P3–226: Mitochondrial ubiquitination is associated with mitochondrial stress leading to neuronal degeneration in the pathogenesis of Alzheimer's disease

Background: Abnormalities of the ubiquitin(Ub)-proteasome system (UPS) with accumulation of ubiquitinated protein aggregates and mitochondrial dysfunction have been consistently demonstrated in Alzheimer’s disease (AD) as well as other neurodegenerative disorders. Recent studies suggest that inhibition of UPS function might directly affect mitochondrial homeostasis in cells. However, the relationship between UPS dysregulation and mitochondrial stress as well as its contribution to neuronal degeneration in AD remains poorly understood. Objective(s): This study investigated molecular basis of Ub-/ubiquitinated protein species-mitochondria interaction in the central nervous system (CNS) undergoing Alzheimer’s pathogenesis. Methods: Either primary hippocampal neurons from newborn 3xTg AD or C57Bl mice or hippocampal cells were treated with pre-aggregated beta-amyloid (1-42) or proteasome inhibitor (MG115) or vehicle followed by mitochondria/cytosol fractionation at specific times between 8–40 hr after treatment. Some of normal C57Bl neurons were also transfected with vectors that express Ub-EGFP-MOM (mitochondrial outer membrane domain) or EGFP-MOM (control). Western blotting, co-immunoprecipitation (co-IP), and immunocytochemical (IHC) microscopy were performed to examine mitochondrial ubiquitination, release of cytochrome c, and neuronal cell death as well. To further evaluate the functional consequence of mitochondrial ubiquitination, Ub-proteasome-mediated degradation of mitochondria was assayed in a reconstituted cell free system. Results: Co-IP and Western blot revealed a significant increase in polyubiquitinated proteins in mitochondrial fraction, which was corroborated by colocalization of polyubiquitin species and mitochondrial reporter from IHC microscopic observations. Interestingly, increased Ub-binding proteins, p62/sequestosome-1 and ubiquilin-1, as well as proteasomal subunits were detected in association with mitochondria. Reconstitution assay demonstrated disruption of mitochondrial structure, release of cytochrome c as well as inhibition of mitochondrial function following incubation with Ub-proteasome components. Transfection of Ub-EGFP-MOM resulted in apoptotic neuronal cell death. Conclusions: These findings suggest mitochondrial ubiquitination that might be associated with accumulation of Ub-binding proteins in mitochondria and play an important role in the signal transduction of neuronal cell death during the Alzheimer’s pathogenesis.