This is the competitive renewal of a grant which has been continuously funded for the past 23 years. During this time, we have made novel discoveries in the area of mitochondrial DNA damage and repair and how these processes play an important role in the pathogenesis of disease. The present application uses this work as a foundation to design new translational studies intended to develop innovative protocols to enhance the protection of this DNA in mitochondria. These strategies will be designed to block or delay the onset of type I diabetes and to protect islets used for transplantation against the loss of function and viability that occurs during islet isolation and in the early transplantation period.
The first aim i s designed to mechanistically evaluate and optimize the delivery of recombinant DNA repair enzymes and antioxidants to mitochondria in ?-cells by protein transduction. These studies are structured to develop a more thorough understanding of the mechanisms involved in the protection of ?-cells by protein transduction and to optimize the delivery of recombinant DNA repair and antioxidant proteins into mitochondria of ?-cells by the TAT peptide.
The second aim will evaluate the effects of the recombinant proteins developed in the first aim on the pathogenesis of diabetes in two animal models of type 1 diabetes. These studies are designed to determine whether the most effective recombinant protein or proteins, identified in the first aim to block ?-cell toxicity caused by ROS, RNS and cytokines, can block the onset of diabetes in two animal models of type I diabetes when delivered into these animals by protein transduction. One animal model to be studied is the NOD mouse because diabetes in these animals appears to result from purely autoimmune mechanisms and recent work with these animals has indicated that there is a role for mtDNA in the pathogenesis of their disease. The second animal model to be studied is that produced by subdiabetogenic doses of the ?-cell toxin streptozotocin (STZ). Transgenic mice which over-express the recombinant proteins in mitochondria of ?-cells will be used for proof of principle. This model was chosen because diabetes is initiated by a ?-cell toxin, STZ, and is associated with islet inflammation which resembles that seen in human type I diabetes.
The final aim will evaluate the ability of fusion proteins to enhance the viability of islets to be used for transplantation. Although recently islet transplantation has proven to be a promising approach for the treatment of type I diabetes, this procedure has been plagued with problems relating to islet viability. The studies in this aim will explore whether the fusion proteins developed in the first aim can be used to help overcome this problem.
|Alexeyev, Mikhail; Shokolenko, Inna; Wilson, Glenn et al. (2013) The maintenance of mitochondrial DNA integrity--critical analysis and update. Cold Spring Harb Perspect Biol 5:a012641|
|Shokolenko, Inna N; Fayzulin, Rafik Z; Katyal, Sachin et al. (2013) Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance. J Biol Chem 288:26594-605|
|Yuzefovych, Larysa; Wilson, Glenn; Rachek, Lyudmila (2010) Different effects of oleate vs. palmitate on mitochondrial function, apoptosis, and insulin signaling in L6 skeletal muscle cells: role of oxidative stress. Am J Physiol Endocrinol Metab 299:E1096-105|
|Shokolenko, Inna; Venediktova, Natalia; Bochkareva, Alexandra et al. (2009) Oxidative stress induces degradation of mitochondrial DNA. Nucleic Acids Res 37:2539-48|
|Grishko, V I; Ho, R; Wilson, G L et al. (2009) Diminished mitochondrial DNA integrity and repair capacity in OA chondrocytes. Osteoarthritis Cartilage 17:107-13|
|Rachek, Lyudmila I; Yuzefovych, Larysa V; Ledoux, Susan P et al. (2009) Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes. Toxicol Appl Pharmacol 240:348-54|
|Druzhyna, Nadiya M; Wilson, Glenn L; LeDoux, Susan P (2008) Mitochondrial DNA repair in aging and disease. Mech Ageing Dev 129:383-90|
|Pastukh, Viktoriya; Shokolenko, Inna N; Wilson, Glenn L et al. (2008) Mutations in the passenger polypeptide can affect its partitioning between mitochondria and cytoplasm: mutations can impair the mitochondrial import of DsRed. Mol Biol Rep 35:215-23|
|Alexeyev, M F; Venediktova, N; Pastukh, V et al. (2008) Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther 15:516-23|
|Harrison, Jason F; Rinne, Mikael L; Kelley, Mark R et al. (2007) Altering DNA base excision repair: use of nuclear and mitochondrial-targeted N-methylpurine DNA glycosylase to sensitize astroglia to chemotherapeutic agents. Glia 55:1416-25|
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