Extended Tracking of Single Synaptic Proteins with Upconverting Nanoparticles

描述（由申请人提供）：神经元通过突触之间的化学信号相互交流，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数据发现，突触形成发生在发育过程中，突触前神经元通过特定的粘附蛋白附着在突触后细胞上，确保两个细胞之间足够接近以允许突触成熟。特异性粘附蛋白的天然表达变化与小鼠的自闭症谱系障碍有关，一些患有自闭症谱系障碍的个体的DNA中存在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。为了在突触形成过程中跟踪单个事件，有必要使用一种成像探针，这种探针必须足够明亮，光稳定性足以在单分子跟踪所需的强度下持续几分钟到几小时。探针还需要足够小，以免干扰粘附蛋白的相互作用。尽管有多种成像探针可用于跟踪活细胞中的单个蛋白质，但没有一种成像探针在突触发育的时间尺度上具有稳定性。这项提议的研究详细介绍了使用上转换纳米颗粒的生物成像探针的发展，这些纳米颗粒明亮，不闪烁，并且在数小时内具有光稳定性，这些特性将允许我们实时跟踪突触发育的事件。组合方法将用于合成小（直径小于10 nm）且在近红外（NIR）激发后显示明亮可见发光的纳米颗粒。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颗粒对神经元中的单个蛋白质进行活细胞成像，以确定有关特定蛋白质如何参与突触成熟的更多信息，并揭示该过程中的扰动如何导致神经系统疾病（如自闭症）的细节。

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.