- Email: [email protected]
98 Mutant huntingtin leads to increased levels of mitochondrial DNA damage and mitochondrial dysfunction in mouse striatal cells
Abstracts / Mitochondrion 10 (2010) 200–242
p140 catalytic subunit. The aim of the current work is to biochemically characterize p55 proteins that individually harbor these seven mutations. Biochemical assays have been set up to address: (1) whether the seven p55 variants bind to and stimulate p140, (2) whether these mutations affect folding or stability of p55; and (3) whether altered DNA binding is associated with any of the p55 variants? Preliminary results suggest that at least some of the variants are defective at stimulating p140 by monitoring the extension of a 0 5 -end-labeled primer hybridized to M13 DNA. The ultimate goal of this work is to discover biochemical defects associated with novel POLG2 mutations that may give insights into the pathogenesis of mitochondrial disease. doi:10.1016/j.mito.2009.12.088
97 Light therapy—A promising new treatment for Parkinson’s disease and other mitochondrial diseases P.A. Trimmer *, K.M. Schwartz, Emily N. Cronin-Furman Morris K. Udall Parkinson’s Research Center of Excellence and Department of Neurology, University of Virginia, Charlottesville, VA 22908, USA
Light therapy (LT) is a novel, safe, non-invasive treatment. Far red to infrared light (600–980 nm) is used world-wide to treat a wide range of human disease conditions. LT induces cellular changes by interacting with photo-acceptors in mitochondria and stimulating primary molecular processes by activating cytochrome c oxidase, stimulating ATP synthesis, increasing energy metabolism and improving cell survival. We are developing LT for the treatment of complex neurodegenerative conditions such as Parkinson’s disease (PD). We have used human transmitochondrial cybrid cell lines to explore the role that mitochondrial dysfunction plays in the pathogenesis of PD. PD cybrid cell lines exhibit altered mitochondrial morphology, reduced oxygen utilization, altered assembly of the electron transport chain, Lewy body formation, and reduced axonal transport of mitochondria. Low level LT with 810 nm (50 mW/cm2 for a total of 2 J) or 980 nm (15 mW/cm2 for a total of 0.6 J) was able to normalize the movement of mitochondria by axonal transport and improve oxygen utilization in many but not all PD cybrid cell lines. The differential response of PD cybrid lines to LT was correlated with the degree of mitochondrial dysfunction. Cell lines with substantial mitochondrial dysfunction were less responsive to LT than cell lines with more intact mitochondrial function. Our results thus far suggest that LT has the potential to become a new treatment for PD and other neurodegenerative diseases characterized by mitochondrial dysfunction. Funds and/or equipment were provided by the NIH, PhotoThera and LiteCure. doi:10.1016/j.mito.2009.12.089
98 Mutant huntingtin leads to increased levels of mitochondrial DNA damage and mitochondrial dysfunction in mouse striatal cells Sylvette Ayala-Torres a,*, Julie K. Andersen b, Carlos A. Torres-Ramos b, David G. Nicholls d, Karina Acevedo-Torres b, Almas Siddiqui d, Sulay Rivera c a Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; b Department of Physiology and Biophysics, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; c Department of Biochemistry, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; d Buck Institute for Age Research, Novato, CA, USA
Huntington’s disease (HD) is an autosomal dominant, typically late-onset neurodegenerative disease characterized by neurodegeneration in the striatum leading to involuntary choreiform movements, behavioral abnormalities, and cognitive impairment.
