Sanjoy K. Bhattacharya - US grants

Glaucoma refers collectively to a group of eye diseases whose molecular basis is poorly understood. Worldwide primary open angle glaucoma (POAG) is one of the leading causes of blindness and is currently incurable. Distinguishing symptoms of POAG are increased intraocular pressure (IOP) and glaucomatous optic neuropathy. Increased resistance to aqueous outflow through the filtering trabecular meshwork (TM) tissue appears to play a key role in the onset and progression of POAG. Blockage at the level of trabecular meshwork leads to increased IOP. Proteomic and Western analyses of normal and glaucomatous TM have revealed cochlin, a secreted protein with unknown function, present exclusively in glaucomatous but not in normal TM. Subsequently we have also observed cochlin containing deposits by immunohistochemistry in glaucomatous TM. In the human cochlea, cochlin is associated with mucopolysaccharide deposits in progressive auditory dysfunction. In the TM, deposition of cochlin and mucopolysaccharides may interfere with regulation of aqueous outflow and may cause slow but progressive elevation of IOP. We have extended these studies to mice and found that cochlin levels were elevated in the DBA/2J model of glaucoma but not in control animals. The studies proposed here will use mouse models to determine the role of cochlin in IOP elevation. The central hypothesis to be tested is that cochlin deposits in the extracellular matrix obstruct aqueous outflow in glaucomatous TM, elevate IOP and contribute to the pathogenesis of POAG. The long- term goals of this project are to establish the mechanistic involvement of cochlin in IOP elevation and the pathogenesis of POAG, and to develop effective therapies for preventing disease progression. Our hypothesis will be tested with following specific aims: (1) To determine whether cochlin over-expression results in elevated IOP;(2) To determine whether DBA/2J mice lacking cochlin maintain normal IOP;(3) To test whether cochlin message down-regulation results in normal IOP in DBA/2J mouse. Methods will include intraocular injections, IOP measurements, viral infections, immunohistochemisty as well as other molecular and cell biological techniques.

ABSTRACT Poor regeneration and reconnection of retinal ganglion cell (RGC) axons is a major obstacle for treating ocular trauma and diseases including glaucoma. There are as yet no therapies to repair optic nerve once the damage is done. Our new studies have discovered cohorts of RGCs that have a very high regenerative capacity. Furthermore, we now uncover previously unrecognized ability of distinct lipids to promote axon growth. In Aim 1, we will use High Content Screening and functionally test various candidate genes for their ability to promote neurite growth. In Aim 2, we will determine lipid profiles in RGCs, and functionally test neurite growth-promoting effects of select lipids. In Aim 3, we will use in vivo optic nerve injury model to determine RGC axon regeneration receiving various treatments. Identifying novel targets that further increase RGC axon regeneration to the brain represents a critical future study. Results obtained from these studies will provide invaluable information on developing future therapies to repair degenerated optic nerve.

PROJECT SUMMARY / ABSTRACT The overall goal of this proposal is to better understand impaired phospholipid metabolism in the trabecular meshwork (TM) tissue in glaucoma. The term phospholipid (PL) here refers to primarily three classes of lipids: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE). A better understanding of PL metabolism in the anterior eye chamber will provide insight into glaucoma pathology and facilitate the development of new, disease-modifying intervention strategies. Our long-term goal is to develop better intervention strategies for glaucoma by understanding the pathological changes in metabolism, and in particular PL metabolism, in the glaucomatous anterior chamber. The absence of a comprehensive knowledge of PL changes in diseased compared to healthy aqueous humor (AH) or TM represents a critical barrier to progress in treating and preventing glaucoma. The proposed research is based on our extensive preliminary studies performed over the past seven years. We discovered a substantial decrease in PS and a general increase in PE lipid species in glaucomatous TM compared to controls. This has followed rigorous analyses of levels and activity of enzymes in a comprehensive and unbiased manner, demonstrating their alteration at critical branch points. Thus, we propose to comprehensively investigate all PL interconversion enzymes in an unbiased manner. We also found vastly different fold changes differing only slightly with respect to acyl-chain length (or structural features) between control and glaucomatous TM or AH suggesting that the functions of transport proteins are potentially aberrant in glaucomatous TM. We propose to investigate selected PL transport proteins in glaucomatous TM tissue. Pathologic TM tissue consistently demonstrates altered biophysical properties, for example an elevated elastic modulus. Our central hypothesis is that phospholipid metabolism is impaired in glaucoma, affecting biophysical properties of the trabecular meshwork and contributing to its pathophysiology. In Aim 1 we will test whether PL interconversion enzymes are altered in glaucoma, In Aim 2 we will evaluate whether PL transport proteins are altered in glaucoma, and finally in Aim 3 we will determine whether restoring specific PLs that become deficient in the diseased state can improve the biophysical properties of glaucomatous TM. We will also evaluate correlation of biophysical properties with IOP lowering in murine models. We will use routine (Western blot, immunosorbent assays) and innovative methods analytical techniques such as kinetic histochemistry employing imaging mass spectrometry. The proposal also has brought forth a conceptual innovation that is PL metabolism impairment in TM in glaucoma. The expected outcome of the proposed research is the identification of aberrations in specific enzymes of PL metabolizing pathways and in PL molecules that dysregulate biophysical properties of TM. These new insights will have an important impact on developing novel glaucoma therapies.

