THE EFFECTS OF MSRA AND MSRB IN ANOXIA TOLERANCE IN AGING

DROSOPHILA MELANOGASTER

by

Nirthieca Suthakaran

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Medicine

In Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, FL

May 2018 Copyright by Nirthieca Suthakaran 2018

ii

ACKNOWLEDGEMENTS

I would like to thank my awesome lab mates whom I had the privilege of knowing over the last few years. You all have made my time in graduate school very smooth and enjoyable. I will definitely miss working with all of you. I especially would like to thank Dr. Binninger for his continuous support and encouragement throughout the years. I have learned so much and grew as a scientist under his guidance. I would also like to thank my committee, Dr. Milton and Dr. Prentice for their insightful input as well as encouraging me to step back from my project and look at the bigger picture. Lastly, I would like to thank my loving family for allowing me to practice my presentations with them countless times and for supporting me throughout this incredible journey.

iv ABSTRACT

Author: Nirthieca Suthakaran

Title: The Effects of MsrA and MsrB in Anoxia Tolerance in Aging Drosophila melanogaster

Institution: Florida Atlantic University

Thesis Advisor: Dr. David Binninger

Degree: Master of Science

Year: 2018

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 age- related 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.

v DEDICATION

To my backbone, my loving family whom have been with me throughout every step of my research journey. Especially my brother, Vithu, for knowing how to always keep a smile on my face.

THE EFFECTS OF MSRA AND MSRB IN ANOXIA TOLERANCE IN AGING

DROSOPHILA MELANOGASTER

TABLES ...... x

FIGURES ...... xi

I. INTRODUCTION ...... 1

Aging, Free Radicals & Oxidative Stress ...... 1

Anoxia and Oxidative Damage ...... 2

Methionine Sulfoxide Reductase (Msr) ...... 3

MsrA ...... 5

MsrB ...... 6

Drosophila melanogaster ...... 7

Stock Lines of Msr ...... 8

Previous Experiments from the Binninger Lab ...... 9

II. AVERAGE RECOVERY TIME FROM ANOXIA AS A FUNCTION OF AGE ...... 16

Background ...... 16

Materials and Methods ...... 17

Creation of Deletion Lines ...... 18

Aging and Classifying Single Deletion Line Flies ...... 18

Anoxia Tank...... 19

Measurement of Average Recovery Time ...... 20

Statistics ...... 20

vii Results ...... 21

III. PERCENT SURVIVAL FROM ANOXIA AS A FUNCTION OF AGE ...... 27

Background ...... 27

Materials and Methods ...... 27

Results ...... 27

IV. EXTENSION OF TIME UNTIL SPREADING DEPRESSION ...... 32

Background ...... 32

Materials and Methods ...... 33

Aging and Classifying Single Deletion Line Flies ...... 33

Age ...... 33

Wildtype Line ...... 33

Msr Double Deletion Line ...... 33

Young Age ...... 33

20-25 Days Old ...... 33

5-10 Days Old ...... 33

Middle Age ...... 33

40-45 Days Old ...... 33

20-25 Days Old ...... 33

Old Age ...... 33

60-65 Days Old ...... 33

40-45 Days Old ...... 33

Anoxia Tank...... 34

Measurement of Average Recovery Time and Survival ...... 34

viii Results ...... 35

V. PROLONGED ANOXIA ...... 43

Background ...... 43

Materials and Methods ...... 44

Results ...... 44

V. DISCUSSION ...... 48

VI. REFERENCES ...... 57

ix TABLES

Table 1: Nomenclature and Genotype ...... 13

Table 2: Nomenclature and Genotype ...... 19

Table 3: Age Classification for Wildtype and Msr Double Deletion Lines ...... 33

x FIGURES

Figure 1: Reactive Oxygen Species (ROS)...... 3

Figure 2: Methionine Sulfoxide Reductase Enzymes ...... 5

Figure 3: MsrA null mutant...... 9

Figure 4: MsrB null mutant ...... 9

Figure 5: Average Activity Levels for Double LOF and Wildtype Flies Following

Anoxic Stress ...... 13

Figure 6: Average Survival Rate for Double LOF and Wildtype Flies Following Anoxic

Stress ...... 14

Figure 7: Average Time of Recovery for Double LOF and Wildtype Flies Following

Anoxic Stress ...... 14

Figure 8: Average Time of Recovery for Double LOF, MsrA LOF, MsrB LOF and

Wildtype Flies Following Anoxic Stress ...... 15

Figure 9: Lifespan of Drosophila is Shortened in the Absence of Msr Activity ...... 17

Figure 10: Average Time of Recovery for Msr Double LOF and Wildtype Flies

Following Anoxic Stress ...... 22

Figure 11: Average Recovery Time for Wildtype, MsrA LOF, MsrB LOF, and Msr

Double LOF Flies Following Anoxic Stress ...... 26

Figure 12: Average Percent Survival for Msr Double LOF and Wildtype Flies Following

Anoxic Stress ...... 28

xi Figure 13: Average Percent Survival for Double LOF, MsrA LOF, MsrB LOF and

Wildtype Flies Following Anoxic Stress ...... 31

Figure 14: Average Time to Fall into Spreading Depression for Wildtype and Msr

Double LOF Flies Following Slower Flow Rate of Anoxic Stress ...... 37

Figure 15: Average Time of Recovery for Wildtype and Msr Double LOF Flies

Following Slower Flow Rate of Anoxic Stress ...... 39

Figure 16: Average Percent Survival for Wildtype and Msr Double LOF Flies Following

Slower Flow Rate of Anoxic Stress ...... 41

Figure 17: Percent of Oxygen at a Flow Rate of 0.025 L/sec ...... 42

Figure 18: Percent of Oxygen at a Flow Rate of 1.348 L/sec ...... 42

Figure 19: Average Recovery Time for 5-10 Day Old Wildtype and Msr Double LOF

Flies Following 3 Hours and 6 Hours of Anoxic Stress ...... 45

Figure 20: Average Recovery Time for 40-45 Day Old Wildtype and Msr Double LOF

Flies Following 3 Hours and 6 Hours of Anoxic Stress...... 45

Figure 21: Average Percent of Survival for 5-10 Day Old Wildtype and Msr Double

LOF Flies Following 3 Hours and 6 Hours of Anoxic Stress ...... 46

Figure 22: Average Percent of Survival for 40-45 Day Old Wildtype and Msr Double

LOF Flies Following 3 Hours and 6 Hours of Anoxic Stress ...... 46

Figure 23: Serotonin Synthesis and Release ...... 53

xii I. INTRODUCTION

Aging, Free Radicals & Oxidative Stress

At first glance, we as humans may exhibit differences, whether it be in physical characteristics, personality, cultural background, or even in how we leave an impact in this world. Despite our prominent qualities of diversity, we all share the commonality of undergoing aging. Aging can be defined as deterioration of the physiological functions necessary for survival and fertility with time [28]. Although the process of aging is inevitable, the question of how we age still remains until this day. Aging has become more prevalent in industrialized societies with longer life expectancies. When populations get older, age-related neurodegenerative diseases such as Alzheimer’s

Disease (AD) [14, 35, 63], Parkinson’s Disease (PD) [82], Amyotrophic Lateral Sclerosis

(ALS) [32, 27, 30, 45] as well as cardiovascular disease [4], Atherosclerosis [66, 80],

Rheumatoid Arthritis [39], Diabetes [21, 29, 1], Schizophrenia [64] and motor neuron disorders [18] develop. By studying the fruit fly, a genetically tractable model system, we have begun to see that aging and disease progression might be influenced and regulated by specific genes [65].

According to the Free Radical Theory of Aging, the accumulation of oxidative damage over time from the action of free radicals is a major contributor to the aging process [36]. A free radical is an uncharged molecule with an unpaired valence electron that is highly reactive. Free radicals are unavoidably produced through aerobic respiration. When electrons move through the electron transport chain, they react with O2

1 to create H2O. In a small proportion of these reactions, O2 accepts one electron instead of

- two, which allows for the creation of highly reactive superoxide anion, O2 . [56]. The superoxide anion is a source of hydrogen peroxide (H2O2) and a hydroxyl free radical is made when the Fenton reaction occurs. The superoxide anion, hydrogen peroxide, and hydroxyl radical are included in the group of molecules known as reactive oxygen species (ROS) [56]. Other than aerobic respiration, extracellular components such as UV light, toxins in the environment, and ionizing radiation can lead to ROS. ROS is capable of oxidizing cell macromolecules such as lipids, protein, RNA and DNA, causing them to lose functionality [6]. When these macromolecules are oxidized, the partial or full loss of function contributes to the aging process. [5, 6, 8, 78].

Anoxia and Oxidative Damage

Previously, it was mentioned that certain environmental insults are sources for ROS

[Figure 1]. This study focuses on the environmental stress of anoxia. Anoxia is the response of organisms to oxygen deprivation and it differs throughout the animal kingdom.

Mammals only tolerate anoxia for a few minutes before undergoing irreversible brain damage [61]. In contrast, the fruit fly, Drosophila melanogaster enters a protective coma called spreading depression, which allows it to withstand hours of anoxia [77]. In spreading depression, there is a suppression of overall metabolic rate and prevention of cellular injury during anoxia and reoxygenation conditions. Interestingly, the most severe damage from anoxia occurs during reoxygenation. This is the period when oxygen is reintroduced into the system, which leads to a burst of reactive oxygen species (ROS) [9]. ROS and their oxidized protein products are found to be more common in older individuals, especially those with age-related neurodegenerative disorders such as Alzheimer’s disease [14, 35,

2 63], cardiovascular disease [4,24], Amyotrophic Lateral Sclerosis [27], Parkinson’s disease

[82], and Huntington’s disease [75].

Figure 1: Reactive Oxygen Species (ROS) are produced as a byproduct of aerobic respiration or through extracellular sources. ROS can oxidize crucial macromolecules in the cell.

Methionine Sulfoxide Reductase (Msr)

The vast majority of species have cellular defenses against oxidative damage from

ROS. The levels of ROS present in the cells must be carefully regulated because low levels of ROS can function as signaling molecules that protect cells against oxidative damage, whereas an excess amount can lead to cell death [79]. An antioxidant enzyme repair system known as the methionine sulfoxide reductase (Msr) family reverses protein damage caused by oxidation of methionine residues in proteins to methionine sulfoxide (Met-(o)). ROS is capable of oxidizing several different amino acids, although the two sulfur containing amino acids, methionine and cysteine, are most sensitive. Methionine is hypothesized to function as an endogenous antioxidant that is found in the catalytic sites of proteins [50].

The sulfur in the side chain of methionine makes it susceptible to oxidation to methionine 3 sulfoxide [11, 72]. Methionine and cysteine are distinct from other amino acids because their oxidation is reversible [77]. Oxidation of methionine may cause significant structural and functional changes in proteins, resulting in either reduced or a complete loss of function

[43].

When methionine is oxidized by ROS to form methionine sulfoxide, it can be reduced back to functional methionine by methionine sulfoxide reductase (Msr).

Methionine sulfoxide, has two different enantiomers since the sulfur atom is asymmetrical within the methionine [75]. Two Msr genes were found and designated MsrA (discovered in 1981) and MsrB (discovered in 2001). As shown in Figure 2, the enzyme encoded by the MsrA gene specifically reduces the S enantiomer (met-S-(o)) while the enzyme encoded by the MsrB gene specifically reduces the R enantiomer (met-R-(o)) of methionine sulfoxide [72]. Both genes were originally found in the bacterium E. coli [31]. In addition to repairing oxidative damage to methionine, both the MsrA and MsrB enzymes can function as efficient antioxidants by reducing methionine sulfoxide created by a reaction with ROS back to functional methionine [77]. In this process, Msr uses thioredoxin as the reductant to reduce the disulfide bond and regenerate Msr [2]. Thus, the ROS can be destroyed before they are able to damage any cellular components.

