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

Análisis de la fisiología mitocondrial y la bioenergética en moscas de la fruta

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.

Autor destacado: Explorando la función mitocondrial y la toxicidad química usando Drosophila melanogaster 

Análisis de la función mitocondrial en un mutante PINK1B9-nulo de Drosophila melanogaster mediante respirometría de alta resolución

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  

Calibración de sensores polarográficos de oxígeno para análisis de respirometría de alta resolución en Drosophila melanogaster

Para comenzar, agregue dos mililitros de tampón MIRO five a la cámara de Oroboros. Cubra la cámara con un tapón sin introducir burbujas de aire. A continuación, tire con cuidado del tapón para formar una sola burbuja de aire.Abra el software Oroboros DatLab en el campo de temperatura del bloque. Ingrese una temperatura de 25 grados Celsius y haga clic en Conectar a Oxygraph-2k. Haga clic y seleccione la carpeta donde se guardará la calibración.Y haz clic en guardar. Cuando se abra una nueva ventana, asigne un nombre al experimento y a las muestras. A continuación, haga clic en guardar.A continuación, vaya al menú de diseño y elija la primera opción, experimento de calibración. Gráfico tres, temp. Cuando la línea roja muestre una línea recta estándar con puntos alrededor del picomol cero, seleccione dos puntos, uno para la cámara A y el otro para la cámara B.Seleccione la concentración de oxígeno a la derecha de la pantalla.Seleccione el punto marcado y copie los valores de temperatura y presión barométrica relativos al punto. Pegue estos valores en los cuadros de abajo y haga clic en calibrar y copiar en el portapapeles. A continuación, ve al menú de archivos y selecciona guardar y desconectar.

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 ...