Mechanisms of direct dopaminergic neuronal reprogramming to study aging

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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.