Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Summary

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1^B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Abstract

Neurodegenerative diseases, including Parkinson's Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.

This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1^B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Introduction

Mitochondria are cellular organelles that control important functions, including apoptotic regulation, calcium homeostasis, and participation in biosynthetic pathways. By possessing autonomous genetic material, they are capable of contributing to cellular maintenance and repair processes. Their structure houses the electron transport chain and oxidative phosphorylation, both crucial for cellular energy^1,2,3. In particular, energy control is achieved through adenosine triphosphate (ATP) production via oxidative phosphorylation (OXPHOS)². Disruption of energy metabolism with impairment of mitochondrial functions occurs both in cell survival and death^4,5, frequently associated with a wide range of human pathologies, such as cancer, and neurodegenerative diseases such as Parkinson's Disease (PD)^3,6.

PD is a chronic, progressive, and neurological disorder. The primary cause of this disease is the death of brain cells, especially in the substantia nigra, which are responsible for the production of the neurotransmitter dopamine, which controls movement^6,7,8. The earliest observation that linked Parkinsonism to mitochondrial dysfunction was made in 1988, in experimental models using toxins that inhibit the respiratory chain Complex I⁹.

Currently, there are several methods to evaluate mitochondrial dysfunction^10,11,12,13; however, compared to conventional approaches, high-resolution respirometry (HRR) presents superior sensitivity and advantages^13,14. For example, HRR protocols allow the evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes^14,15. Mitochondrial dysfunctions can be assessed in intact cells, isolated mitochondria, or even ex vivo^10,11,13,14.

Mitochondrial dysfunctions are closely associated with many pathological and physiological processes. It is therefore important to study mitochondrial physiology and bioenergetics using genetically and experimentally tractable model systems. In this regard, research on Drosophila melanogaster, the fruit fly, has several advantages. This model shares fundamental cellular characteristics and processes with humans, including the use of DNA as genetic material, common organelles, and conserved molecular pathways involved in development, immunity, and cell signaling. In addition, fruit flies have a rapid life cycle, easy maintenance, low cost, high throughput, and fewer ethical concerns, thus constituting an invaluable tool for dissecting complex cellular processes^{16,17,18,19,20}.

Furthermore, a homolog of the PTEN-induced putative kinase 1 (pink1) gene is expressed in D. melanogaster. It plays a crucial role in the removal of damaged mitochondria through the process of mitophagy⁸. In humans, mutations in this gene predispose individuals to an autosomal recessive familial form of PD associated with mitochondrial dysfunction^8,21,22,23. Consequently, the fruit fly is a powerful animal model for studies on the pathophysiology of PD and screening of drug candidates focusing on mitochondrial dysfunction and bioenergetics. Therefore, the present work explains how to analyze mitochondrial function in a model of PD from D. melanogaster using the HRR technique in the OROBOROS with the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Protocol

We used the strains w¹¹¹⁸ (white) and w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3 (referred to as Pink1^B9) (FlyBase ID: FBgn0029891) from the Bloomington Drosophila stock center (ID number: 34749). In this study, male D. melanogaster PINK1^B9-null mutants are compared with male D. melanogaster from the w¹¹¹⁸ strain, which is used as a control group (genetic background). Other parameters must be analyzed concomitantly with the respirometry experiments to ensure that the flies have the correct genotype (Pink1^B9/Y), such as thorax deformities and locomotion problems, which are well described for pink1^B9 mutant flies^24,25,26.

1. Animals and housing

1. Keep the flies in glass bottles containing 10 mL of standard diet (yeast 1.73%, soy flour 0.9%, corn flour 7.3%, agar 0.5%, corn syrup 7.6%, and propionic acid 0.48%) at constant temperature and humidity (25 °C and 60%, respectively), with a 12 h:12 h light/dark cycle.
2. For each experiment, use two flies aged 1-3 days post-eclosion.
   ​NOTE: The PINK1^B9-null mutant virgin female flies were crossed with w¹¹¹⁸ males to obtain the F1 males with genotype Pink1^B9/Y.

2. Sample preparation

1. Prepare the MiR05 buffer as described in Table 1.
2. Anesthetize the flies on ice and transfer them to microcentrifuge tubes (two flies per tube).
3. In each tube, add 200 µL of chilled MiR05 buffer and homogenize the flies. Homogenize manually, applying gentle pressure (approximately 4-6 strokes of the pestle) to prevent the disintegration of mitochondria.

3. High-resolution respirometry calibration of polarographic oxygen sensors

NOTE: The OROBOROS chambers have a total volume of approximately 2 mL. Calibration is required to ensure the oxygen flux is close to 0 pmol to start the assay.

