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.

项目概要 昼夜节律系统是一种古老的机制，在生物体中进化来适应性地调整内部状态 它由每个细胞中的分子振荡器组成，通过有节奏的基因。 表达，调节由节律性突触功能产生的可预测的行为可塑性。 突触与体体的时空分离阻止了分子振荡器成为 有节奏的突触过程的机械起源，并有必要对局部突触进行研究 Lipton 实验室发现 BMAL1（昼夜节律机制的核心组成部分）具有节律性。 定位于突触，与突触激酶 Ca2+/钙调蛋白依赖性激酶 (CaMKII⍺) 相互作用 并组织突触小泡池的昼夜节律组装 BMAL1 的昼夜定位。 磷酸化无能小鼠模型（Bmal-S42A）中突触丢失，该模型也丢失了昼夜节律动态 这些发现将昼夜节律系统与受损的学习和记忆相结合。 突触可塑性和突触生成行为的调节，以独立于 核心转录时钟如何通过生化和/或生物物理学仍然未知。 机制，与昼夜节律时间同步有节奏地组装突触小泡池。 这项拟议工作的目标是深入了解生物钟如何生化和 以生物物理方式组装突触小泡，以调节其昼夜节律 出于两个相关但独立的目标，本提案研究了划分和动态。 BMAL1 与突触激酶 CaMKII⍺ 产生的生化相互作用，即生物分子凝聚 目标 1 提出了这种相互作用和突触前信号的可塑性依赖于 BMAL1。 定义 BMAL1 和 CaMKII⍺ 蛋白质序列中的结构元件，这些元件是其 这将通过在细胞和细胞中进行一系列互补的体外测定来实现。 与重组蛋白一起评估这些蛋白质的相互作用以及这些蛋白质的缩合 这些生物化学和生物物理相互作用对突触的影响。 然后使用非神经元系统评估囊泡，该系统重建突触囊泡样 目标 2 将识别突触前神经调节系统的形式和功能。 需要并招募 pBMAL1(S42) 来控制突触小泡的急性突触前可塑性。 将进行依赖于 pBMAL1(S42) 进行突触小泡控制的神经调节剂。 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 血清素是节律和睡眠/觉醒的关键调节剂，可增强 pBMAL1 (S42) 的集体知识。 从该提案中获得的成果有可能发现用于修改时间的可药物靶点 共享昼夜节律和突触功能的神经综合征中突触前可塑性的组织。