The goal for this project is to determine how cleavage of Bcl-xL by caspases contributes to cell death in vivo. Caspase cleavage of cellular substrates is a key event in mediating apoptosis. The Bcl-2 family member Bcl-xL is an inhibitor of apoptotic cell death, and can also be a substrate of caspases. Cleavage of Bcl-xL [not only abolishes its anti-death activity] but also convert Bcl-xL from a potent anti-apoptotic protein to potent pro-death molecule. Cells transfected with the N-terminally truncated Bcl-xL cleavage fragment (?N-Bcl-xL) display accelerated cell death. To probe the in vivo effects of Bcl-xL cleavage, a caspase-uncleavable Bcl-xL knock-in mouse was generated by mutating the two caspase cleavage sites within Bcl-xL. Interestingly, caspase-resistant Bcl-xL mice retain a healthy thymus in old age suggesting that cleavage of Bcl-xL leads to cell death that results in thymic atrophy. My preliminary data suggest that these mice may have defective positive selection (generation of a viable T-cell receptor). Therefore, we will explore the possibility that caspase-cleavage of Bcl-xL kills developing thymocytes that fail the positive selection checkpoint. By doing so, we will provide mechanistic insights into what is termed death by neglect, the failure of thymocytes to receive survival cues. Because these mice maintain a morphologically normal thymus throughout life, I will determine if they also maintain normal immune responses to influenza virus. Influenza particularly affects the elderly, who display poor T-cell responses to the virus. This is presumed to be because of immunosenescence, in part attributed to thymic atrophy. A long-standing assumption is that thymic atrophy contributes to waning immune responses due to decreased T-cell production, but mouse models to definitively test this hypothesis have been lacking. Therefore, we are in a unique position to determine if preserved thymic function also confers protection from influenza infection in aged animals. We will critically evaluate whether thymic atrophy compromises the immune responses needed to control influenza. These studies will also address the role of thymic selection and T-cell contributions to influenza virus immunity.
The anti-apoptotic role of Bcl-xL is well established, yet its conversion to a pro-apoptotic molecule via caspase cleavage is less appreciated and understudied. Using a caspase-resistant Bcl-xL knock-in mouse which fails to undergo thymic atrophy during aging, will study the impact of cleaved Bcl-xL on T-cell development and the effects of thymic atrophy on waning immune responses during aging.
