (PRs) our vision is lost. PR degeneration underlies the major cause of blindness in the elderly – Age-related Macular Degeneration (AMD). According to the European Federation of Pharmaceutical Industries and Association (EFPIA), 20 million Europeans are affected by AMD. This project studies the maintenance of PR function during health, and what goes wrong in causing their degeneration - as in AMD. Specifically, the aim of the project was to understand the role of Protein S as an activator of TAM receptors in the retina.
Signaling through TAM receptors in essential for maintaining a healthy, functional retina. Dysregulated TAM signalling manifests as autoimmune disease, sterility and neuronal degeneration. Specifically in the eye, blindness develops due to degeneration of photoreceptors. Signaling through the Mer receptor is crucial to retinal health, as mutations in the single Mer gene result in unbalanced retinal homeostasis in which the fundamental process of photoreceptor phagocytosis by the underlying RPE cell layer is perturbed, leading to photoreceptor degeneration. Thus, Mer function is key to photoreceptor viability, and mutations in Mer cause blindness in mice and rat animal models, and in Man. Despite its critical role in maintaining retinal health and vision, the in-vivo bioactivity and bio-relevance of the TAM ligands Protein S (ProS) and Gas6 is not yet known, nor is their impact on photoreceptor viability. Moreover, mechanistic aspects of TAM signaling in-vivo are also mostly unclear.
Our first goal in studying the role of ProS and Gas6 was to characterize their presence and expression pattern in the retina. For this, we isolated various parts of the mouse retina and quantified ProS, Gas6 and TAM proteins. We found that among the ligands Gas6 concentration is 20-fold that of ProS, suggesting it’s greater importance. This result was surprising, since deletion of Gas6 did not result in retinal degeneration.
Next, we aimed to understand whether ProS expression is important for retinal health. For this, we generated genetically engineered mice deleted from ProS expression in the retina, and tested the effect of ProS removal on PR viability. We found that deletion of ProS alone was not sufficient to caouse retinal degeneration in mice. This raised the possibility that ProS and Gas6 may be functionally redundant. To test possible functional redundancy we generated mice deficient for both Gas6 and ProS in the retina. These mice exhibited severe retinal degeneration, to the same extent caused by deletion of the TAM receptor Mer. Our results indicate, for the first time, that ProS is a functional ligand for TAM receptors in-vivo, and that ProS is crucial for PR viability and retinal health. Moreover, the fact that ProS is present at much lower levels than Gas6 (see previous aim) shows it has greater bioactivity as a TAM ligand in the retina.
After having established that ProS is important for retinal health, our next objective was to evaluate the levels of ProS in the eye as a biomarker for retinal degenerative disease. For this we tested for the presence of ProS in aqueous humor – the fluid present in the anterior chamber of the eye. This fluid is secreted by ciliary body cells, which we found secrete high amounts of ProS. We obtained aqueous humor samples from patients with retinal degenerative disease (AMD, Retinitis Pigmentosa) and compared to those obtained from healthy individuals (cataract surgery patients, without any signs of any inflammatory eye disease). Out pilot assay indicates that aqueous humor from 3/3 healthy controls contained high levels of ProS, as expected. In contrast, only 1/5 diseased samples had detectable levels of ProS, indicating that 4/5 patients had significantly lower levels of ProS in the aqueous humor. This suggests that low levels of ProS are correlative with retinal degenerative disease, and may be used as a diagnostic marker for such diseases. While this study has layed the basis for this hypothesis, analysis of more samples is needed before a final conclusion is made.
This study is of high relevance to the general community, and to health-related policy makers. Retinal degenerative diseases in general, and AMD in particular are prevailing diseases which impose a heavy personal, social and economical burden. Being the most common cause of vision loss in individuals aged 50 and older, understanding the causes will help developing new treatments and preventive measures.
