一种新型基于 H129 的顺行单突触示踪剂具有标记强度强、示踪效率高、逆行标记减少的特点。
A novel H129-based anterograde monosynaptic tracer exhibits features of strong labeling intensity, high tracing efficiency, and reduced retrograde labeling.
机构信息
State Key Laboratory of Virology, CAS Center for Excellence in Brain Science and Intelligence Technology, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
University of Chinese Academy of Sciences, Beijing, China.
出版信息
Mol Neurodegener. 2022 Jan 10;17(1):6. doi: 10.1186/s13024-021-00508-6.
BACKGROUND
Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK.
METHODS
Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gK) or the mutant gK (gK, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gK pseudotyped and gK coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties.
RESULTS
H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gK pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gK, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer's disease and control mouse brains.
CONCLUSIONS
gK pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.
背景
病毒示踪剂是绘制脑连接组图谱的重要工具。单纯疱疹病毒 1 型（HSV-1）株 H129（HSV1-H129）具有主要的顺行转导性，是一种很有前途的可被开发为顺行病毒示踪剂的候选物。在我们之前的研究中，我们开发了源自 H129 的顺行多突触示踪剂和 TK 缺失（H129-dTK）单突触示踪剂。然而，由于 H129-dTK 示踪剂存在一些固有缺陷，其广泛应用受到限制，例如由于 TK 缺失导致的标记强度低和轴突末端入侵引起的潜在逆行标记。HSV-1 的糖蛋白 K（gK）在病毒进入、出芽和病毒诱导的细胞融合中发挥重要作用。其缺失严重削弱了病毒的出芽和传播能力，而对病毒基因组复制和病毒蛋白表达的限制则较小。因此，我们通过靶向 gK 构建了一种新型源自 H129 的顺行单突触示踪剂（H129-dgK），克服了 H129-dTK 的局限性。
方法
我们使用建立的开发病毒示踪剂的平台和流水线，从 H129-G4 中删除 gK 基因，生成一种新型示踪剂。gK 缺失的病毒（H129-dgK-G4）在表达野生型 H129 gK（gK）或突变 gK（gK、A40V、C82S、M223I、L224V、V309M）的 Vero 细胞中重新构建和繁殖。然后，在体外和体内测试获得的 gK 假型和 gK 包被的 H129-dgK-G4 病毒示踪剂，以表征其示踪特性。
结果
H129-dgK-G4 表达高水平的荧光蛋白，消除了免疫染色进行成像检测的需要。与 TK 缺失的单突触示踪剂 H129-dTK-G4 相比，H129-dgK-G4 标记的神经元荧光强度强 1.76 倍，下游脑区标记的突触后神经元数量多 2.00 倍。gK 假型通过减少轴突末端入侵，将逆行标记减少 77%，从而显著提高了 H129-dgK-G4 的顺行特异性示踪。此外，在辅助性 AAV 辅助转染表达 gK 的情况下，H129-dgK-G4 允许对阿尔茨海默病和对照小鼠大脑中的单突触连接进行映射和量化，并比较其回路连接的差异。
结论
gK 假型 H129-dgK-G4 是一种新型的顺行单突触示踪剂，克服了 H129-dTK 示踪剂的局限性，具有标记强度强、示踪效率高、顺行特异性提高等理想特性。
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