4 Reduction

Oxidation

Reduction

Figure 2: Methionine Sulfoxide Reductase Enzymes: The enzyme encoded by the MsrA gene specifically reduces the S enantiomer (met-S-(o)) while the enzyme encoded by the MsrB gene specifically reduces the

R enantiomer (met-R-(o)) of methionine sulfoxide.

The second function of Msr is to act as a catalytic antioxidant. Although it is based on the same mechanism of the methionine redox process discussed before, the product is different. The Met-(o) made through ROS oxidation can be reduced to methionine using

Msr. For each methionine residue that is oxidized, there is one ROS molecule destroyed

[50]. There is more evidence of the catalytic antioxidant role of Msr in human lens cells. It was shown that an ROS-mediated cell death followed from a lack of MsrA, any of the three mammalian MsrB proteins, or all Msr enzymes. In contrast, an overexpression of MsrA was seen to decrease ROS levels [55].

MsrA

The MsrA gene was discovered in 1981 [11]. Through the investigation of protein synthesis in E..coli and the L12 ribosomal protein, it was found that L12 could be inactivated if any of its methionine residues were oxidized. Weissbach and his colleagues

5 discovered that the MsrA enzyme reduced methionine sulfoxide in L12 [11, 83]. While the

MsrA gene is found in E. coli, mice, yeast, Drosophila, and humans; only mammals have one MsrA gene [49]. In Drosophila, MsrA was initially identified as an ecdysone-induced protein 28/29kD (Dmel/Eip71CD) because it was discovered as a polypeptide generated in response to ecdysone in Drosophila cell culture [74]. The MsrA mutants in E. coli demonstrated an increased sensitivity to oxidative stress [59]. When the MsrA gene was knocked out in mice, an increased sensitivity to oxidative stress, a reduction by 40% in lifespan and a neurological disorder were seen in the original study [59]. In a follow-up study, only increased sensitivity to oxidative stress was found in the MsrA gene knockout mice [73]. Contrarily, overexpression of the bovine MsrA in the Drosophila central nervous system (CNS) extended the lifespan of the fly [72]. MsrA is also found in high concentrations in retinal pigmented epithelial (RPE) cells and the retina is particularly sensitive to oxidative damage. Oxidative damage can cause the loss of photoreceptor cells involved in diseases such as age related macular degeneration, retinitis pigmentosa, and diabetic retinopathy [19]. Lastly, there is evidence linking MsrA to Alzheimer’s disease because of decreased levels of MsrA in the brain tissue of Alzheimer patients [26]. When the methionine residue is oxidized, the amyloid beta peptide exhibits toxic effects in

Alzheimer patients [13].

MsrB

The MsrB gene encodes a selenoprotein, which are a group of proteins that contain selenium. Selenium is part of the formation of selenocysteine residues that often exhibit catalytic roles in proteins [59]. Uniquely, Drosophila has only one MsrB gene but mammals have three MsrB genes (MsrB1, MsrB2 and MsrB3) [42]. Yet, their main gene

6 is MsrB1. In mouse studies, an MsrB1 knockout exhibited an increase sensitivity to oxidative stress [73]. A study of MsrB in human lens cells resulted in increased oxidative stress-induced death upon silencing MsrB’s expression [55]. Due to the recent discovery of MsrB in comparison to MsrA, there are fewer studies available of MsrB.

Drosophila melanogaster

With over a century of genetic studies, Drosophila melanogaster stands as a widely used genetic model organism. This eukaryotic model has its genome fully sequenced on www.flybase.org and is ideal for studying genetics and developmental biology [23]. D. melanogaster is easy to genetically manipulate and has become an ideal model for examining roles of specific genes in disease, especially under anoxia. [22]. This model also has a highly homologous genome to humans, both genetically and physiologically at a cellular level. For example, 70% of all genetic diseases present in humans contain a homologous gene in Drosophila [15]. Drosophila have one MsrA gene and one MsrB gene that reside about eight map units apart on the third chromosome [48]. Another name for

MsrA is Ecdysone-induced protein 28/29 kD (Dmel/Eip71CD) and there are six known isoforms in the fruit fly. Studies have indicated that ecdysone induction of MsrA protects

Drosophila KC cells from oxidative stress [70]. KC cells are a heterogeneous Drosophila cell culture made from Drosophila embryos. In Drosophila, MsrB is known as SelR

(DmelSelR) has eight known isoforms, although it does not contain selenium [48]. Due to differential exon one splicing, three transcripts code for proteins targeted for the mitochondria. The other five isoforms code for proteins that reside in the cytoplasm [42].

Studies indicate that overexpression of MsrB in D. melanogaster’s nervous system had no dramatic or consistent lifespan effect on cornmeal food [76].

7 Stock Lines of Msr

The genesis research interest of the lab was the demonstration that overexpression of the bovine MsrA gene selectively within the central nervous system (CNS) of

Drosophila resulted in a substantial increase in lifespan [72]. It was previously predicted that overexpression of MsrB should have a similar effect. However, an in-depth analysis showed that the overexpression of MsrB in Drosophila had no effect on lifespan or any other phenotype that was examined [76]. Our lab found that overexpression studies of

MsrA and MsrB were unproductive. Therefore, we changed our experimental approach to use classical genetic mutations.

Our lab created a series of MsrA and MsrB deletion mutations using imprecise excision of P-element transposons that resided within the two genes. These mutations are fairly large deletions (1.5Kb for MsrA and 2.5Kb for MsrB) of the promoter and portions of the transcription unit, thereby leading to complete loss-of-function (LOF) alleles

(unpublished data). The stock lines we work with in our lab were created by Kelli Robbins,

Lindsay Bruce, and several other students from the Binninger lab. The genetic lines used in this study were isogenized by repeated backcrosses to a lab wild-type strain designed yw to minimize effects due to differences in genetic backgrounds. This allows for a significant reduction in variable behavior and complements previous experiments done on stress tolerance [7].

The MsrA null mutant resulted from the loss of exon 1, the promoter, and a portion of exon 2. [Figure 3]. The MsrB null mutant was created through the loss of exon 1, exon

2, and exon 3 which included the promoter and nearly 2 Kb of the transcribed region

[Figure 4]. In summary, the MsrA and MsrB P-element excisions created a knockout of the

8 proximal promoter region, 5’UTR in the mRNA and a large part of the open reading frame

(ORF). The double mutant was created by conventional genetic recombination since both genes reside on the third chromosome. All lines created were molecularly tested through genomic PCR, reverse transcription PCR (RT-PCR), and western blotting to confirm their genotype. More specifically, strains of Drosophila are available that are homozygous for wild-type alleles of both MsrA and MsrB, homozygous for the MsrA LOF allele, homozygous for the MsrB LOF and homozygous for both MsrA and MsrB LOF alleles.

This last strain is, to our knowledge, the only developmentally complex eukaryotic model organism that is completely deficient in all known Msr activity. Not surprisingly, the phenotype of MsrA and MsrB mutants is usually more severe in the presence of oxidative stress.

Figure 3: MsrA null mutant. MsrA p-element excision between -300 and 1172 bp of Eip71CD gene on

3L arm.

Figure 4: MsrB null mutant. MsrB p-element excision between -364 and 2163 bp of SelR gene on 3R arm.

Previous Experiments from the Binninger Lab

A recent study found that overexpression of endogenous MsrA in Drosophila neurons extended their lifespan and led to protection against oxidative stress [17], while 9 another study found that overexpression of human MsrB3 in Drosophila neurons also extended lifespan [51]. Drosophila’s tolerance of anoxic conditions, combined with the fact that both MsrA and MsrB are present in its genome, made it an ideal model organism to study our hypothesis that MsrA and MsrB play a role in recovery from anoxic stress. The study of this animal model’s anoxia tolerance, with brains adapted through millions of years of evolution, may suggest protective pathways that could possibly serve as therapeutic targets for neurodegenerative diseases characterized by oxygen deprivation and the aftermath of reperfusion.

The experiments completed in this thesis are a continuation of this investigation initiated by Danielle Howard in the Binninger lab. The goal was to use the genetic and molecular tools available for Drosophila to better understand the underlying molecular mechanisms of their Msr genes. Previous work by Danielle Howard showed that overall, single loss-of-function mutations in either MsrA or MsrB or mutations in both genes resulted in a longer recovery from the protective coma induced by anoxia. Being that these experiments were not done by me, I do not know the exact percentage of anoxia used.

However, the experiments completed in my thesis utilized an oxygen sensor and measured the percentage of anoxia present. The average movements of both the wild-type and

MsrA/MsrB double LOF flies were seen to increase with age, but the MsrA/MsrB double

LOF flies showed varying age-dependent mobility [Figure 5]. The double LOF flies, however, displayed a higher mortality rate than wildtype line that contained both MsrA and

MsrB genes [Figure 6]. Overall, the MsrA/MsrB double LOF flies had a longer average recovery time in comparison to wildtype flies right after anoxic stress was applied [Figure

7].

10 Danielle originally hypothesized that MsrA and MsrB are involved in anoxia tolerance from the increased ROS production seen following a period of anoxic stress. The strain designations and genotypes used in Danielle’s studies are summarized in Table 1.

Her double LOF flies (AB113) were initially found to have a significantly lengthened average time of recovery when compared to wildtype flies (WT77) for all age groups

[Figure 7]. Lastly, analysis of single LOF flies (A3, B22) showed variable age-dependent recovery time when compared to the wildtype (WT77) and double LOF (AB113) lines.

Due to the success seen in the double LOF studies, MsrA LOF (A3) and MsrB LOF

(B22) flies were also stressed with one hour of anoxia and their recovery times were recorded. The data were plotted along with the wildtype and double LOF data shown in

Figure 7. In 5-9 day old (young) flies, all three LOF lines (MsrA LOF, MsrB LOF, and double LOF) recovered on average at a significantly later time than the wildtype flies (59 minutes for MsrA LOF, 69 minutes for MsrB LOF, and 69 minutes for MsrA-MsrB LOF), which recovered in 51 minutes [Figure 8.A]. However, in 15-20 day old (middle-age) flies, only the double LOF line recovered significantly later (78 minutes) than the wildtype (60 minutes), with the MsrA LOF line (59 minutes) and MsrB LOF (66 minutes) showed no significant difference [Figure 8.B]. In the 35-39 day old (old age) flies, the double LOF line recovered significantly (87 minutes, p < 0.05) later than the wildtype line (66 minutes) while the MsrA LOF line recovered significantly earlier (57 minutes) and the MsrB LOF line showed no phenotype (64 minutes) [Figure 8.C].

It is worth noting the increased levels of motility in both the wildtype and the Msr double LOF flies as they age. As flies age, they become more active [Figure 5], but then activity suddenly declines when the flies are near the end of their lifespan (Dr. Bill Ja,

11 Scripps Florida, personal correspondence). To explore this observation further, a

Drosophila Activity Monitor (Trikentics, Waltham, MA) was used to measure the mobility in the flies. Previous lifespan experiments in our lab showed that loss of all Msr activity drastically reduced lifespan in comparison to the wildtype lines at all age groups. In

Danielle’s study, because the double LOF flies were shown to live to only 40-50 days, instead of the usual 70 for wildtype flies, the oldest age group examined was 35-39 days old. The single deletion lines also have a lifespan of 70 days. During the time these experiments were performed, the level of understanding we now have of lifespan was not there before. Hence, this is why I am aging the wildtype and Msr single deletion to senescence (60-65 days) for my experiments. Overall, the decline in activity was observed only for the Msr double LOF flies, and not the wildtype flies [Figure 5]. These findings expanded the scope of Danielle’s hypothesis by showing that the MsrA and MsrB genes play a protective role in oxidative stress.

