Exploring Apoptosome-Independent Mechanisms for Casp9 activation in Axon Pruning

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Project Summary/Abstract Neurons have the capability to activate pathways that cause degeneration of either the entire cell by apoptosis or only the axons. Physiological axon-specific degeneration, also known as pruning, is important as it allows neurons to remove excessive or misguided axon branches and permit plasticity in neuronal connections. However, exactly how a neuron can activate and compartmentalize this pathway to degenerate its axon without putting the rest of the cell at risk is unclear. This is particularly interesting since recent studies identified Bax and caspases, key proteins in the apoptosis pathway, to also regulate axon pruning We are using a microfluidic chamber-based model of sympathetic neurons where deprivation of nerve growth factor (NGF) can induce either apoptosis (when NGF is deprived from both soma and axon compartments) or axon pruning (when NGF is deprived from only the axon compartment). Using this model, we identified substantial overlap but also distinct differences between the apoptosis and axon pruning pathways. Specifically, we found that while caspase-9 (Casp9) is required for both pathways, Casp9 activation is dependent of Apaf-1 during apoptosis but, surprisingly, independent of Apaf-1 during axon pruning. These results were unexpected and point to a novel mechanism of Casp9 activation during axon pruning. In this proposal, our goals are to examine specific aspects of how Casp9 is activated during axon pruning. In Aim 1, we will focus on Bax function during axon pruning. Since Apaf-1 is not needed for axon pruning, we propose that the essential function of Bax during axon pruning is not the release of cyt c (which activates Apaf- 1 in the context of apoptosis), but instead is the release of the Smac, an inhibitor of XIAP, from mitochondria. In Aim 2, we will examine how Casp9 is activated during pruning. Our focus is on identifying key features of Casp9 function that are important for axon pruning. Additionally, we will examine whether components of a dependosome-like complex are important for activating Casp9 during axon pruning. These studies will help uncover critical aspects of how neurons utilize many of the same components for apoptosis and axon pruning yet with distinct differences.