Browsing by Subject "Mitochondria"

Now showing 1 - 15 of 15

Biochemical and genetic studies of mitochondrial protein synthesis in Saccharomyces cerevisiae : characterization of the AEP3 and TRM5 gene products
(2008-08) Lee, Changkeun, 1971-; Appling, Dean Ramsay
Protein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.
Characterization of MTHFD2L expression and alternative splicing and loss of MTHFD1L activity in murine embryos and adults
(2017-05) Bryant, Joshua Dale; Appling, Dean Ramsay; Drew, Michael; Finnell, Richard; Hoffman, David; Mills, Edward; Tiziani, Stefano
In Eukaryotes, folate-dependent one-carbon (1C) metabolism is a highly compartmentalized process in which mitochondria play a central role. Defects in folate metabolism are associated with diseases such as cancer, Alzheimer's disease, and neural tube defects (NTDs). 1C units are attached to tetrahydrofolate (THF) and carried in various oxidation states between folate-dependent enzymes. There is an exchange of 1C units across the mitochondrial membrane, with 1C donors such as serine and glycine being oxidized to formate in the mitochondria, which is then released into the cytoplasm. 1C units in the cytoplasm can be used for the synthesis of purines, thymidylate, and methionine for the methyl cycle. The core of the pathway in both compartments is catalyzed by the methylene-tetrahydrofolate (MTHFD) gene family. These enzymes catalyze the reversible interconversion between CH₂-THF, CH⁺-THF, CHO-THF, and formate. The cytoplasmic protein MTHFD1 is trifunctional and carries the CH₂-THF dehydrogenase, CH⁺-THF cyclohydrolase, and 10-CHO-THF synthetase activities necessary to carry out these interconversions. In the mitochondria, two bifunctional isozymes, MTHFD2 and MTHFD2L, carry the dehydrogenase/cyclohydrolase (D/C) activities. The monofunctional enzyme MTHFD1L is responsible for the synthetase activity. MTHFD2 is only expressed in embryos and transformed cells, and the enzyme responsible for the D/C activity in adults was unknown until the recent discovery of MTHFD2L. In this work, characterization of the expression of MTHFD2L in mouse embryos and adults is described. Expression of MTHFD2L in embryos was found to be switched on between embryonic days 8.5-10.5, and remains high throughout development. MTHFD2L is also widely expressed in adults, with highest expression in brain and lung. A splice variant of MTHFD2L lacking exon 8 was found to be abundant in embryos but was not catalytically active in vitro or in vivo. MTHFD1L is an essential protein, and SNPs in MTHFD1L are associated with increased risk for Alzheimer's disease and NTDs in humans. Loss of MTHFD1L activity in adult mice with and without a folate deficient diet was investigated. Indications of sex-dependent behavioral anomalies were found, with evidence for genotype-dependent hyperactivity in male mice and diet-dependent anxiety in female mice, but further investigation of these findings is warranted. Finally, metabolic defects associated with NTDs and growth restriction in MTHFD1L-null (Mthfd1l [superscript z/z]) embryos were identified. Glycolysis, the TCA cycle, and the metabolism of methionine, purines, and multiple amino acids were found to be disrupted in Mthfd1l [superscript z/z] embryos. These altered metabolic pathways suggest potential future therapies for preventing NTDs in humans.
