Extended Tracking of Single Synaptic Proteins with Upconverting Nanoparticles

使用上转换纳米颗粒对单个突触蛋白进行扩展追踪

• 批准号: 8256087
• 负责人: Alexis D Ostrowski
• 金额: $ 1.08万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-02 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256087
• 关键词: Address; Adhesions; Affect; Antibodies; Autistic Disorder; Binding; Biochemical Genetics; Biocompatible; Birth; Blinking; Brain; Caliber; Cells; Chemicals; Code; Color; DNA; Data; Development; Diagnosis; Diagnostic; Ensure; Event; Foundations; Gene Mutation; Genes; Genetic; Goals; Hippocampus (Brain); Hour; Image; Individual; Interruption; Kinetics; Knowledge; Lead; Life; Light; Link; Location; Mediating; Methods; Mind; Mus; Mutation; Nature; Neurons; Optics; Photobleaching; Play; Process; Property; Proteins; Protocols documentation; Rare Earth Metals; Research; Role; Signal Transduction; Staining method; Stains; Synapses; Time; Water; absorption; autism spectrum disorder; autistic behaviour; behavior change; bioimaging; cellular imaging; combinatorial; imaging probe; interest; luminescence; nanoparticle; nervous system disorder; neuroligin 1; particle; relating to nervous system; research study; single molecule; stoichiometry; synaptogenesis

DESCRIPTION (provided by applicant): Neurons communicate with each other via chemical signals across synapses, a fundamental process that underlies all brain function. Even small aberrations during synaptic development may lead to neurological disease. A growing body of biochemical and genetic data has found that synapse formation occurs during development as the pre-synaptic neuron attaches to the post-synaptic cell via specific adhesion proteins, ensuring that there is close enough proximity between the two cells to permit synapse maturation. Changes in the native expression of specific adhesion proteins have been linked to autism-spectrum disorders in mice, and some individuals with autism spectrum disorders have genetic mutations in their DNA that codes for expression of these specific adhesion proteins. While it is known that these adhesion proteins control the interaction of the pre-synaptic and post-synaptic neurons, a number of questions remain about the mechanism of neuron-neuron interaction, including kinetics, the sequence of binding events, and reversibility or permanence of the interactions. In order to follow individual events over the course of synapse formations, it is necessary to use an imaging probe that is bright and photostable enough to last minutes to hours under the intensity required for single molecule tracking. The probe will also need to be small enough so as not to perturb the interaction of the adhesions proteins. Although a variety of imaging probes are available for single protein tracking in live cells, none are have the stability over the timescale of synapse development. The proposed research details the development of bioimaging probes using upconverting nanoparticles that are bright, non-blinking and photostable over hours-properties that will permit us to follow the events of synapse development in real time. Combinatorial methods will be employed to synthesize nanoparticles that are small (sub-10 nm diameter) and show bright, visible luminescence after near-infrared (NIR) excitation. After synthesis, the nanoparticles with be functionalized to interact with specific neuronal adhesion proteins. Subsequent experiments wil use the developed nanoparticles in live-cell imaging of single proteins in neurons to determine more information about how the specific proteins are involved in synapse maturation and to uncover details as to how perturbations in this process can lead to neurological diseases such as autism. PUBLIC HEALTH RELEVANCE: The described research efforts are focused on understanding how specific neuronal proteins affect synapse formation and maturation. These proteins are of interest because individuals with genetic mutations that cause changes in these proteins have been diagnosed with autism spectrum disorders. A more detailed knowledge of the role of these proteins is critical for the development of diagnostics and treatment for autism.

描述（由申请人提供）：神经元通过突触之间的化学信号相互通信，这是所有大脑功能的基础过程。在突触发育过程中，即使是很小的畸变也可能导致神经系统疾病。越来越多的生物化学和遗传学数据发现，突触形成发生在发育过程中，因为突触前神经元通过特定的粘附蛋白附着到突触后细胞，确保两个细胞之间有足够的接近度以允许突触成熟。特异性粘附蛋白的天然表达的变化与小鼠的自闭症谱系障碍有关，并且一些患有自闭症谱系障碍的个体在其DNA中具有编码这些特异性粘附蛋白表达的基因突变。 虽然已知这些粘附蛋白控制突触前和突触后神经元的相互作用，但关于神经元-神经元相互作用的机制仍存在许多问题，包括动力学、结合事件的顺序以及相互作用的可逆性或持久性。为了跟踪突触形成过程中的单个事件，有必要使用成像探针，该探针足够明亮且光稳定，能够在单分子跟踪所需的强度下持续数分钟至数小时。探针还需要足够小，以便不干扰粘附蛋白的相互作用。虽然有多种成像探针可用于活细胞中的单个蛋白质跟踪，但没有一种在突触发育的时间尺度上具有稳定性。 这项研究计划详细描述了生物成像探针的发展，这种探针使用上转换纳米粒子，这种纳米粒子在数小时内是明亮的、不闪烁的、光稳定的，这些特性将使我们能够真实的跟踪突触发育的事件。将采用组合方法来合成小的纳米颗粒（直径小于10 nm），并且在近红外（NIR）激发后显示明亮的可见光发光。合成后，纳米颗粒被功能化以与特定的神经元粘附蛋白相互作用。随后的实验将使用开发的纳米粒子在神经元中的单个蛋白质的活细胞成像中确定更多关于特定蛋白质如何参与突触成熟的信息，并揭示这个过程中的扰动如何导致自闭症等神经疾病的细节。 公共卫生相关性：所描述的研究工作集中在了解特定的神经元蛋白质如何影响突触的形成和成熟。这些蛋白质是令人感兴趣的，因为具有导致这些蛋白质变化的基因突变的个体已被诊断患有自闭症谱系障碍。更详细地了解这些蛋白质的作用对于自闭症诊断和治疗的发展至关重要。