Biochemical and Biophysical Tuning of Presynaptic Function by the Clock Protein BMAL1

项目摘要 昼夜节律系统是一种古老的机制，在生物体中进化以适应性地调整内部状态 与环境线索联系起来它由每个细胞中的分子振荡器组成， 表达，调节由节律性突触功能产生的可预测的行为可塑性。的 突触与索马的时空分离排除了分子振荡器作为 机械起源的节律性突触过程，并有必要调查当地的突触 时钟利普顿实验室发现，BMAL 1-昼夜节律机制的核心组成部分-是有节奏的， 定位于突触，与突触激酶Ca 2 +/钙调蛋白依赖性激酶（CaMKII）相互作用 并组织突触囊泡池的昼夜节律组装。BMAL 1在植物中的昼夜定位 突触在磷酸无能小鼠模型（Bmal-S42 A）中丢失，该模型也丢失了 突触囊泡簇与受损的学习和记忆配对。这些发现将昼夜节律系统与 突触可塑性和突触产生的行为的调节，以一种独立于 核心转录时钟目前还不清楚BMAL 1是如何通过生物化学和/或生物物理学途径， 机制，有节奏地组装突触囊泡池的相位与昼夜节律的时间。总体 这项工作的目标是深入了解生物钟是如何从生物化学和 以调节它们的昼夜节律的方式生物地组装突触囊泡 划分和动态。有两个相关但独立的目标，这项建议调查了 BMAL 1与突触激酶CaMKII的生物化学相互作用，即生物分子的缩合， 这种相互作用和突触前信号的可塑性依赖于BMAL 1。目标1建议， 定义了BMAL 1和CaMKII β蛋白序列中的结构元件，这些结构元件是它们的功能所需的。 交互.这将通过在细胞中进行一系列互补的体外测定来实现， 用重组蛋白评估这些蛋白的相互作用和这些蛋白的缩合， 相分离的液体状液滴。这些生物化学和生物物理学相互作用对突触的影响， 然后将使用非神经元系统来评估囊泡， 结构的形式和功能。目标2将确定突触前神经调质系统， 需要和募集pBMAL 1（S42）用于它们对突触小泡的急性突触前可塑性控制。的屏幕 将进行依赖于pBMAL 1（S42）用于突触囊泡控制的神经调节剂。信令 然后将在培养的神经元和体内进行实验以确定神经调节剂是否 5-羟色胺是节律和睡眠/觉醒的关键调节剂，增强pBMAL 1（S42）。这样的集体知识 从这个提议中获得的信息有可能发现用于改变时间的药物靶点。 神经系统综合征中突触前可塑性的组织具有昼夜节律和突触功能。

Project Summary The circadian system is an ancient mechanism which evolved in organisms to adaptively align internal state with environmental cues. It is comprised of a molecular oscillator in every cell which, through rhythmic gene expression, regulates predictable behavioral plasticity that is engendered by rhythmic synaptic function. The spatiotemporal segregation of the synapse from the soma precludes the molecular oscillator from being the mechanistic provenance for rhythmic synaptic processes and has warranted investigation for a local synaptic clock. The Lipton lab identified that BMAL1 – a core component of the circadian mechanism – is rhythmically localized to synapses where it interacts with the synaptic kinase Ca2+/calmodulin-dependent kinase (CaMKII⍺) and organizes the circadian assembly of synaptic vesicle pools. The diurnal localization of BMAL1 to the synapse is lost in a phosphoincompetent mouse model (Bmal-S42A) which also loses circadian dynamics of synaptic vesicle clusters paired with impaired learning and memory. These findings link the circadian system to the regulation of synaptic plasticity and synapse generated behavior, in a manner which is independent of the core transcriptional clock. It remains unknown how BMAL1, either through biochemical and/or biophysical mechanisms, rhythmically assembles synaptic vesicle pools in phase with circadian time. The overarching goal of this proposed work is to gain insight into how the circadian clock biochemically and biophysically assembles synaptic vesicles in a manner which regulates their circadian compartmentalization and dynamics. With two related but independent aims, this proposal investigates the biochemical interactions that BMAL1 makes with the synaptic kinase CaMKII⍺, the biomolecular condensation of that interaction and the presynaptic signals that are BMAL1-dependent for their plasticity. Aim 1 proposes to define the structural elements in the BMAL1 and CaMKII⍺ protein sequences which are required for their interactions. This will be accomplished by conducting a series of complementary in vitro assays in cells and with recombinant protein that assess interaction of these proteins and condensation of these proteins into phase separated liquid-like droplets. The effect of these biochemical and biophysical interactions on synaptic vesicles will then be evaluated using a non-neuronal system which reconstitutes synaptic vesicle-like structures in both form and function. Aim 2 will then identify the presynaptic, neuromodulator systems which require and recruit pBMAL1(S42) for their acute presynaptic plasticity control on synaptic vesicles. A screen for neuromodulators which depend on pBMAL1(S42) for synaptic vesicle control will be conducted. Signaling experiments will then be performed in cultured neurons and in vivo to determine if the neuromodulator serotonin, a key regulator of rhythms and sleep/wake, potentiates pBMAL1 (S42). Such collective knowledge gained from this proposal has the potential to uncover druggable targets for modifying the temporal organization of presynaptic plasticity in neurological syndromes which share circadian and synaptic function.