Project Summary Uveitis is a difficult-to-treat inflammatory eye disorder. Treatment options for uveitis are limited by an incomplete understanding of molecular causes of the disease. This proposal builds on our exciting discovery that Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV), an inherited uveitis, is caused by mutations to the gene CAPN5. The CAPN5 gene produces the Calpain-5 (CAPN5) protease, which cleaves off regions of other proteins to regulate their functions. Our data show that CAPN5 becomes hyperactive in ADNIV. We identified Iroquois-3 (IRX3) as a new cleavage target of CAPN5 in the retina. IRX3 is a potent transcription factor implicated in inflammatory disease states. Identification of CAPN5 uveitis and a new CAPN5 target make possible a highly innovative molecular-genetic approach to identifying factors responsible for uveitis. The long-term goal of our research is to identify new therapeutic targets for the treatment of uveitis. This proposal’s objective is to determine the role of IRX3 cleavage in ADNIV. Our central hypothesis is that hyperactive ADNIV CAPN5 hyper-cleaves regulatory domains from IRX3, over-activating IRX3 transcriptional activity at inflammatory genes and driving destructive inflammation in ADNIV. Our rationale is that by studying the newly-identified CAPN5-IRX3 pathway, we will identify new disease mechanisms and therapeutic targets. Our data indicate that CAPN5 cleaves IRX3 toward its C-terminus. Additionally, we generated peptide substrates and a potential inhibitor compound for CAPN5 based on IRX3. Our data also show that IRX3 regulates a subset of ADNIV inflammatory genes in vitro. When comparing wild-type IRX3 to CAPN5-cleaved (“cut”) IRX3, our assays found that cut-IRX3 shows greater nuclear localization and increased binding to the IL- 6 promoter (a key ADNIV inflammatory gene). Our specific aims are to use biochemical, molecular, and physiologic studies in cells and animals to test the hypotheses that: (1) CAPN5 targets a specific domain on IRX3 for cleavage and physical interaction which can be isolated and developed into peptide substrates; (2) CAPN5 cleavage of IRX3 increases IRX3 nuclear localization and activity, contributing to uveitis in mice; and (3) Irx3-null mice will show resistance to developing ADNIV and intraocular inflammation phenotypes. We expect this work to have a significant positive impact on identifying new molecular mechanisms for therapeutic intervention in uveitis. At the completion of this project, we expect to have: (1) Isolated the IRX3 cleavage site and developed peptide substrates and potential inhibitor compounds; (2) Determined the consequences of CAPN5 cleavage on IRX3, identifying new molecular pathways which might be targeted therapeutically; and (3) Determined the significance of the Irx3 pathway in intraocular inflammation. As ADNIV mimics other eye diseases, our work promises to have a broad impact beyond ADNIV and uveitis.