227
Substantial evidence suggests that oxidative stress and mitochondrial dysfunction may play a role in the neurodegeneration associated with HD. We have recently demonstrated that levels of mitochondrial DNA (mtDNA) damage increase in an age-dependent fashion in two in vivo models of HD. However, the mechanisms by which mutant huntingtin is modulating the levels of mitochondrial oxidative damage that subsequently result in mitochondrial dysfunction are not known. Our hypothesis states that mutant huntingtin causes mitochondrial dysfunction by increasing the levels of oxidative mtDNA damage. To test our hypothesis we employed immortalized striatal neuronal progenitor cell lines derived from the striatum of HD knock-in HdhQ111 mice (expressing 111 glutamine repeats) and wild type Q7 mice (expressing seven glutamine repeats). We determined basal levels of mtDNA damage using quantitative PCR and found that mutant Q111 cells exhibited significantly higher basal levels of mtDNA lesions than wild type Q7 cells (0.51 lesions/10-kb/strand vs. 0.1 lesions/10-kb/strand, respectively). We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)/glycolysis in Q7 and Q111 cells and found that Q111 cells exhibit a decreased OCR (50% lower) and an increased ECAR (52% higher) as compared to Q7 cells. Finally, viability studies after exposure of cells to hydrogen peroxide (H2O2) revealed that mutant Q111 cells were more sensitive to H2O2 mediated cell death than wild type Q7 cells (87% vs. 20% cell survival 24 h after treatment with 200 lM H2O2). We conclude that the increased cell vulnerability to H2O2 caused by mutant huntingtin may result from increased levels of mtDNA lesions and that mtDNA damage may lead to lower OCR/mitochondrial dysfunction in HD. Supported by NIH grants U54-NS039408 and G12RR-03051. doi:10.1016/j.mito.2009.12.090
99 Antibiotic effects on mitochondrial translation and in patients with mitochondrial translational defects Christie N. Jones a,*, Chaya Miller b, Ariel Tenenbaum c, Linda L. Spremulli a, Ann Saada b a Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA; b Metabolic Disease Unit, Hadassah Medical Center, Jerusalem, Israel; c Department of Pediatrics, Hadassah Medical Center, Jerusalem, Israel
The infantile presentation of mitochondrial respiratory chain defects frequently simulates acute bacterial infection and sepsis. Consequently, broad spectrum antibiotic therapy is often initiated before definitive diagnosis is reached and without taking into consideration the potential harm of antibiotics that affect mitochondrial translation. We have demonstrated that translation targeted antibiotics (gentamicin, streptomycin, tetracycline, doxycycline and chloramphenicol) adversely affect the growth of fibroblasts from patients with defective mitochondrial translation systems. These defects arise from mutations in nuclear encoded mitochondrial elongation factor Ts and mitochondrial ribosomal proteins S16 and S22. Our findings show that the cumulative effect of the translation defect and antibiotic is greater than the effect of either alone. The effect was especially prominent for the cells of patients with a defect in the mitochondrial ribosome. In addition, we have shown that these antibiotics inhibit mitochondrial translation in vitro and that, in the case of aminoglycosides, they cause the mis-incorporation of a near cognate amino acid. Our results suggest that patients with mitochondrial translation defects may be more vulnerable to toxicside effects of certain translation-targeted antibiotics. Consequently, these antibiotics should be used with caution in patients with mitochondrial translation defects or undefined mitochondrial disorders. doi:10.1016/j.mito.2009.12.091
p140 catalytic subunit. The aim of the current work is to biochemically characterize p55 proteins that individually harbor these seven mutations. Biochemical assays have been set up to address: (1) whether the seven p55 variants bind to and stimulate p140, (2) whether these mutations affect folding or stability of p55; and (3) whether altered DNA binding is associated with any of the p55 variants? Preliminary results suggest that at least some of the variants are defective at stimulating p140 by monitoring the extension of a 0 5 -end-labeled primer hybridized to M13 DNA. The ultimate goal of this work is to discover biochemical defects associated with novel POLG2 mutations that may give insights into the pathogenesis of mitochondrial disease. doi:10.1016/j.mito.2009.12.088
97 Light therapy—A promising new treatment for Parkinson’s disease and other mitochondrial diseases P.A. Trimmer *, K.M. Schwartz, Emily N. Cronin-Furman Morris K. Udall Parkinson’s Research Center of Excellence and Department of Neurology, University of Virginia, Charlottesville, VA 22908, USA
Light therapy (LT) is a novel, safe, non-invasive treatment. Far red to infrared light (600–980 nm) is used world-wide to treat a wide range of human disease conditions. LT induces cellular changes by interacting with photo-acceptors in mitochondria and stimulating primary molecular processes by activating cytochrome c oxidase, stimulating ATP synthesis, increasing energy metabolism and improving cell survival. We are developing LT for the treatment of complex neurodegenerative conditions such as Parkinson’s disease (PD). We have used human transmitochondrial cybrid cell lines to explore the role that mitochondrial dysfunction plays in the pathogenesis of PD. PD cybrid cell lines exhibit altered mitochondrial morphology, reduced oxygen utilization, altered assembly of the electron transport chain, Lewy body formation, and reduced axonal transport of mitochondria. Low level LT with 810 nm (50 mW/cm2 for a total of 2 J) or 980 nm (15 mW/cm2 for a total of 0.6 J) was able to normalize the movement of mitochondria by axonal transport and improve oxygen utilization in many but not all PD cybrid cell lines. The differential response of PD cybrid lines to LT was correlated with the degree of mitochondrial dysfunction. Cell lines with substantial mitochondrial dysfunction were less responsive to LT than cell lines with more intact mitochondrial function. Our results thus far suggest that LT has the potential to become a new treatment for PD and other neurodegenerative diseases characterized by mitochondrial dysfunction. Funds and/or equipment were provided by the NIH, PhotoThera and LiteCure. doi:10.1016/j.mito.2009.12.089
98 Mutant huntingtin leads to increased levels of mitochondrial DNA damage and mitochondrial dysfunction in mouse striatal cells Sylvette Ayala-Torres a,*, Julie K. Andersen b, Carlos A. Torres-Ramos b, David G. Nicholls d, Karina Acevedo-Torres b, Almas Siddiqui d, Sulay Rivera c a Department of Pharmacology and Toxicology, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; b Department of Physiology and Biophysics, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; c Department of Biochemistry, University of Puerto Rico Medical Sciences Campus, San Juan, PR, USA; d Buck Institute for Age Research, Novato, CA, USA
Huntington’s disease (HD) is an autosomal dominant, typically late-onset neurodegenerative disease characterized by neurodegeneration in the striatum leading to involuntary choreiform movements, behavioral abnormalities, and cognitive impairment.
