P2‐157: Apolipoprotein E4 impairs mitochondrial function and increases reactive oxygen species in neuronal cells

Apolipoprotein (apo) E4 is genetically linked to Alzheimer's disease (AD) and has a gene-dose effect on risk and age of onset. ApoE4 can be proteolytically cleaved into C-terminal-truncated fragments, which are found in AD brains, are neurotoxic in vitro, and cause neurodegeneration and behavioral deficits in transgenic mice. ApoE4 fragments escape the secretory pathway and mislocate to mitochondria in neuronal cells, leading to mitochondrial dysfunction and potentially contributing to the apoE4 association with AD. To study the effects of apoE4 on mitochondrial function and the generation of reactive oxygen species (ROS) in neuronal cells. We measured ROS in PC12 cells using a cell-permeable indicator (CM-H2DCFDA) that is non-fluorescent until oxidation occurs within the cell. Undifferentiated PC12 cells expressing apoE4 (0.2 ng/μg protein) had ~3-4-fold higher ROS levels after 24h than cells expressing matched levels of apoE3 or control PC12 cells not expressing apoE. The lactose levels in medium of apoE4-expressing cells were also elevated by ~4 fold after 24 h, indicating an increase in glycolysis. The generation of ATP via glycolysis rather than mitochondrial oxidative phosphorylation may be a compensatory response to impaired mitochondrial function. Oxidation of galactose to pyruvate via glycolysis yields no net ATP, forcing cells to rely on mitochondrial oxidative phosphorylation to generate sufficient ATP. When glucose in the medium was replaced by galactose, comparable ~3-4-fold differences in ROS levels between apoE4 and apoE3 or control cells were apparent after 4 h. The lactose levels in the medium of control, apoE3, and apoE4 cells showed no significant difference in the presence of galactose. Further, PC12 cells expressing apoE4 with an R61T mutation, which abolishes domain interaction, a structural feature characteristic of apoE4, and decreases susceptibility to proteolysis, had ROS and lactose levels similar to those in apoE3-expressing and control PC12 cells. The impairment of mitochondrial function by apoE4 is isoform specific and dependent on domain interaction and leads to increased ROS levels and glycolysis in neuronal cells. Thus, disruption of domain interaction in apoE4 by small molecules is a potential strategy for the prevention and treatment of AD associated with apoE4.