Autophagy is essential for mouse sense of balance
Guillermo Mariño, … , José M.P. Freije, Carlos López-Otín
Guillermo Mariño, … , José M.P. Freije, Carlos López-Otín
Published June 23, 2010
Citation Information: J Clin Invest. 2010;120(7):2331-2344. https://doi.org/10.1172/JCI42601.
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Research Article

Autophagy is essential for mouse sense of balance

Abstract

Autophagy is an evolutionarily conserved process that is essential for cellular homeostasis and organismal viability in eukaryotes. However, the extent of its functions in higher-order processes of organismal physiology and behavior is still unknown. Here, we report that autophagy is essential for the maintenance of balance in mice and that its deficiency leads to severe balance disorders. We generated mice deficient in autophagin-1 protease (Atg4b) and showed that they had substantial systemic reduction of autophagic activity. Autophagy reduction occurred through defective proteolytic processing of the autophagosome component LC3 and its paralogs, which compromised the rate of autophagosome maturation. Despite their viability, Atg4b-null mice showed unusual patterns of behavior that are common features of inner ear pathologies. Consistent with this, Atg4b-null mice showed defects in the development of otoconia, organic calcium carbonate crystals essential for sense of balance (equilibrioception). Furthermore, these abnormalities were exacerbated in Atg5–/– mice, which completely lack the ability to perform autophagy, confirming that autophagic activity is necessary for otoconial biogenesis. Autophagy deficiency also led to impaired secretion and assembly of otoconial core proteins, thus hampering otoconial development. Taken together, these results describe an essential role for autophagy in inner ear development and equilibrioception and open new possibilities for understanding and treating human balance disorders, which are of growing relevance among the elderly population.

Authors

Guillermo Mariño, Alvaro F. Fernández, Sandra Cabrera, Yunxia W. Lundberg, Rubén Cabanillas, Francisco Rodríguez, Natalia Salvador-Montoliu, José A. Vega, Antonino Germanà, Antonio Fueyo, José M.P. Freije, Carlos López-Otín

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Figure 3

Analysis of Lc3, Gabarap, Gate-16, Atg8l, and Lc3a status in WT and Atg4b–/– mice.

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Analysis of Lc3, Gabarap, Gate-16, Atg8l, and Lc3a status in WT and Atg4...
(A) Representative immunoblots of endogenous Atg4b putative substrates. The analyzed tissues correspond to protein extracts from age-matched control and mutant mice fed ad libitum. β-Actin was used as a loading control in all tissues except heart and skeletal muscle (α-tubulin). (B) Quantitative RT-PCR analysis of Lc3, Gabarap, and Atg8l mRNA in liver, showing that the alterations in A were not due to an increase in mRNA synthesis. Transcript expression levels are relative to the expression levels detected in WT mice, which were set at 100%. Black bars, WT; gray bars, Atg4b–/–. (C) Representative immunofluorescence images of endogenous Lc3, Gabarap, Atg8l, and Gate-16 in fed and 4-hour-starved MEFs. Atg4b–/– MEFs showed a reduced number of punctate structures containing Lc3, Gabarap, or Atg8l under both fed and starved conditions. In contrast, Atg4b–/– MEFs exhibit an increase in punctate Gate-16–containing structures. (D) Representative immunofluorescence images of endogenous Atg5, showing an accumulation of immature autophagosomes in Atg4b–/– MEFs. Scale bars: 10 μm. (E) The number of endogenous Lc3, Gabarap, Atg8l, Gate-16, and Atg5 dots was counted and divided by the corresponding cellular area (μm2 × 100). Results shown represent the mean quantification values of 5 images in each depicted condition for each depicted protein. *P < 0.05 for Atg4b–/– values vs. the corresponding (fed or starved) Atg4b+/+ values.