We have studied programmed cell death in the nervous system and the biochemical mechanism of apoptosis. Recently we have focused on the Bcl-2 family of proteins that regulates the survival of neurons during development. One member of this family, Bax, is required for the normal death of neurons and elimination of the Bax gene results in excess neurons in adult animals. How Bax and other Bcl-2 family members regulate cell survival is unknown. We developed a series of monoclonal antibodies against Bcl-2, Bcl-xl, and Bax to probe their mechanism of action in vitro and in vivo. The antibodies revealed that Bax migrates from the cytosol to cell membranes during apoptosis. To explore Bax, Bcl-2 and Bcl-xl movement in neurons the green fluorescent protein has been used to tag and follow the proteins in living cells. We discovered that Bax and Bcl-xl move from the cytosol to the mitochondria as an essential step in their mechanism of apoptosis control. Point mutants of Bax that insert better into mitochondria are more toxic and mutants with less mitochondrial insertion are less toxic indicating that the C-terminus of Bax regulates the mitochondrial association. Therefore, we determined the three dimensional structure of Bax and found that the C-terminal tail fits into a hydrophobic pocket that has been thought to regulate hetero- and homo-dimer formation among Bcl-2 family members. This suggests a new model that dimer formation and mitochondrial docking are structurally linked to disengagement of the C-terminus. In contrast to Bax, Bcl-xl prevents neuron cell death due to many neurotoxic insults. Like Bax, Bcl-xl inserts into mitochondria during apoptosis. We have found one regulatory step that controls Bcl-xl subcellular location. Bad, a pro-apoptotic Bcl-2 family member, binds to Bcl-xl and triggers its docking to mitochondria. Bad binding to Bcl-xl is in turn regulated by phosphorylation and several neurotrophic factors that stimulate kinases prevent Bad from binding to Bcl-xl and thus prevent neuron death. The consequences of Bcl-2 family member binding to mitochondria have been also studied. Bax coalesces into large aggregates on the mitochondrial surface that co-localize with machinery for mitochondrial division and can activate mitochondrial division. Thus, the cell machinery that governs mitochondrial division participates in apoptosis. We also study the role of mitochondria in Parkinson's disease. At least two genes mutated in familial PD are now known to mediate autophagic removal of defective mitochondria suggesting that one cause of PD is an impairment of mitochondrial quality control. This model is being investigated by assessing mitochondrial DNA mutation accumulation in PD models. This model is being investigated by assessing mitochondrial DNA mutation accumulation in PD models. Molecular steps in this process such as how the mitochondrial kinase Pink1 regulates the cytosolic E3 ligase Parkin and how Parkin signals autophagosomes to engulf and destroy certain mitochondria is under investigation.

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Yao, Yong; Nisan, Danielle; Fujimoto, Lynn M et al. (2016) Characterization of the membrane-inserted C-terminus of cytoprotective BCL-XL. Protein Expr Purif 122:56-63
Yao, Yong; Fujimoto, Lynn M; Hirshman, Nathan et al. (2015) Conformation of BCL-XL upon Membrane Integration. J Mol Biol 427:2262-70
Lazarou, Michael; Sliter, Danielle A; Kane, Lesley A et al. (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524:309-314
Pickrell, Alicia M; Youle, Richard J (2015) The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron 85:257-73
Pickrell, Alicia M; Huang, Chiu-Hui; Kennedy, Scott R et al. (2015) Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress. Neuron 87:371-81
Pickrell, Alicia M; Youle, Richard J (2013) Mitochondrial disease: mtDNA and protein segregation mysteries in iPSCs. Curr Biol 23:R1052-4
Ma, Junhe; Edlich, Frank; Bermejo, Guillermo A et al. (2012) Structural mechanism of Bax inhibition by cytomegalovirus protein vMIA. Proc Natl Acad Sci U S A 109:20901-6
Cooper, Julia Promisel; Youle, Richard J (2012) Balancing cell growth and death. Curr Opin Cell Biol 24:802-3
Wang, C; Youle, R J (2012) Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak. Oncogene 31:3177-89
Edlich, Frank; Banerjee, Soojay; Suzuki, Motoshi et al. (2011) Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. Cell 145:104-16

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