Measuring In Vivo Mitophagy

Abstract

Summary Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

“…To measure basal mitophagy, we crossed the Gba L444P/WT mice to a transgenic mouse model expressing mitochondrial-targeted Keima (mt-Keima), a coral-derived acid-stable fluorescent protein that exhibits a pH-dependent bimodal excitation spectrum [22]. Protonated Keima in acidic lysosomes has stronger 561-nm excitation, whereas neutral Keima is predominantly excited by 458-nm wavelength light [23,24].…”

“…Protonated Keima in acidic lysosomes has stronger 561-nm excitation, whereas neutral Keima is predominantly excited by 458-nm wavelength light [23,24]. The slow turnover of lysosomal Keima allows for the qualitative measurement of mitophagic flux in live cell cultures [22], and the ratio of 561:458 nm-excited Keima fluorescence intensity indicates the delivery of mitochondria to lysosomes. As in Figure 4(A), mt-Keima within lysosomes was mainly excited by the 561-nm (red) wavelength, but retained some modest capacity for 458-nm (green) excitation, which made the mt-Keima fluorescence yellow.…”

New methods for monitoring mitochondrial biogenesis and mitophagy in vitro and in vivo

“…Isolated MEFs from these mice had green tubular mitochondria when examined at 458 nm excitation and had red puncta that overlapped with labeled lysosomes at 561 nm excitation. 27 Sun et al performed several experiments to ensure mt-Keima red signal was specific to degradation of mitochondria in lysosomes. When these mice were treated with chloroquine to block lysosome degradation, red mt-Keima fluorescence decreased, suggesting decreased degradation of mitochondria in lysosomes.…”

New Insight in The Molecular Mechanisms of Neurodegenerative Disease

“…(142) Mitochondrial dysfunction is considered a hallmark of aging (143), and is implicated in apoptosis, senescence, genome instability, inflammation, and metabolic disorders (142,144). The term "mitophagy" was first coined by Dr. Lemasters (149), as well as normal physiological aging (150).…”