SULFOXIDE REDUCTASE DEFICIENCY LEADS TO

MITOCHONDRIAL DYSFUNCTION IN MELANOGASTER

by

Jennifer Verriotto

A Thesis Submitted to the Faculty of

The Charles E. Schmidt College of Science

in Partial Fulfillment of the Requirements for the Degree of

Master of Science

Florida Atlantic University

Boca Raton, Florida

March 2011 REDUCTASE DEFICIENCY LEADS TO

MITOCHONDRIAL DYSFUNCTION IN DROSOPHILA MELANOGASTER

by

Jennifer Verriotto

This thesis was prepared under the direction of the candidate's thesis advisor, Dr. David Binninger, Department of Biological Sciences, and has been approved by the members of her supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science.

SUPERVIS

Gary W. Pe ,Ph.D. Dean, The Charles E. chmidt allege of Science £~r%-/7?~

ii ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. David Binninger for his guidance and constant confidence in my work and me. I would also like to thank everyone in the lab who helped me along the way.

iv ABSTRACT

Author: Jennifer Verriotto

Title: Methionine Sulfoxide Reductase Deficiency Leads to Mitochondrial Dysfunction in Drosophila melanogaster

Institution: Florida Atlantic University

Thesis Advisor: Dr. David Binninger

Degree: Master of Science

Year: 2011

Mitochondria are a major source of reactive oxygen species and are particularly vulnerable to . Mitochondrial dysfunction, methionine oxidation, and oxidative stress are thought to play a role in both the aging process and several neurodegenerative diseases. Two major classes of methionine sulfoxide reductases, designated MsrA and MsrB are that function to repair the enatiomers of methionine sulfoxide, met-(o)-S and met-(o)-

R, respectively. This study focuses on the effect of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. The data show that loss of only one form of Msr in the mitochondria does not completely impair the function of the mitochondria. However, loss of both Msr proteins within the mitochondria leads to an increased ROS production and a diminished energy output of the mitochondria. These results support the hypothesis that Msr plays a key role in proper mitochondrial function.

v METHIONINE SULFOXIDE REDUCTASE DEFICIENCY LEADS TO

MITOCHONDRIAL DYSFUNCTION IN DROSOPHILA MELANOGASTER

List of Tables ...... vii

List of Figures ...... viii

Chapter One – Background and Introduction ...... 1

Aging and Oxidative Damage ...... 1

Oxidative Damage and Mitochondrial Function ...... 2

Mitochondria and Neurodegenerative Diseases ...... 4

Methionine Sulfoxide Reductases (Msrs) History, Function and

Characteristics ...... 5

The Msr System and the Mitochondria ...... 7

Binninger Lab Studies MsrA and MsrB in Drosophila ...... …..10

Chapter Two – Materials and Methods……………………………………………...12

Msr Deficient Drosophila melanogaster Lines…………………………...…12

Creation of MsrB Deficiency Cross ...... 12

Creation of Control and Msr Null Mutants ...... 13

Creation of MsrA Deficiency Cross ...... 14

Molecular and Biochemical Characterization ...... 15

Protein Isolation ……………………………………………………….16

vi Mitochondrial Isolation ...... 16

SDS- PAGE ...... 16

Mitochondrial ATP Synthesis ...... 17

Mitochondrial ROS Production ...... 18

Phenotypic Characterization ...... 18

Climbing Assay ...... 18

Chapter Three – Results ...... 20

Protein Expression Results ...... 22

Climbing Assay Results ...... 23

Mitochondrial ATP Production Results ...... 24

Mitochondrial Expression Results ...... 27

Mitochondrial ROS Production Results ...... 28

Chapter Four- Discussion ...... 29

Creation of Mutant Lines ...... 30

ATP and ROS Production ...... 31

Negative Geotaxis Response ...... 32

Summary and Future Studies ...... 33

Reference………………………………………………………………………………35

vi TABLES

Table 1. Genotype of Stocks ...... 14

Table 2. Percentage Difference Between Mutant Strains and Control………...29

vii FIGURES

Figure 1. Creation of the MsrB deficient cross ...... 13

Figure 2. Morphology of Msr-deficient animals and control ...... 21

Figure 3. Western blot analysis ...... 22

Figure 4. Locomotor assay…………………………………………………………23

Figure 5. ATP production ...... 25

Figure 6. ATP synthase ...... 26

Figure 7. Mitochondrial Western blot analysis ...... 27

Figure 8. ROS-production in flies lacking Msr activity ...... 28

viii I. BACKGROUND AND INTRODUCTION

Aging and Oxidative Damage. Aging is a time-dependent phenomenon characterized by a decline in physiological function among different organisms.

Several theories have been proposed to explain the aging process. One of the most important theories is known as the free radical theory [1, 2]. It hypothesizes that free radicals produced during aerobic respiration cause oxidative damage to macromolecules (e.g., proteins, nucleic acids, lipids) resulting in aging and ultimately death. Considerable evidence suggests that oxidative damage might be related to neurodegenerative diseases and might contribute to a shortened lifespan [3- 5]. This damage may occur from imbalances between oxidant production and repair processes in the different cellular compartments of eukaryotic cells, especially in mitochondria [6]. Mitochondria consume up to 90% of cellular oxygen to support oxidative phosphorylation and synthesis of ATP.

