Title: Elucidating endothelial Caspase-9 signaling pathways in retinal vein occlusion
Description: Central nervous system (CNS) tissues are highly metabolically active which makes them particularly susceptible to vascular injury. Disruption to the supply of oxygen and nutrients by damaged vasculature can result in neurodegeneration in both the eye and brain. The retina is an accessible part of the CNS that can be taken advantage of to study neurovascular diseases through live, non-invasive visualization of vascular and neuronal conditions upon injury. Retinal vein occlusion (RVO) is a common neurovascular disease of the eye and is the second leading cause of blindness in working age adults. While pathophysiology is well described and can be determined by retinal edema, breakdown of the blood-retina-barrier (BRB), inflammation, and neurodegeneration, the underlying signaling pathways behind the pathology is not well understood. To understand the mechanism of disease in RVO, the Troy lab has employed a mouse model to investigate pathways. Previous studies in the lab determined that as early as 1 hour post RVO, there was a large induction of caspase-9, a known cell death protease, in endothelial cells.

When further investigated, it was confirmed that these cells were not dying despite the high expression of caspase-9, implying a non-apoptotic role. Deletion of endothelial caspase-9 was sufficient to protect against the development of retinal edema, capillary ischemia, and neuronal death, indicating caspase-9 is a key player in the mechanism of disease. This thesis work aims to investigate which signaling events drive non-apoptotic endothelial caspase-9 signaling by investigating upstream and downstream mechanisms of endothelial caspase-9. To interrogate this question, the mouse model of RVO was optimized, limiting the variability previously observed to ensure accurate and reproducible results. Then, we used a tamoxifen inducible endothelial cell Apaf-1 (apoptosis protease activating factor-1) knock out (Apaf-1 iECKO) mouse line in order to investigate the contribution of upstream activation of non-apoptotic endothelial caspase-9 signaling. Apaf-1 iECKO mice and WT littermates were subjected to RVO.

Then, expression of caspase-9 and -7, retinal edema, capillary ischemia, neuronal death, vision dysfunction, and BRB integrity were measured. The deletion of endothelial Apaf-1 resulted in reduced expression of cl-caspase-9 and caspase-7, indicating endothelial caspase-9 was activated by Apaf-1. Apaf-1 deletion also resulted in protection against some of the pathologies seen after RVO including retinal edema, capillary ischemia, and neurodegeneration. Lastly, in order to elucidate the signaling pathway further, experiments using endothelial cell-specific AAVs (adeno-associated virus) packaged with a downstream caspase-7 inhibitor were proposed and described. In sum, this thesis work reveals that endothelial caspase-9 is canonically activated by Apaf-1, but still leads to non-apoptotic signaling, indicating downstream caspase-9 substrates could be the source for non-apoptotic function within endothelial cells.