Prohibiting Cell Death in Human Keratocytes: New Insights for Non-surgical Keratoconus Treatment

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Project Summary/Abstract Keratoconus (KC) is a progressing cornea disorder that causes one in every 500 to 2000 Americans to see distorted images with a cone-shaped cornea. 10-20% of KC patients will ultimately require cornea transplant surgeries. Keratocyte apoptosis and collagen degeneration in the cornea lead to stromal thinning, which further induces KC. Up to today, there is no therapy targeted to keratocyte apoptosis. This proposal aims to search for non-surgical treatments for KC cases by inhibiting extrinsic apoptotic in keratocytes under extracellular stress. The human tumor necrosis factor – alpha (TNF-α), a cytokine released in the inflammation response, will bind its cell surface receptors with the assistance of heparan sulfate (HS) proteoglycan (PG). Upon binding with TNF-α, the TNF receptors will start protein recruitment intracellularly, which leads to the self-cleavage of procaspase-8 dimer and the activation of caspase-8 (CASP8). CASP8 will cleave the executive caspases downstream and initiate the cascade of extrinsic apoptosis. This proposal aims to find small molecule compounds that inhibit activated CASP8 (AIM 1) and HS compounds that inhibit interactions between TNF-α and TNF receptors (AIM 3). To accomplish CASP8 inhibition, I propose unbiased proteomics approaches facilitated by sample multiplexing mass spectrometry and antibody-based assays for markers of apoptosis. To find HS compounds that can interrupt TNF-α and TNF receptors interaction, I will utilize glycan microarray technology to screen over 90 HS compounds with different sulfation patterns and chain lengths. I will use 2D-solution nuclear magnetic resonance (NMR) spectroscopy to obtain kinetics of associated protein-ligand interactions (AIM 1) and validate keratoconic effects of the apoptotic inhibitions in 3D-KC cell models (AIM 2&3). This proposal will benefit from my analytical instrumentation skills, including mass spectrometry and solution NMR, and my knowledge about characterizing biomacromolecules, including proteins (proteomics) and glycosaminoglycans (glycomics). I will obtain further training in KC biology and pathology and advanced drug screening pipeline development. During the mentored K99 phase, I will 1) develop an advanced pipeline of drug discovery targeting keratoconic stromal cell apoptosis, 2) strengthen my knowledge in KC biology, 3) build connections between proteomics and glycomics around KC research, and 4) enhance my professional skills needed to be a successful independent investigator. The project will be conducted under the advisory of my mentor Dr. Steven Gygi and my advisory committee: Dr. Dimitrios Karamichos, Dr. Yutao Liu, Dr. Jian Liu, and Lianchun Wang. Harvard Medical School Cell Biology Department will also provide excellent opportunities for my training. The proposed research strategy, combined with the career development training, will guide me to become an independent investigator in the field of Keratoconus biology.