Only 1 -2% of the oxygen consumed by the mitochondrial respiratory chain leads to oxygen radical production and hydrogen peroxide formation [7]. This reactive oxygen species (ROS) production could induce a change in cellular function by damaging the cellular macromolecules that accumulate during aging and neurodegenerative diseases progression.

1 ROS Production in Mitochondria. The generation of mitochondrial ROS (including

superoxide anion radical (O2 ·), hydroxyl radical (·OH), and hydrogen peroxide

H2O2)) is the result of oxidative phosphorylation in the electron transport chain, a process that uses the controlled oxidation of NADH or FADH to generate a potential energy for protons across the mitochondrial inner membrane through complexes I, III, and IV. This potential energy is then coupled to ATP synthesis by complex V. During this process, oxygen normally serves as the ultimate electron acceptor and is reduced to water. However, electrons that leak to oxygen through complexes I and III could generate superoxide anion for ROS production [8]. There is considerable experimental evidence that the two major sites for ROS production are complexes I and III where large changes between the potential energy of the electrons and oxygen reduction take place [9,10].

Consequently, mitochondria are a major source of cellular ROS production and a target for oxidative damage.

ROS and Mitochondrial Function. In aging and disease conditions, cellular ROS levels increase, leading to accumulation of oxidative damage to the mitochondria, impaired calcium buffering, and decrease of ATP production. ATP is indispensable for the body because it maintains chemical gradients by driving channels such as ion pumps, reuptake of neurotransmitters from the synaptic cleft between neurons, as well as acting as the driving force of a multitude of cellular processes. When ATP declines to harmful levels, a massive outflow of excitatory neurotransmitters occurs leading to an increase of ROS [11, 12]. In 2 addition to ATP synthesis, mitochondria are the site of other important metabolic reactions, including steroid hormone and porphyrin synthesis, the urea cycle, lipid metabolism, and interconversion of amino acids. Mitochondria also play central roles in glucose sensing/ regulation, and cellular Ca+2 homeostasis, which affects numerous other cell signaling pathways [13-16]. Despite the harmful cellular damage, ROS at low levels are necessary for some biological processes including cell signaling pathways such as cell proliferation, apoptosis, metalloproteinase function, oxygen sensing, protein kinases, phosphatases, and transcription factors [17-19].

Aging and Mitochondrial Function. Mitochondrial integrity declines with aging and oxidative stress. Oxidative damage to mitochondrial DNA (mtDNA) is caused by its proximity to the source of oxidants and the lack of any protective histone covering, resulting in respiratory chain dysfunction. As the respiratory chain loses efficiency, more ROS are formed, causing more damage to the mitochondria.

The mtDNA and the overall mitochondrial dysfunction have been associated with aging and neurodegenerative diseases [20]. Conversely, several experiments on involved over-expression of mitochondrial . Genes such as

SOD, catalase, MsrA, and INDY play a role in reducing the oxidative damage in the cell by either decreasing the levels of ROS or increasing the repair mechanisms [21-24,31].

3

Associating Mitochondria in Neurodegenerative Diseases. The deleterious effects resulting from the formation of ROS in the could be minimized by various antioxidant systems such as superoxide dismutases, glutathione peroxidase, catalase, methionine sulfoxide reductases, and a variety of DNA-repairing enzymes [25]. There is a stabilizing balance between oxidant production, antioxidant defense, and repair process under normal conditions.

However, in several pathological scenarios the antioxidant defenses become insufficient resulting in oxidative stress often leading to neurodegenerative diseases. One cause of Alzheimer’s disease (AD) may be related to inherited mtDNA mutations that have been found in the control region from AD brains [26] or due to impaired axonal transport [27, 28]. Several studies also showed that mtDNA abnormality might contribute to Parkinson’s disease (PD) due to a chronic infusion of the mitochondrial complex I inhibitor rotenone. The infusions produce a selective loss of substantia nigra and the mechanism of neurotoxicity appears to involve oxidative damage [29, 30]. Amyotrophic Lateral Sclerosis

(ALS) is another neurological disease that has been associated with oxidative stress. ALS cases have been linked to a mutation in the SOD1 that is localized in the mitochondria, where it aggregates and causes mitochondrial dysfunction [31-33]. Huntington’s disease is characterized by cognitive impairment, emotional disturbance, and a movement disorder. Mutant Huntingtin protein binds to the outer mitochondrial membrane and reduces mitochondrial calcium uptake capacity [34]. Other neurodegenerative diseases with correlations to mitochondrial dysfunction are Hereditary Spastic Paraplegia [35], and 4

Friedreich’s Ataxia [36]. Although mitochondria express a variety of protective defenses, these studies have proposed that the oxidation of proteins and the slow accumulation of DNA lesions resulting from the continuous formation of

ROS may contribute to the aging process and neurodegenerative disease progression.