Characterization of the role of the mitochondrial one-carbon metabolism during embryonic development
(2016-12) Shin, Minhye; Appling, Dean Ramsay; Finnell, Richard; Hoffman, David; Fast, Walter; Vokes, Steven
Neural tube defects (NTDs) are one of serious structural birth defects resulting from failure of neural tube closure. Folic acid supplementation is the essential factor for prevention of NTDs. Folate-dependent one-carbon metabolism is a central metabolic pathway participating in a diverse range of metabolic reactions. Mitochondrial one-carbon metabolism is crucial for production of formate as a major 1C donor to the cytoplasm and regeneration of redox cofactors. Mitochondrial MTHFD family enzymes, MTHFD2, MTHFD2L, and MTHFD1L, are major contributors for formate production in mammalian mitochondria. The MTHFD2 and MTHFD2L isozymes possess both CH₂-THF dehydrogenase and CH⁺-THF cyclohydrolase activities, catalyzing the reaction of 10-CHO-THF production in mitochondria. The dehydrogenase activity of these bifunctional enzymes can use either NAD⁺ or NADP⁺ with dual redox cofactor specificity, but requires both phosphate and Mg²⁺ when using NAD⁺. The NADP⁺-dependent dehydrogenase activity is inhibited by inorganic phosphate. With polyglutamylated THF substrate, both of MTHFD2 and MTHFD2L show higher NADP⁺-dependent activity than the monoglutamylated substrate. Phylogenetic analysis indicates that MTHFD2L may be evolved from invertebrate MTHFD2 which is homologous to a primitive fungal MTHFD1. MTHFD1L is expressed ubiquitously throughout the embryogenesis during neural tube closure, and significantly detected at the basal surface of the dorsal neuroepithelium. Lacking Mthfd1l causes retardation in growth and developmental progression. Mthfd1l knockout mouse embryos show defects in proliferation during late neural tube closure and head mesenchyme development during early neural tube closure. However, proliferation during early neural tube closure, apoptosis, and neural crest cell migration were not affected by the loss of Mthfd1l. Finally, we show that maternal formate supplementation significantly improves the dysregulated cellular processes in Mthfd1l [superscript z/z] embryos. This study elucidates the specific metabolic mechanisms underlying folate-associated birth defects, including NTDs.
Defining the role of Mtf1 and N-terminal domain of Rpo41 in transcription initiation and replication
(2012-05) Chang, Hae Ryung; Yin, Yuhui Whitney; Hackert, Marvin L.; Jayaram, Makkuni; Johnson, Kenneth; Molineux, Ian
Mitochondrion is an organelle found in the eukaryotic cell. It is responsible for essential metabolic processes as well as ATP production via oxidative phosphorylation (OXPHOS). The mitochondrion contains DNA that encodes for several subunits in the OXPHOS system as well as rRNA and tRNA for translation. It also has its own replication, transcription and translation machinery. Proper maintenance of the mitochondrial DNA is critical for the cell’s health. Saccharomyces cerevisiae mitochondrial transcription system has been a great model system for its ease of genetic manipulation as well as having conserved RNA polymerases across species. The polymerases are homologues to T7 RNA polymerase, but have longer N-terminal domain and require transcription factor(s). The reason for the extra domain as well as the need for an accessory factor is still unclear. This study reveals the role of Rpo41 N-terminal domain (NTD) as well as clarifies the role of Mtf1, the transcription factor, in transcription initiation. Rpo41 is the 153 kDa catalytic subunit, and Mtf1 is 40 kDa, the transcription factor of the yeast mitochondria. We have shown that Mtf1 is required for correct promoter sequence recognition as well as inhibition of incorrect initiation. Although it was thought that Rpo41 has intrinsic promoter recognition capability, we have shown that Rpo41 can initiate transcription on a pre-melted DNA, even if it is not the consensus promoter sequence. N-terminal truncation mutant studies showed that the NTD of Rpo41 is also required for correct transcription initiation. On linear duplex DNA, N-terminal truncation of 321 amino acids has little effect when Mtf1 is present. On pre-melted DNA, it shows opposite trend from the wild-type. 160 N-terminal amino acid residue truncation shows little activity, whereas Mtf1 increases activity, even on non-promoter initiation sites. We further investigated properties of Rpo41 in replication. A link between mitochondrial transcription and replication has been suggested before, where Rpo41 functions as the leading strand primase. Our studies show that Rpo41 can indeed function as the leading and lagging strand primase, and explains why Rpo41 is able to initiate transcription on non-promoter sites. N-terminal truncation resulted in loss of primase activity, which shows that NTD is required for replication.
Electrical effects and computer simulations of PhotoBioModulation of the brain
(2023-08-21) Huang, Li-Da; Abraham, Jacob A.; González-Lima, Francisco, 1955-; Pan, David Zhigang; Akinwande, Deji; Liu, Hanli
Brain functions have been shown to be affected by external stimuli. Low- Level-Light Therapy (LLLT) using nearinfrared photons is one of the effective ways to modulate the hemodynamic activities in the brain. However, the biphasic hormetic dose-response where bioenergetics are stimulated at a low dose and inhibited at a high dose is well observed in all photon stimulations. The amount of photon energy delivered to the brain are affected by the wavelength as well as the multilayered head structure with variations of optical parameters (OPs). A real 3D volume head model is built for each participant in this study, and the boundary conditions of each OP in each layer is considered. The Monte Carlo simulation with wavelengths ranging from 650 nm to 1064 nm is implemented to investigate the energy delivered to the brain under different radiation profiles. Results show that 1064-nm photons penetrate deeper than 810-nm photons except for scalp absorption at the lower bound due to low melanin content. Collimated-beam radiation is better than diverging-beam due to a more uniform intensity distribution at the scalp surface. Further research to optimize LLLT dosage for each individual is imperative due to the high inter-person variability in structure and OPs. In addition, EEG measures voltage fluctuations resulting from ionic current within the neurons of the brain. Because ATP is the major energy unit consumed by the ion pump and gate, PhotoBioModulation(PBM) will theoretically affect EEG. We establish the relationship between PBM and EEG demonstrated that PBM could increase resting-state alpha, beta, and gamma power by an increment of prefrontal blood oxygen level.
