Establishment of a human, age-specific model for axon growth and regeneration studies

人类脊髓损伤导致终身残疾，治疗选择有限，尽管数十年来 对啮齿动物的研究。最近的证据表明，基因表达存在根本性的差异。 人和老鼠之间的差异，表明迫切需要识别新的或人类特有的候选对象 作为促进脊髓损伤后轴突再生的靶点。此外，轴突的体外研究 生长通常在胚胎或出生后幼年啮齿动物的神经元中进行，随着年龄的增加 与生俱来的生长能力，以及与成年神经元截然不同的转录格局。因此，我们建议 创建一个与年龄相关的人类轴突生长和再生模型。 最近的技术进步现在允许人类成年体细胞被重新编程为干细胞 细胞，然后分化成神经元。然而，通过多能性对这些细胞进行重新编程 被证明抹掉了原始细胞的“年龄”，导致产生的神经元具有更具胚胎特征的特性。 使用直接重新编程协议，我们将对~35岁和~70岁的新生儿进行转分化 人类体细胞通过过度表达特定的神经元因子直接转化为神经元，从而允许 维持细胞的生物学年龄身份。我们将在这些细胞中确定年龄的维持 表观遗传年龄特征、基因表达和细胞老化标志物的水平。作为对轴突的首次研究 在年龄维持的人类神经元中的生长，我们将表征允许的基础轴突生长行为 以及抑制底物，以确定年龄是否在人类轴突生长能力中发挥作用。为了测试这一新资源， 我们将针对之前在其他物种中发现的20种不同的基因和途径来调节轴突生长 它们在人类神经元中的功能守恒。在啮齿动物中，最近发现某些轴突生长 诸如PTEN基因敲除的修饰物在老年动物中的作用不同于幼年动物(Geoffroy等人， 2016)。因此，我们将利用我们的年龄维持的人类INS来确定年龄是否推动轴突生长的变化。 表型。 这项研究的完成将1)建立一种新的与年龄相关的人类轴突细胞培养系统 生长研究，2)确定在人类神经元中具有保守功能的基因及其调节因子，以及3) 确定年龄在轴突生长能力中的作用。识别人类特有的翻译靶点 神经元最终可能导致开发有效的治疗人类脊髓损伤的靶点。

Human spinal cord injury leads to life-long disability, with limited treatment options despite decades of research in rodents. Recent evidence indicates that there are fundamental differences in gene expression between human and mouse, suggesting a critical need for identification of novel or human-specific candidates as targets to increase axon regeneration following spinal cord injury. Additionally, in vitro studies of axon growth are typically performed in neurons from embryonic or young postnatal rodents, ages with increased intrinsic growth ability, and a very different transcriptional landscape from adult neurons. Thus, we propose to create an age-relevant, human model for axon growth and regeneration. Recent technological advances now allow for human adult somatic cells to be reprogrammed into stem cells, and then differentiated into neurons. However, reprogramming these cells through pluripotency has been shown to erase the “age” of the original cell, resulting in a more embryonic identity of the resulting neuron. Using a direct reprogramming protocol, we will transdifferentiate neonatal, ~35 year old, and ~70 year old human somatic cells directly into neurons through overexpression of specific neuronal factors, allowing for maintenance of the cell’s biological age identity. We will determine the maintenance of age in these cells at the level of the epigenetic age signature, gene expression, and markers of cellular aging. As the first study of axon growth in age-maintained human neurons, we will characterize the basal axon growth behavior on permissive and inhibitory substrates to identify if age plays a role in human axon growth ability. To test this new resource, we will target 20 different genes and pathways previously identified in other species to regulate axon growth for their conservation in function in human neurons. In rodents it has recently been shown that certain axon growth modifiers such as PTEN knockout, do not function similarly in aged animals as young animals (Geoffroy et al, 2016). Thus, we will utilize our age-maintained human iNs to determine if age drives changes in axon growth phenotypes. Completion of this research will 1) establish a new human, age-relevant cell culture system for axon growth studies, 2) identify genes and their regulators that have conserved functions in human neurons, and 3) determine the role age plays in axon growth ability. Identification of translational targets specifically in human neurons ultimately may lead to the development of effective therapeutic targets for human spinal cord injury.