Caroline Whitehouse

Autophagy: A new player in skeletal maintenance?

Imbalances between bone resorption and formation lie at the root of disorders such as osteoporosis, Pagets disease of bone (PDB), and osteopetrosis. Recently, genetic and functional studies have implicated proteins involved in autophagic protein degradation as important mediators of bone cell function in normal physiology and in pathology. Autophagy is the conserved process whereby aggregated proteins, intracellular pathogens, and damaged organelles are degraded and recycled. This process is important both for normal cellular quality control and in response to environmental or internal stressors, particularly in terminally‐differentiated cells. Autophagic structures can also act as hubs for the spatial organization of recycling and synthetic process in secretory cells. Alterations to autophagy (reduction, hyperactivation, or impairment) are associated with a number of disorders, including neurodegenerative diseases and cancers, and are now being implicated in maintenance of skeletal homoeostasis. Here, we introduce the topic of autophagy, describe the new findings that are starting to emerge from the bone field, and consider the therapeutic potential of modifying this pathway for the treatment of age‐related bone disorders.

Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover

MINT‐7034220: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with ube2o (uniprotkb:Q6ZPJ3) by two hybrid (MI:0018)

Boning up on autophagy: The role of autophagy in skeletal biology

From an evolutionary perspective, the major function of bone is to provide stable sites for muscle attachment and affording protection of vital organs, especially the heart and lungs (ribs) and spinal cord (vertebrae and intervertebral discs). However, bone has a considerable number of other functions: serving as a store for mineral ions, providing a site for blood cell synthesis and participating in a complex system-wide endocrine system. Not surprisingly, bone and cartilage cell homeostasis is tightly controlled, as is the maintenance of tissue structure and mass. While a great deal of new information is accruing concerning skeletal cell homeostasis, one relatively new observation is that the cells of bone (osteoclasts osteoblasts and osteocytes) and cartilage (chondrocytes) exhibit autophagy. The focus of this review is to examine the significance of this process in terms of the functional demands of the skeleton in health and during growth and to provide evidence that dysregulation of the autophagic response is involved in the pathogenesis of diseases of bone (Paget disease of bone) and cartilage (osteoarthritis and the mucopolysaccharidoses). Delineation of molecular changes in the autophagic process is uncovering new approaches for the treatment of diseases that affect the axial and appendicular skeleton.

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1tr/tr) have ~50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

The G2 chromosomal radiosensitivity assay

Although requiring stringent experimental conditions to achieve good reproducibility, the G2 assay has potential as a sensitive marker for cancer susceptibility, and is particularly useful in population studies. Immediate culture of blood is preferable, but overnight storage of blood either at ambient temperature or at 4 degrees C does not appear significantly to affect G2 scores. Transport of blood may lead to additional variability in assay results and should be well controlled. Although reproducibility is generally good, G2 scores on blood from certain individuals appear to show significant variability in repeat samples. Thus, determination of an individuals radiosensitivity may require multiple assays on different occasions. While it is recognized that the distinction between aligned and mis-aligned discontinuities has no scientific basis, some laboratories have decided for the purpose of record-keeping to score all aligned discontinuities as gaps, and mis-aligned discontinuities as breaks. In all cases the final G2 score should comprise the sum of all gaps and breaks.

Chromosome analysis of workers occupationally exposed to radiation at the Sellafield nuclear facility

PURPOSE To investigate the relationship between stable chromosome aberration frequency in peripheral blood lymphocytes and occupational cumulative radiation exposure. MATERIALS AND METHODS Cytogenetic analysis using G-banding was performed on peripheral blood lymphocyte cultures from 104 workers from the British Nuclear Fuels PLC facility at Sellafield, UK. The study group comprised 61 men with lifetime cumulative doses > 500 mSv, 39 men with minimal exposure (i.e. < 50 mSv) who formed a control group and 4 men with intermediate doses. RESULTS The slope of the dose-response, adjusted for smoking status, for translocations and insertions was 0.55+/-0.31 x 10(-2)/cell/Sv. Consideration of chromosome breakpoints for all aberrations combined in the radiation workers revealed an excess in the C group chromosomes and a deficit in the F group chromosomes with breakpoints being concentrated in the terminal regions whereas the distribution in the control group did not deviate from expectation. CONCLUSIONS The dose-response was not significantly different from the parallel FISH analysis (Tucker et al. 1997) and confirms that chronic radiation exposure appears to be substantially less effective at inducing stable chromosome aberrations in comparison with acute exposure.Purpose : To investigate the relationship between stable chromosome aberration frequency in peripheral blood lymphocytes and occupational cumulative radiation exposure. Materials and methods : Cytogenetic analysis using G-banding was performed on peripheral blood lymphocyte cultures from 104 workers from the British Nuclear Fuels PLC facility at Sellafield, UK. The study group comprised 61 men with lifetime cumulative doses > 500mSv, 39 men with minimal exposure (i.e. < 50 mSv) who formed a control group and 4 men with intermediate doses. Results : The slope of the dose-response, adjusted for smoking status, for translocations and insertions was 0.55 +/- 0.31 x 10 -2 /cell/Sv. Consideration of chromosome breakpoints for all aberrations combined in the radiation workers revealed an excess in the C group chromosomes and a deficit in the F group chromosomes with breakpoints being concentrated in the terminal regions whereas the distribution in the control group did not deviate from expectation. Conclusions : The dose-response was not significantly different from the parallel FISH analysis (Tucker et al. 1997) and confirms that chronic radiation exposure appears to be substantially less effective at inducing stable chromosome aberrations in comparison with acute exposure.

