Role of APLP1 and APLP2 expressed in astrocytes on astrocyte/synapse interactions and synaptic plasticity

为了研究星形胶质细胞APP和APLPs在神经元-星形胶质细胞相互作用中的作用，我们通过western blot分析大鼠海马培养物证实了星形胶质细胞APP、APLP1和APLP2的表达。海马培养免疫荧光分析显示APP、APLP1和APLP2在神经元中的表达与预期一致。与神经元相比，我们还在星形胶质细胞中检测到较强的APLP1信号和较弱的APP和APLP2信号，这与RNA-seq分析中在星形胶质细胞中检测到相对较高水平的APLP1 mRNA相一致（未发表）。通过在HEK细胞中过表达APP、APLP1和APLP2，并在其上培养星形胶质细胞，研究APP和APLPs作为底物对星形胶质细胞形态的影响。已知能增加星形胶质细胞形态复杂性的Neurexin-1-α和mGFP分别作为阳性和阴性对照。shl分析显示，APLP1和APLP2作为底物略微增加了星形胶质细胞的形态复杂性，而APP作为底物没有增加。当星形胶质细胞中的APP、APLP1或APLP2被shRNA敲低时（Young-Pearse et al., 2008），星形胶质细胞中的APP和APLP1 KD较对照组降低了星形胶质细胞的复杂性。此外，星形胶质细胞APLP1 KD减少了星形胶质细胞面积和分支长度。星形胶质细胞中APP、APLP1或APLP2 KD对兴奋性或抑制性突触前钮扣密度及其突触囊泡蛋白水平无显著影响。未来的工作将描述星形胶质细胞APP和APLPs对突触功能的作用，并将分析扩展到完整的大脑。

To study a role for astrocyte APP and APLPs on neuron-astrocyte interactions, we confirmed astrocytic APP, APLP1 and APLP2 expression by western blot analysis of rat hippocampal culture lysates. Immunofluorescence analysis of hippocampal cultures showed the expression of APP, APLP1 and APLP2 in neurons, as expected. We also detected a strong APLP1 signal and a more limited signal for APP and APLP2 in astrocytes compared to neurons, consistent with the detection of relatively higher levels of APLP1 mRNA compared to APP or APLP2 mRNA in astrocytes in the RNA-seq analysis (unpublished). The impact of APP and APLPs as a substrate on astrocyte morphology was studied by overexpressing APP, APLP1 and APLP2 in HEK cells and culturing astrocytes over them. Neurexin-1-α, known to increase astrocyte morphological complexity, and mGFP were used as positive and negative controls, respectively. Sholl analysis showed that APLP1 and APLP2 but not APP as a substrate slightly increased the astrocyte morphological complexity. When APP, APLP1 or APLP2 in astrocytes was knocked down by shRNA (Young-Pearse et al., 2008), APP and APLP1 KD in astrocytes decreased astrocyte complexity compared to the controls. Moreover, astrocyte APLP1 KD reduced astrocyte area and branch length. No significant impact of APP, APLP1 or APLP2 KD in astrocytes on the density of excitatory or inhibitory presynaptic boutons or in their level of synaptic vesicle proteins was detected. Future work will characterize the roles of astrocyte APP and APLPs on synaptic function and also extend the analysis to the intact brain.