Identification of active ingredients' targets to prevent skin ageing

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Objective: To investigate causal molecular mechanism of human skin senescence with under-used pharmacological intervention potential. Overall project aim is to determine specific key pathways leading to senescence, such as mTOR/autophagy/mitophagy and how their targeting impacts on mitochondrial dysfunction and cell senescence. To translate these results, we will identify small molecules that can be used to target the pathways under investigation and have a potential to postpone or revert human skin ageing. Research plan: Year 1: We will investigate how autophagy network changes with senescence in skin fibroblasts.We will perform careful assessment of age-related changes in the mTORC1/autophagy/mitophagy network using young and senescing skin cells. We will assess how senescence induction in dermal fibroblasts by multiple mechanisms (replicative- and stress-induced senescence) impacts on this network. mTORC1 activity and general autophagic flux will be analysed using a range of established markers The data generated will inform the design of interventions to be investigated in Year 2 and become the basis for investigations in 3D models during Year 3 (see below).Milestones: 1. Establishment of the autophagy network as a central signalling hub in skin cell senescence in vitro;2. Identification of autophagy targets suitable for drug interventions.Year 2: We will determine how modulation of autophagy affects mitochondrial function in skin cells.We will modulate mTORC1/autophagy/mitophagy by CRISPR/Cas9-mediated knockout of key regulatory genes to investigate their contribution to mitochondrial phenotypes in cell senescence. Additionally, we will use pharmacological means to modulate autophagy/mitophagy using several commercially available drugs as well as FDA-approved small molecules recently identified by us as autophagy modulators in a high throughput screen. Next, both genetic and pharmacological interventions will be used to measure their impact on mitochondrial function in cultured young and senescent skin fibroblasts. Milestones: 1. Identification of the role of autophagy network in the maintenance of mitochondrial function/metabolism;2. Determination of the consequences of autophagy network perturbation in cell senescence on mitochondrial function.3. Identification of interventions with a potential to rescue autophagy and mitochondrial dysfunction in human skin.Year 3: We will determine how modulation of autophagy affects other features of senescence such as DNA damage and SASP.To understand the interactions between mTOR/autophagy/mitophagy and DNA damage, we will apply the same interventions as above and measure cell proliferation (growth curves, KI67 etc), DNA damage response (DNA damage foci frequencies, frequencies of telomere-associated DNA damage foci, NF-kB activity (RelA nuclear/cytoplasmic ratio), expression of SASP factors. To elucidate this signalling network further, we will combine autophagy modulators as above with interventions into different arms of the signalling network. Milestones: 1. Identification of the role of autophagy network perturbation in senescence-associated cellular phenotypes.Year 4: We will validate our findings in 3D skin models.In the last year we will also apply our findings in the fibroblast cell culture to 3D human skin models. Specifically, we will validate the relevance of autophagy and mitochondrial changes investigated above using histochemistry and functional assays. We will test a set of small molecules that will be identified to have the strongest effect on senescence-associated mitochondrial dysfunction and other phenotypes for their ability to rescue senescence phenotypes and structure of the collagen matrix in 3D cultures. Milestones: 1. Establishment of the relevance of autophagy network perturbation to skin function using 3D skin models;2. Identification of small molecules with a potential to improve skin function using 3D models.