12 Nomenclature Genotype

WT77 MsrA+/+MsrB+/+

-/- +/+ A3 MsrA MsrB

B22 MsrA+/+MsrB-/-

AB113 MsrA-/-MsrB-/-

Table 1: Nomenclature and Genotype. All four genotypes of fruit flies used in preliminary data.

Figure 5: Average Activity Levels for Double LOF (AB113) and Wildtype (WT77) Flies Following

Anoxic Stress. Double LOF flies (AB113) and wildtype flies (WT77) were stressed with one hour of anoxic conditions, after which their movements were recorded every minute with the DAM system.

Significance (p < 0.05) is marked with an asterisk. 64 flies that were 5-9 days old (5.A), 15-20 days old

(5.B), and 35-39 days old (5.C) were tested for each genotype. Unpublished data from Danielle Howard.

13

Figure 6: Average Survival Rate for Double LOF (AB46) and Wildtype (WT60) Flies Following

Anoxic Stress. Double LOF flies (AB46) and wildtype flies (WT60) were stressed with one hour of anoxic conditions, after which they were monitored with the DAM system. Flies that failed to move in the five hour period following the anoxic stress were determined dead. Significance (p < 0.05) is marked with an asterisk. 64 flies that were 5-9 days old (6.A), 15-20 days old (6.B), and 35-39 days old (6.C) were tested for each genotype. Unpublished data from Danielle Howard.

Figure 7: Average Time of Recovery for Double LOF (AB113) and Wildtype (WT77) Flies Following

Anoxic Stress. Double LOF flies (AB113) and wildtype flies (WT77) were stressed with one hour of anoxic conditions, after which their recovery times were recorded with the DAM system. Significance (p <

0.05) is marked with an asterisk. 64 flies that were 5-9 days old (7.A), 15-20 days old (7.B), and 35-39 days old (7.C) were tested for each genotype. Unpublished data from Danielle Howard.

14

Figure 8: Average Time of Recovery for Double LOF (AB113), MsrA LOF (A3), MsrB LOF (B22) and Wildtype (WT77) Flies Following Anoxic Stress. Double LOF flies (AB113), MrsA LOF flies (A3),

MsrB LOF flies (B22) and wildtype flies (WT77) were stressed with one hour of anoxic conditions, after which their recovery times were recorded with the DAM system and then averaged. Significance (p < 0.05) is marked with an asterisk. 64 male flies that were 5-9 days old (8.A), 15-20 days old (8.B), and 35-39 days old (8.C) were tested for each genotype. Unpublished data from Danielle Howard.

15 II. AVERAGE RECOVERY TIME FROM ANOXIA AS A FUNCTION OF AGE

Background

Numerous studies provide evidence that Msr genes play an important role in aging.

An accumulation of oxidized and modified proteins, increased sensitivity to oxidative stress, and a shortened or unaffected lifespan all result from the knock-out of the MsrA gene [76]. Previous experiments completed by Lindsay Bruce in our lab confirmed that homozygous loss of both Msr loci significantly decreased lifespan compared to wildtype controls whereas homozygous single Msr deletions for either MsrA or MsrB showed no significant difference [Figure 9]. The mean lifespan for all lines was between 64 and 75 days, whereas the Msr double deletion mean lifespan was around 40 days [Figure 9].

Since there was no evidence in the literature of anoxia tolerance past 40 days, I extended these studies by using single deletion lines of either the MsrA or MsrB mutants to determine if these genes continue to play a role in anoxia tolerance as they approach senescence (60-65 days). Tolerance to anoxia is measured by the average time of recovery and survival of the fruit flies from the anoxic coma. This was the first time our lab performed experiments on Drosophila aged past 35-39 days. Since the double mutants do not survive past 40 days, the lines used in this study were wildtype (WT60),

MsrA deletion (A90), and MsrB deletion (B54). As the flies age, it is assumed that their susceptibility to anoxia increases. It is therefore expected for all lines to gradually increase in recovery time as the flies age. Since the wildtype line has both MsrA and

16 MsrB genes, we anticipate that the wildtype strain will recover faster than either Msr single deletion strain at all age groups.

Figure 9: Lifespan of Drosophila is Shortened in the Absence of Msr Activity. Approximately 100 flies per strain were sorted into groups of 10 and kept in a 25°C incubator on a 12h day/night cycle. The log- rank test was used to determine significant differences between the survival curves. Panel A. One copy of

MsrA clearly rescues the life span in comparison to the double deletion line (p < 0.001), although it does not restore it to the wildtype life span (p < 0.001). Two copies of MsrA fully rescues the lifespan (p = 0.78 vs. wildtype). Panel B. In a similar manner, one copy of MsrB almost entirely rescues the lifespan (p =

0.02 vs. wildtype and p <0.001 vs. Msr double deletion). Two copies of MsrB results in a full rescue (p =

0.08).

Materials and Methods

The groups of flies that were used have the following genotypes: MsrA and MsrB present (WT60; MsrA+/+B+/+) line, MsrA single LOF line (A90; MsrA-/-B+/+), MsrB single

LOF line (B54; MsrA+/+B-/-), and MsrA/MsrB double LOF (AB46; MsrA-/-B-/-) line (Table

17 2). The following procedures were done to determine the average recovery time after anoxic stress for these single deletion lines during normoxia, anoxia, and reoxygenation.

Creation of Deletion Lines

Lines with a p-element insertion in the second exon of MsrA (#16671, y1 w67c23; P{EPgy2}Eip71CDEY05753) and the 5’UTR region of MsrB (#17116, w1118; P{EP}EP3340EP3340/TM6B, Tb1) were obtained from the Bloomington Stock

Center (Bloomington, IN). Imprecise excision of both p-elements was obtained through the introduction of transposase (w; Trasposase-Gla/Cy2-3). The MsrA and MsrB loci are both located on the third chromosome. Genetic recombination between the

MsrA-/-B+/+ and MsrA+/+B-/- lines yielded the MsrA-/-B-/- line. The MsrA-/-B-/- was backcrossed to YW to minimize genetic differences in the progeny, and four genotypes were recovered: MsrA+/+B+/+, MsrA-/-B+/+, MsrA+/+B-/-, and MsrA-/-B-/-.

Aging and Classifying Single Deletion Line Flies

In Danielle’s study, six genotypes of Drosophila were used. Two wildtype lines

(WT77, WT60), one MsrA Loss-of-Function (LOF) line (A3), one MsrB LOF line (B22), and two double MsrA-MsrB LOF lines (AB113, AB46), which were all created from yellow-white background flies by Lindsay Bruce. The males were kept in vials of 50 flies per vial and tested 5-9 days, 15-20 days, or 35-39 days. For my study, four genotypes of flies were used in this experiment. One wildtype line (WT60), one MsrA Loss-of-Function

(LOF) line (A90), one MsrB LOF line (B54), and MsrA/MsrB double LOF (AB46) line.

Flies for each genotype were put into bottles and allowed to grow for about ten days. The flies were then cleared. After five days, the male flies were collected and their age was determined to be one to five days old. To be consistent with previous anoxia studies in the

18 literature, males were used for experiments at 5-10 days old, 20-25 days old, 30-35 days old, 40-45 days old, 50-55, and 60-65 days old. Flies used in the “normoxia” samples were not exposed to anoxia or reoxygenation conditions. Flies used as “anoxia” and

“reoxygenation” samples were exposed to 1 hour of 100% N2 in the anoxia tank. Average recovery time for each fly line was determined using the Drosophila Activity Monitor.

Nomenclature Genotype

WT60 MsrA+/+MsrB+/+

A90 MsrA-/-MsrB+/+

B54 MsrA+/+MsrB-/-

AB46 MsrA-/-MsrB-/-

Table 2: Nomenclature and Genotype. All four genotypes of Drosophila were used in my study.

Anoxia Tank

One fly was placed into each individual tube of the Drosophila Activity Monitor holder, which contained 32 tubes. The purpose of the small holes is for the gases to freely flow through the tube. The Drosophila Activity Monitor holders were placed into a sealed anoxia tank. Anoxia was induced by pumping 100% nitrogen gas for 1 hour into the tank, while displacing oxygen out of the tank through a second pipe. 32 flies of each genotype 19 were exposed to a nitrogen gas flow rate of 1.961 L/sec. Oxygen sensing tests ran displayed at least 95% oxygen-free conditions.

Measurement of Average Recovery Time

The Drosophila Activity Monitoring (DAM system, Trikinetics) was used to monitor mobility. The DAM system contains the Drosophila Activity Monitor Holder. As briefly mentioned before, this holder is composed of 32 small tubes with holes drilled on one of the sides. Eight flies of each genotype were loaded into each row of tubes so that one genotype was present in each row, allowing all four genotypes to be tested at once.

This pattern of loading was rotated after each trial to ensure that each genotype was placed in a different row during the course of testing. When the holder is connected to the DAM system, the infrared beam measured activity in each tube. As each fly broke the beam, the system recorded the activity. The Drosophila Activity Monitor holder was placed into the

Drosophila Activity Monitoring System for 10 minutes of normoxia. Afterwards, the holder was taken out of the Drosophila Activity Monitor and placed into the anoxia tank for one hour. After one hour of anoxia, the holder was removed from the tank and placed into the Drosophila Activity Monitor. All trials occurred during the hours of 12:00pm and

5:00pm to avoid behavior modification due to circadian rhythms. The first ten minutes of each trial were omitted from the analysis to allow for a stabilization period. Flies were monitored for 5 hours of reoxygenation. The first time during reoxygenation when a fly breaks through the IR beam, that individual fly is considered “recovered.”

Statistics

Statistical analysis of the data was conducted using GraphPad Prism. Average recovery time was determined for each trial. N= number of trials, each trial had 32 flies

20 per genotype. The statistical test performed was a t-test. The average recovery time for the MsrA and MsrB single deletion mutants as well as the MsrA/MsrB double deletion strains were individually compared with the wildtype line.

Results

Msr double loss-of-function (AB46) flies recovered significantly later and at a slower rate than wildtype (WT60) flies.

Since ROS concentration significantly increases following a period of anoxic stress

[9], Danielle had previously began investigating and finding that MsrA and MsrB play a role in recovering from anoxic stress. I repeated these experiments by stressing the wildtype and MsrA-MsrB double LOF flies at three age groups with an hour of anoxia, after which they were monitored to determine their average recovery times. From the data collected, I continued to find that Msr double LOF flies displayed a significantly longer average recovery time when compared to wildtype flies at 5-10 days old (42 minutes for wildtype and 62 minutes for MsrA-MsrB double LOF) [Figure 10.A], 20-25 days old (44 minutes for wildtype and 62 minutes for MsrA-MsrB LOF) [Figure 10.B], 30-35 days old

(51 minutes for wildtype and 74 minutes for MsrA-MsrB LOF [Figure 10.C], and 40-45 days old. (67 minutes for wildtype and 113 minutes for MsrA-MsrB LOF) [Figure 10.D].

All p-values were less than 0.0001.

