Mitochondrial pathology

The central premise of this dissertation is that mitochondrial antioxidant enzymes
are essential to lens cell viability by preserving lens cell mitochondria and protecting
and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes,
Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored.
In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization
to the lens cell mitochondria, its ability to be induced by H20 2-oxidative
stress, and speculate how PRDX3 function/sf could affect the lens. We will also examine
the reduced levels of MsrA by targeted gene silencing and its effect on reactive oxygen
species production and mitochondrial membrane potential in human lens cells to
determine its role in mitochondrial function in the lens. Lastly, we will examine the
ability of MsrA to repair and restore function to a critical mitochondrial protein,
Cytochrome c. The collective evidence strongly indicates that the loss of mitochondrial-specific enzymes, such as PRDX3 and MsrA, are responsible for increased reactive
oxygen species levels, decreased mitochondrial membrane potential, protein aggregation
and lens cell death, and further indicates that mitochondrial repair, protective, and
reducing systems play key roles in the progression of age-related cataract and other agerelated
diseases.

The excess generation of Reactive oxygen species (ROS) can damage
cell components and disrupt cellular functions. Methionine in proteins is easily
oxidized by ROS and converted to methionine sulfoxide. The enzyme peptide
Methionine Sulfoxide Reductase reduces methionine sulfoxide back to methionine.
We report here that MsrA over expression in rat cardiac myocytes prevents damage
from ROS and increases cell viability after hypoxic/reoxygenation events. The nonsteroidal
anti-inflamatory drug (NSAID) sulindac contains a methyl sulfoxide moiety
that can scavenge ROS. Sulindac can be reduced by MsrA and contribute as an
antioxidant in the cell. Our results demonstrate that 1 OOuM sulindac can reduce cell
death in rat cardiac myocytes during hypoxia/reoxygenation, and
ischemia/reperfusion in Langendorf[ perfusions. The BNIP proteins are pro-apoptotic
members of the Bcl-2 family of apoptosis regulating proteins. Hypoxia/acidosis
stabilizes BNIP-3 and increases its association with the mitochondria, causing the
release of cytochrome C and cell death. We report the retrograde perfusion
Langendorffmodel is inconclusive in mouse hearts.

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.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS.

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.

Diabetic retinopathy is an ischemic retinal neovascular disease causing vision loss among adults. The studies presented involve the design and testing of a gene therapy vector to inhibit retinal revascularization, similar to that found in diabetic retinopathy. Gene therapy has proven to be an effective method to introduce therapeutic proteins to treat retinal diseases. Targeting a specific cell type and expression of therapeutic proteins according to the tissue microenvironment should have an advantage over traditional gene therapy by avoiding unwanted transgene expression. Hypoxia plays a significant role in the pathophysiology of many retinal ischemic diseases. Retinal Mèuller cells provide structural and functional support to retinal neurons, as well as playing a significant role in retinal neovascularization. Targeting Mèuller cells may be an effective strategy to prevent retinal neovascularization under pathological conditions. ... The hypoxia regulated, glial specific vector successfully reduced the abnormal neovascularization in the periphery by 93% and reduced the central vasobliterated area by 90%. A substantial amount of exogenous endostatin was produced in the retinas of P17 OIR mice. A significant increase in human endostatin protein and reduced vascular endothelial growth factor (VEGF) were identified by Western blot and ELISA, respectively. These findings suggest hypoxia-regulated, glial cell-specific scAAV mediated gene expression may be useful to prevent blindness found in devastating retinal diseases involving neovascularization.

The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.