AAV8-Syn-Cre

The angiomotin (Amot)-Yes-associated protein 1 (Yap1) complex plays an important role in regulating the inhibition of cell contact, cellular polarity, and cell growth in many cell types. However, the roles of Amot and the Hippo pathway transcriptional coactivator Yap1 in the central nervous system remain unclear. Here, researchers show that Amot is a key mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. The function of Amot in developing neurons depends on interaction with Yap1, which is also essential for dendritic growth and arborization in vitro. Conditional deletion of Amot and Yap1 in neurons results in reduced complexity of Purkinje cell dendritic trees, abnormal cerebellar morphology, and impaired motor coordination. These results suggest that the role of Amot and Yap1 in dendritic growth does not depend on interaction with TEA domain (TEAD) transcription factors or expression of Hippo pathway-dependent genes. Instead, Amot and Yap1 regulate dendritic development by affecting phosphorylation of S6 kinase and its target S6 ribosomal protein.

To be able to observe the morphology of individual Amot^−/− Purkinje cells, the researchers intracerebrally injected newborn Amot fl/fl;STOP-Tom pups and control STOP-Tom pups with low-titer serotype 8 adeno-associated virus (AAV8) that expressed Cre under the neuron-specific synapsin 1 promoter (AAV8-Syn-Cre; Figure 1A). AAV8 has previously been shown to have high tropism for Purkinje cells, allowing cell type-specific expression of Cre and deletion of Amot in Purkinje cells. Three weeks after virus injection, animals were humanely killed, and the morphology of Purkinje cell dendritic trees was analyzed using high-power images of 100 μm sagittal sections of the cerebellum (Figure 1A and B). The researchers measured Purkinje cell dendritic tree height and width, primary and secondary branch lengths, dendritic field area and the number of dendritic branch points. All these measurements were significantly reduced in Amot^−/− Purkinje cells compared with control cells, indicating impaired dendritic branching of Purkinje cells in vivo (Figure 1B–G). Amot ablation clearly impaired dendritic tree morphological complexity but not the distribution of synaptic markers within the molecular layer, including vesicular gamma-aminobutyric acid transporter (VGAT), vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 (Figure 1H-M). These findings indicate that Amot knockdown in cultured hippocampal neurons did not affect the number of synapses.

Figure 1. Amot deletion in neurons impairs dendritic tree morphology of Purkinje cells. (Rojek K O, et al., 2019)