Cell Weight: Mitochondrial autophagy research sharp weapon, to help neuroscience research.

Learn about the latest progress in neuroscience ● click the blue word to pay attention to us ● it is mentioned in the biology textbook of senior high school that mitochondria are the structure for producing energy in cells and the main place for cells to carry out aerobic respiration, which is called energy factory.mitochondria are one of the most sensitive organelles to various damages. Therefore, the process of clearing damaged mitochondria is necessary for the stability and health of mitochondria. Mitochondrial autophagy is a way to remove damaged mitochondria by autophagy selective degradation.fluorescent protein, photo Citation: although it is theoretically possible to monitor mitochondrial autophagy by labeling mitochondria with autophagy probes, in fact, it is difficult for probes to achieve accurate localization of mitochondria.if the autophagy probe is located in advance or incorrectly, it will lead to nonselective autophagy and lead to false positive signal of mitochondria.Professor Atsushi Miyawaki of RIKEN comprehensive research center of brain science, Institute of physics and chemistry of Japan, developed a mitochondrial probe MT mkeima in 2011, which improved the specificity of mitochondrial localization by turning off the gene one day before observing the expression of mitochondrial autophagy.however, the fluorescent protein carried by MT mkeima is reversible sensitive to acid and can only be detected in living samples (1).after 9 years, Professor Atsushi Miyawaki's research team returned strongly. On May 28, 2020, he published an article in cell magazine to screen a kind of almost acid resistant fluorescent protein Tolles, and constructed mitochondrion probe mitochondria (Mito srai), which can be reliably labeled in vivo and in vitro (2).a schematic diagram of the composition of autophagy probe, cited from ref. 2. Most fluorescent proteins are denatured more or less by strong acid, and their fluorescence cannot be recovered under neutral pH. therefore, it is necessary to monitor autophagy probes with special fluorescent proteins that are completely acid resistant.through high-throughput screening experiments, the researchers found that a fluorescent protein, called Tolles protein, could fully express fluorescence not only in neutral pH 7, but also in acidic pH 4 – 5. In vitro experiments found that this fluorescent protein still retained its full fluorescence after incubation at 37 ℃ for 18 hours at pH 4.0, In order to obtain the mitochondrial autophagy probe, the researchers fused Tolles protein with green fluorescent protein with relatively low pH sensitivity and localized it into mitochondrial matrix to construct Mito srai plasmid.the plasmid can increase the mitochondrial localization specificity by using tetracycline (TET) - induced gene expression system.cyanocarbonyl-3-chlorophenylhydrazone (CCCP) induces mitochondrial degradation by destroying the potential cyanide m-chlorophenylhydrazine in mitochondrial membrane.CCCP was incubated with Mito srai in vitro. It was found that mitochondrial autophagy can be observed and quantified under confocal microscope. Mito srai can also be used in flow cytometry. These results indicate that Mito srai can monitor mitochondrial autophagy and conduct quantitative research.what scenarios can Mito srai be applied to? The first is the development of new drugs. At present, the research and development of new drugs that induce mitochondrial autophagy and damage mitochondria is in the blank period.the researchers used high-throughput screening technology combined with Mito srai to screen nearly 76000 compounds, looking for small molecules that can induce mitochondrial autophagy.a compound named t-271 was found to enhance mitochondrial autophagy by relying on ubiquitin ligase parkin without directly damaging normal mitochondria.although the chemical structure of t-271 may still need to be optimized to enhance drug activity, t-271 and its derivatives may be potential compounds for the treatment of mitochondrial related diseases. Secondly, it is applied to central nervous system diseases. Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra compacta (SNC).recent studies have found that abnormal mitochondrial autophagy in brain neurons is closely related to the occurrence of Parkinson's disease.PD like behavior was simulated by injecting neurotoxin 6-hydroxydopamine (6-OHDA) into the SNC of mice.the researchers injected AAV virus carrying Mito srai plasmid into SNC brain region, and injected 6-OHDA one week later. The results showed that a large number of Mito srai fluorescent signals were detected in neurons of midbrain region, and most of them were non dopaminergic neurons, and a small part were dopaminergic neurons.this suggests that dopaminergic neurons in SNC cause pathological degeneration, which may be due to their inability to respond to mitochondrial damage induced by 6-OHDA.pictures are cited from reference 2. In general, Mito srai, a mitochondrial autophagy probe that can respond in both in vitro and in vivo tissues, was firstly developed. Secondly, compound t-271, which can induce mitochondrial autophagy, was found by this probe combined with high-throughput screening technology, which is likely to become a new drug for inducing mitochondrial autophagy.finally, it further revealed that dopaminergic neurons in PD can not effectively deal with mitochondrial damage, so it may lead to degeneration and death.References: 1. Katayama, H., Kogure, T., Mizushima, N., yoshimori, T., and Miyawaki, A. (2011) delivery.Chem . Biol. 18, 1042–1052.2. Katayama et al.,Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration, Cell,(2020),