Spatiotemporal Dynamics of Isozyme-Specific PKC Activity during Plasticity

描述（由申请人提供）：突触可塑性是与学习和记忆相关的细胞，由高度调节的信号级联介导，在小树突棘中区室化。参与这些级联反应的数百种蛋白质在空间和时间上的调节使短暂的突触输入能够被转导成持久的结构和功能突触修饰。蛋白激酶C（PKC）家族由12种以上的同工酶组成，在突触可塑性的诱导、表达和维持中起重要作用。然而，在目前的实验方法，包括差的同工酶歧视，时空分辨率和灵敏度的局限性，有限的理解PKC同工酶在介导这些变化的信号级联的确切作用。为了克服这些问题，本研究的第一个目的是开发新的，高度优化的，基于荧光的PKC同工酶的荧光共振能量转移（FRET）和双光子荧光寿命成像（2 pFLIM）的传感器。这些高度敏感的传感器将报告同工酶特异性PKC活性与亚微米空间和亚秒级时间分辨率刺激后的一个单一的脊椎。利用多个激活步骤的PKC同工酶，几个传感器将被开发为8个PKC同工酶中的每一个显示有脊柱可塑性的作用。通过使用这些传感器，2 pFLIM，谷氨酸uncaging，在单脊椎结构可塑性的同工酶特异性PKC活性的时空分布将阐明在目标二的拟议研究。然后通过抑制特异性同工酶来检查在结构可塑性的诱导、表达和维持中特异性同工酶的需求。最后，将检查PKC同工酶的上游激活，以确定受体激活可塑性的要求，以及同工酶活性对各种输入的异质性。实验 这项研究提出的新方法将有助于深入了解PKC如何适应介导可塑性的复杂信号网络。这将增强对突触可塑性的分子机制的理解;突触可塑性的功能障碍是许多神经精神疾病的特征。特别是这些研究可以提供洞察疾病，如阿尔茨海默病和双相情感障碍，其中PKC功能障碍已牵连。最后，由于PKC参与了许多细胞过程的调节，因此本文开发的工具将对更广泛的细胞生物学社区有用，包括与PKC功能相关的其他疾病（如癌症）的研究。

DESCRIPTION (provided by applicant): Synaptic plasticity, the cellular correlate to learning and memory, is mediated by highly regulated signaling cascades, compartmentalized in small dendritic spines. The regulation in space and time of the hundreds of proteins involved in these cascades enables short-lived synaptic inputs to be transduced into long-lasting structural and functional synaptic modifications. The protein kinase C (PKC) family, consisting of more than 12 isozymes, has been implicated to play an essential role in the induction, expression and maintenance of synaptic plasticity. However, limitations in current experimental approaches, including poor isozyme discrimination, spatiotemporal resolution, and sensitivity, have limited understanding of the precise role of PKC isozymes in the signaling cascades that mediate these changes. To overcome these problems, the first aim of this study is the development of new, highly-optimized, fluorescence-based sensors for PKC isozymes based on fluorescence resonance energy transfer (FRET) and 2-photon fluorescence lifetime imaging (2pFLIM). These highly sensitive sensors will report isozyme-specific PKC activity with submicron spatial and subsecond temporal resolution upon stimulation of a single spine. Taking advantage of multiple activation steps of PKC isozymes, several sensors will be developed for each of eight PKC isozymes shown to have a role in spine plasticity. Through the use of these sensors, 2pFLIM, and glutamate uncaging, the spatiotemporal profile of isozyme-specific PKC activity during single spine structural plasticity will be elucidated in aim two of the proposed study. The requirement of specific isozymes in the induction, expression and maintenance of structural plasticity will then be examined by inhibition of specific isozymes. Finally, the upstream activation of PKC isozymes will be examined to determine the requirement of receptor activation for plasticity, as well as the heterogeneity of isozyme activity to various inputs. The experiments proposed in this study will provide insight into how PKC fits into the complex signaling networks that mediate plasticity. This will enhance understanding of the molecular mechanisms of synaptic plasticity; the dysfunction of which is a feature of many neuropsychiatric disorders. In particular these studies may provide insight into disorders such as Alzheimer's disease and bipolar disorder, in which PKC dysfunction has been implicated. Finally, since PKC is involved in the regulation of numerous cell processes, the tools developed here will be useful for the broader cell biological community, including the study of other diseases related to PKC function such as cancer.