LOCATER: Large-scale Observation of Cellular Activity Through Exosomal Reporters

 描述(申请人提供)：电生理学和荧光成像被广泛用于研究活体神经活动。然而，这些方法需要侵入性的程序，只能对少量神经元进行采样，活动指示器的全脑荧光成像只能捕获安乐死前大脑最后状态的快照。W提议定位器：一种将超越这些先前技术的限制的变革性系统。其中，我们将使用报告活动的RNA条形码捕获大量神经元中的单细胞活动痕迹，然后将这些活动痕迹从大脑输出到外体中，以便通过循环血液进行非侵入性捕获。外切体是由内体衍生的囊泡，含有蛋白质和RNA货物，由神经元分泌，很容易穿过血脑屏障。因此，外切体为捕获神经元中的核酸货物并将这些货物输出到循环血液中提供了理想的底物。利用定位器技术捕获循环血液中神经元群体分泌的报告活性的单细胞RNA条形码，并使用下一代测序(NGS)量化单个条形码序列的丰度，将有助于对活动的可扩展纵向监控 来自小的神经元群体或整个大脑。为了开发这个系统，我们将创建一种AAV载体，它使用即刻早期基因(IEG)启动子来驱动受定位器监视的每个神经元特有的RNA条形码的转录。我们将在每个活性报告条形码上包括噬菌体RNA茎环，以促进RNA条形码与噬菌体外壳蛋白连接的外体跨膜蛋白的结合，以便通过正常的内体蛋白运输进行条形码的外体定位。表位标签也将被添加到胞外体跨膜蛋白的胞外域，以实现从循环血液中高保真地捕获含有条形码的胞外体。为了使用定位器监测体内的神经活动，我们将在小鼠大脑的不同区域表达标记的外体膜蛋白和报告活动的细胞RNA条形码，并使动物受到刺激，以捕获伴随行为的单细胞神经元活动轮廓。除了捕捉大脑中单个神经元的活动痕迹外，定位器系统还可以绘制大脑中报告活动的细胞条形码的空间来源。在纵向活动监测后，动物将被处死，随后对每个大脑进行切片和清除组织 使用清晰度技术。我们将对澄清的组织进行FISH，以识别包括在每个AAV载体上的多重FISH条形码，这些条形码指定唯一的活动报告RNA条形码。通过这种方式，连续几轮鱼类的比色读数将被用来识别大脑中每个报告活动的条形码的细胞来源。使用定位器非侵入性地监测动物行为中的单细胞活动状态，并将这些状态映射回它们在大脑中的细胞起源，将改变我们对行为、感觉和 神经发育、精神和神经退行性疾病。

 DESCRIPTION (provided by applicant): Electrophysiology and fluorescent imaging are widely used to study neural activity in vivo. These methods, however, require invasive procedures and can only sample small populations of neurons, and whole-brain fluorescent imaging of activity indicators is only able to capture a snapshot of the last state of the brain prior to euthanasia. W propose LOCATER: a transformative system that will transcend the limitations of these previous techniques. Therein, we will capture single-cell activity traces in large populations of neurons using activity-reporting RNA barcodes, and then export these activity traces from the brain in exosomes for non- invasive capture via circulating blood. Exosomes are endosome-derived vesicles containing protein and RNA cargo that are secreted from neurons and that readily cross the blood-brain barrier. Hence, exosomes provide an ideal substrate for capturing nucleic acid cargo in neurons and exporting these cargoes into circulating blood. The use of LOCATER technology to capture activity-reporting single-cell RNA barcodes secreted by neuronal populations in circulating blood and quantify the abundance of individual barcode sequences using next generation sequencing (NGS) will facilitate scalable longitudinal monitoring of activity from small neuronal populations or the entire brain. To develop this system we will create an AAV-vector that uses an immediate early gene (IEG) promoter to drive transcription of an RNA barcode unique to each neuron under LOCATER surveillance. We will include bacteriophage RNA stem loops on each activity-reporting barcode to facilitate binding of RNA barcodes to bacteriophage coat protein-linked exosomal transmembrane proteins for exosomal localization of barcodes via normal endosomal protein trafficking. Epitope tags will also be added to the extracellular domains of exosomal transmembrane proteins to enable high-fidelity capture of barcode- containing exosomes from circulating blood. To monitor neural activity in vivo using LOCATER, we will express tagged exosomal membrane proteins and activity-reporting cellular RNA barcodes in sub-regions of the mouse brain and subject animals to stimulation to capture single cell neuronal activity profiles accompanying behavior. Beyond capturing activity traces from single neurons in the brain, the LOCATER system also makes it possible to map the spatial origins of activity-reporting cellular barcodes in the brain. Following longitudinal activiy monitoring, animals will be sacrificed, followed by sectioning of each brain and clearing of tissue using the CLARITY technique. We will subject the clarified tissue to FISH to identify multiplexed FISH barcodes included on each AAV-vector that specify a unique activity-reporting RNA barcode. In this manner, colorimetric readout from serial rounds of FISH will be used to identify the cellular origins of each activity-reporting barcode in the brain. The use of LOCATER to monitor single-cell activity states non-invasively in behaving animals and map these states back to their cellular origins in the brain will transform our understanding of behavior, sensation, and neurodevelopmental, psychiatric, and neurodegenerative disease.