Maintenance, regeneration, and repair of skeletal systems: molecular regulation of autophagy in the joint

Our skeletons constantly adapt throughout life so they are strong enough to withstand our needs, while remaining relatively light to facilitate movement. This is achieved by balanced activity of bone building cells called osteoblasts, and bone resorbing cells called osteoclasts which remove old or poor quality bone. In ageing and in some genetic conditions, this balance can often be lost. If resorption exceeds the amount of bone being made it can lead to osteoporosis, where bones are less dense, more fragile and prone to fracture. In other cases, bone making can exceed resorption and this can lead to bone being made in places it normally wouldn't be found. This can include spurs on the edges of the bone that can impede joint movement in the hips, spine or knees, or mineralization of soft tissues like cartilage and ligaments, which normally help to facilitate smooth joint movement, leading to joint stiffness. However, we still don't fully understand how the cells of the skeletal system, the osteoblasts and osteoclasts of the bone and the chondrocytes that maintain joint cartilage, are affected by ageing. One hypothesis is that autophagy, which is a process by which cells break down and recycle parts of their own working machinery, becomes impaired in the ageing skeleton. This causes cells to become less healthy over time and less able to adapt to the needs to of the organism. Evidence for this idea comes from genetic studies which have linked some autophagy genes to osteoarthritis and osteoporosis, which suggests if autophagy is impaired it might lead to earlier onset of skeletal ageing. We want to use zebrafish as an animal model to test whether this is happening.

Zebrafish are a model system which make bones in the same ways humans and other animals do, but are also translucent. This means that we can label the cells of the skeletal system with fluorescent markers and watch their behaviour in the living fish in response to injury, to ageing. We can also monitor the effects of the addition of drugs, such as steroids, or bisphosphonates which are also used clinically. Indeed, some of the drugs in the clinic were first identified using zebrafish. We have generated some lines of zebrafish in which some of the regulators of autophagy are switched off, and we have seen that these fish have abnormal skeletons, with changes both to cartilage and to bone. These changes become more severe as the fish age. We will use live imaging of fluorescently tagged cells in the living fish to monitor autophagy during skeletal development in normal and mutant fish, and to watch how skeletal cells behave in young and old fish in response to injury so that we can identify which cells might cause these differences. When we better understand which cells are causing these changes, we will test which proteins and processes in those cells are disrupted and see whether we can identify ways to change this, by working in tissue culture, in which cells are grown in petri dishes to allow us to study these processes in more detail.

Although skeletal pathologies are prevalent during ageing, our understanding of the key drivers at the cellular and molecular levels remains poor. Autophagy dysregulation is implicated in skeletal diseases of ageing, including osteoarthritis and osteoporosis, arguing that a more complete picture of autophagy regulation in skeletal cell-types is essential. The LIM homeodomain transcription factor, LMX1B, is a key regulator of skeletal development and homeostasis. It also contributes to autophagy transcriptional control. Crucially, we found that LMX1B interacts with the autophagy machinery via the ATG8 protein family, with these acting as co-factors to boost LMX1B-mediated transcription for cell stress protection. This regulatory interface merits further investigation in a skeletal context.

Loss of function LMX1B mutations cause the rare condition, Nail-Patella syndrome (NPS). This is associated with a range of skeletal phenotypes, including missing and misshapen bones and joints. Lmx1b null mice die at birth and heterozygotes do not recapitulate NPS phenotypes, diminishing them as Lmx1b skeletal models. In zebrafish, lmx1b is duplicated and we have generated mutant lines for lmx1ba and lmx1bb. Phenotypic analysis shows lmx1ba to be the predominant driver of skeletal phenotypes: fish lacking lmx1ba are viable but show severe, progressive skeletal abnormalities. Leveraging the imaging capabilities of zebrafish, we will use transgenic reporter lines to define how lmx1ba and autophagy cooperate during development, regeneration, fracture repair, and ageing. This will be functionally correlated with laser capture RNAseq and proteomic comparisons of wild type and lmx1ba null fish undergoing skeletal repair. Working in MSC-derived skeletal lineages, we will test how the ATG8-LMX1B interface shapes skeletal and autophagic gene expression for accurate differentiation, with its impact assessed in vivo using wild type and Atg8-binding mutant Lmx1ba rescue zebrafish lines.