Fidelity of nucleotide incorporation by the human mitochondrial DNA polymerase
(2005) Lee, Harold Ray, 1978-; Johnson, Kenneth A. (Kenneth Allen)
The human mitochondrial DNA polymerase (pol γ) is a nuclearly encoded enzyme, imported to the mitochondria, solely responsible for the replication of the mitochondrial genome. I have characterized the kinetics of nucleotide incorporation, determined the discrimination constants for misincorporation, and calculated the overall fidelity of this enzyme. Additionally, I have investigated the dependence of these parameters on the concentration of magnesium ion present in the reaction. There have been reports in the literature of pol γ having reverse transcriptase activity, and of attempts to determine a physiological role for this activity. Indeed, many steady-state kinetic assays of this enzyme reported in the literature are reverse transcriptase assays. I have characterized the kinetics of incorporation of the reverse transcriptase activity of polymerase γ, both in single turnover and processive polymerization assays. Additionally, I have characterized the activity of the 3′-5′ exonuclease domain on a DNA/RNA heteroduplex. For many years there has been research into the factors that contribute to polymerase discrimination. Base pair hydrogen bonding, base stacking, steric interactions, active site tightness, and factors unknown are all believed to play a role. However, recent attempts to investigate the contribution to discrimination afforded by base pair hydrogen bonding in Klenow fragment, using “shape mimic” nucleoside analogs have led to the supposition that base pairing plays little, if any, role in discrimination. In order to investigate the contribution of base pair hydrogen bonding to discrimination in pol γ, I have characterized the incorporation of natural nucleotides opposite the dT analog 2, 4-difluorotoluene deoxynucleoside (dF) and the dA analog 9- (1-aza-4-methyl-benzimidazolyl)-1′-β-2′-deoxyriboside (dQ). Additionally, the kinetic parameters of incorporation of dF opposite dT, and of dQ and 4-methylbenzimidazole (dZ) opposite dA have been determined. The rates of 3′-5′ exonuclease removal of natural nucleosides paired opposite dF and dQ have been determined as well. The mitochondrial polymerase is the sole enzyme responsible for replication of the mitochondrial genome. In the absence of accurate and efficient replication by polymerase γ, several clinical pathologies are observed including cardiac and neural myopathy, mitochondrial myopathy, anemia, and potentially fatal lactic acidosis. These symptoms are known effects of oxidative damage to the mitochondrial genome and of toxicity associated with the treatment of HIV infection with nucleotide reverse transcriptase inhibitors. Additionally, several diseases have been associated with mitochondrial genome mutation and depletion including Parkinson’s disease and Alzheimer’s disease. A full understanding of the fidelity of the mitochondrial DNA polymerase and the mechanisms by which this fidelity is insured will aid in the understanding of diseases associated with mitochondrial damage and in the design of drugs used to fight HIV, lacking the potentially fatal mitochondria based toxicities.
