Researchers from the Laboratory for Cell Function Dynamics in Japan have developed a new method for detecting mitophagy, the process of recycling damaged mitochondria.
The Necessity of Mitophagy
Our mitochondria are the powerhouses of our cells, turning the food we eat into ATP, the form of chemical energy that our cells use. However, over time, our mitochondria become damaged, thus harming our cells’ basic ability to function. One method that our cells use to stave off this problem is called mitophagy, which destroys and recycles these damaged mitochondria so that new ones can take their place. The failure of cells to perform mitophagy leads to mitochondrial dysfunction, which is one of the hallmarks of aging.
Therefore, much research has been devoted to stimulating mitophagy, as researchers attempt to rid the cells of bad mitochondria without harming healthy ones.
Fixed Cells Make High Throughput Possible
Fixation is any process that keeps cells in an immobile state, whether through the use of a chemical such as formaldehyde or sub-freezing temperatures. However, fixation affects cellular chemistry, and, as the researchers of this study explain, mt-mKeima, a commonly used compound that is currently used to detect the occurrence of mitophagy, is sensitive to fixation. Therefore, they sought out and developed mito-SRAI, a new method of detecting mitophagy that was insensitive to fixation, making it suitable for high-throughput cell assays, which often use fixed cells.
The researchers also demonstrated that mito-SRAI is not susceptible to being destroyed by the cell’s proteosome, making it ideal for use in living samples.
Finally, and probably most interestingly, the researchers used their novel method along with a host of related techniques to screen 76,000 compounds for potential therapeutic use, testing them for specificity and toxicity. A compound called T-271 was the researchers’ top choice after this selection process, as it appears to have the unique property of inducing mitophagy in damaged mitochondria without harming healthy ones, although considerably more refinement and analysis of this compound is required in order to progress to in vivo and human trials.
Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
This paper did not go into extensive detail about T-271, and it is possible that the researchers will further analyze this compound in a future paper. The fact that the researchers were able to screen 76,000 compounds once developing this novel detection method shows how far we have come in our ability to perform biological research.
It also shows that such fundamental research into creating better biomarkers is the sort of basic research that is necessary for the development of mitophagic and other therapies. Being able to productively use fixed cells in high-throughput assays to directly analyze the effectiveness of a therapy is an enormous boon for the rapid and successful deployment of therapies that target not only this particular hallmark of aging but other disorders as well.
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