Mechanisms of direct dopaminergic neuronal reprogramming to study aging

大脑形成我们的思想，它控制我们的运动，我们的认知和其他器官的功能，并解释感觉。然而，由于其非常有限的神经元再生能力，活体组织的可用性受到阻碍，因此这一非凡的器官对研究来说是一个挑战。因此，旨在理解神经生理学的工作在很大程度上依赖于动物模型。然而，很少有物种能像人类一样长寿。因此，随着我们年龄的增长，神经元功能发生了什么变化，这个问题仍然非常难以捉摸。中枢神经系统中的多巴胺能（DA）神经元控制着一系列行为，包括自主运动、动机和奖励，以及认知。DA神经元反映了这种广泛的行为，一直是神经科学领域关注的对象。然而，年龄如何影响多巴胺驱动的神经元功能，还有待在人类系统中研究。该提案旨在开发一个人类神经元系统，可以研究衰老，这将代表该领域的重大进展，并可能导致更好地理解神经元衰老的生理学。神经元直接重编程是指将一个终末分化的细胞直接转化为一个新的终末分化状态。这产生了功能性神经元，被称为诱导神经元。我们和其他人已经证明，来自老年供体的人类皮肤成纤维细胞可以转化为特定的神经元亚型。重要的是，直接重编程的细胞保留了亲代成纤维细胞年龄特征的许多重要方面。这使得直接细胞重编程成为在细胞水平上研究人类大脑衰老的理想方法。最近，我们开发了一种从老年人中产生表达DA神经元（iDANs）主要特征的诱导神经元的方法。我们现在的目标是1)改进这个工具，使方法更有效，细胞产物更接近于在人类中脑中发现的，2)研究这个系统中的衰老生理学。更具体地说，以下目标将被解决：1)内在DA神经元需求的调查。我们将通过基因表达谱、单细胞测序以及计算模型来筛选和研究中脑DA命运决定因素的作用，以更清晰地描绘细胞重编程要求，从而从皮肤成纤维细胞中产生人类DA神经元。2)外源性DA神经元需求的研究。为了给DA神经元的成熟和功能提供最佳的环境，我们将使用中脑DA命运决定分泌分子，并通过与星形胶质细胞共培养促进突触成熟。3) iDANs的衰老生理研究。我们将对来自不同年龄供体的成纤维细胞重编程为idan。我们将研究衰老过程中发生的神经元生理变化，以更好地了解衰老对DA神经元功能的影响。

The brain forms our thoughts, it controls our movements, our cognition and the function of other organs, and interprets senses. This remarkable organ, however, represents a challenge to study as live tissue availability is prevented by its very limited neuronal regenerative capacity. As such, work aiming at understanding neuronal physiology has vastly relied on animal models. However, few species live for as long as humans do. Therefore, the question as to what happens to neuronal functions as we age remains very elusive. Dopaminergic (DA) neurons in the CNS control a spectrum of behaviors, including voluntary movements, motivation and reward, as well as cognition. Reflective of this wide range of behaviors, DA neurons have been the object of much attention in the field of neuroscience. However, how old age affects dopamine-driven neuronal functions has yet to be studied in a human-based system. This proposal aims at developing a human neuronal system in which aging can be studied, which will represent a major advance in the field and may lead to a better understanding of the physiology of neuronal aging. Direct neuronal reprogramming consists in converting a terminally differentiated cell directly to a new terminal state of differentiation. This generates functional neurons which are referred to as induced neurons. We and others have demonstrated that human skin fibroblasts from elderly donors can be converted into specific neuronal subtypes. Importantly, directly reprogrammed cells retain many important aspects of the age signature of the parental fibroblasts. This makes direct cell reprogramming an ideal approach to study aging of the human brain at a cellular level. Recently, we have developed a method to generate induced neurons from elderly individuals that express the main characteristics of DA neurons (iDANs). We now aim to i) refine this tool to make the method more efficient and the cell product more closely resembling that found in the human midbrain and ii) study the physiology of aging in this system. More specifically, the following objectives will be addressed: 1) Investigation of intrinsic DA neuronal requirements. We will screen and study the role of mesencephalic DA fate determinants by gene expression profiling, single cell sequencing as well as computational modeling to dress a clearer portrait of the cell reprogramming requirements to generate a human DA neuron from a skin fibroblast. 2) Investigation of the extrinsic DA neuronal requirements. To provide the optimal environment for the DA neuron to mature and function in, we will use mesencephalic DA fate determinant secreted molecules, as well as promote synaptic maturation though co-cultures with astrocytes. 3) Study of the physiology of aging in iDANs. We will reprogram fibroblasts from donors of different ages to iDANs. We will investigate changes in neuronal physiology occurring during aging to better understand the impact of aging on DA neuronal functions.