Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:752684 |
| Date | January 2018 |
| Creators | Tran, Anh Nhi |
| Contributors | Helfrich, Miep ; Coxon, Fraser ; Rochford, Justin |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=238273 |
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