Aberrant intraneuronal calcium (Ca2+) handling triggers mitochondrially-mediated cell death in multiple neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, and stroke. These pathological elevations in mitochondrial Ca2+ cause transiently elevated mitochondrial respiration, mitochondria hyperpolarization, pathological reactive oxygen species production, and then membrane potential collapse and apoptosis. The L-type Ca2+ channel (LTCC) Cav1.2 is a prominent regulator of intraneuronal Ca2+, and its disrupted activity has been implicated in death of newborn neurons as well as an array of neurological disorders, revealing a critical need to clarify Cav1.2's pathological contribution to neurodegeneration. We have discovered that in addition to its established localization to the plasma membrane (pm), Cav1.2 also resides in mitochondria (mito). Our preliminary data shows that LTCC activation promotes mitochondrial Ca2+ efflux in isolated mitochondria and, correspondingly, brain-specific knockout of Cav1.2 increases reactive oxygen species damage in intact cells. Together, these data suggest that novel mitochondrial Cav1.2 is a major regulator of mitochondrial energetics and Ca2+ homeostasis. Our central hypothesis is that excessive pmCav1.2 activity potentiates mitochondrial Ca2+ overload and apoptotic neuron death, whereas mitoCav1.2 divergently protects neurons by acting as a transient mitochondrial Ca2+ release valve. To test this model of mitochondrial regulation, we propose the following aims: 1) Define the physiological and pathological contributions of Cav1.2 in neuronal mitochondria function. I will test the hypothesis that disruption of Cav1.2 in neurons in vivo increases mitochondria-mediated apoptosis after ischemia-reperfusion injury, and that in both ex vivo cell culture and isolated mitochondria, disruption of Cav1.2 increases mitochondrial respiration, membrane potential, and ROS production. My hypothesis is consistent with the mitoCav1.2 fraction as a major regulator of mitochondrial energetics. 2) Determine the mechanism by which mitoCav1.2 protects from neurotoxicity. I will use Ca2+ imaging in intact cells and isolated mitochondria from brain-Cacna1c KO and WT mice to examine the respective contributions of pmCav1.2 and mitoCav1.2 to calcium dynamics. Finally, using plasma membrane- and mitochondria- targeted Cav1.2 constructs in a synthetic lethality cell culture screen, I will test the differential effects of pmCav1.2 and mitoCav1.2 on cell survival during ischemia-like Ca2+ overload. At the conclusion of this project, I will have revealed mechanisms of a novel mitochondrial Ca2+ channel in mitochondrial function and neuron death, and also distinguished roles of pmCav1.2 and mitoCav1.2. These studies are positioned to unveil a new paradigm for mitochondrial Ca2+ regulation in neurons, thereby contributing to our understanding of neurological disorders in a direction that could identify new treatment opportunities for patients suffering for neurodegenerative conditions.
Changes in the way that neurons use calcium is a well-established trigger for cell death in devastating neurodegenerative conditions. Cav1.2 is a channel that mediates neuronal calcium levels and is known be in the cell membrane. Our findings show that functional Cav1.2 is also in mitochondria, and we thus propose to determine the respective functions of plasma membrane and mitochondrial Cav1.2 in protection from neuron cell death.