Mechanisms of impaired mitochondrial one-carbon metabolism and drug exposures to valproic acid or dolutegravir in neural tube defects
(2020-05-14) Steele, John William; Finnell, Richard H.; Appling, Dean Ramsay; Agarwala, Seema; Wallingford, John B; Gross, Steven S
Neural tube defects (NTDs) are among the most severe and prevalent human congenital malformations. Their etiology is complex and multifactorial, influenced by dynamically interacting genetic and environmental factors. It is well known that maternal dietary folate status is the greatest modifying factor associated with risk for NTD-affected pregnancies, and that dietary fortification of folic acid (FA) can prevent a significant proportion of NTDs. However, many NTDs have proven to be FA-resistant, presenting a need to understand mechanisms underlying these FA-resistant defects and develop novel intervention strategies targeting this population. A class of FA-resistant NTD mouse models have been developed by inactivating genes associated with mitochondrial one-carbon metabolism (mOCM). Thus, Part One of this work sought to elucidate mechanisms by which impaired mOCM results in FA-resistant NTDs. By crossing mice heterozygous for loss of Slc25a32, a gene coding for the mitochondrial folate transporter, with mice heterozygous for the Crooked Tail (Cd) allele of Lrp6, a gene coding for a Wnt co-receptor, it was discovered that a proportion of resulting co-heterozygous offspring presented with NTDs, suggesting a novel digenic interaction between Lrp6 and mOCM. Further experiments demonstrated that Lrp6 regulates expression of mOCM genes in mouse embryos, while CHO cells lacking Slc25a32 demonstrated impaired Wnt signaling rescued by the one-carbon donor, glycine. Building on those data, it was discovered that maternal glycine supplementation could prevent NTDs in Slc25a32 null embryos, and that glycine or serine supplementation may reduce NTDs in Lrp6 Cd mice. Part One experiments also demonstrated that embryonic stem cells lacking Mthfd1l, another mOCM gene, have proliferation defects and are sensitive to hypoxia. Environmental exposure to certain pharmaceutical compounds also increases risk for NTDs. Part Two of this work sought to identify mechanisms of NTD pathology associated with two common pharmaceuticals, the anticonvulsant, valproic acid (VPA), and the HIV integrase inhibitor, dolutegravir (DTG). Untargeted metabolic profiling was performed on VPA-treated mouse embryos, and predictive biomarkers of VPA sensitivity were identified by comparing VPA-affected and unaffected embryos. Other experiments identified a novel, calcium-enhanced interaction between DTG, folate, and folate receptor, suggesting a plausible mechanism by which DTG may enhance NTD risk.
Mechanistic insights into the function of the mitochondrial uncoupling protein in Caenorhabditis elegans
(2010-08) Pfeiffer, Matthew Edwin; Mills, Edward M.; Bratton, Shawn B.; Wright, Casey W.; Combs, Alan B.; Morgan, Philip G.
The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics.
Mitochondrial uncoupling links lipid catabolism to Akt inhibition and blockade of skin tumorigenesis
(2014-08) Nowinski, Sara Marie; Mills, Edward Michael
In order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling.
Neurodegeneration caused by mitochondrial complex I dysfunction in the mouse retina
(2005) Zhang, Xian; González-Lima, Francisco
Neurodegenerative diseases have been closely linked to dysfunction of mitochondria. For example, Leber's hereditary optic neuropathy is associated with mutations in mitochondrial complex I. To create an in vivo animal model of neurodegeneration for studying the mechanisms and treatments of neurodegenerative diseases, the mouse eye was injected with the pesticide rotenone, a specific mitochondrial complex I inhibitor. Then, the neurotoxicity of rotenone on the retina was characterized at the cellular level. Finally, a therapeutic intervention was tested using the model. A dose of methylene blue was found to effectively prevent the neurodegeneration caused by rotenone. Following intravitreal injection of rotenone, the retinal ganglion cell layer (GCL) and the retinal nerve fiber layer (RNFL) showed degeneration as indicated by viii the reduction of their thicknesses. The maximum reduction in the GCL and RNFL thickness in complex I staining was around 40% and 89% respectively at 24 h. The GCL thickness reduction was also verified with cresyl violet staining. The number of GCL cells was reduced by 21% (cell profile counts) and 23% (unbiased stereological cell counts) in rotenone-treated eyes. There was a preferential reduction in the proportion of larger cells, while no overall cellular morphometric changes (soma area, perimeter, and diameter) were observed. Therefore, the reduction in GCL thickness 24 h after rotenone microinjection could be accounted for by cell loss and nerve fiber shrinkage, but not by overall soma size change. This optic neuropathy model was used to test the hypothesis that methylene blue, a reduction–oxidation agent that can act as a powerful antioxidant, may be protective against rotenone. Rotenone-induced neurodegeneration in the retinal ganglion cell layer 24 h after injection was completely prevented by the injection of methylene blue along with rotenone, as indicated by both the GCL thickness and cell numbers. This is the first animal model of optic neuropathy resulting from mitochondrial dysfunction, and our studies suggest that it could be used as a convenient means to test new treatments to prevent neurodegeneration. It was concluded that methylene blue may be a promising therapeutic agent in optic neuropathy and perhaps other neurodegenerative diseases caused by mitochondrial dysfunction
Regulation of DIAP1 function by Dropsophila Omi and the N-end rule pathway
(2007-12) Malladi, Madhavi, 1976-; Bratton, Shawn B.