21

A. B.

* *

C. D.

* *

Figure 10: Average Time of Recovery for Msr Double LOF (AB46) and Wildtype (WT60) Flies

Following Anoxic Stress. Double LOF flies (AB46) and wildtype flies (WT60) were stressed with one hour of anoxic conditions, after which their recovery times were recorded with the DAM system. An asterisk indicates a p-value <0.0001. 32 flies that were 5-10 days old (10.A), 20-25 days old (10.B), 30-35 days old

(10.C), and 40-45 days old (10.D) were tested for each genotype.

22 Analysis of single loss-of-function flies (A90, B54) showed variable age-dependent recovery time data when compared to the wildtype (WT60) and Msr double loss-of- function (AB46) flies.

Due to the interesting results seen with the double LOF experiments, MsrA LOF

(A90) and MsrB LOF (B54) flies were also stressed with an hour of anoxia and their recovery times were recorded. I wanted to determine if a similar pattern of longer recovery behavior was seen in the single deletion lines up until the same time period of 40-45 days.

The data were plotted along with the wildtype and the double LOF data shown in Figure

9. In 5-10 day old flies, the MsrB LOF, and double LOF recovered at a significantly later time (52 minutes for the MsrB LOF and 62 minutes for MsrA-MsrB LOF), on average, than the wildtype line (42 minutes) [Figure 11.A]. Likewise, in 20-25 day old flies, the double

LOF (62 minutes) and MsrB LOF (50 minutes) recovered significantly later than the wildtype (44 minutes) [Figure 11.B], while no significant difference in average recovery time was seen between the wildtype and the MsrA single deletion mutant (47 minutes)

[Figure 11.B]. This pattern was also seen in the 30-35 day old flies. (50 minutes for wildtype, 53 minutes for MsrA LOF, 60 minutes for MsrB LOF, and 74 minutes for MsrA-

MsrB LOF) [Figure 11.C].

23 The wildtype line (WT60) recovered significantly later as they approached senescence, while the MsrA and MsrB mutants reached maximum recovery time at middle age (40-45 days old).

Since these studies indicated that MsrA and MsrB genes seem to affect how fast

Drosophila are recovering from anoxic stress, I wanted to see if this was evident as the flies approached senescence. The same lines containing both MsrA and MsrB genes

(WT60) and single LOF (A90 and B54) were stressed with anoxia for one hour. Their average recovery time was analyzed. In 40-45 day old flies, all three LOF lines, the MsrA

LOF (84 minutes), the MsrB LOF (85 minutes), and double LOF (113 minutes) recovered at a significantly later time, on average, than the wildtype line (67 minutes) [Figure 11.D].

In 50-55 day old flies, there was a surprising drop in recovery time among the single deletion mutants (67 minutes for MsrA LOF, 68 minutes for MsrB LOF) and no significant difference in recovery time seen between them and the wildtype line (61 minutes) [Figure

11.E]. At senescence, all lines displayed a slight increase in recovery time in comparison to those that were 50-55 days old. However, there was no significant difference in average recovery time among the Msr single deletion lines (79 minutes for MsrA LOF, 77 minutes for MsrB LOF) and the wildtype line (71 minutes) [Figure 11.F].

24

A. B.

* * * *

Genotype p value Genotype p value

MsrA+MsrB+ vs MsrA LOF 0.1493 MsrA+MsrB+ vs MsrA LOF 0.1449

MsrA+MsrB+ vs MsrB LOF 0.0242 MsrA+MsrB+ vs MsrB LOF 0.0276

MsrA+MsrB+ vs MsrA-MsrB- <0.0001 MsrA+MsrB+ vs MsrA-MsrB- <0.0001

C. D.

* * * * *

Genotype p value Genotype p value MsrA+MsrB+ vs MsrA LOF 0.4955 MsrA+MsrB+ vs MsrA LOF <0.0001

MsrA+MsrB+ vs MsrB LOF 0.0055 MsrA+MsrB+ vs MsrB LOF 0.0025

MsrA+MsrB+ vs MsrA-MsrB- <0.0001 MsrA+MsrB+ vs MsrA-MsrB- <0.0001

25

E. F.

50-55 Day Old Average Recovery Time 60-65 Day Old Average Recovery Time

180 180

y

y r

160 r 160 e

+ + e v

MsrA MsrB v + + o

140 o 140 MsrA MsrB

c

c e MsrA LOF e MsrA LOF

) 120

120 )

R

R

s

s f

MsrB LOF f

e

e o

o MsrB LOF

t

t

100 100

u

u

e

e

n

n

i

i

m

m i

i 80 80

M

M

T

T

(

(

e

e 60 60

g

g

a

a r

r 40 40

e

e

v

v A A 20 20 0 0 + F + F B F B F r O O r O O s L L s L L + M rA B + M rA B s r s r rA s rA s s M M s M M M M Genotype Genotype

Genotype p value Genotype p value MsrA+MsrB+ vs MsrA LOF 0.2931 MsrA+MsrB+ vs MsrA LOF 0.2181

MsrA+MsrB+ vs MsrB LOF 0.3315 MsrA+MsrB+ vs MsrB LOF 0.2689

Figure 11: Average Recovery Time for Wildtype (WT60), MsrA Loss of Function (A90), MsrB Loss of

Function (B54), and Msr Double Loss of Function (AB46) Flies Following Anoxic Stress. All lines were stressed with one hour of anoxia, after which their recovery times were recorded with the DAM system and then averaged. Significance (p<0.05) is marked with an asterisk. 32 flies that were 5-10 days old (11.A), 20-

25 days old (11.B), 30-35 days old (11.C), 40-45 days old (11.D), 50-55 days old (11.E), and 60-65 days old

(11.F) were tested for each genotype.

26 III. PERCENT SURVIVAL FROM ANOXIA AS A FUNCTION OF AGE

Background

In the previous study, I examined the recovery time from the protective coma induced by the anoxia. In the course of those experiments, I observed that some animals died as a result of the anoxic stress. Therefore, I wanted to examine more carefully how well the Msr mutant flies were able to survive the anoxia throughout their lifespan.

Materials and Methods

The groups of flies that were used have the following genotypes: MsrA and MsrB present (wild-type) line, MsrA single LOF line, MsrB single LOF line, and MsrA-MsrB double LOF line. The procedures for collecting fly lines, exposing them to anoxic stress at a nitrogen gas flow rate of 1.961 L/sec, monitoring their movement using the Drosophila

Activity Monitor and utilizing statistical analysis were the same as Chapter 2. Animals whose movement failed to be detected after five hours of reoxygenation were presumed

“dead.”

Results

Msr double LOF flies (AB46) continued to have a significantly lower percent survival than wildtype flies (WT60) immediately following anoxic stress.

Due to increased recovery time seen in double LOF flies (AB46), we began to look at how well the flies survived the hour of anoxic stress. The flies were stressed and recorded with the DAM system. If no movement was recorded during five hours following the anoxic stress, the flies were considered dead. There was a 100% survival seen in the 5-10

27 and 20-25 day old double LOF flies and wildtype flies [Figures 12.A- 12.B]. However, the double LOF line had a significantly lower percent of survival than the wildtype line in the

30-35 (81% survival) and 40-45 day old group (38% survival) [Figures 12.C- 12.D].

A. B.

C. D.

* *

Figure 12: Average Percent Survival for Msr Double LOF (AB46) and Wildtype (WT60) Flies

Following Anoxic Stress. Double LOF flies (AB46) and wildtype flies (WT60) were stressed with one

hour of anoxic conditions, after which they were monitored with the DAM system. Flies that failed to

move in the five hour period following anoxic stress were determined dead. Significance (p < 0.05) is

marked with an asterisk. 32 flies that were 5-10 days old (12.A), 20-25 days old (12.B), 30-35 days old

(12.C), and 40-45 days old (12.D). 32 flies were tested for each genotype.

28 Wildtype flies (WT60) had a 100% survival as they approached senescence, while the single deletion flies (A90, B54) had an age dependent percent survival following anoxic stress.

There was a 100% survival seen in all four genotypes only at 5-10 days old [Figure

13.A]. The wildtype flies continued to show 100% survival at all age groups tested.

[Figures 13.A- 13.F]. There was a 97% survival in the MsrB deletion flies at 20-25 days and 30-35 days [Figures 13.B –13.C], while a 100% survival was seen from 40-45 days onto senescence [Figures 13.D –13.F]. There was a 94% survival in the MsrA deletion flies at 50-55 days [Figure 13.E] and a 91% survival at 60-65 days [Figure 13.F]. In Msr double mutant flies, there was a significantly lower percent of survival (81%) in comparison to the wildtype line at 30-35 days. In fact, as the double mutants approached senescence (40-45 days), only 38% survived [Figures 13.C- 13.D].

29

A. B.

C. D.

* *

30

E. F.

Figure 13: Average Percent Survival for Double LOF (AB46), MsrA LOF (A90), MsrB LOF (B54) and

Wildtype (WT60) Flies Following Anoxic Stress. All lines were stressed with one hour of anoxic conditions, after which they were monitored with the DAM system. Flies that failed to move in the five hour period following anoxic stress were determined dead. Significance (p < 0.05) is marked with an asterisk. 32 flies that were 5-10 days old (13.A), 20-25 days old (13.B), 30-35 days old (13.C), 40-45 days old (13.D), 50-55 days old

(13.E), and 60-65 days old (13.F). 32 flies were tested for each genotype.

31 IV. EXTENSION OF TIME UNTIL SPREADING DEPRESSION

Background

Fruit flies enter a protective coma called spreading depression after losing their locomotive activity under anoxia. This protective quiescent phase is a temporary cessation of neuronal and muscular activity [34, 62]. The onset of this coma preserves energy to avoid the consequences of full energy loss while reflecting a greater survival over long- term anoxia [7]. In the previous experiments, a higher flow rate of nitrogen gas was used to mimic anoxic conditions. Within a minute, flies of all genotypes (wildtype, MsrA deletion, MsrB deletion, and Msr double LOF) were comatose as evident by no further movement. An alarming mass death in the Msr double deletion flies occurred near senescence (40-45 days), where only 38% survived. This lethality seen among Msr double mutants raised the question of whether the induction of anoxia at a higher flow rate of nitrogen gas caused this. Through this study, the anoxia tolerance of wildtype and double deletion flies were tested under a significantly decreased flow rate of nitrogen gas (0.025

L/sec or 1.348 L/sec). The flies were stressed with an hour of anoxia and their average recovery time was recorded through the DAM system. It is expected that the wildtype flies will continue to recover significantly faster than the Msr double deletion flies. However, both genotypes are expected to recover faster and have improved survival since the time to fall into spreading depression is gradually increased among the lines. If our prediction is proven to be correct, these results may possibly suggest that spreading depression affects anoxia tolerance.

32 Materials and Methods

The experimental strategy and statistical analysis utilized were the same as in previous chapters. However, the flow rate of nitrogen gas was modulated. The Pasco

PASPort oxygen sensor device was utilized to measure the percentage of oxygen in the anoxia tank. Average recovery time for each fly line as well as their percent of survival were found through the Drosophila Activity Monitor.

Aging and Classifying Single Deletion Line Flies

For this study, only wildtype (WT60) and MsrA-MsrB double LOF (AB46) lines were used. Only males are used for experiments at 5-10 days, 20-25 days, 40-45 days, and 60-

65 days. Being that the lifespan for wildtype is until 60-65 days while the Msr double mutants neared 40-45 days, the “young age,” “middle age,” and “old age” were classified differently. In the wildtype line, young age was between 20-25 days old, middle age was between 40-45 days old, while 60-65 day old flies were under old age [Table 3]. In the

Msr double deletion line, young age was between 5-10 days old, middle age was 20-25 days old, and old age was 40-45 days old [Table 3].

