| 研究実績の概要 |
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of relatively higher levels of APLP1 mRNA compared to APP or APLP2 mRNA in astrocytes in the RNA-seq analysis (unpublished).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.
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