The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.
The role of the human mitochondrial polymerase in the toxicity of nucleoside analogs and aging
(2004) Hanes, Jeremiah Wayne, 1977-; Johnson, Kenneth A. (Kenneth Allen)
The toxic side effects associated with the administration of nucleoside analogs used to treat HIV are correlated with the kinetics of incorporation by the human mitochondrial polymerase γ (Pol γ). The reconstitution of recombinant human enzyme has allowed for a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. One nucleoside analog in particular, zidovudine (AZT), exhibits unique kinetics of incorporation by Pol γ. Evidence is presented supporting a model in which the kinetics of incorporation of AZT differ from that of natural nucleotides and other nucleoside analogs, in that phosphoryl transfer is reversibly linked to binding by way of a slower than normal enzyme isomerization following phosphoryl transfer. The implication of the unique reaction kinetics on the toxicity associated with the use of AZT is also discussed. The most toxic nucleoside analog approved for treatment of HIV is zalcitabine (ddC). It was shown previously that the incorporation of ddC by Pol γ is relatively efficient and that the removal by the proofreading exonuclease was too slow to measure under the conditions used. Both efficient incorporation and slow removal are likely to contribute to the observed clinical toxicity. I revisited this phenomenon by measuring the rate of excision more accurately and under various conditions in order to better understand the mechanistic basis for the slow removal of ddC. In addition to a role in the toxicity of nucleoside analogs used to treat HIV, Pol γ may play an important part in the process of aging. The mitochondrial theory of aging states that electrons derived from the electron transport chain during normal respiration produce especially high levels of reactive oxygen species (ROS) in the mitochondria. These ROS can damage the mitochondrial genome, compromising its integrity. One of the most common products of oxidative damage to DNA is 8-oxodG. I have examined the kinetic parameters governing the replication of oxidatively damaged DNA by Pol γ and show that replication fidelity is reduced when incorporation is performed when 8-oxodG is the templating base. I provide evidence suggesting that oxidized free nucleotide (8-oxodGTP) may also be mutagenic.
Targeting mitochondria via methylene blue : implications in memory enhancement and neuroprotection
(2015-05) Auchter, Allison Michelle; González-Lima, Francisco, 1955-; Monfils, Marie-H.; Schallert, Timothy; Haley, Andreana; Barea-Rodriguez, Edwin
Memory—though seemingly simple in concept—is altogether a notoriously elusive and difficult process to understand. While some memories are fragile and changeable under certain circumstances, others are persistent and difficult to erase. This work is a comprehensive investigation into memories: how to change persistent ones, enhance complicated ones and protect delicate ones. The first objective of this work was to explore a strategy for manipulating fear memories, which are notoriously difficult to erase. This was done by testing different parameters for attenuation of fear using extinction as a strategy for interrupting fear memory reconsolidation. Briefly, by manipulating extinction parameters such that subjects were unable to predict the occurrence of fearful stimuli, we were able to maximize the degree of memory updating. Since memory processing is contingent upon specific neuronal activity, and neuronal activity is primarily fueled by products of mitochondrial respiration, the second and third objectives of this work focused largely on how manipulating mitochondrial activity enhances and protects memory processes respectively. This was done using mitochondrial enhancer methylene blue (USP grade). Methylene blue (MB) is a synthetic dye with a unique ability to cross the blood-brain barrier and diffuse into neuronal mitochondria. There, it serves as a redox electron cycler, increasing neurons’ capacity for mitochondrial respiration. The second objective of this work examined the effect of post-extinction administration of MB and its interaction with reconsolidation update mechanisms in the persistent attenuation of fear memories. Building on findings from the first objective, we found that administering MB after extinction helped subjects maintain the fear attenuation induced by extinction. The third objective explored the ability of MB to prevent the cognitive deficits arising from chronic cerebral hypoperfusion, a risk factor for mild cognitive impairment and Alzheimer’s disease. Finally, quantitative cytochrome oxidase histochemistry (a marker for mitochondrial activity) was used to map the effects of MB on brain mitochondria in the hypoperfusion model. Methylene blue attenuated some of the cognitive deficits that arose from chronic cerebral hypoperfusion, which was reflected in enhanced brain mitochondrial activity.
