Function of reactive astrocytes in aging and neurodegenerative disease

反应性星形胶质细胞(RAS)是不同病因的正常衰老和神经退行性变的特征，提示了潜在的共同机制。然而，RAS在神经退行性变中的作用是复杂的，因为它们可以由许多不同的刺激触发，对中枢神经系统(CNS)既有有利的影响，也有有害的影响。因此，更好地了解不同反应性星形胶质细胞的原因和功能对于设计能够选择性地增强保护功能和抑制毒性效应的疗法是必要的。还迫切需要扩展可用的工具来生成反应性星形胶质细胞并描述不同的反应性星形胶质细胞功能。这一应用的长期研究目标是从机制上深入了解引起反应性星形胶质细胞的因素以及不同类型反应性星形胶质细胞的功能。对于K00阶段，RAS的形成和在阿尔茨海默病(AD)和衰老中的作用将在Mel Feany博士的实验室进行询问。在包括阿尔茨海默病(AD)、癌症和衰老在内的各种中枢神经系统损伤中，可以观察到细胞外基质(ECM)和脑僵硬的急剧变化。然而，虽然已知在硬基质上生长星形胶质细胞可以促进反应性星形胶质细胞表型，但机械激活的星形胶质细胞的生理效应尚未被探索。在目标1中，通过在不同硬度的基质上培养星形胶质细胞来研究RAS，以了解机械变化对星形胶质细胞的影响。这个模型将被用来研究RA诱导毒性的潜在机制。蛋白多糖(PG)是衰老和RAS中表达最高的基因之一。来自费尼博士实验室的初步数据证实了前列腺素和果蝇神经变性模型之间的遗传相互作用。我假设RAS会在ECM中产生PG的失衡，从而创造出一种神经毒性的环境，抑制神经元的生长和重塑。为了测试这一点，候选PG将在IPSCs中被敲除，这些细胞将被分化为反应性和非反应性星形胶质细胞，并与神经元共同培养，以确定每个PG在星形胶质细胞介导的神经毒性中的作用。将对野生型反应性和非反应性iPSC-星形胶质细胞进行蛋白质组学研究，以确定机械激活星形胶质细胞的反应性星形胶质细胞分泌组，并确定新的候选基因。在目标2中，将使用体内苍蝇模型来研究PGs在RAS在衰老和神经退行性变背景下的毒性中的作用。为了模拟衰老，PG候选人将被选择性地击倒在野生型苍蝇中，这些苍蝇将老化长达2个月。此外，一个双二聚体表达系统将被用来选择性地敲除胶质细胞中的PG候选基因，同时独立地操纵神经细胞来建立AD模型。神经退行性变将在PG基因敲除后进行评估，以确定PG在每种情况下的作用。拟议的研究将揭示反应性星形胶质细胞的新功能，最终目标是激发创新的星形胶质细胞靶向干预，可用于改善老龄化人口的健康和福祉。该研究计划将为申请者提供胶质细胞、衰老和神经变性领域的培训。

Reactive astrocytes (RAs) are a feature of normal aging and neurodegeneration of varying etiologies, suggesting a potential common mechanism. However, the role of RAs in neurodegeneration is complex because they can be triggered by many different stimuli and can have both beneficial and harmful effects to the Central Nervous System (CNS). Thus, a better understanding of the causes and functions of different reactive astrocytes is necessary to design therapeutics that can selectively bolster protective functions and inhibit toxic effects. There is also a vital need to expand the tools available to generate reactive astrocytes and to describe diverse reactive astrocyte functions. The long-term research goal of this application is to gain deep mechanistic understanding of the factors that elicit reactive astrocytes and the functions of different types of reactive astrocytes. For the K00 phase, the formation and the role of RAs in Alzheimer’s Disease (AD) and aging will be interrogated in the lab of Dr. Mel Feany. Dramatic changes in the extracellular matrix (ECM) and brain stiffness are observed in various CNS injuries, including Alzheimer’s disease (AD), cancer, and aging. However, while growing astrocytes on stiff substrates is known to promote a reactive astrocyte phenotype, the physiological effects of mechanically activated astrocytes have not been explored. In Aim 1, a novel model will be developed to study RAs by culturing iPSCastrocytes on substrates of different stiffness to understand the effects of mechanical changes on astrocytes. This model will be utilized to examine potential mechanisms of RA-induced toxicity. Proteoglycans (PGs) are among the most highly upregulated genes in aging and RAs. Preliminary data from Dr. Feany’s lab identified genetic interactions between PGs and models of neurodegeneration in the fly. I hypothesize that RAs produce an imbalance of PGs in the ECM, which creates an environment that is neurotoxic and inhibitory to neuronal growth and remodeling. To test this, candidate PGs will be knocked out in iPSCs, which will be differentiated to reactive and non-reactive astrocytes and co-cultured with neurons to determine the role of each PG in astrocyte-mediated neurotoxicity. Proteomics will be performed on wild-type reactive and non-reactive iPSC-astrocytes to define the reactive astrocyte secretome of mechanically activated astrocytes and identify new candidate genes. In Aim 2, in vivo fly models will be used to examine the role of PGs in toxicity of RAs in the contexts of aging and neurodegeneration. To model aging, PG candidates will be selectively knocked down in wild-type flies, which will be aged up to 2 months. Additionally, a dual bipartite expression system will be used to selectively knockdown PG candidates in glial cells while manipulating neuronal cells independently to model AD. Neurodegeneration will be assessed upon PG knockdown to determine the role of PGs in each context. The proposed studies will reveal new functions of reactive astrocytes with the ultimate goal of inspiring innovative astrocyte-targeted interventions that can be used to improve the health and well-being of the aging population. The research plan will equip the applicant with training in the fields of glia, aging and neurodegeneration.