Skip to Content
Merck
  • VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle.

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

Autophagy (2019-01-19)
Khalid Arhzaouy, Chrisovalantis Papadopoulos, Nina Schulze, Sara K Pittman, Hemmo Meyer, Conrad C Weihl
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-p62/SQSTM1 antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Adenosine 5′-[γ-thio]triphosphate tetralithium salt, ≥75% (HPLC), powder
Sigma-Aldrich
Paraformaldehyde, reagent grade, crystalline
Sigma-Aldrich
Leu-Leu methyl ester hydrobromide, ≥97% (TLC)
Sigma-Aldrich
SIGMAFAST Protease Inhibitor Tablets, For General Use
Sigma-Aldrich
Saponin, Molecular Biology, used as non-ionic surfactant
Sigma-Aldrich
IGEPAL® CA-630, viscous liquid
Sigma-Aldrich
Dimethyl sulfoxide, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma, ≥99.7%
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, ascites fluid, clone B-5-1-2
Sigma-Aldrich
Anti-Myosin (Skeletal, Fast) antibody, Mouse monoclonal, clone MY-32, purified from hybridoma cell culture
Sigma-Aldrich
Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Sodium deoxycholate, BioXtra, ≥98.0% (dry matter, NT)
Roche
KAPA Express Extract
Sigma-Aldrich
NMS-873, ≥98% (HPLC)