Figure 1. The fluorescent reporter mt-Keima allows for the in vivo analysis of mitophagy. The green signal indicates mt-Keima localized in mitochondria and the red signal indicates mt-Keima that has been targeted for degradation in lysosomes.
Mitophagy is a cellular process that selectively removes damaged, old, or dysfunctional mitochondria. It is an essential mechanism to maintain mitochondrial quality and integrity. Mitochondria are responsible for sustaining cellular bioenergetics, regulating intracellular calcium dynamics and modulating the threshold for cell death. Various age-related pathologies have been associated with changes in mitochondrial function. Studying the intersection of mitochondrial biology, metabolism and aging represents an important focus of research with important implications to improve public health.
Changes in mitophagy have been associated to both aging and age-related illnesses. Before the publication of this study, the methods utilized to detect both autophagy (the controlled degradation of cellular components) and mitophagy in vivo were cumbersome, not sensitive enough and difficult to quantify. To circumvent these limitations, we created a transgenic mouse model that produces the fluorescent coral protein Keima. We targeted Keima to the mitochondrial matrix to produce mt-Keima. There are two properties of mt-Keima that make it particularly well suited to measure mitophagic flux. First, its fluorescence is dependent on pH and thus it is easy to distinguish whether mt-Keima is located in the mitochondria (green signal at pH 8.0) or has been delivered to the lysosome (red signal at pH 4.5) for degradation (Figure 1). Moreover, while the lysosome is filled with various proteases, Keima appears relatively resistant to lysosomal degradation and is stable for at least 24 hours after delivery to the lysosome. This allows for an integrative measure of mitophagy, giving some measurement of the flux of the system.
Analysis of the mt-Keima mice revealed that mitophagic flux varied among different tissues and even within tissues. For instance, in the brain, high levels of mitophagy were observed in the dentate gyrus region of the brain, an area that is enriched in stem cells and the presumed seat for learning and memory. The mitophagic flux in this region was markedly diminished in older mice (Figure 2). Further analysis of this mouse model revealed that alterations in mitophagy could be observed in various relevant genetic models such as mice expressing the HTT (Huntingtin) transgene, which is a model for Huntington’s disease, or in mice with altered rates of mitochondrial DNA mutations (POLGγ knockin mice). In addition, environmental stresses such as high fat diet also markedly altered mitophagic flux (Fig 3).
Figure 2. Mitophagic flux in the dentate gyrus region of the mouse brain. A) mt-Keima signal in young and B) old mice. C-D) Corresponding control DAPI staining to visualize cell nuclei in these animals. E) Quantification of mt-Keima signal from young and old mice (n=5 per group).
Figure 3. Analysis of liver sections from mice fed normal chow (NC) or a high fat diet (HFD). A) mt-Keima section are on top and liver sections stained with Oil Red O to assess lipid content are below. Scale bar, 20 mm. B) Quantification of hepatic mitophagy as measured by relative mt-Keima signal intensity (n=5 per group).
This study also took advantage of recent improvements in light microscopy techniques by using stimulated emission depletion (STED) microscopy. STED is a type of super-resolution microscopy that allows us to visualize subcellular structures in live cells. Its developer, Dr. Stephan W. Hell, received the Nobel Prize in Chemistry for this technique in 2014. We were able to successfully obtain high resolution images of mt-Keima expression within both lysosomes and structures believed to be autolysosomes (Figure 4). Thus, the mt-Keima mouse appears to provide a valuable tool to assess how mitophagy is altered by genetic, environmental or pharmacological interventions.
Figure 4. (A) Representative STED image of mt-Keima mouse embryonic fibroblasts (MEFs) showing merged red and green fluorescence. (B) The red only mt-Keima signal. (C) Merged red mt-Keima signal with LysoSensor fluorescence (shown in cyan). Scale bar, 2 mm. (D) Higher magnification of the mt-Keima red signal within lysosomes. Arrows indicate what appears to be a recently ingested autophagosome decorated with mt-Keima and contained within what is presumed to be a newly formed autolysosome. Scale bar, 1 mm.
The mt-Keima transgenic mouse is a novel reporter mouse strain that allows for a fast and convenient strategy to assess mitophagy within tissues that should have widespread utility in the field. Ongoing studies include further characterization of autophagy-deficient mice in the endothelium and smooth cell compartment, as well as genetic and pharmacological strategies to induce mitophagy to further explore its role in age-related diseases.
Nuo Sun, Jeanho Yun, Jie Liu, Daniela Malide, Chengyu Liu, Ilsa I. Rovira, Kira M. Holmström,
Maria M. Fergusson, Young Hyun Yoo, Christian A. Combs, and Toren Finkel.
Molecular Cell. 2015 Nov 19;60(4):685-96
[Text Abstract on PubMed]