Methionine Oxidation and Oxidative Damage. Oxidation of proteins by ROS has been associated with aging, oxidative stress, and neurodegenerative diseases

[37, 38]. All amino acids are susceptible to oxidation, although, their levels of susceptibility vary greatly. This oxidation may result in structural and functional changes in proteins [39]. Free and protein-bound methionine residues are among the most susceptible to oxidation resulting in the formation of methionine sulfoxide (met-(o)) residues by H2O2, hydroxyl radicals, hypochlorite, chloramines, and peroxynitrite, all of which are produced in biological systems

[40]. However, unlike the oxidation of other amino acid residues, the effect on this sulfur containing amino acid is reversible by the action of methionine sulfoxide reductases (Msr) which catalyze the -dependent reduction of free and protein-bound met-(o) to methionine [41,42]. Oxidation of methionine residues leads to the formation of both R- and S- enantiomers of met-(o), designated met-R-(o) and met-S-(o), respectively, due to the chiral center of the sulfur [43].

5

The History of the Msr Repair System. MsrA was first identified by Weissbach and colleagues in 1981 while they were conducting studies on the biological activity of ribosomal protein L12 in Eschericia coli [44]. Previous studies showed that this protein contained methionine residues that, when oxidized to met-(o), would cause the protein to lose its biological activity, as well as lead to the conversion of a hydrophilic residue from an originally hydrophobic one [43, 45].

However, E.coli extracts revealed a novel that reduced met-(o), and thus repaired the protein and restored its function [44]. This enzyme was designated peptide methionine sulfoxide reductase (pMsr), later renamed MsrA. Even though MsrA is highly conserved in both its sequence and structure throughout nearly all of the species that have been examined, it only reduces the met-S-(o) enatiomer [41, 43]. Later, MsrB was identified as the E.coli YeaA protein, which has Msr activity and specifically repairs met-R-(o) [46].

Methionine Sulfoxide Reductases: Function and Characteristics. Methionine sulfoxide reductases catalyze the reduction of free and protein-bond met-(o) to methionine [47]. All Msr proteins are ubiquitously expressed in cells and together with their substrates and cofactors form repair systems that protect cells from oxidative stress, and maintain cellular redox homeostasis. The Msr enzymes are classified according to their substrate specificity into two types: MsrA which is specific for the reduction of met-S-(o) in both protein-bound and free forms, and

MsrB which catalyzes only the reduction of met-R-(o), primarily in proteins [48].

The three dimensional structures of MsrA and MsrB have been determined, 6 showing that these proteins have different configurations, yet their active sites resemble mirror images of each other [49]. Most living organisms contain homologues of MsrA and MsrB genes and their evolution throughout different species led to a different genetic organization and copy number per organism

[50]. For instance, prokaryotes and unicellular eukaryotes usually have a single

MsrA and MsrB, while plants and vertebrates often have multiple forms of MsrA and MsrB. Multiple locations of Msr indicates that various cellular compartments independently maintain the system for repair of oxidized methionine residues

[51]. Due to its function in reducing methionine sulfoxides, Msr has being implicated in the repair of damaged proteins, antioxidant repair by scavenging

ROS, and regulation of protein functions. Additionally, it is thought to influence the aging process and to be directly associated in aging related neurodegenerative diseases including Alzheimer’s and Parkinson’s diseases [3].

The Msr System and the Mitochondria. The mitochondrial respiratory chain constitutes the main intracellular source of ROS in most tissues. The steady state concentration of these oxidants is maintained at nontoxic levels by a variety of antioxidant defenses and repair enzymes including MsrA and MsrB [52].

Previous studies have shown that over-expression of MsrA in the central nervous system of Drosophila was found to extend their lifespan by 70% [3]. This protein is usually detected in both the cytosol and mitochondria [53-54]. Other studies demonstrated that over-expression of mammalian MsrB2 and MsrB3 protect yeast cells against H2O2 mediated cell death [55]. Hence, the overexpression of 7 methionine sulfoxide reductases could lead to the preservation of mitochondrial integrity by decreasing the intracellular ROS build-up through its scavenging role, hence contributing to cell survival, protein maintenance, and life span.

The Role of Msr in Aging and Neurodegenerative Diseases. Aging is affected by genetic predisposition and environmental factors that may contribute to eventual

DNA degradation and cellular [56]. Additionally, there is a large impact by ROS that could lead to a decrease in lifespan and an acceleration of the aging process. Research conducted in Drosophila show that an increase in lifespan might be accomplished by the over-expression of MsrA [3]. However, knockdowns of this enzyme in mouse lead to an increase sensitivity to oxidative stress. These changes may be due in part to changes in Msr activity further confirming the need for ROS scavengers to increase the lifespan of an organism

[57,58]. Research has also shown that there is a relationship between Msr and neurodegenerative diseases. A studied conducted in AD brain regions showed a decrease in MsrA activity and an increase in carbonyl levels when compared to control subjects [59]. Additionally, oxidation of α-synuclein in

Parkinson’s disease could inhibit its fibrillation if Msr activity is impaired [60].

Overexpression and knockout of the Msr genes. To understand the relationship between oxidative stress, protein oxidative damage, and lifespan, the

Msr genes have been either over-expressed or disrupted in organisms ranging

8 from bacteria to humans. For instance, MsrA over-expressing cells have been shown to protect WI-38 SV40 human fibroblasts, eye lens cells, and T- lymphocytes against H2O2-mediated oxidative stress [61-63]. MsrA over- expression also protected cardiac myocytes and neuronal PC12 against hypoxia/ reoxygenation induced cell death [64, 65]. On the other hand, lack of MsrA in different models has given differing results. For instance, MsrA null mice showed increased sensitivity to oxidative stress, but it did not decrease the lifespan [66].

