Identification of active ingredients' targets to prevent skin ageing

目的：探讨人类皮肤衰老的分子机制及药物干预潜力。总体项目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。为了转化这些结果，我们将确定可用于靶向正在研究的途径的小分子，并有可能推迟或逆转人类皮肤衰老。研究计划：第一年：我们将研究自噬网络如何随着皮肤成纤维细胞的衰老而变化。我们将使用年轻和衰老的皮肤细胞仔细评估mTORC 1/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（复制性和应激诱导的衰老）如何诱导真皮成纤维细胞衰老对该网络的影响。将使用一系列已建立的标记物分析mTORC 1活性和一般自噬通量。所产生的数据将为第2年研究的干预措施的设计提供信息，并成为第3年3D模型研究的基础（见下文）。建立自噬网络作为体外皮肤细胞衰老的中心信号枢纽;2.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敲除来调节mTORC 1/自噬/线粒体自噬，以研究它们对细胞衰老中线粒体表型的贡献。此外，我们将使用药理学手段来调节自噬/线粒体自噬，使用几种市售药物以及FDA批准的小分子，我们最近在高通量筛选中鉴定为自噬调节剂。接下来，遗传和药理学干预将用于测量它们对培养的年轻和衰老皮肤成纤维细胞中线粒体功能的影响。军事：1。鉴定自噬网络在维持线粒体功能/代谢中的作用;2.确定细胞衰老中自噬网络扰动对线粒体功能的影响.识别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/线粒体自噬和DNA损伤之间的相互作用，我们将应用与上述相同的干预措施并测量细胞增殖（生长曲线、KI 67等）、DNA损伤反应（DNA损伤灶频率、端粒相关DNA损伤灶频率、NF-κ B活性（RelA核/胞质比）、SASP因子表达。为了进一步阐明该信号网络，我们将联合收割机将上述自噬调节剂与干预结合到信号网络的不同臂中。军事：1。自噬网络扰动在衰老相关细胞表型中的作用的鉴定。第四年：我们将在3D皮肤模型中验证我们的发现。在最后一年，我们还将把我们在成纤维细胞培养中的发现应用于3D人类皮肤模型。具体来说，我们将使用组织化学和功能测定来验证上述研究的自噬和线粒体变化的相关性。我们将测试一组小分子，这些小分子将被鉴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作用，因为它们能够在3D培养物中挽救衰老表型和胶原基质结构。军事：1。使用3D皮肤模型建立自噬网络扰动与皮肤功能的相关性;2.使用3D模型识别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.