Age Wildtype Line Msr Double Deletion Line

Young Age 20-25 Days Old 5-10 Days Old

Middle Age 40-45 Days Old 20-25 Days Old

Old Age 60-65 Days Old 40-45 Days Old

Table 3: Age Classification for Wildtype and Msr Double Deletion Lines. All age groups were used in this study.

33 Anoxia Tank

The flies in each respective tube were placed into the anoxia tank. The flies were also stressed in an anoxia tank containing 100% nitrogen gas for 1 hour. To increase the time to fall into spreading depression, the flow rate of nitrogen gas was reduced to either

0.025 L/sec or 1.348 L/sec.

Measurement of Average Recovery Time and Survival

The Drosophila Activity Monitoring (DAM system, Trikinetics) was used again to monitor mobility. The experimental design was modified in order for mobility to be measured during the one-hour period of anoxia as well. The Drosophila Activity Monitor holder was first placed into the Drosophila Activity Monitoring System. Both were then placed in the opened anoxia tank for 10 minutes of normoxia. Afterwards, the anoxia tank’s lid was sealed shut and induction of anoxia was done by pumping nitrogen gas either at

0.025 L/sec or 1.348 L/sec for one hour. After one hour of anoxia, the lid of the anoxia tank was opened and five hours of reoxygenation commenced. All trials occurred during the hours of 12:00pm and 5:00pm to avoid behavior modification due to circadian rhythms.

The first ten minutes of each trial were omitted from the analysis to allow for a stabilization period. The first time during reoxygenation when a fly breaks through the IR beam, that individual fly is considered “recovered.” Animals whose movement failed to be detected after five hours of reoxygenation were presumed “dead.”

34 Results

Only at a young age, Msr double LOF flies (AB46) fell into spreading depression significantly later than wildtype flies (WT60) following anoxic stress.

Since my previous studies indicate that MsrA and MsrB genes seem to affect how fast Drosophila are recovering from anoxic stress, I wanted to see if these genes also affected how fast the double deletion and wildtype flies fell into spreading depression.

For both flow rates, the wildtype line takes significantly longer to fall into spreading depression in comparison to the Msr double deletion line at a young age [Figures 14.A-

14.B]. This significant difference was not seen at either flow rate in middle and old age.

35 Young Age

A. Flow Rate of 0.025 L/sec B. Flow Rate of 1.348 L/sec

Young Age: Time to Fall into Spreading Depression Young Age: Time to Fall into Spreading Depression * *

Genotype Time Genotype Time MsrA+MsrB+ 6.526 minutes MsrA+MsrB+ 5.526 minutes MsrA-MsrB- 5.781 minutes MsrA-MsrB- 3.531 minutes

Middle Age

C. D. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Middle Age: Time to Fall into Spreading Depression Middle Age: Time to Fall into Spreading Depression

Genotype Time Genotype Time + + + + MsrA MsrB 5.571 minutes MsrA MsrB 4.094 minutes - - - - MsrA MsrB 5.375 minutes MsrA MsrB 3.750 minutes

36 Old Age

E. F. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Old Age: Time to Fall into Spreading Depression Old Age: Time to Fall into Spreading Depression

Genotype Time Genotype Time MsrA+MsrB+ 4.094 minutes MsrA+MsrB+ 3.844 minutes MsrA-MsrB- 3.750 minutes MsrA-MsrB- 3.656 minutes

Figure 14: Average Time to Fall into Spreading Depression for Wildtype (WT60) and Msr

Double LOF (AB46) Flies Following Slower Flow Rate of Anoxic Stress. All lines were stressed with both a flow rate of 0.025 L/sec and 1.348 L/sec of nitrogen gas and were monitored with the

DAM system. Significance (p < 0.05) is marked with an asterisk. Flies used were young (14.A,

14.B), middle age (14.C, 14.D), and old age (14.E, 14.F). 64 flies were tested for each genotype.

As they reached senescence, Msr double LOF flies (AB46) recovered significantly earlier than wildtype flies (WT60) immediately following anoxic stress.

Based on my findings from previous experiments that utilized a higher flow rate of nitrogen gas, I predicted that the double LOF flies would also take significantly longer than the wildtype flies to recover from anoxic stress. To increase the time to fall into spreading depression, I slowed the flow rate of nitrogen gas to 0.025 L/sec and 1.348 L/sec. The flies were stressed for an hour of anoxia and their average recovery time was recorded with the

DAM system. At both flow rates, the double LOF flies recovered significantly earlier than wildtype flies (p < 0.0001) at all age groups [Figures 15.A-15.F].

37 Young Age

A. B. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Young Age Average Recovery Time Young Age Average Recovery Time

* *

Middle Age

C. D. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec Middle Age Average Recovery Time Middle Age Average Recovery Time

* *

38 Old Age

E. F. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Old Age Average Recovery Time Old Age Average Recovery Time

* *

Figure 15: Average Time of Recovery for Wildtype (WT60) and Msr Double LOF (AB46) Flies

Following Slower Flow Rate of Anoxic Stress. All lines were stressed with both a flow rate of 0.025 L/sec

and 1.348 L/sec for one hour of anoxic conditions, after which they were monitored with the DAM system. Significance (p < 0.05) is marked with an asterisk. Flies used were young (15.A, 15.B), middle age (15.C,

15.D), and old age (15.E, 15.F). 64 flies were tested for each genotype.

Prior to reaching senescence, double LOF flies (AB46) had 100% percent survival following anoxic stress.

It was observed that following a slower flow rate of nitrogen gas, the double LOF flies recovered significantly faster than wildtype flies. In my next experiments, I wanted to see how their survival was affected by this parameter. The flies were stressed with a nitrogen gas flow rate of 0.025 L/sec and 1.348 L/sec. If no movement was recorded in the

DAM system during five hours following the anoxic stress, the flies were considered dead.

There was a 100% survival seen in the young and middle age double LOF flies, while a

97% survival (flow rate of 0.025 L/sec) and a 91% survival (flow rate of 1.348 L/sec) were observed towards the end of their lifespan (40-45 days) [Figures 16.E– 16.F].

39 Young Age

A. Flow Rate of 0.025 L/sec B. Flow Rate of 1.348 L/sec

Young Age Immediate Survival Young Age Immediate Survival

Middle Age C. D. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Middle Age Immediate Survival Middle Age Immediate Survival

40

Old Age

E. F. Flow Rate of 0.025 L/sec Flow Rate of 1.348 L/sec

Old Age Immediate Survival Old Age Immediate Survival

Figure 16: Average Percent Survival for Wildtype (WT60) and Msr Double LOF (AB46) Flies

Following Slower Flow Rate of Anoxic Stress. All lines were stressed with one hour of anoxic conditions with a flow rate of 0.025 L/sec and 1.348 L/sec, after which they were monitored with the DAM system.

Flies that failed to move in the five hour period following anoxic stress were determined dead. Significance

(p < 0.05) is marked with an asterisk. 64 flies that were young (16.A-16.B), middle age (16.C-16.D), and old age (16.E-16.F). 64 flies were tested for each genotype.

Verification of the induction of hypoxic conditions

After running oxygen sensor testing at the two slow flow rates of nitrogen gas, we ruled out the possibility of leaking occurring in the tank and confirmed that the tank is at least 95% oxygen free. At a flow rate of 0.025 L/sec, it takes about 6 minutes to reach hypoxia [Figure 17]. However, at a flow rate of 1.348 L/sec, hypoxia is reached at about 4 minutes [Figure 18]. From these tests and sources from the literature, it is concluded that these conditions were hypoxic since there was 5% oxygen in the tank [85]. After seeing the noticeable fluctuations between 0% and 5% upon approaching 20 minutes for both flow rates, it has been decided that the time period for these tests will be extended in the future.

41

Percent of Oxygen at a Flow Rate of 0.025 L/sec 100

90

80

70

(%) 60 2

of O of 50

40 Percent 30 20 10

0 0 5 10 15 20 Time (Minutes) Figure 17: Percent of Oxygen at a Flow Rate of 0.025 L/sec

Percent of Oxygen at a Flow Rate of 1.348 L/sec 100

90

80

70

(%) 60 2 50 40

Percent of O Percentof 30

20

10 0 0 5 10 15 20

Time (Minutes)

Figure 18: Percent of Oxygen at a Flow Rate of 1.348 L/sec

42 V. PROLONGED ANOXIA

Background

Through my previous studies and all of the work that has been done in our lab, D. melanogaster have shown that they tolerate one hour of exposure to anoxia. In the literature, it has been reported that fruit flies can tolerate several hours of exposure to hypoxic and/or anoxic conditions without any apparent adverse effects [47]. Unlike mammals, insects do have the ability to spontaneously recover from extended periods of anoxia [47]. To survive hypoxia, Drosophila have the ability to greatly reduce metabolic rate during spreading depression. Since my previous experiments demonstrated that the

Msr double deletion flies recovered significantly faster than the wildtype flies after exposure to one hour of anoxia, I wanted to determine if this effect was still observed in

5-10 day old and 40-45 day old flies after three or even six hours (chronic) of anoxia. I utilized the same slow flow rate of nitrogen gas (0.025 L/sec) done in the previous study.

I anticipated that the Msr double LOF flies will take significantly longer to recover compared to the wildtype flies. However, I predicted a significantly lower survival rate for both fly lines after chronic anoxic stress. I also expected the recovery time to be significantly extended for both the wildtype and Msr double LOF fly strains, since the anoxia time period was prolonged.

43 Materials and Methods

The groups of flies that were used have the following genotypes: MsrA and MsrB present (wildtype) line and MsrA-MsrB double LOF (AB46) line. In this study, the average time to fall into spreading depression as well as the average recovery time and percent of survival following anoxic stress were determined.

The experimental procedures were the same as described previously except the duration of the anoxia stress was extended to three as well as six hours. Lastly, I utilized the slowest flow rate of 0.025 L/sec for this entire study.

Results

When exposed to three hours of anoxia, the young wildtype flies recovered significantly later than the Msr double deletion flies.

Based on my previous studies, I unexpectedly found that wildtype flies took significantly longer than Msr double deletion flies to recover when a slower flow rate of

N2 was used for the one hour period of anoxia. I wanted to see if the extension of anoxia to three and six hours would affect the recovery rates of both fly lines. Using the same slowest flow rate of 0.025 L/sec, the WT60 line took significantly longer (103.22 minutes) to recover from three hours of anoxia in comparison to the Msr double deletion line (77.84 minutes) at 5-10 days [Figure 17.A]. At the same flow rate, the young wildtype flies took 255.04 minutes to recover from six hours of anoxia in comparison to the Msr double deletion mutants, who took 202.65 minutes [Figure 17.B]. After 3 hours, the 40-45 day old wildtype flies recovered at 196.47 minutes [Figure 18.A].

44 A. B.

*

Genotype p value Genotype p value + + - - MsrA+MsrB + vs MsrA-MsrB- < 0.0001 MsrA MsrB vs MsrA MsrB 0.1417

Figure 19: Average Recovery Time for 5-10 Day Old Wildtype (WT60) and Msr Double LOF (AB46)

Flies Following 3 Hours and 6 Hours of Anoxic Stress. All lines were stressed with a flow rate of 0.025

L/sec of nitrogen gas and were monitored with the DAM system. Significance (p < 0.05) is marked with an asterisk. 32 flies were tested for each genotype.

C.