227
Substantial evidence suggests that oxidative stress and mitochondrial dysfunction may play a role in the neurodegeneration associated with HD. We have recently demonstrated that levels of mitochondrial DNA (mtDNA) damage increase in an age-dependent fashion in two in vivo models of HD. However, the mechanisms by which mutant huntingtin is modulating the levels of mitochondrial oxidative damage that subsequently result in mitochondrial dysfunction are not known. Our hypothesis states that mutant huntingtin causes mitochondrial dysfunction by increasing the levels of oxidative mtDNA damage. To test our hypothesis we employed immortalized striatal neuronal progenitor cell lines derived from the striatum of HD knock-in HdhQ111 mice (expressing 111 glutamine repeats) and wild type Q7 mice (expressing seven glutamine repeats). We determined basal levels of mtDNA damage using quantitative PCR and found that mutant Q111 cells exhibited significantly higher basal levels of mtDNA lesions than wild type Q7 cells (0.51 lesions/10-kb/strand vs. 0.1 lesions/10-kb/strand, respectively). We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)/glycolysis in Q7 and Q111 cells and found that Q111 cells exhibit a decreased OCR (50% lower) and an increased ECAR (52% higher) as compared to Q7 cells. Finally, viability studies after exposure of cells to hydrogen peroxide (H2O2) revealed that mutant Q111 cells were more sensitive to H2O2 mediated cell death than wild type Q7 cells (87% vs. 20% cell survival 24 h after treatment with 200 lM H2O2). We conclude that the increased cell vulnerability to H2O2 caused by mutant huntingtin may result from increased levels of mtDNA lesions and that mtDNA damage may lead to lower OCR/mitochondrial dysfunction in HD. Supported by NIH grants U54-NS039408 and G12RR-03051. doi:10.1016/j.mito.2009.12.090
99 Antibiotic effects on mitochondrial translation and in patients with mitochondrial translational defects Christie N. Jones a,*, Chaya Miller b, Ariel Tenenbaum c, Linda L. Spremulli a, Ann Saada b a Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA; b Metabolic Disease Unit, Hadassah Medical Center, Jerusalem, Israel; c Department of Pediatrics, Hadassah Medical Center, Jerusalem, Israel
The infantile presentation of mitochondrial respiratory chain defects frequently simulates acute bacterial infection and sepsis. Consequently, broad spectrum antibiotic therapy is often initiated before definitive diagnosis is reached and without taking into consideration the potential harm of antibiotics that affect mitochondrial translation. We have demonstrated that translation targeted antibiotics (gentamicin, streptomycin, tetracycline, doxycycline and chloramphenicol) adversely affect the growth of fibroblasts from patients with defective mitochondrial translation systems. These defects arise from mutations in nuclear encoded mitochondrial elongation factor Ts and mitochondrial ribosomal proteins S16 and S22. Our findings show that the cumulative effect of the translation defect and antibiotic is greater than the effect of either alone. The effect was especially prominent for the cells of patients with a defect in the mitochondrial ribosome. In addition, we have shown that these antibiotics inhibit mitochondrial translation in vitro and that, in the case of aminoglycosides, they cause the mis-incorporation of a near cognate amino acid. Our results suggest that patients with mitochondrial translation defects may be more vulnerable to toxicside effects of certain translation-targeted antibiotics. Consequently, these antibiotics should be used with caution in patients with mitochondrial translation defects or undefined mitochondrial disorders. doi:10.1016/j.mito.2009.12.091