Retinal Pigment Epithelium Secreted Matrix Metalloproteinase 2 as a Novel Target in Choroideremia

PROJECT SUMMARY: Choroideremia is a blinding inherited retinal disease in which retinal pigment epithelial (RPE) cells contribute significantly to chorioretinal degeneration, but the underlying molecular mechanisms remain largely unclear. A critical barrier to progress in developing therapies is understanding how RPE cells contribute to death of neighboring tissues in choroideremia. Our long-term goal is to determine how RPE dysfunction contributes to chorioretinal degeneration and to develop novel therapies. Choroideremia is caused by mutations in CHM, encoding Rab escort protein 1, which facilitates prenylation of Rab GTPases (Rabs). Prenylation is required for Rabs to associate with lipid membranes and direct protein trafficking. Our preliminary studies implicate Rab-mediated mTORC1 signaling and downstream MMP2 secretion in choroideremia. The overall objective of this proposal is to identify pathways downstream of defective Rab prenylation that lead to increased MMP2 secretion and choroidal degeneration. We hypothesize that CHM deficiency leads to increased RPE-secreted MMP2 via increased mTORC1 signaling, contributing to choroidal degeneration. The rationale for this proposal is that identification of specific defects in RPE function will inform future targeted interventions for this untreatable disease and will reveal important mechanisms that may be relevant to other chorioretinal degenerative diseases. The central hypothesis will be tested by pursuing two specific aims: 1) Determine how CHM deficiency impacts MMP2 secretion; and 2) Demonstrate the effects of RPE-secreted MMP2 on choroidal degeneration. Under the first aim, we will use CHM-/- iPSC-RPE cells to investigate the role of candidate Rabs and mTORC1 signaling on MMP2 secretion, using shRNA knockdown studies, transduction with a CHM-independent Rab, and pharmacologic mTORC1 modulation. Additionally, we will use an established Chm knockout mouse model to investigate levels of MMP2 activity to validate the mouse model for use in future translational studies targeting the MMP2 pathway. For the second aim, we will study the effects of secreted MMP2 on choroidal endothelial cells in culture as well as effects on choroidal vascular density, blood flow, and ECM content in vivo in a Chm Mmp2 double knockout mouse. This proposal is innovative because it seeks to identify specific RPE membrane trafficking abnormalities in choroideremia and the effects on neighboring tissues, building on our preliminary data supporting the role of MMP2, to support future targeted therapies based on a detailed understanding of disease mechanism. The contribution from this research is expected to be identification of key pathologic pathways downstream of CHM deficiency in choroideremia that lead both to RPE cell dysfunction and choroidal atrophy. Additionally, these results may lead to translational studies in other chorioretinal degenerative diseases with evidence for RPE dysfunction and altered protease activity, such as age-related macular degeneration. This contribution will be significant because it will advance therapeutic efforts by identifying targets for this currently untreatable blinding disease.