MsrA was shown to not to be a major virulence determinant for the oral pathogen

Actinobacillus actinomycetemcomitans [67], and a decrease in growth patterns of

MsrA null yeast was manifested when it was exposed to H2O2 treatment [68].

MsrA null mice also exhibited abnormal phenotype, additional studies were able to show increased sensitivity to oxidative stress [69]. In contrast to MsrA, few

MsrB over-expression and disruption studies have been conducted. For instance, over-expression of the MsrB and MsrB3 proteins protected leukemia cells and yeast cells against oxidative stress mediated cell death [70, 55]. Studies performed on Drosophila suggested that over-expression of MsrB has no influence on fruit fly aging [6]. Also, disrupting each of the MsrB genes in humans lens cells lead to increased oxidative stress-induced cell death [71]. Few studies have been done to understand the relationship between oxidative stress, protein oxidative damage, and lifespan in cells or organisms fully deficient for both MsrA and MsrB activities. For instance, double loss-of-function (null) MsrA/ MsrB yeast showed increased sensitivity to oxidative stress and an abnormal phenotype [72].

Mycobacterium tuberculosis also lacking both MsrA and MsrB genes decreased 9 its defenses against reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) and it was readily killed by nitrite or hypochlorite [73].

Drosophila melanogaster and Msr genes. The model organism for this study is

Drosophila melanogaster (Drosophila). It is an excellent model organism for investigating ROS production and its relationship to aging and neurodegenerative diseases. They are easy to culture, have a short life span, and have a fully sequenced genome [74]. Additionally, the Drosophila mitochondrial genome is similar to that of mammals [75]. Its mtDNA is transcribed polycistronically and its basal transcriptional machinery is highly conserved when compared to that of humans [76]. Drosophila has one MsrA and one MsrB gene. In mammals, MsrA is encoded by one gene [77] while MsrB is encoded by three genes designated

MsrB1-B3 [78]. The Drosophila MsrA (dMsrA) gene is located on 3 and the MsrA protein has a molecular mass of 28-29kD. Moreover, it expresses four isoforms generated through differential splicing (designated A-D) [79]. The

Drosophila MsrB (dMsrB) gene is also found on chromosome 3; the dMsrA and dMsrB loci on chromosome 3 are separated by approximately 8 m.u. The dMsrB protein has a molecular mass of 17kD and is expressed as at least seven isoforms (designated A,B,C,E,G,H,I) [80].

Binninger lab Studies on the function of dMsrA and dMsrB in Oxidative Damage to Protein and Aging. My working hypothesis is based on previous studies done by former graduate students Katie Foss and Kelli Robbins. Katie showed loss of 10

MsrA gene in flies does not influence lifespan or sensitivity to paraquat-induced oxidative stress when compared to wild-type revertant controls. Kelli demonstrated through molecular and phenotypic characterization that MsrA/MsrB double-mutant strains of Drosophila have dramatically reduced lifespan, severe locomotor dysfunction and increased sensitivity to oxidative stress.

My goal for this project was to determine whether loss of Msr activity within the mitochondria would lead to an increased ROS production and a diminished energy output of the mitochondria. The preliminary results suggest that the Msr null mutants may have mitochondrial dysfunction due to increased ROS production that reduces energy input and affects locomotor activity

11

II. MATERIALS AND METHODS

Msr Deficient Drosophila melanogaster lines

Background

To understand the effects caused by mutating the MsrA and MsrB genes in

Drosophila melanogaster, a series of crosses were established in our lab. The spontaneous mutations and chromosomal rearrangements were obtained through P-element transposon insertions that lead to genetic mutations and altered gene expression [81]. Prior experiments performed by former students

Katie Foss and Kelli Robbins suggested that flies having mutations in only one of the Msr genes lacked any apparent phenotype and only strains of flies with mutations in both MsrA and MsrB genes would exhibit an obvious phenotype when assessing the role of these genes in lifespan, neuromuscular activity and oxidative stress.

This chapter describes how the stocks and a series of genetic crosses of

Drosophila were generated in our lab to obtain the Msr mutants. The fly lines will be referenced as the strain designations listed in Table 1 (Summary of

Genotypes).

Creation of the MsrB Deficiency cross

Kelli Robbins created a MsrB deficient line (MsrA+ MsrB-) by crossing the stock,

12

17116, which contains a P-element (EP3340) in the promoter a few bases upstream of the transcriptional start site of MsrB, to the deficiency line 7956

(W[1118]; Df(3R) Exel 7305 / TM6B, tb[1]), both obtained from the Bloomington

Stock Center (Indiana University). The stock 7956 contains a deletion on chromosome 3R between 6606276-6697983, which includes the MsrB gene at

86C7 (6,689,071-6,694,379). It is also homozygous lethal, so the stock was maintained with the third chromosome balancer, TM6B. Flies that lacked the

TM6B markers were collected and PCR was performed to confirm their genotype

(Fig. 1).

