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，它在生物体中进化来适应内部状态 有了环境线索。它由每个细胞中的分子振荡器组成，通过节律基因 表达，调节由节律性突触功能产生的可预测的行为可塑性。这个 突触与胞体的时空分离排除了分子振荡器成为 节律性突触过程的机制起源，并证明了对局部突触的研究 钟。利普顿实验室发现，BMAL1--昼夜节律机制的核心成分--是有节奏的 定位于突触，与突触激酶钙/钙调蛋白依赖的激酶(CaMKII⍺)相互作用 并组织突触小泡池的昼夜节律集合。BMAL1的昼夜定位 突触在磷缺乏小鼠模型(BMAL-S42A)中丢失，该模型也失去了昼夜节律动力学 突触囊泡团与学习和记忆受损配对。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。目前尚不清楚BMAL1是如何通过生化和/或生物物理 机制，有节奏地组装突触囊泡池与昼夜节律同步。最重要的是 这项拟议工作的目标是深入了解生物钟是如何通过生物化学和 生物物理学以调节突触小泡昼夜节律的方式组装突触小泡 分门别类和动态。有两个相互关联但独立的目标，这项提案调查 BMAL1与突触蛋白激酶CaMKII⍺的生物分子缩合作用 这种相互作用和突触前信号的可塑性依赖于BMAL1。目标1建议 确定BMAL1和CaMKII⍺蛋白序列中的结构元件，这些结构元件是其 互动。这将通过在细胞和细胞中进行一系列补充的体外测试来实现 用重组蛋白评估这些蛋白的相互作用和这些蛋白缩合成 相分离的类液滴。这些生化和生物物理相互作用对突触的影响 然后，将使用重建突触小泡样结构的非神经元系统来评估小泡 形式上和功能上的结构。然后，目标2将识别突触前、神经调节系统 需要并招募pBMAL1(S42)用于其对突触小泡的急性突触前可塑性控制。一个屏幕，用来 神经调节剂依赖于pBMAL1(S42)来控制突触小泡。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/清醒的关键调节器，它增强了pBMAL1(S42)。这样的集体知识 从这项提议中获得的有可能发现可用于改变时间的可用药靶点 具有昼夜节律和突触功能的神经综合征突触前可塑性的组织。

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.