Activity levels of SOD mutations in Drosophila mitochondria

using small scale enrichment

Janelle Burgos*, Ariel Chasipanta

*Janelle Burgos, jburg006@plattsburgh.edu.

Ariel Chasipanta, achas001@plattsburgh.edu.

Abstract

This experiment was conducted to determine the relationship between superoxide dismutase
(SOD) mutation location in the cell and the amount of mitochondrial activity/mg protein with
mutations in the three types of SOD. An enrichment procedure of Drosophila mitochondria with
and without the 50% SOD mutations was adapted using a suspension in a sugar gradient and
centrifugation; the activity/mg protein levels of the isolated mitochondria were calculated with
mean and SEM. The flies with SOD1 and SOD3 mutations showed lower levels of activity in
comparison to the wild type and SOD2 mutation suggesting that SOD1 and SOD3 are resulting
in higher levels of oxidative stress which may result in macro-autophagy because of
mitochondrial and cellular location.

Keywords: Drosophila, SOD1, SOD2, SOD3, oxidative stress, mitochondria, SDH assay
INTRODUCTION

Cell energy comes in various forms but mostly as ATP which is a product formed in the
mitochondria. The mitochondrion creates the energy in a cell by means of a proton gradient
created by the electron transport chain, a series of complexes that move electron. The gradient is
then used to produce ATP through an F-class ATPase (Kim, 2008). The ATP can be used for a
variety of processes and reactions throughout the cell.

In aerobic organisms, the mitochondrias energy production can also result in the production of
reactive oxygen species (ROS). These ROSs are produced when there is a high ratio of
NADH/NAD+ and a low proton motive force (p) or a low concentration of Coenzyme Q (CoQ)
and a high p. ROSs can lead to oxidative stress and DNA damage if left unchecked in the cell
(Murphy, 2009). This damage can lead to various symptoms and diseases such as cancer,
cardiovascular and neurodegenerative diseases, chronic fatigue, and heart attack. Regulation of
high ROS concentration is paramount to the homeostasis of an organism (Guo et al., 2013).

The ROS we will focus on in this report is superoxide (Blackney et al., 2014). Superoxide is a
proximal ROS and is toxic to an organism (Murphy, 2009). It is most commonly produced by
NADH oxidase which is a complex in the electron transport chain (Muller et al., 2007). When
the concentration of superoxide get too high, the enzyme superoxide dismutase (SOD) binds to it
and forms hydrogen peroxide (H2O2) (Huang and Manton, 2004). SOD has three forms that
result in a reduction in superoxide concentration in various locations in the cell: SOD1, SOD2,
and SOD3. In research, SOD1 has been found primarily in the inter membrane space (IMS) of
the mitochondria while SOD2 is found in side of the cristae, and SOD3 accumulates outside of
the mitochondria in the extracellular space (Zelko et al., 2002, Higgins et al., 2003, Weyemi et
al. 2012, Tafuri et.al, 2015). Mutations in these three forms can lead to a reduction in SOD
activity which can directly result in an increase in superoxide concentration in the cell and high
oxidative stress.

In this report, the relationship between SOD mutations and mitochondria activity was explored in
Drosophila. In order to accurately enrich live mitochondria on a small scale, adaptation of a lab
procedure created by Hyun-Ju Kim was necessary in (Kim, 2008). By taking advantage of cell
compartmentalization, isolation of the mitochondria from the other organelles and complexes in
the cell allows for clear and precise data about activity in various locations within the
mitochondria without interference by the complexities of other organelles and proteins.

MATERIALS AND METHODS

Fly stocks

Oregon R, SOD3 KG06029, and SOD2 n283/CyO Drosophila were obtained from the Bloomington
Stock Center. SOD1X39/TM3 Drosophila were obtained from the John Phillips lab. The flies were
maintained at 25C at 60% humidity and were run on a 12hr light:dark cycle. They were fed the
Nutri-Fly Bloomington formulation (NF-B) (Genesee Scientific).

Differential centrifugation

Whole flies, approximately 60 for each trial, were homogenized using 2ml of Isolation Buffer
(IB) (40ml of 210mM solid mannitol, 70mM solid sucrose, 1mM EGTA, and 5mM HEPES ph
7.2). The homogenized flies were then centrifuged and collected in intervals while remaining on
ice as much as possible for preservation (Figure.1). 1.5ml of the fly mixture was collected and
transferred to an eppendorf tube which was then centrifuged at 1000xG for 5 minutes at 4C.
200ul of the supernatant was collected and put in a separate eppendorf, labeled S1. The
remaining supernatant was measured and put in an eppendorf, labeled P2, and centrifuged at
1000xG for 5 minutes at 4C.