Females Males Cross 1: w-; + ; 17116 w-; + ; 7956 w-; + ; TM6B ; + ; TM6B

Figure 1. Creation of the MsrB deficient cross. These lines are homozygous lethal; therefore, we selected for recombinants that were homozygous viable to insure that the recessive lethal mutation on the chromosome with the P-element insertion was removed by the recombination. Only one cross was perform to maintain the stock. Progeny had either balanced alleles or 17116/7956.

Creation of the Control and Msr Null Mutant Lines

In addition to generating the MsrB deficient line, Kelli Robbins also created the

Msr null mutant lines (MsrA- MsrB-) and control lines (MsrA+ MsrB+) by

13 recombining two separate P-element stocks 16671 and 17116. The 16671 line contained a P-element (EY05753) that resides in exon 2 of MsrA. Both of these

P-elements are located on the third chromosome and disrupt transcription of the

MsrA and MsrB genes respectively. Recombination events produced reciprocal recombinants (MsrA+ MsrB+) and strains of Drosophila deficient for any Msr activity (MsrA- MsrB-). From the resulting progeny, three double-mutant lines

(MsrA-MsrB-) and four wild-type control lines (MsrA+MsrB+) were established and screened using PCR to confirm that they contained P-elements in their expected locations. All of the double-mutant lines were balanced over TM6B, while the controls were homozygous.

Creation of the MsrA null mutant line

Katie Foss created the MsrA null mutant line (MsrA- MsrB+) by using the P- element stock 16671. By jumping out the P-element residing in Exon 2 of the

MsrA locus in this strain, a deletion null mutant was produced. It begins 300 bp upstream of the TATA box in the promoter and extends 1,172 bp downstream of the transcriptional start site. PCR was performed to determine its phenotype and showed that there was no evidence of an MsrA cDNA in the new strain (Katie

Foss’s FAU thesis)

14

Table 1: Genotype of Stocks

Strain Genotype Description

- + 90 Deletion MsrA MsrB MsrA null MsrA-MsrB+

17116/7956 MsrA+MsrB- Parental MsrB null MsrA+MsrB-

N MsrA-MsrB- MsrA/MsrB null mutants MsrA-MsrB-

R MsrA+MsrB+ Wild-type, reciprocal + + MsrA MsrB recombinant controls

15

Molecular and Biochemical Characterization

Background

The overall goal of this project was to determine the effects of Msr deficiencies on mitochondrial function by utilizing mutant alleles of MsrA and MsrB. Therefore, characterization of the mutant strains was performed using Western blot analysis to show the presence or absence of the Msr proteins. Additionally, mitochondrial

ATP and H2O2 synthesis were used to verify energy and ROS production, respectively. This chapter also explains the different molecular and biochemical techniques utilized for determining the ubiquitous and subcellular expression and activity of the MsrA and MsrB proteins in Drosophila.

Protein Isolation

Procedure:

For total protein expression using Western blot analysis, approximately 50 homozygous and heterozygous adult flies were collected using CO2, placed in a

2.0 mL microcentrifuge tube and frozen at -80˚C. To prepare the extracts, the frozen flies were kept on ice and homogenized in 400µL of Buffer B (50mM

HEPES, potassium salt/pH 7.6, 50 mM KCl, 1mM EGTA, 1mM MgCl2, 10% glycerol), for 10 sec. Next, the samples were centrifuged at 16,300 x g for 20 min at 4˚C [6]. The supernatant was carefully collected and the protein concentration determined using a standard Bradford assay following the manufacturer’s instructions (Bio-Rad). The protein was stored at -80˚C until needed 16

Mitochondrial preparation

Procedure:

The preparation method was modified and developed from an existing protocol to determine ATP synthesis and H2O2 production [8, 82]. The samples were also used for detecting protein expression through Western blot analysis. About 150 adult flies were immobilized by chilling on ice; then ground in 600 µl of isolation

Buffer A (100 mM KCl, 50 mM Tris, pH?, 5mM MgCl2, and 1mM EDTA) and centrifuged until clear at 800x g for 10 min at 4˚C. The supernatant was immediately transferred and centrifuged at 10,000 x g for 15 min at 4˚C. Next, the supernatant was discarded and the pellet resuspended in 100 µl Buffer B containing 225 mM sucrose, 44 mM KH2PO4, 12.5 mM Mg acetate, and 6 mM

EDTA to a final concentration of approximately 15mg protein/ml and kept on ice.

Protein concentration was measured using a standard Bradford assay (Bio-Rad).

Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Procedure:

Protein extracts (5 µg) obtained by homogenization were resolved by SDS-PAGE and transferred onto PVDF membranes. The concentration of polyacrylamide ranged from 15-20% to maximize separation of the small MsrA (28kDa) and

MsrB (17kDa) proteins from a group of higher molecular weight proteins (55-

100kDa) proteins that were causing backround problems on the Western blots

(see results below). Next, the membranes were preincubated for 1 hour with 5% skim milk in 1X TTBS (10X TBS and 0.05% Tween 20), followed by incubation 17 for 1 hour with the corresponding antibody. Polyclonal antibodies against dMsrA and dMsrB were used at 1:500 and 1:1000 dilution, respectively. As a loading control, an antibody against Drosophila β-tubulin and cytochrome C (Santa Cruz) were used at a 1:200 dilution. Immunoblot signals were visualized using the

Immun-Star chemilumiscent system (Bio-Rad).

