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，在小树突棘中划分。这些级联反应中涉及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转化为持久的结构和功能突触修饰。蛋白激酶C （PKC）家族由超过12种同工酶组成，在突触可塑性的诱导、表达和维持中发挥重要作用。然而，目前实验方法的局限性，包括同工酶识别能力差、时空分辨率和灵敏度，限制了对PKC同工酶在介导这些变化的信号级联中的确切作用的理解。为了克服这些问题，本研究的第一个目标是基于荧光共振能量转移（FRET）和2光子荧光寿命成像（2pFLIM）开发新的、高度优化的PKC同工酶荧光传感器。这些高度敏感的传感器将报告同工酶特异性PKC活性，在亚微米空间和亚秒的时间分辨率下刺激单个脊柱。利用PKC同工酶的多个激活步骤，将为8种PKC同工酶中的每一种开发几种传感器，这些PKC同工酶显示在脊柱可塑性中起作用。通过使用这些传感器、2pFLIM和谷氨酸释放，将在本研究的目标二中阐明单脊柱结构可塑性过程中同工酶特异性PKC活性的时空分布。特异性同工酶在诱导、表达和维持结构可塑性中的需求将通过抑制特异性同工酶来检验。最后，我们将研究PKC同工酶的上游激活，以确定受体激活对可塑性的要求，以及同工酶活性对不同输入的异质性。实验

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.