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.
View: Text | PDF
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

×

Figure 4

Loss of INPP5K impairs lysosome homeostasis by suppressing autophagic lysosome reformation.

Options: View larger image (or click on image) Download as PowerPoint
Loss of INPP5K impairs lysosome homeostasis by suppressing autophagic ly...
(A) Control or Inpp5k knockdown (KD) cells expressing LAMP1-RFP were used to monitor the formation of reformation tubules at autolysosomes under live-cell imaging conditions. Cells were cultured in nutrient-free EBSS (8 hours) to activate prolonged autophagy, and a subset of cells were then also treated with 10% FBS (0–60 minutes) to stimulate robust ALR. After treatments, cells were subjected to live-cell imaging to monitor the formation of membrane reformation tubules. n = 5 independent experiments. Dotted boxed regions are shown at high magnification in inset. Yellow arrows indicate LAMP1-RFP–positive reformation tubules. Scale bars: 5 μm. (B) Quantification of the percentage of total cells showing the presence of LAMP1-RFP–positive reformation tubules. Data are representative of n = 5 independent experiments in which 20–25 cells were imaged and counted/cell line/treatment in each of the experiments. Mean ± SEM, 2-way ANOVA followed by Bonferroni’s post hoc multiple-comparisons test, ***P < 0.0001, ###P = 0.0002. (C) In fixed-cell experiments to monitor reformation tubules, cells were treated as described above (A), followed by rapid fixation under microtubule stabilizing conditions and immunostaining for LAMP1 to identify tubules (arrows). Scale bars: 5 μm. Images from n = 3 independent experiments and used to quantify (D) the percentage of cells with LAMP1+ tubules (n = 200 cells/cell line/experiment) and (E) tubule length (n = 30 cells/cell line/experiment). Data are mean ± SEM, 1-way (E) or 2-way (B and D) ANOVA followed by Bonferroni’s post hoc multiple-comparisons test, ***P = 0.00053, ##P = 0.00177, †††P = 0.000572, ‡‡‡P = 0.000661.