Autophagy-monitoring and autophagy-deficient mice

Abstract

Discovery of yeast autophagy-related (ATG) genes and subsequent identification of their homologs in other organisms have enabled researchers to investigate physiological functions of macroautophagy/autophagy using genetic techniques. Specific identification of autophagy-related structures is important to evaluate autophagic activity, and specific ablation of autophagy-related genes is a critical means to determine the requirements of autophagy. Here, we review currently available mouse models, particularly focusing on autophagy (and mitophagy) indicator models and systemic autophagy-related gene-knockout mouse models.

Keywords: autophagy; knockout mouse; mitophagy; reporter mouse; selective autophagy.

Figure 1.

Transgenic mice expressing autophagy probes for monitoring autophagy. (1) GFP-LC3 mice express EGFP-LC3 for labeling phagophores and autophagosomes. The number of GFP puncta is counted before and after stimulation. (2) tfLC3 mice express RFP-EGFP-LC3 or mCherry-EGFP-LC3 for measuring autophagy flux. The numbers of GFP⁺ RFP⁺ puncta (yellow) and RFP puncta (red) are counted. Yellow puncta represent phagophores and autophagosomes, and red puncta represent autolysosomes. (3) GFP-LC3-RFP-LC3ΔG mice express GFP-LC3-RFP-LC3ΔG for measuring autophagy flux. The reduction of the fluorescence intensity of GFP indicates autophagy flux because GFP-LC3 is degraded though autophagy (and quenched) as a substrate. The reduction of the fluorescence intensity of GFP-LC3 in the total cells is measured and corrected by the fluorescence intensity of RFP-LC3∆G in the total cells as an internal control. (4) Mito-QC mice express mCherry-EGFP fused with the FIS1 mitochondrial targeting sequence for monitoring mitophagy. The numbers of GFP⁺ RFP⁺ (yellow) puncta and RFP puncta (red) are counted. Yellow puncta represent mitochondria, and red puncta represent mitochondria in the lysosomes. (5) Mt-Keima mice express Keima (pH-dependent fluorescent protein) fused with the COX8 mitochondrial-targeting sequence for monitoring mitophagy. mt-Keima is excited predominantly by 458-nm light in a neutral environment (mitochondria), and by 561-nm light in an acidic environment (lysosome). The ratio of the 561-nm:458-nm excited Keima fluorescence intensity indicates the delivery of mitochondria to the lysosome. Squares with a blue dotted-line show the stages detected with the probes. F.I., fluorescence intensity.

Figure 2.

Timing of lethality in autophagy-deficient mice. Mice deficient in genes involved in the ATG conjugation system (atg3^−/−, atg5^−/−, atg7^−/−, atg12^−/−, atg16l1^−/−) and ulk1/2 DKO mice are born with normal morphology but die within 1 d after birth. Mice deficient in genes functioning upstream of the ATG conjugation system (rb1cc1^−/−, atg13^−/−, becn1^−/−, uvrag^−/−, pik3c3/vps34^−/−, atg9a^−/−) die in utero. Others (atg4b^−/−, atg4c^−/−, lc3b^−/−, gabarap^−/−, ulk1^−/−, ulk2^−/−) show no obvious (or weak) phenotypic effects.