Senescent exosomes in skin ageing

Background
There is compelling evidence that senescence drives ageing and may confer a uni-directionality to ageing. However, we have unexpectedly achieved senescence reversal yielding a highly efficient protocol for human cell rejuvenation (Genome Biology, 2015).
Senescence also leads to increased exosome release. Once liberated into the extracellular milieu, these tiny vesicles (50-90nm) are an elegant means of cell-to-cell communication. miRNAs are packaged into exosomes. We hypothesise that the exosome's cargo alters during senescence and plays a role in ageing phenotypes.
Preliminary data
- Exosomes isolated from senescent fibroblasts (validated by TEM) elevate senescence markers in 'young' proliferating fibroblasts.
- Knockdown of previously identified senescence-associated miRNAs (NAR, 2014), which increase during human ageing, enables senescence reversal (unpublished).
To our knowledge, this is the first data linking exosomes, senescence and rejuvenation. Therefore, we are poised to make significant contributions to this exciting and emerging field.
Key aims
1. Determine the autocrine/paracrine function of senescent exosomes in and between skin cell types (i.e. primary human dermal fibroblasts and keratinocytes), including:
a. characterising the route to senescence induction using established senescence and ageing markers;
b. tracking and quantitating PKH67 fluorescently-labelled exosomes using super resolution SIM in 2D; and c. high-throughput live cell imaging using state-of-the-art microscopy (IN Cell 2200, GE).
2. Explore the autocrine and paracrine function of senescent exosomes and their impact on ageing phenotypes using:
a. 2D co-culture experiments, and
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b. sophisticated 3D living skin equivalent (LSE) models to assess a panel of established skin ageing phenotypes (e.g. thickness, matrix proteins) and senescence markers.
3. Determine miRNA exosome cargo and their intracellular targets using NGS of:
a. the miRNA cargo of young, senescent and rejuvenated exosome;
b. miRNA/mRNA expression profiles of the corresponding exosome producing cells; and
c. miRNA/mRNA expression profiles of target cells following exosome exposure.
Following 1x50bp single end Illumina HiSeq NGS runs, differentially expressed miRNAs will subjected to extensive bioinformatics analysis e.g. target predication, mining of skin ageing array datasets (publically available and from Unilever), and IPA. The other exosome RNA molecules (mRNA, lcRNA and ncRNAs) will form the basis of future collaborative projects.
4. Use miRNA knockdown to enable senescence reversal, and determine if this alters exosome cargo and function.
a. miRNA validation (including miRNA in situ hybridisation in skin tissue bank sections and mRNA target validation).
b. antimiRs transfection, exosome cargo profiling and exosome application to 2D and 3D LSE models followed by ageing phenotype characterisation.
Timescale
Aim 1a Y1Q1-3, 1b Y1Q2-4, 1c Y1Q4-Y2Q2. Aim 2a Y2Q1-3, 2b Y2Q1-4.
Aim3 Y2Q4-Y3Q3 (Placement Y3Q2-3).
Aim 4a Y3Q3-Y4Q1, 4b Y3Q3-Y4Q3.
Thesis and manuscript writing Y4Q1-4.
Strategic Fit
This proposal aligns to the BBSRC's Key Research Priority 3: Bioscience for health (Strategic plan 2013- 2014), under the umbrella of 'the ageing process' and 'new regenerative biology and tissue engineering' research opportunities, and the multidisciplinary RCUK programmes of 'Lifelong health and Wellbeing' and the 'UK Regenerative Medicine Platform'.