Table_1_Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling.xlsx

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5′ end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.

活动调节细胞骨架相关蛋白（activity-regulated cytoskeleton-associated protein, Arc）是突触可塑性与记忆形成过程中不可或缺的关键蛋白。携带来自结构性GAG逆转录转座子序列残迹的Arc基因，其编码的蛋白可自组装为包裹Arc mRNA的衣壳样结构。从神经元释放的Arc衣壳被认为是一种全新的mRNA胞间传递机制。然而，目前仍缺乏Arc在哺乳动物脑内进行胞间运输的相关实验证据。为实现活体状态下单神经元内Arc分子的追踪，我们搭建了腺相关病毒（adeno-associated virus, AAV）介导的实验方案，借助CRISPR/Cas9同源独立靶向整合（homologous independent targeted integration, HITI）技术，将荧光报告标签融合至小鼠Arc蛋白的N末端。我们证实，可将编码mCherry的序列精准敲入Arc开放阅读框（open reading frame, ORF）的5'端。尽管Arc起始密码子周围存在9个spCas9基因编辑靶点，但编辑的精准性高度依赖序列上下文，仅单个靶点可实现报告基因的框内整合。在海马脑区诱导长时程增强（long-term potentiation, LTP）时，我们观测到Arc蛋白水平的上调与荧光强度及mCherry阳性细胞数量的增加显著相关。通过邻近连接分析法（proximity ligation assay, PLA），我们证实mCherry-Arc融合蛋白可通过在突触后棘突与跨膜蛋白stargazin结合，保留Arc的正常功能。最后，我们在编辑神经元的mCherry阳性棘突附近的mCherry阴性周围神经元中，观测到mCherry-Arc与突触前蛋白Bassoon的相互作用。本研究首次为Arc在哺乳动物脑内的神经元间活体转运提供了直接实验证据。