Mitochondrial ATP Synthesis

Procedure:

To determine ATP production, 10 µl of isolated mitochondria were incubated in

90 µl of resuspension buffer containing mitochondrial respiration stimulating substrates. The isolated mitochondria were added simultaneously in duplicates to Buffers 1, 2, and 3 that contained:

Buffer 1 - 10mM glutamate, 5mM malate, 30µM ADP, 1µM pyrophosphate, and

1mg/ml BSA

Buffer 2 - 30µM ADP, 1µM pyrophosphate, and 1mg/ml BSA

Buffer 3 - 1µM pyrophosphate and 1mg/ml BSA.

Each addition was incubated for 1 min, 2 min, and 3 min after which 80µl mitochondria/substrate solution was transferred to 90µl of ATP monitoring lucifarase/luciferin solution (Invitrogen). ATP levels were measured based on luminescence using a Spremax M5e 96 well plate reader. Protein concentration was measured using a standard Bradford assay (Bio-Rad).

18

Mitochondrial ROS production

Procedure:

The rate of mitochondrial H2O2 in isolated mitochondria was detected by using the Amplex Red kit (Invitrogen) following manufacturer’s protocol. A standard curve was prepared to produce H2O2 concentrations of 0-10 µM. Because this assay is continuous, the fluorescence was measured at multiple time points for

30 min

Phenotypic Characterization

Background

Locomotion was utilized to evaluate the effects of Msr deficiency in Drosophila. A modification of a previously described method [83] was used to determine the ability of each strain to climb the side of a vertical tube. Drosophila has a native negative geotactic response and stronger wild-type flies were expected to climb more rapidly than the phenotypic abnormal double Msr mutants.

Climbing assay

Procedure

Groups of five male and five female flies (1 day post-eclosion) were collected in separated vials by anesthesia with CO2 and allowed to fully recover before the assay. Flies were transferred without anesthetization to the test chamber (30 cm tall with 25 cm wide) and tapped to the bottom. The total number of flies that reached at least 15 cm in 10 sec was recorded. This test was repeated three 19 times with the same flies and consisted of males and females in separate tubes.

Flies were given at least a 5 min resting period between those three trials.

20

III. RESULTS

The Msr null flies showed evidence of a severe phenotype when compared to the wild-type and single null Msr mutants. They were unable to fly and they have impaired locomotor function. About 20% of the Msr null larvae die before eclosion. Of those that eclose successfully, about 25% have severe developmental problems, especially related to proper wing development. Another

40% of the flies have a cream-colored opaque abdomen with little segmentation definition. Many of these animals die within a short time because they were unable to climb out of the food. To perform all the assays, only the healthiest and strongest of the Msr null adults were used (Fig. 2)

Figure 2. Morphology of Msr-Deficient Animals and control. (A) About 25% of the

Msr null flies will present with a wing and posturing defect. The flies are unable to unfurl their wings post eclosion and usually die within 3 days. (B) Msr null mutants often have an opaque-like quality about their abdomens, even when aged. (C) Male wild-type abdomen. Picture provided by Kelli Robbin.

21

Protein expression

Western blot analyses were performed to verify the presence of the MsrA and

MsrB proteins in isolated protein. Wild type, single null, and double null mutants were tested. Western blotting confirmed that lack of either MsrA or MsrB alleles do not code for functional proteins. The locations of the P-elements in the Msr null flies also suggested that both transposon-insertion mutations would be loss of function alleles (Fig. 3).

1 2 3 4

MsrA (28kDa)

β-tubulin (55 kDa)

MsrB (17 kDa)

β-tubulin (55 kDa)

Figure 3. Western Blot Analysis. Protein extracts (5µg) from adult flies of the indicated genetic strains were analyzed by Western blotting using antibodies specific for the indicated protein as described in the Materials and Methods section. Lane 1-MsrA+B+; Lane 2-MsrA-B+; Lane 3-MsrA+B-; Lane 4-MsrA-B-. β- tubulin was used as a loading control. Data indicate that the MsrA- and MsrB- alleles are both loss-of-function (null) alleles.

22

Climbing assay

The locomotor ability of the Msr null flies was compared to their reciprocal wild- type control and single Msr null mutants following the climbing assay protocol.

Only 1% of the Msr double null flies were able to climb 15 cm in 10 sec when compared to about 67% performed by the wild type controls and single Msr mutants (Fig. 4)

Locomotor Assay

80

70

60

50 MsrA+MsrB+

40 MsrA-MsrB+ 30 MsrA+MsrB- %Succesful 20 MsrA-MsrB- 10 0 * Strain

Figure 4. Impaired Mobility of Msr Null Mutants. Ten flies (1 day post-eclosion) of the indicated genotype were assayed for their ability to climb 15 cm in 10 sec as described in the text. The results are the average of three trials per sex. The data are expressed as the percentage of flies that were successful in the assay. Error bars are the SEM, * statistically different from control (P< 0.001).

