Oxidative Stress

Oxidation by reactive oxygen species is the major source of RNA damaging insult in living organisms. Increased RNA oxidation has been strongly implicated in a wide range of human diseases; predominantly neurodegeneration. Oxidized RNA should be removed from the cellular system to prevent their deleterious effect to the cells and organisms. In eukaryotic cells, mitochondria are the major intracellular sources of ROS and may cause greater damage to the mitochondrial RNA. In this study, we first investigated the RNA oxidation, by measuring the level of 8-hydroxy-Guanosine (8-oxo-Guo), inside mitochondria and cytoplasm in cultured human cells. We discovered that the mitochondrial 8-oxo-Guo is higher than its cytoplasmic counterparts under both normal growth and oxidative stress condition. Next, we explored the role of human polynucleotide phosphorylase (hPNPase) in controlling RNA oxidation inside mitochondria and cytoplasm. hPNPase binds to oxidized RNA with higher affinity, reduces the 8-oxo-Guo level in total RNA and protects cells against oxidative stress. In this study, the molecular mechanism of hPNPase in 8-oxo-Guo reduction was investigated. First, the effect of hPNPase activities on the 8-oxo-Guo level in mitochondria and cytoplasm was examined. The knockdown of hPNPase increased both the mitochondrial and cytoplasmic 8-oxo-Guo, whereas overexpression had the opposite effect. Second, our study revealed that hSUV3, an RNA helicase that forms a functional complex with hPNPase in mitochondria, was dispensable in reducing 8-oxo-Guo levels.

The detrimental effects of oxidative stress caused by the accumulation of Reactive
Oxygen Species (ROS) have been acknowledged as major factors in aging, senescence and
several neurodegenerative diseases and conditions such as Parkinson’s disease and stroke
(ischemia/reperfusion). Mammalian models are extremely susceptible to these stresses that
follow the restoration of oxygen after anoxia; however, some organisms including the
freshwater turtle Trachemys scripta can withstand several bouts of anoxia and repeated
reoxygenation without any apparent pathology. T. scripta thus provides us with an
alternate vertebrate model in which we can investigate physiological mechanisms of
neuroprotection without the damaging effects that come with oxidative stress. The major
objective of this study was to investigate the protective mechanisms in the turtle brain
under conditions of anoxia and oxidative stress. Specifically, the focus is on the Methionine Sulfoxide Reductase system (Msr), an antioxidant and cellular repair system,
and how it is regulated to protect the brain against such stressors.
Previous studies in my lab have demonstrated that Msr mRNA and protein levels
are differentially upregulated during anoxia and reoxygenation. To investigate the
regulation of Msr, FOXO3a was directly induced by transfecting a human FOXO3a
plasmid into turtle brain cell cultures, as FOXO3a has been shown to regulate MsrA levels
in other animal models. Pharmacological manipulation of FOXO3a was also performed
using the green tea extract Epigallocatechin gallate (EGCG) as it has been shown to
increase expression of FOXO3a during oxidative stress conditions in other models. I found
that an induction of human FOXO3a increased FOXO3a levels and showed protection
against cell death during oxidative stress. Furthermore, treatment of cells with EGCG
increased expression of FOXO3a only when the cells were exposed to oxidative stress and
decreased cell death. Induction of FOXO3a and EGCG treatment did not increase MsrA
levels, however MsrB3 levels were upregulated under both treatments but only in the
presence of oxidative stress. These results suggest that MsrA and MsrB3 protect the cells
from oxidative stress damage through different molecular pathways and that EGCG may
be a therapeutic target to treat diseases related to damage by oxidative stress.

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways.

Stroke is one of the leading causes of human death in the United States. The debilitating effects of an ischemic stroke are due to the fact that mammalian neurons are highly susceptible to hypoxia and subsequent oxygen reperfusion. From studies in Drosophila melanogaster, cGMP-dependent Protein Kinase (PKG) enzyme is thought to affect anoxia tolerance by modifying the electrical current through potassium ion channels. In this research, two animal models were employed: Drosophila melanogaster and mammalian neurons exposed to stroke-like conditions. First, in vivo studies using Drosophila were performed to further our knowledge about the differences between the naturally occurring variants of the Drosophila foraging gene, which shows different protein levels of PKG. Mitochondrial density and metabolic activity between two fly genotypes exposed to anoxia and reoxygenation were compared. It was found that flies with less enzyme potentially showed mitochondrial biogenesis and higher metabolic rates upon reoxygenation. Next, in vivo studies where PKG enzyme was activated pharmacologically were performed; it was found that the activation of the cGMP/PKG pathway led to neuroprotection upon anoxia and reoxygenation. Furthermore, this model was translated into the in vitro model using Drosophila cells. Instead of anoxia and reoxygenation, hypoxia mimetics and hydrogen peroxide were used to induce cellular injury. After showing the cGMP/PKG pathway activation-induced cell protection, the potential downstream targets of the molecular signaling as well as underlying biochemical changes were assessed. It was found that mitochondrial potassium ion channels were involved in the protective signaling and the signaling modulated metabolic function. Furthermore, it was found that acidosis protected Drosophila cells from cell death, metabolic disruption, and oxidative stress. Finally, this research was translated to a mammalian in vitro model of neuronal damage upon stroke-like conditions; there, it was demonstrated that the cGMP/PKG pathway activation in rat primary cortical neurons and human cortical neurons was protective from low oxygen and acute oxidative stress. The results of this study lead to a better understanding of molecular mechanisms taking place during low oxygen and oxidative stresses. Consequently, this knowledge may be used to identify potential therapeutic targets and treatments that may prevent detrimental neurological effects of an ischemic stroke in humans.

