VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle.

ABSTRACT

Differentiated tissue is particularly vulnerable to alterations in protein and organelle homeostasis. The essential protein VCP, mutated in hereditary inclusion body myopathy, amyotrophic lateral sclerosis and frontotemporal dementia, is critical for efficient clearance of misfolded proteins and damaged organelles in dividing cells, but its role in terminally differentiated tissue affected by disease mutations is less clear. To understand the relevance of VCP in differentiated tissue, we inactivated it in skeletal muscle of adult mice. Surprisingly, knockout muscle demonstrated a necrotic myopathy with increased macroautophagic/autophagic proteins and damaged lysosomes. This was not solely due to a defect in autophagic degradation because age-matched mice with muscle inactivation of the autophagy essential protein, ATG5, did not demonstrate a myopathy. Notably, myofiber necrosis was preceded by upregulation of LGALS3/Galectin-3, a marker of damaged lysosomes, and TFEB activation, suggesting early defects in the lysosomal system. Consistent with that, myofiber necrosis was recapitulated by chemical induction of lysosomal membrane permeabilization (LMP) in skeletal muscle. Moreover, TFEB was activated after LMP in cells, but activation and nuclear localization of TFEB persisted upon VCP inactivation or disease mutant expression. Our data identifies VCP as central mediator of both lysosomal clearance and biogenesis in skeletal muscle. Abbreviations AAA ATPases Associated with diverse cellular Activities; TUBA1A/α-tubulin tubulin alpha 1a; ATG5 autophagy related 5; ATG7 autophagy related 7; ACTA1 actin alpha 1, skeletal muscle; CLEAR coordinated lysosomal expression and regulation; CTSB/D cathepsin B/D; Ctrl control; DAPI diamidino-2-phenylindole; EBSS Earle's balanced salt solution; ELDR endolysosomal damage response; ESCRT endosomal sorting complexes required for transport; Gastroc/G gastrocnemius; H&E hematoxylin and eosin; HSPA5/GRP78 heat shock protein family A (Hsp70) member 5; IBMPFD/ALS inclusion body myopathy associated with Paget disease of the bone, frontotemporal dementia and amyotrophic lateral sclerosis; i.p. intraperitoneal; LAMP1/2 lysosomal-associated membrane protein 1/2; LLOMe Leu-Leu methyl ester hydrobromide; LGALS3/Gal3 galectin 3; LMP lysosomal membrane permeabilization; MTOR mechanistic target of rapamycin kinase; MYL1 myosin light chain 1; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MSP multisystem proteinopathy; PBS phosphate-buffered saline; PCR polymerase chain reaction; Quad/Q quadriceps; RHEB Ras homolog, mTORC1 binding; SQSTM1 sequestosome 1; TFEB transcription factor EB; TA tibialis anterior; siRNA small interfering RNA; SQSTM1/p62, sequestosome 1; TARDBP/TDP-43 TAR DNA binding protein; TBS Tris-buffered saline; TXFN, tamoxifen; UBXN6/UBXD1 UBX domain protein 6; VCP valosin containing protein; WT wild-type.