Glutamatergic argonaute2 promotes the formation of the neurovascular unit in mice

Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2) mediates microRNA (miRNA)mediated gene silencing, which is critical for many facets of brain development, including NVU development. Here, we found that Ago2 in glutamatergic neurons was critical for NVU formation in the developing cortices of mice. Glutamatergic neuronspecific loss of Ago2 diminished synaptic formation, neuronal-to-endothelial cell contacts, and morphogenesis of the brain vasculature, ultimately compromising the integrity of the blood-brain barrier. Ago2 facilitated miRNA targeting of phosphatase and tensin homolog (Pten) mRNA, which encodes a phosphatase that modulates reelin-dependent phosphatidylinositol 3-kinase (PI3K)Akt signaling within the glutamatergic subpopulation. Conditionally deleting Pten in Ago2-deficient neurons restored Akt2 phosphorylation as well as postnatal development and survival. Several mutations in AGO2 impair small RNA silencing and are associated with Lessel-Kreienkamp syndrome, a neurodevelopmental disorder. When expressed in a neuronal cell line, these human AGO2 loss-of-function variants failed to suppress PTEN, resulting in attenuated PI3K-Akt signaling, further indicating that dysregulation of Ago2 function may contribute to both impaired development and neurological disorders. Together, these results identify Ago2 as central to the engagement of neurons with blood vessels in the developing brain. To understand the role of endogenous Ago2 in the CNS, we generate a mutant mouse line where Ago2 expression was deleted in Vglut2-expressing excitatory neurons (by crossing the Slc17a6-Cre mouse line with an Ago2 conditional allele, Slc17a6-Cre, Ago2flox/flox mice). The cortical cells will be isolated from mice brain at P14 for scRNAseq analysis复杂神经血管单元（neurovascular unit, NVU）与血脑屏障的正确构建，对于建立并维持健康且功能完备的中枢神经系统至关重要。RNA结合蛋白AGO2（argonaute2, Ago2）介导微小RNA（microRNA, miRNA）依赖的基因沉默过程，该过程对大脑发育的诸多环节均具有关键作用，包括神经血管单元的发育。本研究发现，谷氨酸能神经元中的Ago2对小鼠发育阶段大脑皮层的神经血管单元形成至关重要。特异性敲除谷氨酸能神经元中的Ago2，会削弱突触形成、神经元-内皮细胞连接以及脑血管的形态发生，最终破坏血脑屏障的完整性。Ago2可促进微小RNA靶向调控张力蛋白同源磷酸酶（phosphatase and tensin homolog, Pten）的mRNA，该基因编码一种可在谷氨酸能神经元亚群中调节reelin依赖的磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI3K）-Akt信号通路的磷酸酶。在Ago2缺陷神经元中条件性敲除Pten，可恢复Akt2的磷酸化水平，同时挽救小鼠的出生后发育与存活。AGO2的多种突变会损伤小RNA沉默功能，并与Lessel-Kreienkamp综合征——一种神经发育障碍——相关。当在神经元细胞系中表达这些人类AGO2功能丧失型变异体时，它们无法抑制PTEN的表达，导致PI3K-Akt信号通路活性减弱，进一步表明Ago2功能失调可能既会导致发育受损，也会引发神经系统疾病。综上，本研究证实Ago2是发育大脑中神经元与血管相互作用的核心调控因子。为阐明内源性Ago2在中枢神经系统中的作用，我们构建了一种突变小鼠品系：在表达Vglut2的兴奋性神经元中敲除Ago2（通过将Slc17a6-Cre小鼠与Ago2条件性等位基因小鼠杂交，获得Slc17a6-Cre; Ago2flox/flox小鼠）。我们将从出生后第14天（P14）的小鼠大脑中分离皮层细胞，用于单细胞RNA测序（scRNAseq）分析。