Identification of active ingredients' targets to prevent skin ageing

目的：本项目将利用未充分利用的药物干预潜力，研究人类皮肤衰老的分子机制。该项目的总体目标是确定导致衰老的具体关键途径，如mTOR/自噬/有丝分裂吞噬，以及它们的靶向如何影响线粒体功能障碍和细胞衰老。为了转化这些结果，我们将识别小分子，这些小分子可以用于靶向研究中的途径，并有可能延缓或逆转人类皮肤衰老。研究计划：第一年：我们将研究皮肤成纤维细胞的自噬网络如何随着衰老而变化。我们将使用年轻和衰老的皮肤细胞仔细评估mTORC1/自噬/有丝分裂网络中与年龄相关的变化。我们将评估通过多种机制(复制和应激诱导的衰老)诱导真皮成纤维细胞衰老对这一网络的影响。MTORC1活性和一般自噬通量将使用一系列已建立的标记进行分析，所产生的数据将为第二年要调查的干预措施的设计提供依据，并成为第三年3D模型研究的基础(见下文)。第二年：我们将确定自噬的调节如何影响皮肤细胞中的线粒体功能。我们将通过CRISPR/Cas9介导的关键调控基因的敲除来调节mTORC1/自噬/有丝分裂吞噬，以研究它们在细胞衰老中对线粒体表型的贡献。此外，我们将使用药理学手段来调节自噬/有丝分裂吞噬，使用几种市售药物以及FDA批准的小分子，最近我们在高通量筛选中确定了自噬调节器。下一步，将使用遗传和药物干预来衡量它们对培养的年轻和衰老皮肤成纤维细胞线粒体功能的影响。第3年：我们将确定自噬的调节如何影响衰老的其他特征，如DNA损伤和SASP。为了了解mTOR/自噬/有丝分裂吞噬与DNA损伤之间的相互作用，我们将应用与上面相同的干预措施，并测量细胞增殖(生长曲线，Ki67等)，DNA损伤反应(DNA损伤焦点频率，端粒相关DNA损伤焦点频率，核因子-B活性(relA核质比)，SASP因子的表达。为了进一步阐明这个信号网络，我们将结合上述自噬调节器和对信号网络不同分支的干预。第4年：我们将在3D皮肤模型中验证我们的发现。在去年，我们还将把我们在成纤维细胞培养中的发现应用到3D人类皮肤模型中。具体地说，我们将使用组织化学和功能分析来验证自噬和上面研究的线粒体变化的相关性。我们还将尝试挽救包含衰老细胞的皮肤3D模型中与年龄相关的功能缺陷。因此，我们将测试一组小分子，这些小分子将被确定为对衰老相关的线粒体功能障碍和其他表型(见上文)具有最强的影响，因为它们能够在3D培养中拯救线粒体功能障碍、炎症标志物以及胶原基质的密度和结构。

Objective:This project will investigate causal molecular mechanism of human skin senescence with under-used pharmacological intervention potential. The overall aim of the project 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).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. 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-B 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. 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 also attempt to rescue age-related functional deficits in skin 3D models incorporating senescent cells. Thus, 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 (see above) for their ability to rescue mitochondrial dysfunction, inflammation markers as well as density and structure of the collagen matrix in 3D cultures.