Modelling the neuronal cell biology of human ageing in vitro

目的1 -将人真皮成纤维细胞分化为神经元的方案效率的并排比较（第1年）。目前有两种主要途径可用于获得保留供体衰老特征的成纤维细胞诱导的神经元。途径1涉及将成纤维细胞直接重编程为神经元（PMID：26456686）;对于途径2，成纤维细胞在分化为神经元谱系之前首先转化为诱导的神经元祖细胞（iNPC）（PMID：24379375）。这两种方法都显示出产生的神经元保留了一定程度的供体细胞的分子年龄。然而，尚未对这两种方法进行比较。这是在体外模拟人类衰老的细胞生物学的一个非常重要的步骤，并确定用这两种方法获得的神经元在功能上是否等同。因此，作为试验设置的一部分，我们的目标是对两种方法进行初步比较。分化后，我们将通过特异性神经元标记物的免疫染色来确认神经元身份。将通过RNAseq评估老化表型，以揭示神经元的老化特征。 目的2 -测量年轻和老年神经元的线粒体转运和功能（第2年）。我们将首先测量来自不同年龄的人类供体（“年轻”细胞< 20岁，“老年”细胞> 60岁）的诱导神经元中的线粒体转运。线粒体将用荧光活体染料MitoTracker染色，并通过延时旋转圆盘显微镜记录这些细胞器的轴突运输3-5分钟。我们和其他人已经表明，线粒体转运减少是无脊椎动物和小鼠神经元老化的标志。这组实验对于确定轴突运输缺陷是否实际上是从无脊椎动物到人类的神经元老化的进化保守标志是重要的。有人认为，线粒体可能在模型生物老化过程中显示出功能缺陷，尽管尚不清楚这是否也适用于老化的人类神经元。为了回答这个问题，学生将使用活体染料和报告者的组合来测量这些细胞中的线粒体膜电位和Ca 2+稳态。 目的3 -确定cAMP/PKA通路的激动剂在老年神经元中具有神经保护作用（第3年）。我们发现，慢性激活的cAMP/PKA途径在果蝇神经元在体内强烈刺激线粒体运输在以后的生活（PMID：29606421）。值得注意的是，持续喂食途径激动剂8-Br-cAMP，同时刺激线粒体转运，也减少了蛋白质聚集的出现，表明蛋白质稳态改善。我们将对cAMP/PKA通路的化学激动剂进行试点筛选，这些化学激动剂将增加老年神经元中的线粒体运动，预计这将具有神经保护作用。将在二次筛选中测定途径拮抗剂，具体取决于项目结束时的可用时间。由于cAMP/PKA通路参与基因激活，我们将从筛选中获得最有希望的命中，并在药物处理的老年细胞中进行RNAseq实验。我们的目标是将功能数据与转录组学分析相匹配，以发现衰老过程中通路诱导的神经保护的潜在介质。

Aim 1 - Side-by-side comparison of protocol efficiencies to differentiate human dermal fibroblasts into neurons (YEAR 1). Two main routes are currently available to obtain fibroblast-induced neurons that retain the ageing signature of the donor. Route 1 involves direct reprogramming of the fibroblasts into neurons (PMID: 26456686); with Route 2, fibroblasts are first converted into induced neuronal progenitor cells (iNPCs) before differentiation into a neuronal lineage (PMID: 24379375). Both methods have been shown to yield neurons that retain a degree of the molecular age of the donor cells. However, a side-to-side comparison of the two methods have not yet been undertaken. This is a very important step towards modelling the cell biology of human ageing in vitro and to establish if the neurons obtained with the two methods are functionally equivalent. Therefore, as part of the assay setup, we aim to perform an initial comparison of the two methodologies. After differentiation, we will confirm neuronal identity by immunostaining for specific neuronal markers. The aged phenotype will be assessed via RNAseq to reveal the ageing signature of the neurons. Aim 2 - To measure mitochondrial transport and function in young and old neurons (YEAR 2). We will initially measure mitochondrial transport in induced neurons derived from human donors of different ages ('young' cells < 20 years, 'old' cells > 60 years). Mitochondria will be stained with the fluorescent vital dye MitoTracker and the axonal transport of these organelles recorded by time-lapse spinning disk microscopy for 3-5 minutes. We and others have showed that reduced mitochondrial transport is a hallmark of neuronal ageing in invertebrates and mice. This set of experiments is important to establish whether defective axonal transport is in fact an evolutionary conserved hallmark of neuronal ageing from invertebrates to humans. It has been suggested that mitochondria may display functional defects during ageing of model organisms, although it is not clear whether this is also true in ageing human neurons. To answer this question, the student will use a combination of vital dyes and reporters to measure the mitochondrial membrane potential and Ca2+ homeostasis in these cells. Aim 3 - To identify agonists of the cAMP/PKA pathway with a neuroprotective role in old neurons (YEAR 3). We found that chronic activation of the cAMP/PKA pathway in Drosophila neurons in vivo strongly stimulates mitochondrial transport during later life (PMID: 29606421). Remarkably, sustained feeding with the pathway agonist 8-Br-cAMP, while stimulating mitochondrial transport, also reduces the appearance of protein aggregation, indicative of improved protein homeostasis. We will undertake a pilot screen for chemical agonists of the cAMP/PKA pathway that would increase mitochondrial motility in older neurons, which is predicted to be neuroprotective. Pathway antagonists will be assayed in a secondary screen, depending on time availability towards the end of the project. As the cAMP/PKA pathway is involved in gene activation, we will take the most promising hits from our screen and perform RNAseq experiments in drug-treated old cells. Our aim is to match the functional data to transcriptomic analyses in order to discover potential mediators of pathway-induced neuroprotection during ageing.