Our studies of cerebral ischemia have shown that caspase-9 is a critical mediator of edema, neuronal process loss and neuronal death. We have an RO1 funded project to study the mechanisms of edema in cerebral ischemia, NS081333 Mechanisms and Treatment of Cerebral Edema. In that grant we are examining how caspase-9 regulates cerebral edema due to cerebral ischemia. However, this project was written, reviewed and funded before we knew that there were caspase-9 conditional knockout mice available; Marc Tessier-Lavigne made the mice when he was at Genentech and kindly told us about these mice. We now propose to develop cell specific inducible caspase-9 knockout mice to examine the function of caspase-9 in the neurovascular unit during cerebral ischemia. The neurovascular unit, composed of neurons, glial cells and blood vessels, is a key target in cerebral ischemia. Our prior work suggests that while caspase-9 may function to orchestrate death in some cell types, neurons for example, there may be cell-specific functions of caspase-9 activity, such as a function in endothelial cells leading to edema but not to endothelial cell death. Caspase-9 null mice die in late embryogenesis with overgrowth of the frontal cortices. Thus these mice are not good tools to examine the functions of caspase-9 in adult animals or to study the cell specific functions of caspase-9. Genentech has developed a caspase-9 conditional null mouse to which we now have access. Our preliminary data show that tMCAo (transient middle cerebral artery occlusion) in rat and mouse models activates caspase-9 in neurons and in blood vessels. Moreover, activation in neurons leads to process loss and neuronal death while activation in blood vessels does not activate effector caspases or induce death. This raises the question of how activation of caspase-9 in each of these cell types leads to the development of stroke pathology. To approach this question, we propose to develop cell specific inducible knockouts of caspase-9 in neurons and endothelial cells. After obtaining the MTA for the caspase-9 floxed mice, we investigated the availability of inducible cell specific Cre mice. We are choosing to use inducible Cre because we are studying a model of adult disease and want to avoid any developmental phenotype that might occur with constitutive cell specific knockout of caspase-9. To target endothelial cells, we were able to get an MTA for Cdh5(PAC)-CreERT2 mice from Raif Adams. For neuronal knockout, Synapsin-driven CreERT2 mice are available from Jackson Labs. Due to the sequester, our RO1 project received a 12.5% cut for the entire project, thus there is not funding in the RO1 to develop these new tools that we are now proposing. The current RO3 grant proposal will allow us to generate these mice that will then be used in our funded RO1 project. These cell specific inducible knockout mice will be a valuable tool for our work and in the future will also be useful for other studies of caspase-9 function and caspase-9 cell specific substrates. Identifying the cell specific substrates of caspase-9 will provide additional disease targets for therapeutic interventions.
Stroke is the 3rd largest cause of death and the largest cause of disability in the U.S., yet there are no effective therapies for the vast majority of cases. Stroke-induced cerebral edema, swelling of the brain, due to dysfunction of the blood brain barrier is the major cause of death within the first few days after stroke. We have found that blocking activity of a protease blocks development of cerebral edema and provides substantial neuroprotection following an ischemic insult. This could lead to development of an effective therapeutic intervention for stroke.
