Investigation of novel mitoNEET ligand NL-1 as a therapeutic for cerebral ischemia and reperfusion injury
Date of Graduation
School of Pharmacy
Werner J. Geldenhuys
Paul R. Lockman
Cerebral ischemia reperfusion injury, a common type of stroke, is a neurodegenerative disorder which is the leading cause of permanent disability in the United States. The underlying pathophysiology of ischemic stroke involves an occlusion in the vasculature supplying oxygen and glucose to the brain tissue, leading to infarction and eventual necrosis in the brain. MitoNEET is an outer mitochondrial protein that plays a role in the cellular bioenergetics and its redox physiology. Although its endogenous ligands are under investigation, mitoNEET binds molecules with a thiazolidinedione moiety, such as NL-1. This dissertation investigates NL-1 as a potential cytoprotective agent, and its possible use as a stroke therapeutic. After establishing the binding of NL-1 to mitoNEET, the effects of the drug on cellular bioenergetics as well as the mitochondrial function were studied. Upon the successful preliminary studies, NL-1 was formulated into a polymeric nanoparticle system and its efficacy evaluated in an in vitro stroke model on endothelial cells. The NL-1 loaded nanoparticles were found to reduce the generation of cell damaging peroxide, which is an important component of the oxidative stress causing milieu. The NL-1 nanoparticles also helped improve cell survival by decreasing the apoptotic cell population, one of the principal modes of cell damage following stroke. To evaluate promising in vitro results in vivo, a two-hour transient middle cerebral artery occlusion model was utilized. Analysis of brain sections after 24 hours showed a significantly reduced infarct volume and hemispheric swelling following treatment with NL-1 and NL-1 nanoparticles. Reduced levels of IgG extravasation were seen in treated animals compared to controls, after a 72-hour period following treatment. Qualitative analysis also revealed decreased staining area and intensity for GFAP, nitrotyrosine and 4-hydroxynonenal. Future studies need to be focused on determining the exact mechanism of action of NL-1 in the mitochondrial axis, which could help improve its dosing. A long-term study with focus on cognitive and motor neurological outcomes, following treatment with NL-1 would be the primary translational future aim. In summary, this dissertation establishes the preliminary basis for use of NL-1 as a stroke therapeutic which could compliment the current therapy in alleviating the effects of reperfusion injury.
Saralkar, Pushkar, "Investigation of novel mitoNEET ligand NL-1 as a therapeutic for cerebral ischemia and reperfusion injury" (2021). Graduate Theses, Dissertations, and Problem Reports. 8144.