MULTIOMICS MODULE Summary The Multiomics (MO) module is a new module that allows MVRC researchers to perform cutting- edge high throughput analyses, including mass spectrometry, metabolomics and lipidomics. Specific Aims are: 1) To provide extensive high throughput analyses including mass spectrometry, metabolomics, metabolic flux determinations, ligandomics and lipidomics, 2) To assist investigators with sample preparation specialized for small sample size and laboratory protocols tailored for parts of eye tissue and to provide training in operating the equipment, 3) To provide support and training in analytic and visualization tools for the large data sets generated by these analyses. This module replaces and refines the previous ?Shared Equipment? module and includes multiomics technologies only. The use of the specialized equipment is also complemented by integration of multi-omics analyses, machine-learning and computational biology assistance. The main rationale for the module is that it allows us to house a vast array of high-end equipment in one location with dedicated technical staff for using and maintaining the equipment, in order to maximize use of these new technologies in generating data for existing research projects and for obtaining pilot data for new grant applications. Additionally, housing common reagents and internal and external standards with a shared electronic dashboard enables effective utilization of resources, and promotes sharing reagents, protocols and cross-fertilization of ideas. This module supports innovative research and will enable analysis of global ocular metabolic changes, including metabolic flux determinations. Progressive and complex diseases are now recognized to be associated with incremental changes in the tangents of metabolic concentrations and disruptions in early metabolite-protein interactions can change the course of disease. Therefore, information derived from multiomics can be innovatively utilized to tailor intervention strategies. This module thus sits on the very forefront of such strategies. 1

Project Summary The present application requests funds to support travel for young scientists to attend the XV Biennial Meeting of the Association for Ocular Pharmacology and Therapeutics (AOPT) in Miami from March 4th-7th, 2021. This is a conference that brings together scientists who are developing new ways to treat eye diseases. AOPT is a global not-for-profit 501(c)3 organization for scientists and individuals from all disciplines related to ocular pharmacology and its therapeutic applications to eye disease. AOPT has a diverse, multi-national membership composed of preclinical and clinical scientists, students, and healthcare professionals. Members are from academic institutions, pharma and biotech industries, device companies, clinics and private practice. To achieve its mission, the society organizes biennial conferences and provides editorial leadership for the peer- reviewed Journal of Ocular Pharmacology and Therapeutics. The mission of AOPT is threefold (from AOPT website: www.aopt.org): (1) To serve as a global forum and network for the publication, dissemination and exchange of information and knowledge on treatments of eye diseases, from basic and clinical ocular pharmacology and therapeutics to related disciplines such as pharmacokinetics and dynamics, metabolism, translational research, safety, drug delivery, and pharmaceutics; (2)To promote the advancement of research and scientific progress in these fields; (3) To assist researchers and eye-care professionals in furthering their understanding of the treatment of ocular disorders. In line with the AOPT mission, the specific Aims of the R13 application is: (I) To enhance the emerging careers of 20 young scientists (including women and underrepresented minorities) in ocular therapeutics by providing travel awards; (II) To provide a forum for dissemination of the most recent advances in the state of knowledge on novel therapeutic approaches to ocular disorders and disease; (III) 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 theme of AOPT-2021 is ?Restoring vision through Regeneration? and a number of sessions are scheduled which are focused on regenerative medicine related to anterior as well as posterior eye segments.