John D. Ash, Ph.D - US grants

The overall objective of this research is to develop an in vivo understanding of the molecular events controlling the differentiation of proliferating lens epithelial cells as they become post-mitotic lens fiber cells. In particular the research described in this proposal is aimed at identifying the role of cell cycle regulators in the differentiation process. In situ hybridization and immunohistochemistry will be employed to observe the expression patterns of endogenous cell cycle regulators as lens cells differentiate. Transgenic mice will be generated with expression vectors which will allow the lens specific over expression of cell cycle regulatory proteins.

DESCRIPTION (provided by applicant): Proliferative retinopathies are a classification of diseases that include sickle cell retinopathy, diabetic retinopathy, branch vein occlusion retinopathy, and retinopathy of prematurity. An interesting paradox in these retinopathies is that the hypoxia in the retina triggers growth of blood vessels in the vitreous but not in the retina itself. If we could identify the mechanism of this inhibition we may be able to temporarily suppress the inhibition and stimulate vessel growth within the retina. In order to develop strategies to prevent proliferative retinopathies we must understand the changes in gene expression that are responsible for disease progression. We propose to utilize proteomics and DNA-microarrays to identify differential expressed genes between normal retinas and retinas from two mouse models of proliferative retinopathy. The proposed experiments will determine the gene expression profile of the retinas during the course of these disease models. We anticipate that such knowledge will significantly increase our understanding of human retinopathy of prematurity. Some of the differentially expressed proteins are likely to be essential regulators for normal retinal vascular development, while other proteins may be inhibitors of vascular development. By identifying new candidate proteins, this project would feed into our long-range goals of identifying proteins that are essential regulators of retinal vascular development.

health science research; vision; retina; angiogenesis; laboratory mouse;

6. Project Summary/Abstract Inherited retinal degenerations are characterized by the loss of retinal neurons (most often photoreceptors). More than 150 genes in the Retnet database have been shown to cause some form of retinal degeneration when the gene is mutated, indicating a strong genetic component to these diseases. Often, blindness isn't congenital but is delayed until the 5th, 6th, or 7th decade of life. The genetic mutation is present at conception and yet photoreceptors survive and function for decades with the deleterious mutation. This phenomenon suggests that the retina has an endogenous system of self protection. By learning how this system of endogenous protection functions, we may be able to exploit it to further delay or even prevent blindness altogether. This project was designed to further our knowledge of the endogenous mechanisms by which retinal photoreceptors are protected from cell death. The four aims of this proposal will: Determine whether PIM kinases are required for induced protection of photoreceptors (aim 1). Determine whether induced protection of retinal photoreceptors is brought about through increased mitochondrial biogenesis or mitochondrial repair (aim 2). Determine whether inhibition of mitochondrial biogenesis or repair through genetic mutation of PGC-1 activity prevents preconditioning induced protection (aim 3). Test new cytokines that we developed for enhanced neuroprotective activity (aim 4).

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Molecular Biology Module has supported investigators of the Vision COBRE in their research programs primarily through DNA purification from tissues and PCR-based genotyping. The module also supports investigators through maintenance of equipment used for gene expression analysis by real-time PCR, and Western blots. Dr. Ash is the director of this module and he oversees its day to day operation. Dr Ash is qualified to supervise this module based on his extensive experience in real-time PCR, Western blot analysis, DNA purification, and PCR genotyping. Dr Ash has worked with genetically modified mice since 1994. During this 14 year period, Dr Ash has maintained approximately 30 lines of genetically modified mice. His lab currently genotypes 5 to 10,000 samples per year. Dr. Carr is the assistant director. Dr Carr has extensive experience in animal genotyping, as well as analysis by Real-Time PCR and ELISA. Operations of the Molecular Biology Module are conduced by a full time research assistant Fatemeh Shariati. Projects are initiated by a service request. Following completion of service Mrs. Shariati completes a report describing the completion of the job and all results. The report will contain a gel picture documenting accurate PCR reactions, and an assessment of positive and negative results. All reports are approved by the module director before they are returned to the users. If the results are not satisfactory, Mrs. Shariati will repeat the analysis. The module is operating at a rate of approximately 8,000 reactions per year.

Project Summary: One potentially important approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. To explore the potential of ultimately stimulating the resident Müller glia in the damaged human retina, we will take a comparative approach using zebrafish (regeneration competent), chick (regeneration limited), and mouse (regeneration refractory). We will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin conformation in isolated retinal progenitor cells and Müller glia in developing zebrafish, chick, and mouse retinas. We will also study changes Müller glia from all three model organisms as they are activated/reprogrammed in response to retinal injury (light damage, NMDA) or exposure to extrinsic factors that are capable of inducing their activation in the absence of retinal damage. Aims 1 and 2 will generate transcriptome and chromatin data of genes and chromatin structures that are associated with formation of Müller glia progenitor cells. In Aim 3, we will integrate this data using newly developed bioinformatic analysis to identify transcription factors and transcriptional networks that control neurogenic competence in Müller glia from each organism. In Aim 4, we will validate and test candidate genes in regulating the dedifferentiation of Müller glia in zebrafish, chick, and mice, using a combination of gain- and loss-of-function approaches. This work will begin to identify the transcription factors and miRNAs that regulate the extent of retinal regeneration in the three different model organisms. Understanding how to restore Müller glia to a youthful status will enable targeted regenerative retinal therapies.

