Defective lysosome reformation during autophagy causes skeletal muscle disease
Meagan J. McGrath, … , Catriona A. McLean, Christina A. Mitchell
Meagan J. McGrath, … , Catriona A. McLean, Christina A. Mitchell
Published October 29, 2020
Citation Information: J Clin Invest. 2021;131(1):e135124. https://doi.org/10.1172/JCI135124.
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Research Article Cell biology Muscle biology

Defective lysosome reformation during autophagy causes skeletal muscle disease

Abstract

The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k–/– muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k–/– myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k–/– muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.

Authors

Meagan J. McGrath, Matthew J. Eramo, Rajendra Gurung, Absorn Sriratana, Stefan M. Gehrig, Gordon S. Lynch, Sonia Raveena Lourdes, Frank Koentgen, Sandra J. Feeney, Michael Lazarou, Catriona A. McLean, Christina A. Mitchell

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Figure 3

INPP5K regulates lysosome homeostasis during autophagy.

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INPP5K regulates lysosome homeostasis during autophagy. (A) qRT-PCR vali...
(A) qRT-PCR validation of Inpp5k-KO myoblasts. Myoblasts isolated from n = 3 Inpp5kfl/fl mice and transduced with Cre or LacZ (control) adenovirus. ***P < 0.0001. (B) Cells in growth media or EBSS to activate autophagy, with LAMP1 staining of lysosomes. Cell borders are outlined. Yellow boxed region shown at high magnification in inset. Representative of n = 3 primary myoblast populations and used to quantify (C) number of LAMP1+ puncta/μm2 (n = 40 cells/cell line/treatment), *P = 0.0025, and (D) percentage of cells with enlarged LAMP1-positive organelles (LPOs) (n = 200 cells/cell line/treatment). *P = 0.021. (E) Lysosomal protein expression (actin loading control) after autophagy activation with densitometry analysis (at 4 hours EBSS) (F–H). Representative of n = 3 cell lines/genotype and experiment performed in triplicate. LAMP1 *P = 0.012, LAMP2 *P = 0.012, cathepsin L *P = 0.014. (I) Magic Red fluorescent cathepsin L substrate (Ac-FR-AFC) staining to monitor functional lysosomes. Hoechst staining nuclei. n = 3 experiments and used to quantify (J) functional lysosomes (positive for Ac-FR-AFC staining). n = 30 cells/cell line/treatment for each experiment. ***P = 0.0012, ###P = 0.0002, †P = 0.025, ‡‡‡P = 0.0005, NS not significant. Data presented in all graphs are the mean ± SEM, with Student’s t test (A and F–H) or 2-way ANOVA followed by Bonferroni’s post hoc multiple-comparisons test (C, D, and J) used to determine statistical significance. All scale bars: 20 μm.