Identification of active ingredients' targets to prevent skin ageing

目的：探讨未充分利用药理干预潜力的人皮肤衰老的致病分子机制。整个项目的目标是确定导致衰老的特定关键途径，如mTOR/自噬/线粒体自噬，以及它们的靶向如何影响线粒体功能障碍和细胞衰老。为了翻译这些结果，我们将确定可用于靶向正在研究的途径的小分子，这些小分子有可能延缓或恢复人类皮肤老化。研究计划：第一年：研究皮肤成纤维细胞自噬网络随衰老的变化。我们将使用年轻和衰老的皮肤细胞仔细评估mTORC1/自噬/线粒体自噬网络中与年龄相关的变化。我们将评估多种机制（复制性和应激性衰老）诱导真皮成纤维细胞衰老对该网络的影响。mTORC1活性和一般自噬通量将使用一系列已建立的标记物进行分析，生成的数据将为二年级调查的干预措施设计提供信息，并成为三年级3D模型调查的基础（见下文）。里程碑:1。1 .体外皮肤细胞衰老中自噬网络作为中心信号中枢的建立；确定适合药物干预的自噬靶点。第2年：我们将确定自噬调节如何影响皮肤细胞的线粒体功能。我们将通过CRISPR/ cas9介导的关键调控基因敲除来调节mTORC1/自噬/线粒体自噬，以研究它们在细胞衰老过程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/有丝自噬，使用几种市售药物以及fda批准的小分子，最近由我们在高通量筛选中鉴定为自噬调节剂。接下来，将使用遗传和药物干预来测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。里程碑:1。1 .确定自噬网络在线粒体功能/代谢维持中的作用；细胞衰老过程中自噬网络扰动对线粒体功能影响的测定。鉴定有可能挽救人类皮肤自噬和线粒体功能障碍的干预措施。第三年：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/自噬与DNA损伤之间的相互作用，我们将采用与上述相同的干预措施，测量细胞增殖（生长曲线，KI67等），DNA损伤反应(DNA损伤灶频率，端粒相关DNA损伤灶频率，NF-kB活性（RelA核/细胞质比），SASP因子的表达。为了进一步阐明这一信号网络，我们将上述自噬调节剂与信号网络不同分支的干预措施结合起来。里程碑:1。自噬网络扰动在衰老相关细胞表型中的作用鉴定。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于3D人体皮肤模型。具体来说，我们将通过组织化学和功能分析来验证自噬和线粒体变化的相关性。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具有最强的影响，因为它们能够在3D培养中挽救衰老表型和胶原基质的结构。里程碑:1。1 .利用三维皮肤模型建立自噬网络扰动与皮肤功能的相关性；利用3D模型鉴定具有改善皮肤功能潜力的小分子。

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.