June 2009

Document Type


Degree Name



Dept. of Physiology and Pharmacology


Oregon Health & Science University


Numerous studies have shown that critical aspects of the pathology associated with a wide variety of neurodegenerative diseases may be based in mitochondrial dysfunction, in particular defects in mitochondrial Ca(superscript 2+) homeostasis. A key aspect of the mitochrondrial Ca(superscript 2+) regulation is the permeability transition (PT), which depends on the opening of an inner membrane channel, the mitochorndrial permeability transition pore (PTP). Inappropriate activation of the PTP depletes mitochondrial Ca(superscript 2+) stores, elevates the cytosolic Ca(superscript 2+) and increases the generation of reactive oxygen species, ultimately leading to depletion of cellular ATP and cell death. A key regulator of the PTP is cyclophilin D (CyPD), a cyclophilin found exclusively in mitochondria. Initial studies in mice have demonstrated the inactivation of the nuclear gene encoding CyPD (CyPD-KO) results in neuroprotection following a number of pathologic challenges such as ischemia/reperfusion and experimental autoimmune encephalomyelitis, EAE, a mouse model of multiple sclerosis. Studies on isolated mitochondria prepared from a number of tissues demonstrate that many cell types from CyPD-KO animals are able to accumulate higher levels of Ca(superscript 2+) than WT mitochondria yet the cellular correlate of this in vitro response had not been carefully investigated. To address this question, the present study compared mitochondria Ca(superscript 2+) dynamics in primary cortical neurons isolated from adult CyPD-KO and WT mice using a fluorescent Ca(superscript 2+) indicator, ratiometric pericam, targeted to mitochondria. Mitochondria in the CyPD-KO neurons are able to accumulate significantly higher levels of Ca(superscript 2+) than mitochondria in WT neurons in response to the combined actions of depolarization and receptor activation coupled to Ca(superscript 2+) release from endoplasmic reticulum (ER), while no difference was observed in response to either stimulus alone. This suggests that CyPD inactivation leads to a delay in the PTP opening in CyPD-null neurons in response to elevated cytosolic Ca(superscript 2+) levels created by combined stimuli. Furthermore, the study shows that CyPD inactivation leads to a modulation of PTP opening and mitochondria Ca(superscript 2+) dynamics in response to the oxidative stress. The results also show that CyPD-null adult cortical neurons are significantly less susceptible to oxidative challenges. Therefore, this study demonstrates for the first time mitochondrial Ca(superscript 2+) dynamics in live adult cortical neurons and the modulation of mitochondrial Ca(superscript 2+) and PTP opening by CyPD inactivation under elevated cytosolic Ca(superscript 2+) levels and oxidative stress. Establishing the parameters for the prevention of PTP opening in neurons under elevated cytosolic Ca(superscript 2+) and oxidative stress is essential for the development of neuroprotective strategies. CyPD inactivation may become a key neuroprotective target in the development of therapies for neurodegenerative diseases.




School of Medicine



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