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theses

ETD doctoral

Traumatic brain injury (TBI) is one of the leading causes of death and disability in the world, and there are currently no available therapies to treat TBI-induced cell damage or to prevent TBI-associated cognitive impairments after injury. Diffuse axonal injury (DAI), a common TBI pathology, results from the shear and stretch of neuronal axons by the inertial forces of rotational head motions during TBI. Disruptions in sodium (Na+) and calcium (Ca2+) ion homeostasis significantly contribute to TBI and DAI pathogenesis. This dissertation will explore potential strategies for the development of TBI and DAI therapeutics targeting rising Ca2+ influx and mitochondrial dysfunction post-TBI. We examined target protein expression after injury in an in vivo controlled cortical impact (CCI) model of mild TBI (mTBI), and found alterations in expression of NCX1, the ubiquitously expressed sodium–calcium exchanger, and dynamin-related protein1 (Drp1), a GTPase regulator of mitochondrial fission, in the cortex and hippocampus following mTBI. We also demonstrate that pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation in an in vitro model of DAI. Moreover, using a hippocampal organotypic slice model of oxidative stress, we show that dynasore, but not SN-6, provides neuroprotection from H2O2-induced oxidative stress and cell death. In addition to pharmacological manipulation, we also investigate the use of brain-targeted exosomes, or nano-sized extracellular vesicles, to deliver short interfering RNA (siRNA) as a potential approach for the development of TBI therapeutics. Exosomes can be targeted to the central nervous system (CNS) by expression of a fusion protein, comprised of the CNS-specific rabies viral glycoprotein (RVG) and Lamp2b, a membrane protein expressed in exosomes, in exosome producing cells. We demonstrate that that RVG-exosomes loaded with siRNA via electroporation can deliver fluorescently-labeled siRNA to cultured primary dissociated neurons and to cells within cultured hippocampal organotypic slices. Furthermore, we present data to support that RVG-exosomes loaded with siRNA targeting NCX1 significantly downregulate NCX1 expression in vitro, and that NCX1 knockdown with siRNA-loaded RVG-exosomes attenuates mitochondrial depolarization following NMDA-mediated excitotoxicity. Taken together, the data presented in this dissertation highlight the potential of NCX1 and Drp1 as targets for TBI therapeutics, and underscore the potential of RVG-exosome-RNAi delivery as a promising approach for future development of TBI therapeutics.

ETD_11425

Ph.D.

Includes bibliographical references

doi:10.7282/t3-p72n-g318

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