Drosophila melanogaster tolerates several hours of anoxia (the absence of
oxygen) by entering a protective coma. A burst of reactive oxygen species (ROS) is
produced when oxygen is reintroduced to the cells. ROS causes oxidative damage to
critical cellular molecules, which contribute to aging and development of certain agerelated
conditions. The amino acid, methionine, is susceptible to oxidation, although this
damage can be reversed by methionine sulfoxide reductases (Msr). This project
investigates the effect of Msr-deficiency on anoxia tolerance in Drosophila throughout
the lifespan of the animal. The data show that the time for recovery from the
protective comma as well as the survival of the animals lacking any Msr activity
depends on how quickly the coma is induced by the anoxic conditions. Insight into
the roles(s) of Msr genes under anoxic stress can lead us to a path of designing
therapeutic drugs around these genes in relation to stroke.

Oxidative stress is considered a major factor in the etiology of age related diseases and the aging process itself. Organisms have developed mechanisms to protect against oxidative damage resulting from increased production of reactive oxygen species during aging. One of the major antioxidant systems is the methionine sulfoxide reductase (Msr) enzyme family. The two major Msr enzymes, MsrA and MsrB, can stereospecifically reduce the S and R epimers, respectively, of methionine sulfoxide in proteins back to methionine. This study, using Drosophila melanogaster, decribes the first animal system lacking both MsrA and MsrB. The loss of either MsrA or MsrB had no effect on lifespan in Drosophila, but loss of MsrB results in a slight decrease in locomotor activity from middle age onward. Double mutants lacking both forms of Msr have a significantly decreased lifespan and decreased locomotor activity at all ages examined. The double Msr mutants had no detectable increase in protein oxidation or decrease in mitochondrial function and were not more sensitive to oxidative stress. These results suggested that other cellular antioxidant systems were protecting the flies against oxidative damage and the decreased life span observed in the double knockouts was not due to widespread oxidative damage. However, one cannot exclude limited oxidative damage to a specific locus or cell type. In this regard, it was observed that older animals, lacking both MsrA and MsrB, have significantly reduced levels of dopamine, suggesting there might be oxidative damage to the dopaminergic neurons. Preliminary results also suggest that the ratio of F to G actin is skewed towards G actin in all mutants. The present results could have relevance to the loss of dopaminergic neurons in Parkinson’s disease.

Aging is a biological process that has many detrimental effects due to the
accumulation of oxidative damage to key biomolecules due to the action of free
radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative
damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each
form has been shown to reduce a specific enantiomer of bound and free oxidized
methionine. Effects of Msr have yet to be studied in the major developmental
stages of Drosophila melanogaster despite the enzymes elevated expression
during these stages. A developmental timeline was determined for MsrA mutant,
MsrB mutant, and double null mutants against a wild type control. Results show
that the Msr double mutant is delayed approximately 20 hours in the early/mid
third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon
is due to an issue gaining mass.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.

The turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide reductases (Msrs), primarily MsrA and MsrB. The Msr system is capable of reversing oxidation of methionines in proteins and Msr subtypes have been implicated in protecting tissues against oxidative stress, as well as, enhancing the longevity of organisms from yeast to mammals. Preliminary data, unpublished results, indicate that MsrA protein and transcripts are elevated by anoxia. A recent study on Caenorhabditis elegans demonstrated that FOXO is involved in activation of the MsrA promoter. Using the turtle MsrA promoter sequence we worked to determine which regions in the promoter are necessary for activation by anoxia. The results of the present study were 1) to prepare a TAT-FOXO3a fusion protein which could penetrate animal cells and 2) to construct a FOXO3a expression vector for transcription studies on MsrA expression.