(90) NEUROBLASTOMA CELLS TRANSFORMED WITH MITOCHONDRIA FROM ALZHEIMER'S DISEASE PATIENTS ARE MORE SUSCEPTIBLE TO APOPTOSIS

Amy K. Carroll
MitoKor, 11494 Sorrento Valley Road, San Diego, CA 92121

Mitochondrial dysfunction has been implicated in Alzheimer's disease (AD) and is manifested as a decrease in complex IV (CIV) activity in AD brain and platelets. Cytoplasmic hybrid (cybrid) cells were created by fusing platelets from AD patients with SH-SY5Y neuroblastoma cells that have been previously depleted of mitochondrial DNA. These cybrids provide a valuable in vitro model of AD and recapitulate several important features of the AD phenotype: decreased CIV activity, increased production of reactive oxygen species (ROS), altered calcium homeostasis and upregulation of ROS scavenging enzymes. In this study, AD cybrids have been studied to assess the consequence of a mitochondrial defect on cell viability and the cellular response to apoptotic stimuli.

Control and AD cybrids were treated with apoptogens that act by increasing intracellular calcium levels. Treatment with thapsigargin or carbachol significantly increased caspase-3 activity in AD cybrids over the control cells. This increase was inhibited by cyclosporin A and by specific inhibitors of calcium release, suggesting that the pathway involves opening of the permeability transition pore. Cytochrome c release in response to the calcium ionophore ionomycin was also greater in the AD cybrids and was confirmed by mass spectrometry and western blot analysis. Cell viability was determined by a live-dead assay to confirm apoptotic cell death and to differentiate the process from secondary necrosis.

A reduced calcium buffering capacity in neurons of AD patients may play an important role in the pathology of the disease. This is the first study to demonstrate that a defect in mitochondrial electron transport leads to an increased risk of apoptosis as measured by release of apoptotic factors from mitochondria and a downstream activation of caspases.