Thesis Defense Announcement
College of Arts and Sciences announces the Final Thesis Defense of
for the Degree of Master of Arts
November 06, 2019 at 03:00 PM in Ellington Hall Room 109
Advisor: Michael Kennedy
Role of NIPSNAP1 in oxidative stress
ABSTRACT: NIPSNAP1 (4-nitrophenyl phosphatase domain and non-neuronal SNAP25-like protein homolog1) is an evolutionarily conserved protein that is found in a wide range of species. In mammals, it is highly expressed in liver, kidney, and brain. NIPSNAP1 is localized to the inner mitochondrial membrane and was recently shown to translocate to the outer mitochondrial membrane to initiate mitophagy. Our group has shown that NIPSNAP1 interacts with the cytoplasmic domain of the amyloid precursor protein (APP). Mutations in APP, resulting in its abnormal proteolytic cleavage, play a causative role in pathogenesis of Alzheimer's Disease (AD). We hypothesize that the interaction between NIPSNAP1 and APP may contribute to mitochondrial dysfunction and possibly to neurodegeneration associated with AD. To investigate the molecular and cellular role of NIPSNAP1, the first aim of this study was to develop and validate an shRNA lentiviral-mediated gene silencing approach. Three shRNA constructs, targeting different regions of NIPSNAP1 coding sequence, were designed and tested in Hep1-6 mouse hepatoma cell line. shRNA constructs were delivered to the cells using a lentiviral approach. Immunoblot analysis showed that one shRNA construct reduced NIPSNAP1 protein levels by approximately 85%. Reduction of NIPSNAP1 protein levels required at least 10 days after shRNA lentiviral transduction, suggesting that NIPSNAP1 protein is highly stable in these cells. The second aim of this study was to investigate whether NISNAP1 deficiency contributes to mitochondrial dysfunction and oxidative stress. Reactive oxygen species (ROS) was induced by treating cells with Antimycin A and the levels of ROS was detected using MitoSOX microplate assay. Surprisingly, we found lower levels of ROS in NIPSNAP1 deficient cells compared to normal control cells. These results suggest that NIPSNAP1 regulates mitochondrial function, although additional experiments are needed to investigate the precise role of NIPSNAP1 in regulation of ROS. Taken together, this work establishes a NIPSNAP1 knock-down cell line in which detailed molecular and cellular studies can be conducted in the future. Ultimately, this approach may provide insights into the molecular functions of NIPSNAP1 and its interaction with APP.