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

 描述（由申请人提供）：电生理学和荧光成像广泛用于研究体内神经活动。然而，这些方法需要侵入性程序，并且只能对少量神经元进行采样，并且活动指标的全脑荧光成像只能捕获安乐死前大脑最后状态的快照。我们提出定位器：一个变革性的系统，将超越这些以前的技术的局限性。其中，我们将使用活性报告RNA条形码捕获大量神经元群体中的单细胞活性痕迹，然后将这些活性痕迹从大脑输出到外泌体中，以通过循环血液进行非侵入性捕获。外泌体是内体衍生的囊泡，其含有从神经元分泌的蛋白质和RNA货物，并且容易穿过血脑屏障。因此，外泌体提供了用于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的理想底物。使用LOCATER技术捕获由循环血液中的神经元群体分泌的活性报告单细胞RNA条形码，并使用下一代测序（NGS）定量单个条形码序列的丰度，将促进活性的可扩展纵向监测。 从小的神经元群体或整个大脑。为了开发这个系统，我们将创建一个AAV载体，该载体使用立即早期基因（IEG）启动子来驱动在LOCATER监视下每个神经元特有的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泌体跨膜蛋白的结合，用于通过正常的内体蛋白运输进行条形码的外泌体定位。表位标签也将被添加到外泌体跨膜蛋白的胞外结构域，以使得能够从循环血液高保真捕获含有条形码的外泌体。为了使用LOCATER监测体内神经活性，我们将在小鼠大脑的子区域中表达标记的外泌体膜蛋白和活性报告细胞RNA条形码，并使动物接受刺激以捕获伴随行为的单细胞神经元活性谱。除了捕捉大脑中单个神经元的活动轨迹外，LOCATER系统还可以绘制大脑中活动报告细胞条形码的空间起源。在纵向活动监测后，处死动物，然后对每个脑切片并清除组织 用的是一种技术。我们将使澄清的组织经受FISH以鉴定包含在每个AAV载体上的多重FISH条形码，其指定独特的活性报告RNA条形码。以这种方式，来自连续轮FISH的比色读数将用于鉴定脑中每个活性报告条形码的细胞来源。使用LOCATER非侵入性地监测行为动物的单细胞活动状态，并将这些状态映射回大脑中的细胞起源，将改变我们对行为，感觉和行为的理解。 神经发育、精神和神经变性疾病。

 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.