Genetic regulation

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.

Oxidative damage is an inevitable consequence of aerobic respiration. Methionine
sulfoxide reductases (Msr) are a group of enzymes that function to repair oxidized
methionine residues in both free methionine and methionine in proteins. MsrA was the
first of these enzymes to be discovered and is the most thoroughly studied. It is thought to
play a role in both the aging process and probably several neurodegenerative diseases. I
recently obtained a strain of Drosophila that was reported to have a P-element transposon
located within Exon 2 (part of the open reading frame) of the eip71cd gene, which is the
Drosophila homolog of MsrA. Thus, the transposon insertion should disrupt expression
of the msrA gene. I did a series of experiments to "jump out" the P-element in an effort
to recover two types of isogenic strains. The first would be a null mutation of the MsrA
gene created by deletion of flanking genomic DNA when the P-element excised from the
chromosome. The second would be a precise excision of the P-element, which would
restore the genetic locus to its original structure. This study looks at the effect of a null
mutant of the MsrA gene on aging and resistance to oxidative stress.

One methionine sulfoxide reductase A (TMSRA) and two methionine sulfoxide reductase
Bs (TMSRB 1 and TMSRB2) were isolated from tobacco plants. TMSRA showed
specificity for the reduction of Met-(S)-SO and both TMSRBs were specific for the
reduction of Met-(R)-SO. TMSRA was the cytosolic form and both TMSRBs were
plastid forms based on sequence comparison and expression tests. TMSRA and TMSRB2
could use either thioredoxin (TRX) or dithiothreitol (DTT) as reducing system, while
TMSRB 1 showed little activity with TRX but much more activity with DTT, which was
similar to the mitochondrial MSRB2 from mammals. Ferredoxin (FD) is not the reducing
system for Msrs, but might reflect the redox status in the cell and control the activity of
Msrs indirectly.

Reovirus is a common virus that usually affects children; this infection causes symptoms such as respiratory and or gastrointestinal aliments. Morbidity most often occurs in impoverished countries where supportive hospitalization is not available. In the U.S. and other established countries morbidity is not an issue. When WI-38 cells are infected with reovirus the infection is either resisted by the cells or a persistent latent infection occurs. In this study, gene expression was analyzed by comparing Reovirus-infected WI-38 cells with mock infected cells. P.R.O.M(TM) analysis was performed on RNA sent to Genka Biotechnology Inc. Bioinformatics was used to analyze the data. Reovirus infection of the WI-38 cells resulted in increased mRNA levels for a number of transcription regulatory genes, and possibly more significant, decreased mRNA levels for some very important regulatory genes. These changes may be responsible for establishing the antiviral replication environment observed in these normal cells.

Aging is a multifactoral biological process of progressive and deleterious changes partially attributed to a build up of oxidatively damaged biomolecules resulting from attacks by free radicals. Methionine sulfoxide reductases (Msrs) are enzymes that repair oxidized methionine (Met) residues found in proteins. Oxidized Met produces two enantiomers, Met-S-(o) and Met-R-(o), reduced by MsrA and MsrB respectively. Unlike other model organisms, our MsrA null fly mutant did not display increased sensitivity to oxidative stress or shortened lifespan, suggesting that in Drosophila, having either a functional copy of either Msr is sufficient. Here, two Msr mutant types were phenotypically assayed against isogenic controls. Results suggest that only the loss of both MsrA and MsrB produces increased sensitivity to oxidative stress and shortened lifespan, while locomotor defects became more severe with the full Msr knockout fly.

Experimentally naive rats show variance in their locomotor reactivity to novelty, some displaying higher (HR) while others displaying lower (LR) reactivity, associated with vulnerability to stress. LRHR phenotype is proposed as an antecedent to the development of stress hyper responsiveness. Results presented here show emergence of antidepressive-like behavior following peripubertal-juvenile exposure to chronic variable physical (CVP) and chronic variable social stress (CVS) in HR rats, and depressive-like behavior following CVP in the LRs. The antidepressive-like behavior in HR rats was accompanied by increased levels of acetylated Histone3 (acH3) and acetylated Histone4 (acH4) at the hippocampal brain-derived neurotrophic factor (BDNF) P2 and P4 promoters respectively. This effect may mediate increased mossy fibre (MF) terminal field size, particularly the suprapyramidal mossy fibre projection volume (SP-MF), in the HR animals following both stress regimens. These findings show that chronic variable stress during adolescence induces individual differences in molecular, neuromorphological and behavioral parameters between LRs and HRs, which provides further evidence that individual differences in stress responsiveness is an important factor in resistance or vulnerability to stress-induced depression and/or anxiety.

Sponges are an important source of bioactive marine natural products, or secondary metabolites. The common Caribbean reef sponge, Axinella corrugata, produces an antitumor and antibacterial chemical, stevensine. This study determined whether environmental stressors, such as elevated temperature and exposure to Amphibalanus amphitrite larvae, affect the production of stevensine by A.corrugata and if the stressors caused A.corrugata to exhibit differential gene expression. Temperature stress resulted in no significant change in the production of stevensine; only two genes were significantly differentially expressed, including hsp70. Larval stressed resulted in increased production of stevensine and significant differential gene expression (more than seventy genes). This study suggests that A.corrugata may be resilient to elevations in temperature and that one of stevensine's roles in nature is as an antifoulant.

Mitochondria are a major source of reactive oxygen species and are particularly vulnerable to oxidative stress. Mitochondrial dysfunction, methionine oxidation, and oxidative stress are thought to play a role in both the aging process and several neurodegenerative diseases. Two major classes of methionine sulfoxide reductases, designated MsrA and MsrB are enzymes that function to repair the enatiomers of methionine sulfoxide, met-(o)-S and met-(o)- R, respectively. This study focuses on the effect of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. The data show that loss of only one form of Msr in the mitochondria does not completely impair the function of the mitochondria. However, loss of both Msr proteins within the mitochondria leads to an increased ROS production and a diminished energy output of the mitochondria. These results support the hypothesis that Msr plays a key role in proper mitochondrial function.

Experimentally naive rats exhibit varying degrees of novelty exploration. Some rats display high rates of locomotor reactivity to novelty (high responders; HR), and others display low rates (low responders; LR). The novelty-seeking phenotype (LRHR) is introduced as a model of stress responsiveness. In this thesis I examined effects of chronic variable physical and social stress or control handling on the levels of various neurotrophins in the hippocampus, and changes in mossy fibre terminal fields in LRHR rats. A positive correlation is seen between histone deacetylase 2 and brain-derived neurotrophic factor (BDNF) levels both of which are oppositely regulated in LRHR CA3 fields in response to chronic social stress. Increase in BDNF levels in CA3 field accompanied increase in supra-pyramidal mossy fibre terminal field size (SP-MF) in HRs, and decrease in BDNF levels accompanied decrease in SP-MF volume in LRs. Epigenetic regulation of neurotrophic support underlying these structural changes is discussed.