Figure 20: Average Recovery Time for 40-45 Day Old Wildtype (WT60) and Msr Double LOF

(AB46) Flies Following 3 Hours and 6 Hours of Anoxic Stress. All lines were stressed with a flow

rate of 0.025 L/sec of nitrogen gas and were monitored with the DAM system. Significance (p < 0.05)

is marked with an asterisk. 97% of Msr double mutant flies died post 3 hours of anoxia at 40-45 days.

100% of flies died for both genotypes post 6 hours of anoxia at 40-45 days. 32 flies were tested for

each genotype. 45 A. B.

Figure 21: Average Percent of Survival for 5-10 Day Old Wildtype (WT60) and Msr Double LOF (AB46) Flies Following 3 Hours and 6 Hours of Anoxic Stress. All lines were stressed with a flow rate of

0.025 L/sec of nitrogen gas and were monitored with the DAM system. Significance (p < 0.05) is marked with an asterisk. 32 flies were tested for each genotype.

C.

*

Figure 22: Average Percent of Survival for 40-45 Day Old Wildtype (WT60) and Msr Double LOF

(AB46) Flies Following 3 Hours and 6 Hours of Anoxic Stress. All lines were stressed with a flow rate of

0.025 L/sec of nitrogen gas and were monitored with the DAM system. Significance (p < 0.05) is marked with

an asterisk. 97% of Msr double mutant flies died post 3 hours of anoxia at 40-45 days. 100% of flies in both

genotypes did not survive after 6 hours of anoxic stress. 32 flies were tested for each genotype.

46 The Msr double deletion flies did not survive prolonged anoxia near senescence.

At 5-10 days old, both the wildtype and Msr double deletion flies had a 100% survival at three hours and six hours of anoxia [Figures 19.A –19.B]. However at 40-45 days old, nearly all of the double Msr LOF flies died (only 0.03% survived) while the wildtype had a 100% survival under three hours of anoxia [Figure 20.C]. However, at 40-

45 days, neither the wildtype nor Msr double LOF flies survived after 6 hours of anoxia.

32 flies were used for each genotype.

47 V. DISCUSSION

Although the role of Msr in oxidative stress has been extensively studied, the investigation into how the absence of Msr activity affects anoxia tolerance in Drosophila is a new area of research. In the early 1990s, fruit flies were discovered to be tolerant to acute anoxia (0mm Hg O2). They can survive in this oxygen depleted environment for several hours without any evidence of injury [61]. In our lab, MsrA and MsrB gene deletions were created through imprecise excision of p-element transposons located in each gene. This led to development of Drosophila as the first known in-vivo animal model to lack any known Msr activity. Drosophila has also been a powerful genetic model for human diseases. Sixty-two percent of human genes associated with disease or other health issues likely have homologues in Drosophila including genes for cancer (72%), neurological diseases (64%), metabolic diseases (82%), and renal diseases (69%) [15].

These studies utilized four genotypes of Drosophila - wildtype, strains that lack either MsrA or MsrB as well as a strain completely lacking any known Msr activity due to deletion of both the MsrA and MsrB genes. The key experiments focused on the effect of anoxia under conditions that vary the rate at which the spreading depression coma is induced as well as the length of the anoxia stress. With only a few exceptions, there was little to no significant differences between wildtype and strains lacking one of the two Msr genes. The most interesting data were obtained when the wildtype was compared to the

Msr-deficient strain (also known as the Msr double deletion). Hence, the following discussion will focus on the results obtained utilizing these two genetic strains.

48 What is the effect of altering the rate at which the spreading depression coma is induced?

The time required for the flies to stop moving due to the protective coma induced by anoxia can be varied by altering the rate at which the chamber becomes anoxic (i.e. by controlling the flow rate of the nitrogen gas). For these experiments, the total time of anoxic exposure was one hour. At the highest flow rate tested (1.961 L/sec), both strains were motionless in the spreading depression coma in less than one minute. However, the Msr double deletion strains took significantly longer to recover from the anoxic stress compared to the wildtype throughout the lifespan of the animals [Figure 10]. More interestingly, the

Msr-deficient flies showed a markedly reduced ability to survive the anoxia stress as they approached senescence with only 38% surviving among the flies that were 40-45 days old.

[Figure 13.C]. In contrast, the wildtype strains had nearly 100% survival of the anoxia throughout their entire lifespan including the period of senescence [60-65 days old; Figure

13.F].

According to the oxidative stress theory of aging, as animals age, an increase in

ROS and oxidative stress plays a role in governing lifespan. Previous research suggests that the accumulation of oxidative damage as part of the aging process, in addition to ROS produced during anoxic stress, leads to increased cellular damage, which results in a longer recovery time and decreased percentage of survival [20]. This susceptibility causes the fly to take longer to recover from this anoxic stress with increasing age [3]. This may also be due to the reduction in the levels of both isoforms of Msr enzymatic activity with aging

[12]. These experiments are the first evidence of an age-dependent effect of Msr deficiency in both recovery and survival from anoxic stress.

49 I then asked whether anoxia tolerance (measured through percent survival and/or recovery time) would be altered if the spreading depression was induced more slowly.

Spreading depression is a reversible state where the metabolic rate significantly decreases and allows the fly to preserve cellular ATP while also significantly decreasing total ATP production [81]. As expected, the time to induce the protective coma was longer at the slower nitrogen gas flow rates (1.348L/sec and 0.025L/sec), but there was no significant difference between the wildtype and the Msr-deficient flies [Figures 14.A-14.F]. These data suggest that the onset of spreading depression is not affected by the lack of Msr activity. However, upon reoxygenation, the Msr-deficient flies, in contrast to the previous experiment, recovered faster than wildtype flies throughout their entire lifespan at both nitrogen gas flowrates tested [Figure 15]. More interestingly, the survival of the Msr- deficient flies improved dramatically, especially as the animals approached senescence. As just noted, survival following anoxia where the spreading depression was induced quickly was only 38% at senescence for the Msr-deficient line. However, at the two slower rates of spreading depression induction, survival was nearly 100% throughout the entire lifespan

[Figures 16.A-16.F].

In summary, rapid induction of spreading depression leads to longer recovery times and an age-dependent decrease in survival in the absence of Msr activity. However, inducing anoxia more slowly leads to faster recovery times in the Msr-deficient strains compared to wildtype. Additionally, the flies lacking Msr have wildtype levels of survival, even as they approach senescence.

These unexpected results suggest that the slow induction of spreading depression may give enough time for ischemic preconditioning to commence. Mild ischemic stress

50 triggers late preconditioning by causing the release of chemical signals, referred to as

“triggers.” These triggers in the form of nitric oxide, reactive oxygen species, and adenosine, function as biochemical signals to the heart of a potential threat. When these triggers are released, a signal transduction cascade is activated that includes protein kinase

C (PKC) and Janus-activated kinases 1 and 2 (JAK1/2). This leads to activation of cytoplasmic as well as stress-responsive transcription factors including NF-kB, STAT1, and STAT3. Downstream events result in the upregulation of cardioprotective genes containing the inducible isoform of NOS (iNOS), cyclooxygenase (COX)-2, and antioxidant enzymes such as SOD. Thus, neuroprotective signaling and decreased apoptotic pathway activation as well as gene reprograming and metabolic downregulation may be the reason the Msr-deficient flies recover faster and have markedly improved survival when the induction of anoxia occurs more slowly [57]. Since preconditioning is a genetic reprogramming of the heart caused by exposure to threatening stimuli, such as anoxia and reoxygenation, the extended time to fall into spreading depression may play a role in the activation of stress-responsive and protective genes [10].

Possible interactions between serotonin and Msr

What is the underlying mechanism for the faster recovery and improved survival following anoxia in the Msr-deficient flies when the spreading depression coma is induced more slowly? Recent experiments in our lab suggest that Msr-deficient Drosophila have elevated levels of serotonin. The evidence is still speculative since we have not measured serotonin levels at the time of this writing. Serotonin is a hydrophilic indolamine that acts as a neurotransmitter and neuromodulator in the central nervous system (CNS). In

Drosophila, the serotonergic system begins development in the first instar stage of larval

51 development and reaches full development in viscosity and density in the third instar [40].

Serotonin is released by serotonergic neurons and their excess is transported back into the presynaptic neuron by the serotonin transporter (SerT) in the larval brain [Figure 23]. These serotonergic neurons in D. melanogaster are responsible for the coordination of feeding behaviors and body mass control through the insulin/IGF pathway [71].

If we assume that Msr-deficient flies have elevated serotonin levels, this can lead to “serotonin syndrome” which results in a variety of detrimental effects of serotonergic- induced morphological aberrations (neuritic spheroids) that lead to increased ubiquitination [40]. The altered levels of serotonin can inhibit the insulin/IGF pathway, resulting in developmental delay and growth retardation [40]. Mutations in the insulin/IGF pathway in Drosophila display deleterious effects on longevity, resistance to oxidative stress, and muscle development due to alterations in the mTORC1 pathway [54]. Thus, excess serotonin due to the absence of Msr activity may affect the insulin/IGF, which in turn affects the ability of the animal to resist oxidative stress and possibly allow for faster recovery following anoxia. Future experiments will pursue this hypothesis by pharmacological manipulation of serotonin levels in two ways. First, the culture medium will be supplemented with 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin. The second approach would utilize fluoxetine (Prozac), which effectively raises the serotonin levels by blocking the reuptake of the neurotransmitter. If the hypothesis is correct, wildtype animals would recover faster and have improved survival levels following anoxia in the presence of elevated serotonin levels.

52

Figure 23: Serotonin Synthesis and Release: The initial step in serotonin synthesis is facilitated transport of

L-tryptophan from the blood to the brain. L-tryptophan gets further converted into 5-hydroxytryptophan and then into serotonin. Serotonin is then packaged into vesicles and released into the synaptic cleft via fusion.

Excess serotonin in the synaptic cleft gets transported back into the presynaptic neuron by the serotonin transporter. Image courtesy of https://github.com/research-team/NEUCOGAR and caption courtesy of Kelsey

WhatWilson. are the effects of prolonged anoxic stress?

Fruit flies have been reported to survive up to six hours of anoxia [22]. The anoxia tolerance in fruit flies permits survival of extended anoxia without neuronal deficit, due to the protective coma they entered during anoxia [22]. The previous experiments showed that a slow induction of the spreading depression coma helped Msr-deficient flies recover faster and nearly all of them survived one hour of anoxia. But what would happen if the anoxia were extended to three or even six hours? Using the slowest nitrogen flow rate, very young animals (5-10 days), had 100% survival for both the wild type and Msr-deficient strains even after six hours of anoxia Figure 18]. At 40-45 days old, 100% of the wildtype flies survived three hours of anoxia although none of the animals survived six hours of anoxia. In sharp contrast, only 3% (1 of 32 flies) of the 40-45 day Msr-deficient flies survived the three hours of anoxia. Therefore, there is a clear age-dependent decline in the 53 ability to survive prolonged anoxia in the absence of Msr. The underlying mechanism may be associated with the relationship between the production and depletion of cellular energy during spreading depression. During anoxia, the metabolic rate is known to significantly decrease to allow Drosophila to preserve cellular ATP while also significantly decreasing the total production of ATP [34, 68,69, 81]. When flies start to recover upon reoxygenation, there is less available ATP to restore metabolic deficits due to the ATP depletion during the period of anoxia. Overall, survival is compromised [61,68]. In addition, ATP depletion is known to lead to failure of the Na+/K+ ATPase, which results in dysregulation of ionic homeostasis, protein unfolding and subsequently protein aggregation [37]. Numerous reports in the literature reflect similar trends of a strong inverse correlation between increased stress duration and decreased survival probability, possibly due to deficiency in

ATP production and the inability of the fly to compensate for ATP consumption [34, 81].