Abstract: The retina has high metabolic activity, and retinal degenerations have been associated with mitochondrial dysfunction, dysregulation of metabolism, and toxic oxidative damage. However, little is known about how metabolism is maintained under normal conditions or is dysregulated in degenerating retinas. AMPK (AMP- activated protein kinase) is a key regulator of metabolism in highly metabolic tissues and is a candidate to regulate metabolism in photoreceptors, and its role in retinal metabolism will be rigorously studied in this proposed project using both gain-of-function and loss-of-function approaches. Peroxisome proliferator-activated receptor gamma coactivator-alpha (PGC-1?) and beta (PGC-1?) are key regulators of mitochondrial biogenesis. Adenosine monophosphate dependent kinase (AMPK) is an important regulator of PGC-1 activity. Our goal in this study is to determine the roles of AMPK and PGC-1 activity in retinal photoreceptors.

Abstract: The retina hashigh metabolic activity,and retinal degenerations have been associated with mitochondrial dysfunction, dysregulation of metabolism, and toxic oxidative damage. However, little is known about how metabolism is maintained under normal conditions or is dysregulated in degenerating retinas. AMPK (AMP-activated protein kinase)is a key regulator of metabolism in highly metabolic tissues and is a candidate to regulate metabolism in photoreceptors, and its role in retinal metabolism will be rigorously studied in this proposed project using both gain-of- function and loss-of-function approaches. Peroxisome proliferator-activated receptor gamma coactivator-alpha (PGC-1?) and beta (PGC-1?) are key regulators of mitochondrialbiogenesis. Adenosine monophosphate dependent kinase (AMPK) is an important regulator of PGC-1 activity. Our goal in this study isto determine the rolesof AMPK and PGC-1activity in retinal photoreceptors.

Summary: This application seeks funds to support the travel of young investigators to attend the Biennial International Symposium on Retinal Degeneration (RD meeting) in the years 2020, 2022, and 2024. The meetings are scheduled as satellite meetings of the Biennial Meeting of the International Society for Eye Research (ISER). The location and timing of the RD meetings are coordinated with the location and timing of the ISER meeting. The 2020 RD meeting has been named RD20/20 and will be held in Mendoza, Argentina on October 20th-25th, and will be the 19th successive RD meeting. The locations of meetings in 2022 and 2024 will be in Asia and the US respectively. The Specific Aims of the RD meetings covered by this application are: 1. To enhance the emerging careers of 20 Young Investigators (with equal representation by women and the inclusion of minorities) in retinal degeneration research by providing full travel support. 2. To provide a platform to advance career development of Young Investigators. 3. To provide a forum for dissemination of the most recent advances in the state of knowledge on the pathophysiologic mechanisms of acquired, inherited, and age-related retinal degenerations, and new therapeutic approaches to these diseases. 4. To create an environment that will facilitate the exchange of novel ideas among basic and clinician scientists and generate the opportunity for vision scientists of all ethnic groups and social backgrounds to meet and establish research collaborations. The requested funds will only support the travel, accommodation, and registration fees for students, postdocs, fellows, and junior investigators who otherwise would not have the resources to attend these focused meetings. The RD meetings have a 35-year history of scientific participation by leading retinal degeneration specialists (basic scientists, clinician scientists, and more recently industry scientists), with a focus on macular degeneration and inherited retinal degenerations that affect photoreceptors and the retinal pigment epithelium. All presentations will include fresh, unpublished date covering the latest advances in the field. Topics typically include the latest advances in genetics, mechanisms of disease, drug development, gene therapy and gene editing updates, stem cell therapies, tools to monitor disease progression, and reports on clinical trials. These meetings provide and ideal environment for trainees and young faculty to establish or accelerate their careers in the field by providing exposure to leaders in the field, providing a forum for new avenues for discovery, opportunities to develop collaborations, and will serve to encourage investigators to translate new findings into novel therapies.

Abstract: Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of photoreceptors. The death of these cells results in a permanent loss of vision that can have debilitating impacts on an individual's quality of life. Despite the diversity among triggers for retinal degenerations, the mechanisms surrounding photoreceptor death are often similar, suggesting the possibility of developing gene/mutation-independent approaches to reduce blindness from multiple forms of retinal degeneration. We and others have shown that STAT3 is activated in all retinal cells, including photoreceptors and Müller cells during inherited retinal degeneration. Additional work has shown that activation of STAT3 plays an essential role in promoting a wide array of gene expression changes to increase the cell?s capacity to resist cell death. However, despite these impressive findings, little progress has been made in identifying the mechanisms by which STAT3 regulates protection. In this project, we will use state of the art techniques including single-cell RNA-seq and integrating the data with cell-specific ChIP-seq to comprehensively identify all genes and transcriptional networks regulated by STAT3 in retinal Müller cells and rods.