Evaluating Alzheimer’s Disease Therapeutics on mitoNEET Expression Rachel M. Hemmerlin, Kayle J. Marsh, and Dr. Ashley M. Loe Department of Chemistry, Slippery Rock University of Pennsylvania, Slippery Rock, PA, United States Hallmarks of Alzheimer’s Disease (AD) • Neurodegenerative disease • Common symptoms include dementia, cognitive impairment, and progressive memory loss • Hallmarks of AD include the formation of senile plaques and neurofibrillary tangles Mitochondrial dysfunction and its role in cells • Mitochondria play a role in many cell cycles including energy metabolism, antioxidant production, and cell survival • Mitochondrial dysfunction leads to the formation of reactive oxygen species (ROS) • Impaired mitochondrial pathways include ATP generation, ROS formation and defense, calcium buffering, morphology and dynamics, mPTP opening, and cytochrome c release. Current AD therapeutics • AD therapeutics are cholinesterase inhibitors which inhibit acetylcholinesterase (AChE) and therefore increase the level of available acetylcholine • They are limited to alleviating the symptoms of AD by trying to counterbalance the neurotransmitter disturbance What is mitoNEET and its role in cells • Outer mitochondrial membrane protein with iron sulfur clusters • Plays a role in iron homeostasis, regulating energy metabolism, formation of inter-mitochondrial junctions, and production of ROS • Novel target for treating mitochondrial dysfunction and associated diseases Experiment Scheme Imaging using Fluorescence Microscopy Figure 1. N2a cells expressing mitoNEET-GFP (a) exposed to 5 µM (b), 50 µM (c), or 500 µM (d), donepezil for 24 hours. Expression of mitoNEET in response to donepezil, rivastigmine, and galantamine mitoNEET-GFP ± rivastigmine 200 150 100 50 0 Control 5 µM 50 µM 250 200 150 100 50 0 Control 500 µM mitoNEET-GFP ± donepezil 0.5 uM 5 uM 50 uM 500 uM 800000 600000 400000 200000 0 500 µM * 100 50 0 0.1 uM 1 uM 10 uM 100 uM 1200000 1000000 800000 600000 400000 200000 1000000 800000 600000 * * 400000 200000 0 0 50 µM * 150 mitoNEET-GFP ± galantamine Raw Integrated Density Raw Integrated Density 1000000 5 µM 200 Control 1200000 Control 250 mitoNEET-GFP ± rivastigmine 1200000 Raw Integrated Density mitoNEET-GFP ± galantamine Mean Fluorescence Intensity (a.u.) 250 Mean Fluoresence Intensity (a.u.) Mean Fluorescence Intensity (a.u.) mitoNEET-GFP ± donepepzil Control 0.5 uM 5 uM 50 uM 500 uM Control 0.1 uM 1 uM 10 uM 100 uM Conclusion and Future Direction • Donepezil and rivastigmine did not statistically impact the expression whereas certain concentrations of galantamine slightly statistically down regulated mitoNEET expression. • These finding suggest that current AD drugs do not change mitoNEET expression and therefore do not target or impact mitochondrial dysfunction in cells. • Future studies will explore the expression of mitoNEET in response to Aβ and oxidative stress. References • 2017 Alzheimer's Disease Facts and Figures. www.alz.org/facts/. • Lovell, M. A.; Markesbery, W. R., Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease. Nucelic Acids Res, 2007, 35 (7497-7504) • Fox, N. C.; al., e., Serial magnetic resonance imaging of cerebral atrophy in preclinical Alzheimer's disease. Lancet, 1999, 353, 2125. • LaFerla, F. 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