23

Mitochondrial ATP Production

To verify whether the isolated mitochondria were functional in the mutant flies, the overall ATP production rate was measured by stimulating mitochondrial respiration. Glutamate and malate were used as carbon sources for dehydrogenase reactions in the TCA cycle that generate NADH, which initiates the electron flow through complex I. ADP was used as a substrate for ATP synthase activity (complex V). When glutamate, malate and ADP are incubated together with isolated mitochondria, ATP production could be measured from the entire electron transport chain using a luciferase assay, as described in the

Materials and Methods. ATP synthase activity was measured and subtracted from the overall ATP production to show that ATP production was the result of electron transport chain stimulation and not just a by product of complex V activity (Fig. 5 & 6). Results of these assays indicated that ATP production levels decreased in the Msr null mutants when compared to the wild type controls.

24

ATP Production

150

125

100 MsrA+MsrB+

MsrA-MsrB+

75 MsrA+MsrB-

MsrA-MsrB- 50

g) µ g) (pmol/min/ ATP 25

0 0 1 2 3 Time

Figure 5. ATP Production Decreases in Msr Null Flies. ATP production rate was measured in duplicates using glutamate, malate, and ADP substrates, with ATP formation from ADP subtracted (Fig. 6). Samples were normalized to 1 µg of protein. The Msr double-null mutants showed the lowest level of ATP production when compared to their reciprocal recombinant (wild-type) control and the single

Msr null flies. Control and MsrB single null showed similar results. MsrA single null had lower ATP production than MsrB single null. ATP levels were measured

25 based on luminescence using a Spremax M5e 96 well plate reader. Error bars are the SEM, * statistically different from control (P < 0.005).

ATP Synthase

24 22 20 18

16 MsrA+MsrB+ 14 MsrA-MsrB+ 12 MsrA+MsrB- 10 8 MsrA-MsrB- 6 4 µ g) (pmol/min/ ATP 2 0 0 1 2 3

Time

Figure 6. ATP Synthase activity from ADP to ATP conversion. The ATP synthase activity was measured in duplicates using ADP as substrate. Samples were normalized to 1 µg of protein. Results showed that the ATP synthase activity of each strain is not significantly different from each other. However, the results also reflected a similar pattern to the overall ATP production data (Fig. 5). Error bars are the SEM, * statistically different from control (P < 0.005)

26

Mitochondrial Expression

Western blot analysis was performed to detect the cytochrome c protein in isolated samples used for ATP production assay. The amount of cytochrome c released from the mitochondria extracts would correspond to the amount of mitochondria present in each sample. The results show that reduced ATP production in the Msr null flies (Fig 5) is not the result of fewer mitochondria, based on the visual ratio of cytochrome c to β- tubulin (Fig. 7).

1 2 3 4

Cytochrome c (12 kDa)

β-tubulin (55 kDa)

Figure 7. Mitochondrial Western Blot Analysis. Mitochondrial extract (5µg) from adults of the indicated genetic strains were analyzed by Western blotting.

Cytochrome c antibody was used as an marker to show the presence of mitochondria extract in the samples. β-tubulin was used to evaluate the total amount of cellular protein. Lane 1-MsrA+B+; Lane 2-MsrA-B+; Lane 3-MsrA+B-;

Lane 4-MsrA-B-.

Mitochondrial ROS production 27

The rate of hydrogen peroxide production in mitochondria extracted from adult flies was measured. The amount of ROS production increases over time depending on the amount of oxidative damaged caused in the mutants. The data showed a 40% increase in H2O2 production in the Msr double-null flies as compare with mitochondria isolated from wild-type control flies. The single MsrA and MsrB null flies showed intermediate ROS synthesis. This indicates that the presence of only one form of Msr in the mitochondria might promote protection against reactive oxygen species (Fig 8).

Figure 8. ROS-Production in Flies Lacking Msr Activity. H2O2 production was assayed in mitochondria isolated from adult flies (100) with the indicated genotype as described in the text. H2O2 levels were measured at the indicated

28 time points. Error bars are the SEM, * statistically different from control (P <

0.005)

Table 2. Percentage Difference Between Mutant Strains and Control.

29

IV. DISCUSSION

Our laboratory investigates the function of methionine sulfoxide reductases (Msr) in oxidative damage to proteins and its role in the aging process and development of age-related neurological disorders. The specific goal for this project was to determine whether Msr deficiency leads to mitochondrial dysfunction in the Drosophila model system.

Mitochondria play an important role in cellular function. It is a major site of ATP production as well as the regulator of energy expenditure, apoptosis signaling, and production of ROS. Dysfunctional mitochondria have been related to aging and neurodegenerative diseases. Previous studies have also suggested that the mitochondrial forms of MsrA and MsrB are vital for homeostatic maintenance and the response to oxidative stress [46]. To examine mitochondrial function in Msr null mutants, intact mitochondria were isolated and their ATP and ROS levels measured.

Former graduate students Katie Foss and Kelli Robbins generated the strains used in this research. The mutant flies were MsrA or MsrB single loss-of-function

(null), and Msr double null mutants. A potential caveat in the interpretation of these results is variations in the genetic backgrounds of the parental MsrA null and MsrB null lines [84, 85]. However, the wild-type control and MsrA MsrB double null mutants are sibling stocks and, therefore, have comparable genetic

30 backgrounds.

Prior to beginning the experiments to test for ATP and ROS production in the various mutants, protein expression was confirmed through Western blot analysis. Western blotting was a new technique in the lab and a considerable amount of time and effort were needed to establish it as a routine procedure.

