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

时钟蛋白 BMAL1 对突触前功能的生化和生物物理调节

• 批准号: 10606746
• 负责人: Nicole Marie Gilette
• 金额: $ 3.89万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606746
• 关键词: ARNTL gene; Acute; Alzheimer' s Disease; Amino Acid Sequence; Animals; Behavior; Biochemical; Biological Assay; Biophysical Process; Biophysics; Calcium; Calmodulin; Cells; Circadian Dysregulation; Circadian Rhythms; Circadian desynchrony; Clock protein; Cognition; Cues; Data; Disease; Elements; Exhibits; Feedback; Forskolin; Functional disorder; Gene Expression; Genetic Transcription; Goals; Hour; Impairment; In Vitro; Investigation; Knowledge; Learning; Link; Liquid substance; Longevity; Mammals; Mediating; Mediator of activation protein; Memory; Molecular; Mus; Nerve Degeneration; Nervous system structure; Neurologic; Neuromodulator; Neurons; Neurotransmitters; Norepinephrine; Organism; Periodicity; Pharmacology; Phase; Phosphorylation; Phosphotransferases; Physical condensation; Process; Property; Proteins; Recombinant Proteins; Recycling; Regulation; Research; Role; Schizophrenia; Series; Serotonin; Signal Pathway; Signal Transduction; Sleep; Structure; Synapses; Synaptic Vesicles; Synaptic plasticity; Syndrome; System; Testing; Therapeutic; Time; Translations; Variant; Work; autism spectrum disorder; behavioral plasticity; brain cell; calmodulin-dependent protein kinase II; circadian; circadian pacemaker; controlled release; druggable target; experimental study; feeding; in vitro Assay; in vivo; insight; mouse model; nervous system disorder; neurodevelopment; neuronal cell body; neuropsychiatry; neuroregulation; novel; presynaptic; reconstitution; recruit; response; segregation; serotonin receptor; sleep regulation; spatiotemporal; success; synaptic function; transcription factor

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.

项目摘要 昼夜节律系统是一种古老的机制，它在生物体中演变为适应内部状态 带有环境提示。它由每个细胞中的分子振荡器组成，该细胞通过节奏基因 表达，调节有节奏的突触功能产生的可预测行为可塑性。这 突触的时空隔离从体内隔离阻止了分子振荡器 节奏突触过程的机械出处，并有必要调查当地突触 钟。 Lipton实验室确定BMAL1（昼夜节律机制的核心组成部分）在逻辑上是 与合成激酶Ca2+/钙调蛋白依赖性激酶（CAMKII⍺）相互作用的突触本地化为突触。 并组织突触囊泡池的昼夜节律大会。 BMAL1的昼夜定位 突触在磷酸化的小鼠模型（BMAL-S42A）中丢失，该模型也失去了昼夜动力学 突触囊泡簇与学习和记忆受损配对。这些发现将昼夜节系统与 突触可塑性和突触产生的行为的调节，以独立于 核心转录时钟。尚不清楚BMAL1如何通过生化和/或生物物理学 机理，节奏与昼夜节律相相的合成囊泡池组装。总体 这项拟议工作的目标是深入了解昼夜节律如何在生化和 生物物理以调节昼夜节律的方式组装突触蔬菜 分隔和动态。有两个相关但独立的目标，该提案调查了 BMAL1与突触激酶CAMKII⍺（生物分子缩合）的生化相互作用 该相互作用和突触前信号依赖于它们的可塑性。目标1提案 定义BMAL1和CAMKII⍺蛋白序列中所需的结构元素 互动。这将通过在细胞中进行一系列完成体外评估来实现 用重组蛋白评估这些蛋白质并将这些蛋白质缩合到 相分离的液态液滴。这些生化和生物物理相互作用对突触的影响 然后，将使用非神经元系统评估蔬菜，该系统重构突触囊泡样 形式和功能的结构。 AIM 2然后将确定突触前的神经调节剂系统 要求并招募PBMAL1（S42），以对突触蔬菜进行急性突触前可塑性控制。屏幕 将进行依赖PBMAL1（S42）进行突触囊泡控制的神经调节剂。信号 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是否 5-羟色胺是节奏和睡眠/唤醒的关键调节剂PBMAL1（S42）。这样的集体知识 从该提案中获得的可能有可能发现可药物的目标来修改临时性 神经系统综合症中突触前可塑性的组织，这些神经系统综合症具有共享昼夜节律和突触功能的组织。