Possible interactions between PKG and Msr

While a possible interaction between serotonin and Msr is still highly speculative, our understanding of cGMP-dependent protein kinase (PKG) in anoxia tolerance is more well understood. The PKG cascade is a biochemical pathway that is evidently critical for controlling low oxygen tolerance in the Drosophila. The foraging gene encodes PKG and is important for low-oxygen tolerance in behavior of the fruit fly [22]. Two naturally occurring alleles – rover (forR ) and sitter (fors ) - have been studied in detail. The rover strain is homozygous for the forR allele which expresses high PKG activity while the sitter strain homozygous for fors has low PKG activity [22]. Higher PKG levels (rover allele) allow for better survival under prolonged anoxia, yet there is a reduction in neural function tolerance to acute hypoxia [22]. On the other hand, low PKG activity (sitter allele) protects

54 the fly during anoxic stress by allowing neural function protection during acute hypoxia, however mortality significantly increases [22]. Under slow induction of spreading depression, possibly allowing some level of ischemic preconditioning, the Msr-deficient flies may have lower levels of PKG that would help protect neuronal function. This may allowed them to recover faster than the wildtype flies. However upon prolonged anoxia, these old Msr-deficient flies may have died following the 3 hours of anoxia due to declining

PKG levels as a function of age [Figure 22]. These hypotheses could be tested by using rover and sitter alleles of the foraging gene in the absence of Msr activity. The prediction would be that animals with the sitter allele of the foraging gene will have a higher survival rate and a faster recovery time. Strains with rover allele of the foraging gene would be expected to have higher survival during prolonged anoxia

Interestingly, young (5-10 days) Msr deficient flies recovered significantly faster than the wildtype lines even upon prolonged anoxia. This unusual behavior among animals lacking Msr activity was only observed when spreading depression was induced slowly.

This raises the question of whether the physiology of Msr deficient flies involve longevity pathways. The silent information regulator two proteins (sirtuins or SIRTs) are a group of histone deacetylases that are regulated by nicotinamide adenine dinucleotide (NAD+).

SIRT1 inhibits p53 and reduces its pro-apoptotic effect [52]. In contrast, SIRT1 can also activate a transcriptional coactivator, PGC-1alpha to cause increased glucose levels, insulin sensitivity, and mitochondrial biogenesis [60]. Overall, these effects contribute to their longevity effect of caloric restriction. Recent studies show that the hypothalamus may contribute to longevity effects of SIRT1 and calorie restriction by coordination of neurobehavioral and neuro-endocrinal changes of body temperature, appetite and physical

55 activity. Sirtuins are best known to exert protective effects against neurological disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis [84]. Thus, the improved anoxia tolerance seen in the Msr deficient mutants may possibly be linked to pathways that contribute to longevity.

Overall, the results obtained from this study show that MsrA and MsrB play an age- dependent role in protection against oxidative stress throughout the lifespan of D. melanogaster. MsrA and MsrB are known to behave as antioxidants to reduce methionine sulfoxide (nonfunctional form of methionine from ROS oxidation) back to the functional form of methionine [19]. The original expectation was that Msr-deficient flies would have a compromised ability to tolerate anoxia. As typical of many investigations, the answer is more complicated. The results of experiments using the Msr-deficient flies suggest new lines of inquiry involving ischemic preconditioning and longevity pathways as well as the biochemical pathways of PKG and serotonin. Msr clearly appears to affect how the flies respond to the anoxia and, in most cases, there is an age-dependent affect that is not observed with the wildtype flies. These results support previous studies that suggest the activation of protective mechanisms to defend against oxidative stress, essentially leading us to a better understanding how these Msr genes affect aging. These studies offer possible insight into anoxia-like conditions in humans, such as stroke, that may ultimately contribute to better drug design or other treatments.

56 VI. REFERENCES

1. Afanas'ev, I., Signaling of reactive oxygen and nitrogen species in Diabetes mellitus.

Oxid Med Cell Longev, 2010. 3(6): p. 361-73.

2. Alamuri, Praveen, and Robert J. Maier. “Methionine Sulfoxide Reductase

in Helicobacter Pylori: Interaction with Methionine-Rich Proteins and Stress-Induced

Expression.” Journal of Bacteriology 188.16 (2006): 5839–5850. PMC. Web. 20 Apr.

2018.

3. Ann N Y Acad Sci. 2009 Oct;1177:39-47. doi: 10.1111/j.1749-6632.2009.05045.x.

Review.

4. Aviram, M. (2000). Review of human studies on oxidative damage and antioxidant

protection related to cardiovascular diseases. Free Radic Res, 33 Suppl, S85-97.

5. Beal, M.F., Oxidatively modified proteins in aging and disease. (2002). Free Radic

Biol Med. 32(9): p. 797-803.

6. Beckman, K. B., & Ames, B. N. (1998). The free radical theory of aging matures.

Physiol Rev, 78(2), 547-581.

7. Benasayag-Meszaros, Raquel et al. “Pushing the Limit: Examining Factors That

Affect Anoxia Tolerance in a Single Genotype of Adult D. Melanogaster.” Scientific

Reports 5 (2015): 9204. PMC. Web. 10 Oct. 2017.

8. Berlett, B.S. and E.R. Stadtman, Protein oxidation in aging, disease, and oxidative

stress. J Biol Chem, 1997. 272: p. 20313-20316.

57 9. Blokhina, O., E. Virolainen, and K.V. Fagerstedt, Antioxidants, oxidative damage and

oxygen deprivation stress: a review. Ann Bot, 2003. 91 Spec No: p. 179-94.

10. Bolli, Roberto. “Preconditioning: A Paradigm Shift in the Biology of Myocardial

Ischemia.” American journal of physiology. Heart and circulatory physiology292.1

(2007): H19–H27. PMC. Web. 22 Apr. 2018.

11. Brot, N., & Weissbach, H. (1983). Biochemistry and physiological role of methionine

sulfoxide residues in proteins. Arch Biochem Biophys, 223(1), 271-281. doi: 0003-

9861(83)90592-1 [pii]

12. Bulvik, Baruch E. et al. “Aging Is an Organ-Specific Process: Changes in

Homeostasis of Iron and Redox Proteins in the Rat.” Age 34.3 (2012): 693–704.

PMC. Web. 18 Jan. 2018.

13. Butterfield, D. A. (2006). Oxidative stress in neurodegenerative disorders. Antioxid

Redox Signal, 8(11-12), 1971-1973. doi: 10.1089/ars.2006.8.1971

14. Butterfield, D. A., & Lauderback, C. M. (2002). Lipid peroxidation and protein

oxidation in Alzheimer's disease brain: potential causes and consequences involving

amyloid beta-peptide-associated free radical oxidative stress. Free Radic Biol Med,

32(11), 1050-1060.

15. Chien S, Reiter LT, Bier E, Gribskov M. Homophila: human disease gene cognates

in Drosophila. Nucleic Acids Res. 2002;30(1):149–151.

16. Christen, Y., Oxidative stress and Alzheimer disease. Am J Clin Nutr, 2000. 71(2): p.

621S-629S.

58 17. Chung, H., et al., The Drosophila homolog of methionine sulfoxide reductase A

extends lifespan and increases nuclear localization of FOXO. FEBS Lett. 2010.

584(16): p. 3609-14.

18. Cookson, M.R. and P.J. Shaw, Oxidative stress and motor neurone disease. Brain

Pathol, 1999. 9(1): p. 165-86.

19. Cudic, Predrag et al. “Identification of Activators of Methionine Sulfoxide

Reductases A and B.” Biochemical and biophysical research communications469.4

(2016): 863–867. PMC. Web. 4 Jan. 2018.

20. Das N., Levine R. L., Orr W. C. & Sohal R. S. Selectivity of protein oxidative

damage during aging in Drosophila melanogaster. Biochem J 360, 209–216

(2001). [PMC free article] [PubMed]

21. Davi, G., A. Falco, and C. Patrono, Lipid peroxidation in diabetes mellitus. Antioxid

Redox Signal, 2005. 7(1-2): p. 256-68.

22. Dawson-Scully, K. et al. “Controlling Anoxic Tolerance in Adult Drosophila Viathe

cGMP–PKG Pathway.” The Journal of Experimental Biology 213.14 (2010): 2410–

2416. PMC. Web. 10 Oct. 2017.

23. Drysdale, Rachel A., and Madeline A. Crosby. “FlyBase: Genes and Gene

Models.” Nucleic Acids Research 33.Database Issue (2005): D390–D395. PMC.

Web. 21 Apr. 2018.

24. Finkel, T. (2005). Radical medicine: treating ageing to cure disease. Nat Rev Mol Cell

Biol, 6(12), 971-976. doi: 10.1038/nrm1763

25. Fortini, Mark E. et al. “A Survey of Human Disease Gene Counterparts in

the Drosophila Genome.” The Journal of Cell Biology 150.2 (2000): 23–30. Print.

59 26. Gabbita S. P., Aksenov M. Y., Lovell M. A., Markesbery W. R. Decrease in peptide

methionine sulfoxide reductase in Alzheimer’s disease brain. Journal of

Neurochemistry. 1999;73(4):1660–1666. doi: 10.1046/j.1471-

4159.1999.0731660.x. [PubMed] [Cross Ref]

27. Gibson, G., R. , J.B. Martins, and S. Gandy, Altered oxidation and signal

transduction systems in fibroblasts from Alzheimer patients. Life Sci., 1996. 59: p.

477-89.

28. Gilbert SF. Developmental Biology. 6th edition. Sunderland (MA): Sinauer

Associates; 2000. Aging: The Biology of Senescence.

29. Giugliano, D., A. Ceriello, and G. Paolisso, Oxidative stress and diabetic vascular

complications. Diabetes Care, 1996. 19(3): p. 257-67.

30. Good, P.F., P. Werner, A. Hsu, C.W. Olanow, and D.P. Perl, Evidence of neuronal

oxidative damage in Alzheimer's disease. Am J Pathol., 1996. 149: p. 21-28.

31. Grimaud, R., et al., Repair of oxidized proteins: Identification of a new methionine

sulfoxide reductase. J. Biol. chem., 2001. published October 24, 2001 issue.

32. Gros-Louis, F., Gaspar, C., & Rouleau, G. A. (2006). Genetics of familial and

sporadic amyotrophic lateral sclerosis. Biochim Biophys Acta.

33. Haddad, G.G., et al., Behavioral and Electrophysiologic Responses of Drosophila

melanogaster to Prolonged Periods of Anoxia. J Insect Physiol, 1997. 43(3): p. 203-

210.

34. Haddad, G. G., Sun, Y., Wyman, R. J. & Xu, T. Genetic basis of tolerance to O2

deprivation in Drosophila melanogaster. P Natl Acad Sci USA 94, 10809–10812

(1997).

60 35. Halliwell, B. (2006). Oxidative stress and neurodegeneration: where are we now? J

Neurochem, 97(6), 1634-1658. doi: 10.1111/j.1471-4159.2006.03907.x

36. Harmon, D.J., Free radical theory of aging. Mutation Res., 1992. 275: p. 257-66.

37. Harrison, J. F. & Haddad, G. G. Effects of oxygen on growth and size: synthesis of

molecular, organismal, and evolutionary studies with Drosophila melanogaster. Annu

Rev Physiol 73, 95–113; 10.1146/annurev-physiol-012110-142155 (2011).

38. Hatfield, Dolph L. et al. “Selenium and Selenocysteine: Roles in Cancer, Health and

Development.” Trends in biochemical sciences 39.3 (2014): 112–120. PMC. Web. 21

Apr. 2018.

