Name: Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster
Uploaded: 2023-11-10T12:10:00
Description: Watch this Scientific Journal Video about Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster at JoVE.com

substrate-uncoupler-inhibitor-titration for mitochondrial respiration analysis in drosophila melanogaster

Analysere mitokondriell fysiologi og bioenergetik hos bananfluer

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 energy1,2,3. 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 death4,5, frequently associated with a wide range of human pathologies, such as cancer, and neurodegenerative diseases such as Parkinson&#39;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 movement6,7,8. 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 dysfunction10,11,12,13; however, compared to conventional approaches, high-resolution respirometry (HRR) presents superior sensitivity and advantages13,14. For example, HRR protocols allow the evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes14,15. Mitochondrial dysfunctions can be assessed in intact cells, isolated mitochondria, or even ex vivo10,11,13,14.
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 processes16,17,18,19,20.
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 dysfunction8,21,22,23. 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.

Forfatter i søkelyset: Utforske mitokondriell funksjon og kjemisk toksisitet ved bruk av Drosophila melanogaster 

Analysere mitokondriell funksjon i en Drosophila melanogaster PINK1B9-null mutant ved bruk av høyoppløselig respirometri

Our research uses Drosophila melanogaster as an experimental model to understand mitochondrial function in disease and chemical toxicity. Some questions we want to answer are, how does PINK-1 mutation affect mitochondrial function and how is this linked to Parkinson's Disease? How can we revert, or avert, the mitochondrial dysfunction associated with this mutation?High-resolution respirometry requires specialized training for precise operation and data analysis. In addition, the system we used doesn't allow working with several samples simultaneously and is not educated for high-throughput analysis. Since it's only possible to run two samples at the same time, the experimental design must be well delineated.Our findings indicate progressive impairment of the mitochondrial function we face in PINK1-Null mutants, which is characterized by decrease in OXPHOS CI link, and ETS CI&CII link, and a metabolic metabolic shift in ATP production from oxidative to glycolytic in pathways. These findings are closely related to Parkinson's Disease pathophysiology. One of the key points of high-resolution respirometry is its ability to provide direct and accurate measurements of oxygen consumption, allowing for detailed analysis of mitochondrial function and cellular metabolism.Also, it's versatile. It can be used for wide range of sample types, including isolated mitochondria. Considering that mitochondrial dysfunction is a common feature of neurodegenerative diseases, such as Parkinson's disease, in the future, we will focus on investigating how mitochondrial function and energy elevation could be important targets for therapies directed to neurodegenerative diseases.

Watch this Scientific Journal Video about Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster at JoVE.com

Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster  

Substrat-uncoupler-inhibitor-titrering for mitokondriell respirasjonsanalyse i Drosophila melanogaster

Etter kalibrering av polarografiske oksygensensorer, fjern proppene og åpne kamrene. Vask proppene og kamrene med 100% etanol, 70% etanol og destillert vann. Overfør fluene til et mikrosentrifugerør.Tilsett 200 mikroliter kjølt MiR05-buffer i røret og homogeniser fluene ved å påføre forsiktig trykk med fire til seks slag av pestle. Bruk en mikropipette til å overføre den homogeniserte fluesuspensjonen inn i kammeret. Dekk kammeret med en propp uten å innføre luftbobler.Klikk deretter på layoutmenyen og velg Alternativ 5, Flyt etter korrigert volum. Et nytt nytt vindu åpnes. Her, i rommet, eksperimentell kode, navngi eksperimentet.I prøvefeltet navngir du hver prøve i sitt respektive kammer A og B.Sett deretter enhetsfeltet til Enhet og i konsentrasjonsfeltet definerer du kammervolumet som en per milliliter. For å starte høyoppløselig respirometri, sørg for at oksygenflukssignalet er stabilt på den positive verdien. Legg digitonin for å permeabilisere mitokondriemembranen.Tilsett deretter pyruvat-, malat- og prolinsubstrater til oksygenstrømmen øker og stabiliserer seg. Trykk på F4-tasten på tastaturet og skriv inn navnet på reagenset for å markere hendelsen. Legg ADP til par mitokondriell respiratorisk kjede, og vent på økning og stabilisering av oksygenfluksen.Deretter tilsettes succinat og måles oksygenfluksen. For å hemme ATP-syntase, legg til oligomycin og undersøk for en reduksjon i oksygenfluks. Etter at den røde linjen stabiliserer seg, kobler du fra mitokondriell elektronoverføring ved hjelp av 0,25-mikromolar FCCP til maksimalt oksygenforbruk er nådd, demonstrert av stigningen av den røde linjen.Deretter legger du til rotenonkompleks I-hemmer og venter på reduksjon og stabilisering av oksygenfluksen. Deretter tilsettes malonatkompleks II-hemmer og måler oksygenfluksen. Til slutt, legg til antimycinkompleks III-hemmer og vent på reduksjonen, etterfulgt av økning og stabilisering av oksygenfluksen.Klikk på GraphPad Prism-programvaren for å trekke ut oksygenfluxverdiene fra grafene. OXPHOS CI refererer til verdien av oksygenfluks etter tilsetning av ADP. Bruk deretter den gitte formelen til å beregne OXHPOS CII.Bruk følgende ligning, beregne ETS CI og CII oksygenverdier for uncouples og inhibitorer. Beregn deretter ATP-syntesen som forskjellen mellom OXPHOS og LEAK. Til slutt beregner du OXPHOS ved LEKKASJE-forholdet for å bestemme respirasjonskontrollforholdet.Oksygenfluksen i både OXHPOS CI og OXPHOS CI og CII tilstander var signifikant redusert i PINK1 B9 nullfluer. sammenlignet med kontrollfluer. I tillegg var oksygenfluksen i ETS CI, ETS CII og ETS CI og CII-tilstanden lavere i PINK1 B9 nullfluer, noe som indikerer et svekket elektronoverføringssystem sammenlignet med kontrollfluene.Forstyrrelsen i strømmen av elektroner påvirker OXPHOS-prosessen, noe som fører til redusert ATP-syntese i PINK1 B9 nullfluer. Reduksjonen i respirasjonskontrollforholdet mellom nullfluer antyder mitokondriell frakobling, noe som indikerer at mitokondriene var mindre effektive til å utnytte oksygen og produsere ATP.

Research

JoVE Journal

Biochemistry

Substrate-Uncoupler-Inhibitor-Titration for Mitochondrial Respiration Analysis in Drosophila melanogaster

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

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...