mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy
Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre
Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre
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Concise Communication Bone biology

mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associated Beclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.

Authors

Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre

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Figure 5

Autophagy induction with Tat–Beclin 1 rescues the AV-lysosome fusion defect in MPS chondrocytes.

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Autophagy induction with Tat–Beclin 1 rescues the AV-lysosome fusion def...
(A) Gusb-KO cells were transfected with GFP-2xFYVE and treated or not with Tat–Beclin 1 (10 μM, 2 h). Cells were costained for endosomes (EEA1). Scale bars: 10 μm. (B) Quantitative analysis of PI3P puncta. n = 3 independent transfections; n = 20 cells analyzed. (C) Immunofluorescence of Lamp-1 and LC3 in Gusb-KO cells treated with Tat–Beclin 1 peptide (10 μM; 2 h). Insets show colocalization in selected areas at higher magnification (zoom ×3.5). Scale bars: 10 μm. (D) Quantification of Lamp-1–LC3 colocalization. n = 3 independent treatments; n = 30 cells analyzed. (E) Western blot analysis of the indicated proteins in control and Gusb-KO cells treated with vehicle, Tat–Beclin 1 (20 μM; 2 h), or with an inactive form of Tat–Beclin 1 (mTat-Beclin 1) (20 μM; 2 h). β-Actin was used as a loading control. (F) Quantification of protein levels. n = 3. (G and I) Representative images of GFP–LC3 puncta (AVs), Lamp-1 (G), and SQSTM1/p62 (I) immunostaining in femoral growth plates from Gusb–/– GFP-LC3Tg/+ mice at P6. Tat–Beclin 1 peptide was administered as indicated (2 mg/kg, daily for 6 d). Scale bar: 10 μm (zoom ×2). (H) Quantification of Lamp-1–LC3 colocalization. n = 3 mice per group. Data represent the mean values derived from the indicated number of mice per independent experiment. Error bars indicate the SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.0005, by paired Student’s t test (B, D, and H) and ANOVA followed by Tukey’s post-hoc test (F).