Name: Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster
Uploaded: 2023-11-10T12:10:00
Description: Watch this Scientific Journal Video about Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster at JoVE.com

calibrating polarographic oxygen sensors for high-resolution respirometry analysis in drosophila melanogaster

Analyse van mitochondriale fysiologie en bio-energetica bij fruitvliegen

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.

Auteur in de schijnwerpers: onderzoek naar mitochondriale functie en chemische toxiciteit met behulp van Drosophila melanogaster 

Analyse van de mitochondriale functie in een Drosophila melanogaster PINK1 B9-Null-mutant met behulp van respirometrie met hoge resolutie

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 Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster at JoVE.com

Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry Analysis in Drosophila melanogaster  

Polarografische zuurstofsensoren kalibreren voor respirometrie-analyse met hoge resolutie in Drosophila melanogaster

Voeg om te beginnen twee milliliter MIRO vijf buffer toe aan de Oroboros-kamer. Bedek de kamer met een stop zonder luchtbellen te introduceren. Trek vervolgens voorzichtig aan de stop om een enkele luchtbel te vormen.Open de Oroboros DatLab-software in het veld Bloktemperatuur. Voer een temperatuur van 25 graden Celsius in en klik op Verbinden met Oxygraph-2k. Klik en selecteer de map waarin de kalibratie moet worden opgeslagen.En klik op opslaan. Wanneer een nieuw venster wordt geopend, geeft u het experiment en de voorbeelden een naam. Klik vervolgens op opslaan.Ga vervolgens naar het lay-outmenu en kies de eerste optie, kalibratie-experiment. Grafiek drie, temp. Wanneer de rode lijn een standaard rechte lijn toont met punten rond nul picomol, selecteert u twee punten, één voor kamer A en de andere voor kamer B.Selecteer de zuurstofconcentratie aan de rechterkant van het scherm.Selecteer het gemarkeerde punt en kopieer de temperatuur- en luchtdrukwaarden ten opzichte van het punt. Plak deze waarden in de onderstaande vakken en klik op kalibreren en kopiëren naar klembord. Ga vervolgens naar het bestandsmenu en selecteer opslaan en verbinding verbreken.

Research

JoVE Journal

Biochemistry

Calibrating Polarographic Oxygen Sensors for High-Resolution Respirometry 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 ...