The R740S mutation in the V-ATPase a3 subunit results in osteoclast apoptosis and defective early-stage autophagy

Authors

  • Noelle Ochotny,

    1. Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada
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  • Irina Voronov,

    1. Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada
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  • Celeste Owen,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada
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  • Jane E. Aubin,

    1. Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada
    2. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada
    3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
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  • Morris F. Manolson

    Corresponding author
    1. Faculty of Dentistry, Dental Research Institute, University of Toronto, Toronto, Ontario, Canada
    • Correspondence to: Morris F. Manolson, Faculty of Dentistry, University of Toronto, 124 Edward St., Toronto, Ontario, Canada M5G 1G6.

      E-mail: m.manolson@utoronto.ca

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  • Disclosure Statement: None.
  • Jane E. Aubin and Morris F. Manolson contributed equally to this work.

ABSTRACT

Vacuolar-type H+-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP. J. Cell. Biochem. 114: 2823–2833, 2013. © 2013 Wiley Periodicals, Inc.

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