Investigating senescence in osteoarthritis

"Cellular senescence has physiological roles in tissue repair and during embryogenesis. Increased senescence however causes tissues dysfunction and directly contributes to disease, including osteoarthritis (OA). Genetic and pharmacological clearance of senescent cells has been shown to alleviate OA pathology in mouse models. These studies suggest that compounds that can kill senescent cells (senolytics) represent novel therapeutic options to treat OA. To develop senolytics, we need a better understanding the biology of senescence in vitro and in vivo.
This project build on our work characterising the mTORC1-autophagy pathway, how it contributes to senescence and represents a targetable vulnerability for senescent cell survival. At present, it is not clear whether the phenotypes we observe in multiple in vitro models of senescence are shared in vivo. The aim of this project is to fully characterise the mTORC1-autophagy pathway in senescence, using in vitro and in vivo models of OA. Determining what a senescent cell 'looks like' at the molecular and cellular level in vivo, how the senescent cell number and specific, defined phenotypes change with OA development, and whether the clearance of senescent cells reduces OA severity.
Objectives:Identify the molecular mechanisms of mTORC1-autophagy dysregulation in senescence
Using multiple musculoskeletal cell culture models of senescence, the student will interrogate the mechanisms regulating mTORC1-autophagy, including the ability to sense mitogenic signals such as growth factors involved in musculoskeletal function and regeneration e.g. TGFSS. Genes implicated in OA risk, identified by collaborators in population health will be knocked out using CRISPR/Cas9 and followed up by characterisation of senescence.
A variety of standard molecular cell biology techniques including cell culture, qPCR, Western blot and immunofluorescence microscopy will be utilised.
Characterise senescence in vivo
Immunohistochemical analysis of a large catalogue of fish models (already available) will be used to characterise the number, cell type and location of senescent cells in the musculoskeletal system of animals with evidence of OA. We will cross reporter fish lines for the senescence markers, p21 and p16INK4a (cyclin-dependent kinase inhibitors), with accelerated OA models, autophagy and mTORC1 reporter lines. For proof-of-principle experiments and optimisation of tools, fish will be irradiated to induce premature senescence.
Senescent cells will be isolated from tissue using laser capture microdissection. RNA-sequencing and proteomics analysis will be carried out alongside samples of senescent cells from culture to compare senescence in vitro and in vivo.
Can targeting the mTORC1-autophagy pathway have senolytic potential in vivo?
We have shown in vitro that targeting the mTORC1-autophagy pathway has senolytic activity. In vivo, a screen will be carried out in p21/p16 fish ~3dpf post-irradiation to induce widespread senescence. Published senolytics will be tested alongside mTORC1-autophagy modulators we have recently identified.
These experiments will provide proof-of-principle that in vivo, the mTORC1-autophagy pathway represents a senolytic target. At the same time, we will establish the feasibility of using zebrafish as a platform for screening senolytic compounds. "