1. Starting the calibration, add 2 mL of MiR05 to the chamber. Cover the chamber with the stopper leaving no air bubbles; then, carefully pull the stopper to form one single air bubble.
2. Open the OROBOROS Dat.Lab software. Enter a temperature of 25 °C in the Block temperature field and click on Connect to Oxygraph-2k (Figure 1A).
3. Click and select the folder where the calibration is to be saved and click on Save.
4. When a new window opens, name the experiment and the samples (if calibrating, simply enter the name calibration) and click on Save.
5. Go to the Layout menu and choose the first option: 01 Calibration Exp. Gr.3 - Temp as shown in Figure 1B.
6. Wait for the program to redirect to the calibration layout, wait until the red line shows a standard straight line with points around 0 pmol. Then, select two points: one for chamber A and the other for chamber B (Figure 1C) when the red line is exactly at zero.
   NOTE: The selected points must have oxygen flux (red line) around 0.
   1. To mark the points of both chambers, select O₂ concentration on the right of the screen, select the marked point, and copy both the temperature and barometric pressure values relative to the point. Paste these values in the boxes below and click on Calibrate and Copy to Clipboard in the lower right corner of the screen as shown in Figure 2.
   2. Carry out this process for both chambers (A and B), then go to the File menu and select Save and Disconnect.
      ​NOTE: After the calibration process, the program will redirect the user to the home screen so that the software will be ready to start the HRR test. For this test, we will use the SUIT protocol.

4. SUIT protocol

1. With the calibration completed, remove the stoppers and open the chambers.
2. Wash the stoppers (holding the tip that does not touch the sample) and chambers with 100% ethanol, 70% ethanol, and distilled water, in that order.
   NOTE: Repeat the process for every sample change.
3. Homogenize the flies in 200 µL of buffer, place all sample content in the chamber using a micropipette, and relocate the stopper in the chamber, taking care to not create air bubbles.
   NOTE: If air bubbles form, remove the stopper gently and add 100 µL of MIR05 buffer.
4. Click on the Layout menu and select the option 05 Flow by Corrected Volume as shown in Figure 1B.
5. Wait to be redirected to a new window. Click on Save to select the folder where the experiment is to be saved.
6. Wait for another new window to open. Name the experiment in the space Experimental Code. Then, name each sample in its respective chamber A and B in the Sample field, and set the field Unit to unit.
   NOTE: Other units can also be chosen, for example, Millions of cells or mg.
7. Considering that this protocol uses two flies (2 units) and the chamber volume is 2 mL, in the Concentration field, define 1 per mL (Figure 1D).
   NOTE: Here, the sample amount was normalized by the number of flies; however, this normalization may be performed by the protein content of the sample, mitochondrial DNA quantification, or activity of citrate synthase.
8. When the oxygen flux signal (red line) is steady on the positive value, start the HRR protocol with the titration of substrates, inhibitors, and uncouplers (Figure 3). All the subsequent reagents and equipment are shown in Table 1.
   1. Add digitonin (5 μg/mL) to permeabilize the mitochondrial membrane.
   2. Add pyruvate (5 mM), malate (2 mM), and proline (10 mM) substrates and wait until the oxygen flux increases and stabilizes. To mark the event for each reagent addition, press the F4 key on the keyboard and enter the name of each reagent.
   3. Add ADP (5mM) to couple the mitochondrial respiratory chain and wait for the increase and stabilization of the oxygen flux.
   4. Add succinate (10 mM) and wait for the increase and stabilization of the oxygen flux.
   5. Add Oligomycin (2.5 µM) to inhibit ATP synthase and look for a decrease in oxygen flux.
   6. Wait until the red line stabilizes and uncouple mitochondrial electron transfer using Carbonyl-4-(trifluoromethoxy) phenylhydrazone cyanide (FCCP) with titers of 0.25 µM until the maximum oxygen consumption is reached, demonstrated by the rise of the red line. After stabilization of the oxygen flux, start adding the inhibitors of each complex, one at a time.
   7. Add Rotenone (0.5 µM), a complex I inhibitor. Wait for the decrease and stabilization of the oxygen flux to add the next inhibitor.
   8. Add Malonate (5 mM), a complex II inhibitor. Wait for the decrease and stabilization of the oxygen flux to add the next inhibitor.
   9. Finally, add Antimycin (2.5 µM), a complex III inhibitor. Wait for the decrease followed by the increase and stabilization of the oxygen flux.
      NOTE: The oxygen flux should stabilize at a positive value but below the value for the last inhibition of mitochondrial protein complexes.
9. At the end of the analysis, remove all the content of the chambers using a micropipette. Store at -20 °C for future analysis, when necessary.

5. Data analysis

1. Utilize an appropriate statistical software package for data analysis.
2. Perform t-tests to assess differences between the two groups. For situations involving multiple treatments, use Analysis of Variance (ANOVA) as a statistical test ²⁷.
3. Extract the O₂ flux values from the graph generated by the software. OXPHOS CI refers to the value of O₂ flux after adding ADP. OXPHOS CII is obtained by subtracting OXPHOS CI&CII - OXPHOS CI. OXPHOS CI&CII is the value of O₂ flux extracted after the addition of succinate. ETS CI&CII is the O₂ flux value after adding FCCP. ETS CI = FCCP - Rotenone and ETS CII = Rotenone - Malonate.
4. Calculate ATP synthesis as the difference between the OXPHOS (P) and LEAK (L) (P - L)²⁸.
5. Determine the Respiratory Control Ratio (RCR) by calculating the ratio OXPHOS/LEAK, where RCR = P/L.¹¹

Results

Here, we that O₂ flux in OXPHOS CI (P = 0.0341) and OXPHOS CI&II (P = 0.0392) states is reduced in PINK1^B9 null flies when compared to control flies (Figure 4). This result was also observed in previous findings from our group^29,30.