The effects of inertial load ergometry training and pomegranate juice supplementation on muscle mass and aerobic power in older adults
(2020-04-20) Allen, Jakob Richard; Coyle, Edward F., 1952-; Tanaka, Hirofumi; Kohl, Harold W; Bray, Molly
Skeletal muscle mass is a primary determinant of the ability to maintain autonomy later in life. The reductions in skeletal muscle mass, beginning after the 3 [superscript rd] decade of life influence not only the ability to produce maximal neuromuscular power (P [subscript max]), but possibly aerobic power generation. The primary purpose of study one was to investigate the effect of inertial load ergometry (ILE) training on skeletal muscle mass and cardiovascular function in untrained 50-70 year old subjects (n=30-39). Then secondarily, to determine whether pomegranate juice (POM) supplementation augments those changes compared to a placebo (PLA) group. Over the course of 8 weeks both groups performed exercise training 3 times per week. Each session involved repeated (15-30x) 4s sprints on the ILE, with each sprint designed to elicit maximal power while simultaneously maintaining an elevated oxygen consumption (VO₂) due to a relatively short 30-60 s recovery interval. Training increased thigh muscle volume (TMV) 3.7 ± 0.9% (p<0.001) for the entire population. Furthermore, in the entire population training increased Total Body Mass 1.36 ± 0.33%, Total Body Lean Mass (TBLM) 1.5 ± 0.4%, P [subscript max] 12.0 ± 1.5%, Peak Oxygen Consumption (VO₂peak) 9.8 ± 1.8%, Power at VO₂ [subscript peak] 8.2 ± 1.5%, Ventilatory Threshold (VT) 7.0 ± 2.4%, and functional tests of living 8.5 ± 1.3%, to 17.2 ± 2% (all p<0.05). There were no statistically significant differences in response to training between POM and PLA for any of these measures. These results show that 8 weeks of ILE training was effective at increasing muscle mass, cardiovascular capacity and functional tasks in untrained 50-70 year old adults, with no further effect of POM supplementation
Ultrastructural changes in synaptic and mitochondrial structure throughout postnatal development and long-term potentiation in rat hippocampus
(2015-08) Smith, Heather Lynne; Harris, Kristen M.; Aldrich, Richard; Raab-Graham, Kimberly F.; Schallert, Timothy; Zemelman, Boris
Mitochondria, by providing the vast majority of ATP produced in neurons, fuel many steps of the synaptic vesicle cycle, but little is known about their role in synaptic vesicle clustering and mobilization during synaptic development and plasticity. Long-term potentiation (LTP), a cellular model for learning and memory, has a protein synthesis dependent late phase (L-LTP) that has been shown to recruit both pre- and postsynaptic mechanisms. In spite of their seeming importance in synaptic function, mitochondria are only found in roughly half of all mature presynaptic boutons, which implies that only a subset of boutons are capable of supporting increases in vesicle release. Preliminary exploration of CA1 boutons from perfusion- xed P15 rats, however, demonstrated that while smaller than boutons that contain mitochondria, boutons that are within three microns of mitochondria contain significantly more vesicles than those that are farther away from them, which raises the possibility that diffusion of ATP from nearby mitochondria could be sufficient to fuel LTP. To determine whether this was the case, I prepared 3D reconstructions from serial electron micrographs (3DEM) in order to quantify mitochondrial distance and vesicle counts in CA1 boutons following long-term potentiation in both P15 and adult Long-Evans rats. At both ages, mobilization of reserve pool vesicles following LTP required the presence of mitochondria. Mitochondria themselves also undergo structural changes following LTP. In adults, mitochondria become longer and less frequent along axons, implying that they undergo fusion. Although P15 mitochondria undergo no change in frequency, mitochondrial cristae become wider as mitochondrial matrix becomes more compact at both ages, which are both changes that correlate well with increased rates of respiration. To even further explore ultrastructural components required to support LTP, I used 3DEM to track changes in synaptic and subcellular structure from P8 to P12, during which animals undergo radical changes in their ability to support LTP. I found that spinogenesis may begin at P10 but increases sharply at P12, which coincides perfectly with the onset age of LTP. In conjunction with this increase in dendritic spines, axons built new single synaptic boutons from clusters of dense core and amorphous vesicles previously known to transport proteins and membrane to developing synapses. To fuel the creation of new boutons, mitochondrial division increased at P12, as evidenced by decreased mitochondrial size and increased mitochondrial frequency.