200ul of the P2 supernatant was collected and placed in a new eppendorf, labeled S2. The
remaining supernatant was measured and transferred to a new eppendorf, labeled CM, and
centrifuged at 8,800xG for 10 minutes at 4C. The remaining pellet was suspended with 200ul of
IB, measured. After centrifugation, the remaining supernatant of the CM eppendorf was
measured and transferred to a new eppendorf, labeled S3. The remaining pellet was suspended
with 200ul of IB, volume was measured. Care was taken to ensure there was no increase in
temperature of the supernatant and pellet.

A similar procedure was repeated for the SOD1, SOD2, and SOD3 mutants, ranging
approximately 60 flies per trial. However, the S1 samples were diluted in a series of 100%, 75%,
50%, and 25% S1 concentration after the S1 was made to confirm that the assay was linear.

Enzyme activity determination

The labeled eppendorfs put on ice: S1, S2, S3, P2, and CM were then used in a SDH assay. A
SDH assay buffer (80ml of 10mM Succinate, 10mM Phosphate buffer pH 7.4, and 2mM NaCN)
was created to track enzyme activity over time using DCPIP as a means to track absorption.
Approximately 20ul of DCPIP was added to every 10ml of SDH in a separate conical tube to
reach an absorbance at 600nm of around 0.4 to 0.6 absorbance units. A set of three cuvettes for
each sample were filled with 1ml of SDH assay buffer plus DCPIP. 20ul of the sample fractions
were deposited individually in the cuvette sets; the samples were shaken immediately and then
placed in the LoggerPro SpectroVis Plus to be analyzed.

The resulting slopes for each samples runs were recorded and plotted in Excel. A Bradford
Assay was then performed using the standard manufactures protocol (Sigma-Aldrich) to
determine protein concentrations in each of the sample sets. To calculate the activity/ml from the
SDH data, the average was taken for each sample and multiplied by 50; standard deviation of
each sample was taken as well. The activity/ml data was then divided by the correlating average
protein concentrations for the respective samples to get unit activities/mg of protein.

The SOD1, SOD2, and SOD3 S1 dilution flies underwent a similar assay. Instead of a full
spectrum assay of all of the fractions, only the S1 dilutions were assayed with SDH and data was
collected as a group to include other data values for unit activity/mg of protein for all three
mutations.

Statistical analysis

Data from the class SOD mutant assay was recorded and analyzed to view mitochondrial activity
in Drosophila with the mutation using 4 samples of SOD1, 5 samples of SOD2, and 4 samples of
SOD3. Individual data on wild type Oregon R Drosophila was also analyzed to provide a control
comparison as to wild type mitochondrial activity using 3 weeks of data from both the Bradford
and SDH assays. The means of the unit activity/mg of protein were calculated for each sample,
both class and individual, along with the standard error of the mean (SEM) to provide data for
the hypothesis.

RESULTS

Mitochondrial enrichment in wild type and SOD mutants

In the individual wild type trial, enrichment was attempted through SDH assay occurred via
centrifugation (Figure 2). Average activity levels were high in S2 (1.0997 activity/ml) and CM
(1.4568 activity/ml). The average activity of S3 (0.9290 activity/ml) and P2 (0.5731 activity/ml)
provided inconsistent data due to lower activity levels than S1 and S2 respectively. Enrichment
was successful due to high levels of mitochondrial activity in the CM fraction in comparison to a
0.8367 activity/ml level in the S1 fraction. Enrichment for SOD mutants was not successful due
to inconsistent class data received from the S2, S3, P2, and CM fractions. Data from the SDH
Assay was retrieved solely from the S1fraction and SOD1, SOD2, and SOD3 activities were
0.0174 activity/ml, 0.0260 activity/ml, and 0.0163 activity/ml respectively. Data from the SDH
assay of the SOD mutants reflected similarly to the SOD mutant activity/mg protein data.

Data from the mean activity/mg protein with wild type Drosophila showed gradual increase from
S1 to P2 fractions and reduced in the CM fraction. This trend was reflected in a majority of the
week trials with Week 1 being the major outlier due to a drop in P2 activity. A drastic increase of
activity was found in the P2 fraction of Week 5, with the average being 1.6063 and Week 5 P2
having 2.5830. This resulted in a high standard error of the mean (SEM) whereas all other
fractions have a SEM 0.1.