Results of this project were published by the highly prestigious scientific journal Neuron, in the December 2012 issue. Further press releases generated stimulated many inquiries from all around the world. Finally, according to the BioMedLib service which updates the most relevant and up-to-date publications in the same scientific domain ranked the paper #8 (out of 20) in the January 2013 list – only 3 weeks after the paper was published. In the recent list dated June 2014 the paper was ranked #1, indicating it’s relevance and high impact.
Signaling through TAM receptors in essential for maintaining a healthy, functional retina. Dysregulated TAM signalling manifests as autoimmune disease, sterility and neuronal degeneration. Specifically in the eye, blindness develops due to degeneration of photoreceptors. Signaling through the Mer receptor is crucial to retinal health, as mutations in the single Mer gene result in unbalanced retinal homeostasis in which the fundamental process of photoreceptor phagocytosis by the underlying RPE cell layer is perturbed, leading to photoreceptor degeneration. Thus, Mer function is key to photoreceptor viability, and mutations in Mer cause blindness in mice and rat animal models, and in Man. Despite its critical role in maintaining retinal health and vision, the in-vivo bioactivity and bio-relevance of the TAM ligands Protein S (ProS) and Gas6 is not yet known, nor is their impact on photoreceptor viability. Moreover, mechanistic aspects of TAM signaling in-vivo are also mostly unclear.
Our first goal in studying the role of ProS and Gas6 was to characterize their presence and expression pattern in the retina. For this, we isolated various parts of the mouse retina and quantified ProS, Gas6 and TAM proteins. We found that among the ligands Gas6 concentration is 20-fold that of ProS, suggesting it’s greater importance. This result was surprising, since deletion of Gas6 did not result in retinal degeneration.
Next, we aimed to understand whether ProS expression is important for retinal health. For this, we generated genetically engineered mice deleted from ProS expression in the retina, and tested the effect of ProS removal on PR viability. We found that deletion of ProS alone was not sufficient to caouse retinal degeneration in mice. This raised the possibility that ProS and Gas6 may be functionally redundant. To test possible functional redundancy we generated mice deficient for both Gas6 and ProS in the retina. These mice exhibited severe retinal degeneration, to the same extent caused by deletion of the TAM receptor Mer. Our results indicate, for the first time, that ProS is a functional ligand for TAM receptors in-vivo, and that ProS is crucial for PR viability and retinal health. Moreover, the fact that ProS is present at much lower levels than Gas6 (see previous aim) shows it has greater bioactivity as a TAM ligand in the retina.
After having established that ProS is important for retinal health, our next objective was to evaluate the levels of ProS in the eye as a biomarker for retinal degenerative disease. For this we tested for the presence of ProS in aqueous humor – the fluid present in the anterior chamber of the eye. This fluid is secreted by ciliary body cells, which we found secrete high amounts of ProS. We obtained aqueous humor samples from patients with retinal degenerative disease (AMD, Retinitis Pigmentosa) and compared to those obtained from healthy individuals (cataract surgery patients, without any signs of any inflammatory eye disease). Out pilot assay indicates that aqueous humor from 3/3 healthy controls contained high levels of ProS, as expected. In contrast, only 1/5 diseased samples had detectable levels of ProS, indicating that 4/5 patients had significantly lower levels of ProS in the aqueous humor. This suggests that low levels of ProS are correlative with retinal degenerative disease, and may be used as a diagnostic marker for such diseases. While this study has layed the basis for this hypothesis, analysis of more samples is needed before a final conclusion is made.
This study is of high relevance to the general community, and to health-related policy makers. Retinal degenerative diseases in general, and AMD in particular are prevailing diseases which impose a heavy personal, social and economical burden. Being the most common cause of vision loss in individuals aged 50 and older, understanding the causes will help developing new treatments and preventive measures.
Results of this project were published by the highly prestigious scientific journal Neuron, in the December 2012 issue. Further press releases generated stimulated many inquiries from all around the world. Finally, according to the BioMedLib service which updates the most relevant and up-to-date publications in the same scientific domain ranked the paper #8 (out of 20) in the January 2013 list – only 3 weeks after the paper was published. In the recent list dated June 2014 the paper was ranked #1, indicating it’s relevance and high impact.