Additionally, the polyclonal antisera made prior to when I joined the lab had not been well characterized. Therefore, improving Western blotting to determine whether or not the mutant strains produce MsrA and MsrB proteins was a high priority when this project started. Little, if any, of those preliminary experiments to develop and troubleshoot Westrn blotting for our lab are contained in this thesis.

Both P-element insertions create loss of function alleles in mutants.

The location of both P-elements (described in Chapter I) suggested that these transposon-insertion mutations would be loss-of-function (null) alleles. Western blotting would be the most definitive since all previous efforts to measure Msr activities in extracts of Drosophila tissue had been unsuccessful. As shown in

Fig. 3, each genetic line had the expected protein expression pattern. The wild- type lines clearly synthesized both MsrA and MsrB proteins, the single mutants were missing the corresponding proteins, and there was no evidence that either

Msr protein was being synthesized by the double-null mutants. These results validated previous studies performed through RT-PCR (data not shown) that confirmed that neither allele produces an mRNA encoding a functional protein.

31

ATP production is reduced in the Msr-deficient animals.

The absence of either Msr protein in the double null strains and their severe phenotype including limited flight and impaired climbing ability suggested that the

Msr-deficient flies may have reduced energy capacity due to diminished ATP production. To explore this possibility further, mitochondrial ATP synthesis was measured by stimulating respiration in isolated mitochondria from mutant flies

(described in Materials and Methods). The results showed a reduced ATP production level in the Msr double-null flies (Fig. 5). ATP production was severely impaired, up to 40%, in Msr double-null mitochondria as compared with mitochondria isolated from control flies. The single MsrA null flies showed a moderate level of ATP production while the single MsrB null flies had similar outcome to the wild-type controls; probably due to mitochondria amount and efficiency in the samples. To corroborate these results, Western blot analysis was performed using the same mitochondrial samples. The results show more mitochondria in each of the preparations from the mutant lines compared to the wild-type control. Furthermore, there were more mitochondria in the MsrB null samples than in the MsrA null samples. This means that the high level of ATP produced by the wild-type control was not due to more mitochondria in the sample, but due to the mitochondria efficiency in maintaining energy production

(Fig 7). However, it should also be noted that the ATP synthase activity (Fig 6) reflected overall ATP production levels in mutants, indicating that there may be a

32 dysfunction in complex V for Msr null flies. Future experiments need to be conducted to identify the efficiency of each complex separately.

ROS production is increased in flies lacking Msr activity

The production level of H2O2 release from isolated mitochondria was measured fluorometrically (described in materials and methods). This technique monitors the overall H2O2 production generated in isolated mitochondria by monitoring the increase in fluorescence over time. The results showed a 40% increase in H2O2 synthesis in the Msr double-null flies as compare with mitochondria isolated from wild-type control flies. The single MsrA and MsrB null flies showed intermediate

ROS synthesis (Fig 8). These findings suggest that the decrease of ATP activity observed in flies lacking both Msr genes possibly led to increased H2O2 production rate by increasing the fraction of upstream electron carriers in a reduced state; thus increasing the rate of univalent reduction of oxygen and consequently the generation of H2O2.

Negative Geotaxis Response

Observation of the Msr double-null flies climbing response reveal that only 1% these mutants were able to climb 15 cm in 10 sec when compared to about 67% performed by the wild type controls. In contrast, flies having a mutation in only one of the two Msr genes performed nearly as well as the wild-type control (Fig.

4). The single null flies failed to produce a phenotype when assayed for locomotor functions while the Msr double-null flies present wing and abdominal defects (Fig. 2). Movement is highly restricted, these flies cannot fly or climb and 33 can barely walk. The high metabolic demands of these insect flight muscles cannot tolerate severe mitochondrial dysfunction. Mutants lacking both Msr genes may have experienced an increase in levels of oxidized biomolecules within the mitochondria due to the required ATP energy necessary for normal locomotor activity; revealing itself during the climbing assay. These findings support other phenotypic assays and observations done in previous experiments.

They showed the effects of oxidative stress on the flight muscle of Drosophila and determined these flight defects are a direct result of mitochondrial damage

[86].

In conclusion, the goal of this project was to determine whether Msr deficiency would cause mitochondrial dysfunction in Drosophila melanogaster. Present and previous studies suggest that lack of both Msr genes could cause damage to the mitochondria, which results in decreased ATP production. This reduced activity may also be responsible for the increased ROS production by the upstream electron carriers; these findings further support the link between Msr and ROS- related defects in locomotor function.

The Binninger lab is currently working in the creation of new Msr single and double null mutants with more comparable genetic backgrounds in order to replicate this experiments and confirm mitochondrial dysfunction due to lack of the Msr gene. Additionally, rescue experiments involving Msr mutant flies would reveal whether or not the severe phenotype is directly related to the loss of the mitochondrial isoforms of Msr. They are also generating transgenic lines that contain the putative mitochondrial and cytoplasmic isoforms of dMsrA and dMsrB 34 to create transgenic flies to investigate the effects of over-expression of these isoforms in different Msr mutant backgrounds. Finally, future experiments will include Msr knock-down experiments using RNAi transgenic lines to evaluate to role of tissue-selective loss of MsrA or MsrB

35

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