39. Hitchon, C.A. and H.S. El-Gabalawy, Oxidation in rheumatoid arthritis. Arthritis Res

Ther, 2004. 6(6): p. 265-78.

40. Huser, A., Rohwedder, A., Apostolopoulou, Widmann, A., Pfitzenmaier, J., Maiolo,

E., Thum, A. (2012). The Serotonergic Central Nervous System of the Drosophila

Larva: Anatomy and Behavioral Function. PlosOne, 1-23.

41. Johnson, Ian P. “Age-Related Neurodegenerative Disease Research Needs Aging

Models.” Frontiers in Aging Neuroscience 7 (2015): 168. PMC. Web. 7 Apr. 2018.

42. Kim, H.Y. and V.N. Gladyshev, Alternative first exon splicing regulates subcellular

distribution of methionine sulfoxide reductases. BMC Mol Biol, 2006. 7: p. 11.

43. Kim, Hwa-Young. “The Methionine Sulfoxide Reduction System: Selenium

Utilization and Methionine Sulfoxide Reductase Enzymes and Their

Functions.” Antioxidants & Redox Signaling 19.9 (2013): 958–969. PMC. Web. 18

Jan. 2018.

61 44. Kim, Hwa-Young, and Vadim N. Gladyshev. “Methionine Sulfoxide Reduction in

Mammals: Characterization of Methionine-R-Sulfoxide Reductases.” Ed. Guido

Guidotti. Molecular Biology of the Cell 15.3 (2004): 1055–1064. PMC. Web. 21 Apr.

2018.

45. Knight, J.A., Reactive oxygen species and the neurodegenerative disorders. Annals of

Clinical & Laboratory Science, 1997. 27: p. 11-25.

46. Koc, A., Gasch, A. P., Rutherford, J. C., Kim, H. Y., & Gladyshev, V. N. (2004).

Methionine sulfoxide reductase regulation of yeast lifespan reveals reactive oxygen

species-dependent and -independent components of aging. Proc Natl Acad Sci U S A,

101(21), 7999-8004. doi: 10.1073/pnas.0307929101

47. Kolsch, G., Jakobi, K., Wegener, G. and Braune, H. J. (2002). Energy metabolism

and metabolic rate of the alder leaf beetle Agelastica alni (L.) (Coleoptera,

Chrysomelidae) under aerobic and anaerobic conditions: a microcalorimetric study. J.

Insect. Physiol. 48, 143-151. [PubMed]

48. Kryukov, G.V., et al., Selenoprotein R is a zinc-containing stereo-specific methionine

sulfoxide reductase. Proc Natl Acad Sci U S A, 2002. 99(7): p. 4245-50.

49. Lee, Byung Cheon, Dung Tien Le, and Vadim N. Gladyshev. “Mammals Reduce

Methionine-S-Sulfoxide with MsrA and Are Unable to Reduce Methionine-R-

Sulfoxide, and This Function Can Be Restored with a Yeast Reductase.” The Journal

of Biological Chemistry 283.42 (2008): 28361–28369. PMC. Web. 11 Apr. 2018.

50. Levine, R.L., et al., Methionine residues as endogenous antioxidants in protein. Proc.

natl. Acad. Sci. U.S.A., 1996. 93: p. 15036-15040.

62 51. Lim, D.H., Han, J.Y., Kim, J.R., Lee, Y.S., Kim, H.Y. (2012). Methionine sulfoxide

reductase B in the endoplasmic reticulum is critical for stress resistance and aging in

Drosophila.

52. Luo J, Nikolaev AY, Imai S, Chen D, Su F, Shiloh A, Guarente L, Gu W. Negative

control of p53 by Sir2alpha promotes cell survival under stress. Cell. 2001; 107:137–

148. [PubMed: 11672522]

53. Lutz, P. L., Prentice, H. M. & Milton, S. L. Is turtle longevity linked to enhanced

mechanisms for surviving brain anoxia and reoxygenation? Exp Gerontol 38, 797–

800 (2003).

54. Maor, G., Yearim, A., & Ast, G. (2015). The alternative role of DNA methylation in

splicing regulation. Cell Press, 274-281.

55. Marchetti, Maria A. et al. “Silencing of the Methionine Sulfoxide Reductase A Gene

Results in Loss of Mitochondrial Membrane Potential and Increased ROS Production

in Human Lens Cells.” Experimental eye research 83.5 (2006): 1281–1286. PMC.

Web. 21 Apr. 2018.

56. McCord, J. M. (2000). The evolution of free radicals and oxidative stress. Am J Med,

108(8), 652-659.

57. Milton, Sarah L, and Ken Dawson-Scully. “Alleviating Brain Stress: What

Alternative Animal Models Have Revealed about Therapeutic Targets for Hypoxia

and Anoxia.” Future neurology 8.3 (2013): 287–301. PMC. Web. 17 Apr. 2018.

58. Minniti, A. N., Cataldo, R., Trigo, C., Vasquez, L., Mujica, P., Leighton, F.,

Aldunate, R. (2009). Methionine sulfoxide reductase A expression is regulated by the

63 DAF-16/FOXO pathway in Caenorhabditis elegans. Aging Cell, 8(6), 690-705. doi:

10.1111/j.1474-9726.2009.00521.x

59. Moskovitz, Jackob et al. “Methionine Sulfoxide Reductase (MsrA) Is a Regulator of

Antioxidant Defense and Lifespan in Mammals.” Proceedings of the National

Academy of Sciences of the United States of America 98.23 (2001): 12920–

12925. PMC. Web. 21 Apr. 2018.

60. Nemoto S, Fergusson MM, Finkel T. SIRT1 functionally interacts with the metabolic

regulator and transcriptional coactivator PGC-1{alpha}. J Biol Chem. 2005;

280:16456–16460. [PubMed: 15716268]

61. Nilsson, G.E. and P.L. Lutz, Anoxia tolerant brains. J Cereb Blood Flow Metab,

2004. 24(5): p. 475-86.

62. Nilsson, G. E. & Renshaw, G. M. Hypoxic survival strategies in two fishes: extreme

anoxia tolerance in the North European crucian carp and natural hypoxic

preconditioning in a coral-reef shark. J Exp Biol 207, 3131–3139; 10.1242/jeb.00979

(2004).

63. Nunomura, A., Castellani, R. J., Zhu, X., Moreira, P. I., Perry, G., & Smith, M. A.

(2006). Involvement of oxidative stress in Alzheimer disease. J Neuropathol Exp

Neurol, 65(7), 631-641. doi: 10.1097/01.jnen.0000228136.58062.bf

64. Nunomura, A., T. Tamaoki, and N. Motohashi, [Role of oxidative stress in the

pathophysiology of neuropsychiatric disorders]. Seishin Shinkeigaku Zasshi, 2014.

116(10): p. 842-58.

64 65. Ocorr, Karen, Takeshi Akasaka, and Rolf Bodmer. “Age-Related Cardiac Disease

Model of Drosophila.” Mechanisms of ageing and development 128.1 (2007): 112–

116. PMC. Web. 10 Oct. 2017.

66. Parthasarathy, S., D. Litvinov, K. Selvarajan, and M. Garelnabi, Lipid peroxidation

and decomposition--conflicting roles in plaque vulnerability and stability. Biochim

Biophys Acta, 2008. 1781(5): p. 221-31.

67. Perry, G., A. Nunomura, K. Hirai, X. Zhu, M. Perez, J. Avila, R.J. Castellani, C.S.

Atwood, G. Aliev, L.M. Sayre, A. Takeda, and M.A. Smith, Is oxidative damage the

fundamental pathogenic mechanism of Alzheimer's and other neurodegenerative

diseases? Free Radic Biol Med, 2002. 33(11): p. 1475-9.

68. Rodgers, C. I. et al. Stress preconditioning of spreading depression in the locust CNS.

PloS one 2, e1366; 10.1371/journal.pone.0001366 (2007).

69. Rodriguez, E. C. & Robertson, R. M. Protective effect of hypothermia on brain

potassium homeostasis during repetitive anoxia in Drosophila melanogaster. J Exp

Biol 215, 4157–4165; 10.1242/jeb.074468 (2012).

70. Roesijadi, Guri, Rezvankhah, Saeid, Binninger, David M., and Weissbach,

Herbert. Ecdysone Induction of MsrA Protects Against Oxidative Stress in

Drosophila. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.01.005.

71. Ruaud, A., & Thummel, C. (2008). Serotonin and insulin signaling team up to control

growth in Drosophila. Genes and Development, 1851-1855.

72. Ruan, H., et al., High-quality life extension by the enzyme peptide methionine

sulfoxide reductase. Proc. Natl. Acad. Sci,, USA, 2002. 99(5): p. 2748-2753.

65 73. Salmon, Adam B. et al. “Lack of Methionine Sulfoxide Reductase A in Mice

Increases Sensitivity to Oxidative Stress but Does Not Diminish Life Span.” The

FASEB Journal 23.10 (2009): 3601–3608. PMC. Web. 21 Apr. 2018.

74. Savakis, C., Demetri, G., & Cherbas, P. (1980). Ecdysteroid-inducible polypeptides

in a Drosophila cell line. Cell, 22(3), 665-674.

75. Schoneich, C., Methionine oxidation by reactive oxygen species: reaction

mechanisms and relevance to Alzheimer's disease. Biochim Biophys Acta, 2005.

1703(2): p. 111-9.

76. Shchedrina, V.A., et al., Overexpression of methionine-R-sulfoxide reductases has no

influence on fruit fly aging. Mech Ageing Dev, 2009. 130(7): p. 429-43.

77. Stadtman, E.R., et al., Cyclic oxidation and reduction of protein methionine residues

is an important antioxidant mechanism. Mol Cell Biochem, 2002. 234-235(1-2): p. 3-

9.

78. Stadtman, E.R., et al., Methionine oxidation and aging. Biochim Biophys Acta, 2005.

1703(2): p. 135-40.

79. Stadtman, E.R., Oliver, C.N., Starke-Reed, P.E., and S.G. Rhee, Age-related

oxidation reaction in proteins. Toxicol Ind Health, 1993. 9: p. 187-196.

80. Stocker, R. and J.F. Keaney, Jr., Role of oxidative modifications in atherosclerosis.

Physiol Rev, 2004. 84(4): p. 1381-478.

81. Van Voorhies, Wayne A. “Metabolic Function in Drosophila Melanogaster in

Response to Hypoxia and Pure Oxygen.” The Journal of Experimental Biology212.19

(2009): 3132–3141. PMC. Web. 18 Jan. 2018.

66 82. Wood-Kaczmar, A., Gandhi, S., & Wood, N. W. (2006). Understanding the

molecular causes of Parkinson's disease. Trends Mol Med, 12(11), 521-528. doi:

10.1016/j.molmed.2006.09.007

83. Zhang, X.-H., Baronas-Lowell, D., & Weissbach, H. (2011). Unique metabolic roles

of methionine both free and in proteins. Current Topics in Peptides and Protein

Research, 12, 1-15.

84. Zhang, Feng et al. “Protective Effects and Mechanisms of Sirtuins in the Nervous

System.” Progress in neurobiology 95.3 (2011): 373–395. PMC. Web. 20 Apr. 2018.

85. Zhou D, Xue J, Lai JCK, Schork NJ, White KP, et al. (2008) Mechanisms Underlying

Hypoxia Tolerance in Drosophila melanogaster: hairy as a Metabolic Switch. PLoS

Genet 4(10): e1000221. doi:10.1371/journal.pgen.1000221

67