Stress resistance in neurons from primate iPS cells

描述（由申请人提供）：诱导多能干细胞（iPS细胞）具有与胚胎干细胞相似的特性，但它们可以来源于任何类型的体细胞，如皮肤成纤维细胞。iPS细胞可以提供对专门细胞的访问，例如 神经元，否则将无法用于生物医学研究的物种。在非人类灵长类动物中，黑猩猩在比较生物学中占有独特的地位。在这里，我们建议使用来自三种灵长类动物的iPS细胞：黑猩猩，绒猴和人类。很少有研究利用iPS细胞的独特性质进行哺乳动物衰老的比较研究。我们的假设是，分化的细胞（运动神经元）来自三个灵长类动物物种的非常不同的longevities将表现出不同的抗生理压力，并参与抗应激的关键途径的药理学干预将揭示在何种程度上的抗应激机制不同的神经元之间的这三个灵长类动物物种。具体目标1：验证从黑猩猩、绒猴和人iPS细胞中有效和稳健地衍生运动神经元的条件。我们将从这三种灵长类动物中获得运动神经元，使用三阶段方案（神经外胚层诱导，神经模式化和运动神经元发育/成熟），通过调整已用于人类多能细胞有效分化的方案。我们假设，运动神经元从所有物种将能够形成典型的神经肌肉接头（NMJ）时，与骨骼肌细胞系共培养。具体目标二：评估三种灵长类动物运动神经元的应激抵抗力是否与这三种动物的不同寿命成正比。我们将使用升高的葡萄糖和升高的氧气作为体外条件，模拟神经元在体内可能经历的长期应激。我们假设，神经元在压力条件下的鲁棒性将与灵长类物种的不同寿命成比例变化。我们进一步假设，在线粒体的质量控制机制，在分子和细胞器水平，将是一个主要的决定因素的压力抵抗力。具体目标3。评估药物干预关键通路以改善三种灵长类动物运动神经元应激效应的潜力。虽然抗应激是复杂的，但有三个关键途径：NF-？B、p38 MAP激酶和细胞巯基代谢，这些与氧化应激和高血糖对神经功能的不良影响或细胞保护作用有关。 机制等使用已经建立的作用于这三个途径的药物，我们将评估这些干预措施是否会影响培养的运动神经元应激的不良反应。这些实验将首次允许直接比较来自不同物种的孤立神经元的抗应激能力，并将为物种寿命不同的机制提供新的见解。

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPS cells) have properties similar to those of embryonic stem cells, but they can be derived from any type of somatic cell, such as a skin fibroblast. iPS cells can provide access to specialized cells, such as neurons, in species that otherwise would be unavailable for biomedical research. Among nonhuman primates, the chimpanzee occupies a unique position for comparative biology. Here we propose to use iPS cells from three primates: the chimpanzee, marmoset, and human. Few studies have yet taken advantage of the unique properties of iPS cells for comparative studies of mammalian aging. Our hypothesis is that differentiated cells (motor neurons) derived from three primate species of very different longevities will exhibit differential resistance to physiological stresses, and that pharmacological interventions in key pathways involved in stress resistance will reveal the extent to which mechanisms of stress resistance differ among neurons of these three primate species. Specific Aim 1: To validate conditions for efficient and robust derivation of motor neurons from chimpanzee, marmoset, and human iPS cells. We will derive motor neurons from these three primate species using a three-phase protocol (induction of neuroectoderm, neural patterning, and motor neuron development/maturation), by adapting protocols that have been used for efficient differentiation in human pluripotent cells. We hypothesize that motor neurons from all species will be capable of forming typical neuromuscular junctions (NMJs) when co-cultured with a skeletal muscle cell line. Specific Aim 2: To assess whether stress resistance in motor neurons of three primate species varies proportionally to the different longevities of these three species. We will use elevated glucose and elevated oxygen as in vitro conditions that mimic the long-term stresses that neurons may experience in vivo. We hypothesize that robustness of neurons under conditions of stress will vary in proportion to the differing life spans of the primate species. We further hypothesize that quality control mechanisms in mitochondria, at the molecular and organellar levels, will be a major determinant of stress resistance. Specific Aim 3. To assess the potential for pharmacological interventions in key pathways to ameliorate the effects of stresses in motor neurons of the three primate species. While stress resistance is complex, there are three key pathways: NF-?B, p38 MAP kinase, and cell thiol metabolism, that have been implicated in the adverse effects of oxidative stress and hyperglycemia on neural function, or in cellular protective mechanisms. Using drugs that have been well established to act on these three pathways, we will assess whether these interventions influence the adverse effects of stresses in cultured motor neurons. These experiments will allow, for the first time, direct comparisons of stress resistance in isolated neurons from different species and will provide new insights into the mechanisms by which species differ in longevity.