Constitutive 5-HT7 receptor activity as a target for treatment of tauopathy

多种神经退行性疾病是由蛋白质聚集体的形成和沉积引起的。特别是，微管相关蛋白Tau的聚集导致所谓的Tau病的发展，其特征是神经元内过度磷酸化的Tau蛋白聚集。我们最近发现，通过g12蛋白不依赖cdk5的机制，构成型5-HT7受体（5-HT7R）活性是Tau过度磷酸化和高度捆绑Tau结构形成所必需的。此外，小鼠皮质中选择性的5-HT7R敲除完全消除了与额颞叶痴呆（FTD）相关的病理性人类突变体Tau[R406W]过表达引起的长期增强和记忆损伤。在本提案中，我们的目标是表征5-HT7R-CDK5复合物形成的结构要求。基于这些知识，我们将通过表达5-HT7R的突变形式（不与CDK5结合）特异性地干扰5-HT7R-CDK5信号传导。这将使我们能够研究5-HT7R-CDK5信号在生理条件下以及人类Tau[R406W]或Tau[P301L]突变体神经元过表达诱导的两种形式的Tau病中的小GTPase活性、肌动蛋白丝动力学、脊柱发生和突触可塑性中的作用。由于我们确定了几种临床批准的药物作为潜在的5-HT7R逆转录激动剂，我们将使用高通量双分子荧光互补（BiFC）技术比较它们在防止Tau过度磷酸化和聚集方面的功效。然后研究最有效的药物在体外和体内消除病理性Tau效应的能力，使用参与实验室的尖端技术的协同组合，包括生化和分子生物学分析、病毒操作、定量活体显微镜、FRET测量、电生理记录和行为分析。此外，一项预先注册的双盲随机临床前试验将对最有效的抑制Tau过度磷酸化和聚集的5-TH7R逆激动剂进行研究，以评估其对雄性和雌性Tg4510小鼠多种认知、社交、情感和运动功能的影响，同时控制潜在的锥体外系副作用。最后，我们将利用人类诱导多能干细胞（hiPSCs）衍生的神经元来评估5-HT7R/CDK5信号的作用，这将有助于弥合动物模型与人类之间的遗传和表型差距。因此，本项目的结果有望提高我们对5-HT7R-CDK5信号在神经退行性疾病中的生理和病理生理意义的认识，并识别和表征5- ht7r靶向药物治疗tau病相关疾病的治疗效果。

Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, aggregation of the microtubule-associated protein, Tau, leads to the development of so-called tauopathies, which are characterized by the aggregation of hyperphosphorylated Tau protein within neurons. We recently showed that the constitutive serotonin 5-HT7 receptor (5-HT7R) activity is required for Tau hyperphosphorylation and formation of highly bundled Tau structures through G12-protein-independent, CDK5-dependent mechanism. Moreover, selective 5-HT7R knockdown in mouse cortex fully abrogated impairments in long-term potentiation and memory induced by the overexpression of pathological human mutant Tau[R406W] associated with frontotemporal dementia (FTD). Within this proposal, we aim to characterize structural requirements for 5-HT7R-CDK5 complex formation. Based on this knowledge, we will specifically interfere with 5-HT7R-CDK5 signaling by expressing the mutated form of 5-HT7R (not binding to CDK5). This would enable us to study the role 5-HT7R-CDK5 signaling in small GTPase activities, actin filament dynamics, spinogenesis and synaptic plasticity under physiological conditions and in two forms of tauopathy induced by neuronal overexpression of either human Tau[R406W] or Tau[P301L] mutants. Since we identified several clinically approved drugs to be potential 5-HT7R inverse agonists, we shall compare their efficacies to prevent Tau hyperphosphorylation and aggregation using a high throughput bimolecular fluorescence complementation (BiFC) technique. The most potent drugs will be then investigated for their ability to abrogate pathological Tau effects both in vitro and in vivo using synergistic combination of cutting edge techniques present in participating labs, including biochemical and molecular biological assays, viral manipulations, quantitative intravital microscopy, FRET measurements, electrophysiological recordings, and behavioral analyses. Moreover, a preregistered double-blind randomized preclinical trial of the most efficient 5-TH7R inverse agonist inhibiting Tau hyperphosphorylation and aggregation will be performed to evaluate its effects on multiple cognitive, social, emotional and locomotor functions in male and female Tg4510 mice, also controlling for potential extrapyramidal side effects. Finally, we will evaluate the role of 5-HT7R/CDK5 signaling using human induced pluripotent stem cells (hiPSCs)-derived neurons, which will help to bridge the genetic and phenotypic gap between animal models and humans. Thus, the outcome of this project is expected to improve our understanding of the physiological and pathophysiological significance of 5-HT7R-CDK5 signaling in neurodegenerative diseases and to identify and characterize the therapeutic effects of 5-HT7R-targeting drug for treatment of tauopathy-related disorders.