CI and CII are key components of the electron transport system (ETS), in which CI is responsible for the transfer of el...

Discussion

HRR is a powerful technique for studying mitochondrial respiration and energy metabolism in D. melanogaster and other organisms. It provides a detailed and quantitative assessment of mitochondrial function, allowing researchers to gain insights into the bioenergetics of the cells. The protocol presented here describes the evaluation of mitochondrial respiratory chain function and the activity of specific mitochondrial complexes using the SUIT protocol in D. melanogaster. The SUIT protocol involves syste...

Materials

Name	Company	Catalog Number	Comments
ADP	Sigma-Aldrich	A5285	Adenosine 5′-diphosphate sodium sal (CAS number 72696-48-1); ≥95%; molecular weight = 501.31 g/mol.
Ágar	Kasv	K25-1800	For bacteriologal use
Antimycin-A	Sigma-Aldrich	A8674	Antimycin A from Streptomyces sp. (CAS number 1397-94-0); molecular weight  540 g/mol;
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A7030	Bovine Serum Albumin (CAS number 9048-46-8); pH 7,0 ≥ 98%
Datlab software	Oroboros Instruments, Innsbruck, Austria	20700	Software for data acquisition and analysis
Digitonin	Sigma-Aldrich	D 5628	CAS number 11024-24-1
Distilled water
Drosophila melanogaster strain w[*] Pink1[B9]/FM7i, P{w[+mC]=ActGFP}JMR3			Obtained from Bloomington Drosophila stock center
Drosophila melanogaster strain w1118			Obtained  from the Federal University of Santa Maria
EGTA	Sigma-Aldrich	E8145	Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (CAS number 13638-13-3); ≥97%; molecular weight =468.28 g/mol
FCCP	Sigma-Aldrich	C2920	Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone  (CAS number 370-86-5); ≥98% (TLC), powder
GraphPad Prism version 8.0.1.			Software for data acquisition and analysis
Hepes	Sigma-Aldrich	H4034	4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9); ≥99,5% (titration), cell cultured tested; molecular weight = 238.30 g/mol
High-resolution respirometer Oxygraph O2K	Oroboros Instruments, Innsbruck, Austria	10022-02	Startup O2K respirometer kit
KH2PO4	Sigma-Aldrich	P5379	Monopotassium phosphate (CAS number 7778-77-0); Reagente Plus, molecular weigt = 136.09 g/mol
KOH	Sigma-Aldrich	211473	Potassium hydroxide (CAS number 1310-58-3); ACS reagent, ≥85%, pellets
Malate	Sigma-Aldrich	M1296	Malonic acid (CAS number 141-82-2); 99%, molecular weight = 104.06 g/mol). A solution is pH adjusted to approximately 7.0.
Malic acid	Sigma-Aldrich	M1000	(S)-(−)-2-Hydroxysuccinic acid (CAS number 97-67-6); ≥95% ; molecular weight = 134.09 g/mol
MES	Sigma-Aldrich	M3671	2-(N-Morpholino)ethanesulfonic acid (CAS number 4432-31-9); ≥99% (titration); molecular weight = 195.24 g/mol
MgCl2	Sigma-Aldrich	M8266	Magnesium chloride (CAS number 7786-30-3); anhydrous, ≥98%, molecular weight = 95.21 g/mol
Microcentrifuge tubes	Eppendorf
O2K-Titration Set	Oroboros Instruments, Innsbruck, Austria	20820-03	Hamilton syringes with different volumes
Oligomycin	Sigma-Aldrich	O 4876	Oligomycin from Streptomyces diastatochromogenes (CAS number  1404-19-9); ≥90% total oligomycins basis (HPLC)
Pistil to homogenization
Proline	Sigma-Aldrich	P0380	L-Proline (CAS number 147-85-3); powder; 99%; molecular weight = 115.13 g/mol
Pyruvate	Sigma-Aldrich	P2256	Sodium pyruvate (CAS number 113-24-6), ≥99%; molecular weight = 110.04 g/mol
Rotenone	Sigma-Aldrich	R8875	Rotetone (CAS number 83-79-4); ≥95%, molecular weight 394.42 g/ mol
Succinate	Sigma-Aldrich	S 2378	Sodium succinate dibasic hexahydrate (CAS number 6106-21-4); ≥99%
Sucrose	Merck	107,651,000	Sucrose for microbiology use (CAS number 57-50-1)
Taurine	Sigma-Aldrich	T0625	CAS number 107-35-7