mBAND analysis of chromosome aberrations in lymphocytes exposed in vitro to α-particles and γ-rays

Purpose: To investigate the profiles of chromosome damage induced in vitro by exposure to α-particles and γ-rays. Materials and methods: Human peripheral blood lymphocytes were exposed to three dose regimes: α-particle doses of 0.2 and 0.5 Gy and a γ-ray dose of 1.5 Gy. After culturing for 47 hours, chromosome aberrations involving the number 5 chromosomes were identified using a multi-coloured banding (mBAND) technique. Results: Analysis of the frequencies of chromosome 5 breaks within aberrant cells and within aberrant number 5 chromosomes demonstrated that α-particle irradiation is more likely to result in multiple breaks in a chromosome than γ-irradiation. Additionally, overdispersion was observed for all doses for the distribution of breaks amongst all cells analysed and breaks amongst total number 5 chromosomes, with this being greatest for the 0.2 Gy α-particle dose. The ratio of interchanges to intrachanges (F ratio) was 1.4 and 2.4 for 0.2 and 0.5 Gy α-particles respectively and 5.5 for 1.5 Gy γ-rays. Evaluation of simple versus complex exchanges indicated ratios of 1.9 and 2.7 for 0.2 and 0.5 Gy α-particles respectively and 10.6 for 1.5 Gy γ-rays. The majority of the intrachanges involving chromosomes 5 induced by α-particle radiation were associated with more complex exchanges. Conclusions: This study has confirmed that exchanges induced by exposure to high linear energy transfer (LET) α-particle radiation comprise a greater proportion of intrachanges than those induced by exposure to low LET γ-rays. However, since the majority of these are associated with complex rearrangements and likely to be non-transmissible, this limits their applicability as a marker of past in vivo exposure.

MAP1B Interaction with the FW Domain of the Autophagic Receptor Nbr1 Facilitates Its Association to the Microtubule Network.

Selective autophagy is a process whereby specific targeted cargo proteins, aggregates, or organelles are sequestered into double-membrane-bound phagophores before fusion with the lysosome for protein degradation. It has been demonstrated that the microtubule network is important for the formation and movement of autophagosomes. Nbr1 is a selective cargo receptor that through its interaction with LC3 recruits ubiquitinated proteins for autophagic degradation. This study demonstrates an interaction between the evolutionarily conserved FW domain of Nbr1 with the microtubule-associated protein MAP1B. Upon autophagy induction, MAP1B localisation is focused into discrete vesicles with Nbr1. This colocalisation is dependent upon an intact microtubule network as depolymerisation by nocodazole treatment abolishes starvation-induced MAP1B recruitment to these vesicles. MAP1B is not recruited to autophagosomes for protein degradation as blockage of lysosomal acidification does not result in significant increased MAP1B protein levels. However, the protein levels of phosphorylated MAP1B are significantly increased upon blockage of autophagic degradation. This is the first evidence that links the ubiquitin receptor Nbr1, which shuttles ubiquitinated proteins to be degraded by autophagy, to the microtubule network.

Autophagic receptors Nbr1 and p62 coregulate skeletal remodeling.

Skeletal remodelling is an ongoing process requiring the coordinated action of different cell types to maintain homeostatic control of bone synthesis and degradation. Mutations in p62/SQSTM1 are associated with sporadic and 5q35-linked Paget’s Disease of Bone (PDB), characterized by focal increased bone turnover. These mutations cluster in the ubiquitin associated (UBA) domain and are thought to lead to enhancement of NFκB pathway activation involved in osteoclastogenesis and hyper-responsiveness to receptor activator of nuclear factor-κB ligand (RANKL). The structurally similar selective autophagic receptor, Nbr1, binds to LC3 and p62, and is sequestered into autophagosomes, whereas it accumulates in autophagic-deficient tissues. We have shown that truncation of Nbr1 in a murine model, where it can still interact with p62 but not LC3, leads to increased osteoblast differentiation and activity in vivo. This results in an age-dependent increase in bone mass and bone mineral density. This is a molecular consequence of loss of autophagy receptor function via deletion of its C-terminal UBA domain, and/or modulation of the p38 MAPK cellular signalling pathway.

Characterisation of the FAM69 family of cysteine-rich endoplasmic reticulum proteins

The FAM69 family of cysteine-rich type II transmembrane proteins comprises three members in all vertebrates except fish, and orthologues with a conserved structure are present throughout metazoa. All three murine FAM69 proteins (FAM69A, FAM69B, FAM69C) localise to the endoplasmic reticulum (ER) in cultured cells, probably via N-terminal di-arginine motifs. Mammalian FAM69A is ubiquitously expressed, FAM69B is strongly expressed in the brain and in peripheral endothelial cells, and FAM69C in the brain and eye. Antibodies against mouse FAM69B strongly stain the ER of a subset of neurons in the brain. FAM69 proteins are likely to play a fundamental and highly conserved role in the ER of most metazoan cells, with additional specialised roles in the vertebrate nervous system.