Uncovering the role of proteostasis dysregulation in primary and secondary senescence

Background. Our aim is to understand senescence mechanisms [1-4]. We have identified that
proteasome perturbation can trigger premature senescence (via genome-wide siRNA
screening) and that proteasome dysregulation is a feature of replicative senescence (via
scRNAseq). We also show that sEVs released by senescent cells can generate secondary
paracrine senescence thereby contributing to the SASP [2].
Hypothesis. We hypothesise that proteostasis dysregulation (PD) may act as a primary
senescence trigger which results in unique consequences for neighbouring cell types via
secondary mechanisms.
Aim 1. Determine the temporal kinetics of PD and senescence effector programmes.
We will determine a) which senescence effector programmes are engaged by targeted PD
and b) what are the broader consequences for expression of proteasome components during
senescence initiation and maintenance (see Rotation project for more details). We will also
complement our genetic models of PD with FDA approved proteasome inhibitors (e.g.
PS-341).
Aim 2. Exploring secondary senescence in 2D and 3D co-culture.
Next, we will build on existing co-culture models to establish GFP-fibroblast:RFP-fibroblast
and GFP-fibroblast:RFP-neuron co-culture in 2D and 3D. We will determine if PD senescent
fibroblasts can trigger secondary paracrine senescence in mitotic (fibroblasts) and postmitotic
cells (human iPSC-derived neurons) via soluble and/or sEV SASP. These are virtually
unexplored areas, supported by excellent synergy between the Bishop and Cho labs.
Exploring the secondary senescence response will include physiologically relevant readouts
of cellular function (e.g. neuron electrophysiology - whole cell patch clamp) [5] and
RNA/proteomics profiling.
Aim 3. Uncoupling primary and secondary senescence via senostatic intervention
Finally, we will explore the potential to uncouple secondary senescence using established
'generalise' senostatics and test putative precision senostatics (indicated from Aim 2). This
will enable us to probe the fundamental mechanisms of primary and secondary senescence
and contribute to our overarching interest in understanding the molecular mechanisms of
these distinct endpoints.