Effect of SOD mutants on activity/mg protein

Activity/mg protein in the class data with wild type and SOD mutant Drosophila showed
similarities. SOD2 and wild type averaged similar activity levels suggesting correlation between
the two (Figure 3). The wild type and SOD2 also had activity levels similar to that of the S1 of
the individual data. The SOD1 and SOD3 mutants showed an averaged similar activity level. The
data suggests possible correlation between wild type and SOD2 mutant as well as between SOD3
and SOD1 mutant.

DISCUSSION

This experiment was run under the hypothesis that SOD mutants would result in a 50% reduction
in the enzymatic activity of wild type SOD1, SOD2, and SOD3 as shown in Blackney 2014.
These oxidative stress reducing enzymes play a large role in cancer development, cell growth,
and overall cell homeostasis. The enrichment of mitochondria in the individual trials was overall
successful because of an increase in the activity level in the CM. The reasoning behind the
differential centrifugation to get the CM fraction was to separate and remove all of the complex
proteins and organelles inside the Drosophila that blocked the full read on mitochondrial activity.
The predicted outcome was that S1 would have the lowest activity because it is the least pure and
CM would have the most detected activity due to it being the purest fraction of mitochondria.
The data from the individual set showed a similar gradient. The discrepancies for the S3 and P2
may have been a result of improper centrifugation speeds and times that would allow proper
separation of layers so in the final run; speeds were varied to obtain more viable data. Another
theory is the necessity of a consistently replaced sugar gradient to create a density guide for the
various organelles and proteins to separate into. The sugar gradient (IB buffer) was added in the
beginning for homogenization, but not replaced to the 1.5ml volume after each supernatant was
removed from the centrifuge. This might have created less of a density gradient for separation,
causing a mix in layers, leading to a lack of activity.

Due to data inconsistency, SOD mutant class enrichment was limited to the S1 stage. Data in
both the protein concentration and the activity assay (SDH) showed a relationship between
SOD1 and SOD3 with low levels in both assays. SOD2 remained high in both assays which
gives rise to its connection to the proposed hypothesis. Ideally, all three mutants should have a
reduced activity and protein concentration. The data for SOD2s activity/mg protein show
similar levels between SOD2 and wild type Oregon R. There is a possibility that SOD2, because
it is located in the matrix of the mitochondria, may not be causing as much damage via oxidative
stress as proposed or that its damage is shielded. Its hidden location may allow it the ability to
bypass cell systems that recognize defects in mitochondrial pathways. SOD1 and SOD3,
however, showed a similar lowered activity/mg protein, opening room for discussion in terms of
how their location in the cell might affect their activity and possible interaction between the two.
SOD1 is located in the IMS of the mitochondria and SOD3 is located in the extracellular space,
making them more accessible for recognition. A probable hypothesis is that result of SOD1 and
SOD3 mutations are more likely to be detected by compounds that induce macro-autophagy,
which would in turn, cause a reduction in activity and protein concentration. Further research on
this theory could be tested through the tracking of SOD1 and SOD3interaction with ATG 3 and
ATG8 precursor complexes in macro-autophagy in the cell.

This procedure not only allowed for the ability to look at unknown connections between mutated
SOD enzymes, but allowed for a possible procedure for isolated mitochondria in a small scale
setting. This can prove useful for future researchers, laboratories, and classrooms to conduct
experimental procedures on pure mitochondria that is scaled down so that it does not have the
constraints of high-tech centrifugation machines as well as large lab equipment.

TABLES AND FIGURES

Figure 1
Figure 1 Overview of enrichment procedure

Mitochondria were enriched through several rounds of centrifugation as shown above. Asterisks

indicate fractions assayed. Figure reproduced from (Kim, 2008).

Figure 2

Average Activity/ml of wildtype Oregon

R Drosophila
2
1.5
Activity/ml

1
0.5
0
S1 S2 S3 P2 CM
Fraction

Figure 2 Mitochondrial enrichment of wildtype Oregen R

Enrichment results of mitochondria in wildtype Oregon R via differential centrifugation on the

various fractions. Data averaged and graphed from a 3 week data pool.

Figure 3

Activity/mg of Protein in Oregon R Wild

Type and SOD mutant Drosophila
1.2
activity/mg protein

1
0.8
0.6
0.4
0.2
0
Ore R standard SOD1 SOD2 SOD3
S1 fraction

Figure 3 Class average activity levels for SOD mutants

Class data for S1 SOD mutants (SOD1, SOD2, SOD3) was calculated in order to track

discrepancies between hypothesized activity and resulting activity using class S1 Oregon R

wildtype as a control.
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