Regulation of Actin Dynamics at Functional Subdomains within Dendritic Spines

描述(申请人提供)：树突棘包含大脑中兴奋性突触的突触后成分。突触在本质上是可塑性的，树突棘是维持和修饰突触强度的重要部位，而突触强度被认为是行为学习和发育可塑性的基础。越来越多的证据表明，脊椎中含有相互关联的功能亚域集合。其中最值得注意的是突触后密度，这是一台定位和调节突触后神经递质受体数量的分子机器。此外，脊椎包含一个位于脊膜上的内吞液带，与突触相距甚远。许多神经疾病背后的突触功能障碍与这些功能亚域中发生的突触功能障碍有关。重要的是，其中许多过程需要持续的肌动蛋白聚合。脊髓内肌动蛋白细胞骨架的动态调节对于脊髓的形态可塑性以及突触组成和功能的维持是必要的，并且是突触上神经递质受体插入、稳定和内吞的基础。肌动蛋白聚合和棘内稳定性的变化伴随着并且是诱导长时程增强和其他形式的突触可塑性所必需的。然而，关于脊椎内哪些部位的肌动蛋白重排是启动和维持突触强度变化所特别需要的，我们知之甚少。我认为，肌动蛋白聚合的持续和独立调节发生在脊椎亚区，如突触后密度和内细胞区。我提出的实验将通过使用共聚焦、超分辨率和单颗粒技术测量单个活的树突棘内肌动蛋白的聚合和运动来检验这一假设，并利用荧光蛋白标记的分子来标记突触后密度(PSD)和内细胞区。然后，利用这些分析，我将测试一种广泛提出的机制，即PSD通过皮质蛋白与肌动蛋白细胞骨架相互作用，并检查在LTP诱导过程中，肌动蛋白在脊柱中的组织是如何改变的。这些结果将提供关于单个树突棘内细胞骨架组织和调节的基本新信息，这对于理解脊椎肌动蛋白失调在疾病中的参与至关重要。

DESCRIPTION (provided by applicant): Dendritic spines house the postsynaptic components of excitatory synapses in the brain. Synapses are inherently plastic, and dendritic spines represent an important locus for maintenance and modification of synaptic strength which is thought to underlie behavioral learning and developmental plasticity. Increasing evidence exists that spines contain within them interlinked sets of functional subdomains. Most notable of these is the postsynaptic density, the molecular machine which positions and regulates the number of postsynaptic neurotransmitter receptors. In addition, spines contain an endocytic zone positioned in the spine membrane substantially away from the synapse. The synaptic dysfunction underlying many neurological disorders is associated with impairment of processes occurring at these functional subdomains. Importantly, a number of these processes require ongoing actin polymerization. Dynamic regulation of the actin cytoskeleton within spines is necessary for spine morphological plasticity as well as maintenance of synaptic composition and function, and underlies the insertion, stabilization, and endocytosis of neurotransmitter receptors at the synapse. Changes in actin polymerization and stability within spines accompany and are required for the induction of long term potentiation and other forms of synaptic plasticity. However, little is known about the sites within spines at which actin rearrangement is specifically required for alterations in synaptic strength to be initiated and maintained. I propose that ongoing and independent regulation of actin polymerization occurs at spine subdomains such as the postsynaptic density and endocytic zone. My proposed experiments will test this hypothesis by measuring actin polymerization and movement within individual, live dendritic spines using confocal, super-resolution, and single-particle techniques, and utilizing fluorescent protein-tagged molecules to mark the postsynaptic density (PSD) and endocytic zones. Using these assays, I will then test a widely proposed mechanism that the PSD interacts with the actin cytoskeleton via the protein cortactin, and examine how the organization of actin is altered within spines during induction of LTP. These results will provide fundamental new information about cytoskeletal organization and regulation within individual dendritic spines, critical for understanding the involvement of spine actin dysregulation in disease.