• Depletion of endoplasmic reticulum calcium triggers the loss of ER resident proteins

      Trychta, Kathleen Anne; Harvey, Brandon K. (2019)
      The endoplasmic reticulum (ER) contains proteins that carry out the diverse functions of the ER including calcium storage, protein folding, modification, and trafficking, lipid metabolism, and drug detoxification. When soluble ER resident proteins with an ER retention sequence (ERS) depart from the ER they interact with KDEL receptors in the Golgi membrane and are retrogradely transported to the ER lumen via the KDEL receptor retrieval pathway. ER calcium depletion disrupts this process resulting in the mass departure of ERS-containing proteins into the extracellular space. Such a loss of ER resident proteins has two potential consequences to an affected cell. First, there is a loss of proteins involved in the aforementioned critical ER functions. Second, the relocation of such proteins and their associated functions outside of the cell may cause changes in the extracellular environment. This dissertation describes the identification and characterization of a phenomenon whereby ER resident protein secretion is triggered by pathophysiological ER calcium depletion. By exploiting the enzymatic activity of one of the identified ERS-containing proteins we developed an assay to monitor changes in the ER proteome. We also developed a high-throughput screen that identified drugs that could prevent the release of ER resident proteins following ER calcium depletion and showed that several of these compounds have therapeutic potential in models of ER stress and ischemia. Taken together, the work described in my dissertation identifies a novel molecular mechanism of cellular dysfunction for which I have identified both endogenous biomarkers and possible therapeutics.
    • Measuring Cytosolic and Mitochondrial Labile Zinc Concentrations Following Hypoxia/Hypoglycemia with Fluorescence Biosensors

      McCranor, Bryan; Thompson, Richard B. (2011)
      Zinc is a "trace" metal necessary for proper cellular function, but studies, in multiple cell types, have shown that excess free zinc can be toxic (1, 2). It has also been observed that the intra- and extra-cellular concentrations of labile zinc increase dramatically in models of cerebral ischemia (3, 4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc concentrations have been associated with increased reactive oxygen species (ROS) production and ultimately apoptosis (6, 7). Zinc, specifically, has been shown to inhibit major mitochondrial enzymes of energy production contributing to mitochondrial dysfunction (8). We adapted our expressible fluorescent zinc biosensor (9) to target the mitochondria of PC12 cells, enabling us to ratiometrically image the mitochondrial matrix concentration of labile zinc even at resting (picomolar) levels. This represents the first such development of a sensor with sensitivity for physiological zinc in the mitochondria. We used this biosensor and our previous sensor, in cells which have undergone oxygen/glucose deprivation (OGD), to measure the "free" zinc concentrations following an ischemic-like event. The data suggests that both the intra-cellular and intra-mitochondrial zinc concentrations increase following OGD, albeit at different times, with the mitochondrial increase preceding the cytosolic increase. Our data raises the possibility that an increase in mitochondrial zinc could contribute to cell death in models of ischemia/reperfusion. 1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1-6. 2. Zodl, B., et al. (2003) Journal of Inorganic Biochemistry 97, 324-330. 3. Tonder, N., et al. (1990) Neuroscience Letters 109, 247-252. 4. Frederickson, C. J., et al. (2006) Experimental Neurology 198, 285-293. 5. Fiskum, G. et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129-138 6. Weiss, J. H., et al. (2000) Trends in Pharmacological Sciences 21, 395-401. 7. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524 - 47529. 8. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373-24380. 9. Bozym, R. A., et al. (2006) ACS Chemical Biology 1, 103-111.
    • Microhemorrhages in a Novel Model of Encephalopathy of Prematurity

      Carusillo, Brianna; Simard, J. Marc; 0000-0003-3353-2276 (2016)
      Encephalopathy of prematurity (EP) is a broad designation that encompasses various neurological pathologies common after pre-term birth, including choroid plexus, germinal matrix, and intraventricular hemorrhages, periventricular leukomalacia, neuronal and axonal pathology, and hydrocephalus. EP is common in preterm, low-birth weight infants and is associated with life-long cognitive, behavioral, and motor (cerebral palsy) abnormalities in survivors. Here, a novel clinically-relevant rat model of EP was developed and characterized that combines dual prenatal insults of transient intrauterine ischemia and low-level maternal systemic inflammation (IUI+LPS). Ischemia and inflammation, both potent activators of angiogenesis, led to weakened blood vessels and predisposed to hemorrhage in pups after vaginal delivery. Prominent hemorrhages, white matter damage, and accompanying behavioral deficits were observed in injured pups after vaginal delivery. In pups exposed to the same prenatal insults of IUI+LPS but delivered abdominally, hemorrhages were minimal, neurological function was similar to controls, and myelination and axonal development were significantly better persevered. Additionally, in susceptible animals, preventing or minimizing the impact of microhemorrhages through TLR4 antagonism or genetic deletion, or postnatal iron chelation resulted in improved outcomes. This work is the first to demonstrate the devastating role that neonatal brain microhemorrhages play in the overall pathology of neonatal brain function. This work has shown experimentally that the grave aftereffects of the combined prenatal insults can be significantly ameliorated if microhemorrhages are avoided, for example, by Caesarian-section (C-section) or possibly, by appropriate pharmacological prophylaxis of the high risk mother or affected infant.
    • Neuroprotective role of nicotinamide adenine dinucleotide precursor in modulation of mitochondrial fragmentation and brain energy metabolism

      Klimova, Nina; Kristian, Tibor (2019)
      Nicotinamide adenine dinucleotide (NAD+) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD+ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD+ results in impaired mitochondrial and cellular functions. Administration of NAD+ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age associated physiological decline, and inhibit post-ischemic NAD+ degradation and cellular death. In this work we identified a novel link between NAD+ metabolism and mitochondrial dynamics. A single dose (62.5mg/kg) of NMN, administered in naïve animals and after animals are subjected to transient forebrain ischemia, increases hippocampal mitochondria NAD+ pools and drives a sirtuin 3 (SIRT3) mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species (ROS) levels via SIRT3 driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 (S616)), with mitochondria. In conclusion, manipulation of mitochondrial NAD+ levels by NMN results in metabolic changes that protect mitochondria against ROS and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.