| 研究実績の概要 |
First, we confirmed APP, APLP1 and APLP2 expression in astrocytes. Western blot analysis revealed the presence of APP, APLP1 and APLP2 proteins in lysates from rat hippocampal neuron-astrocyte co-cultures. Immunostaining in hippocampal cultures showed, as previously described, the expression of APP, APLP1 and APLP2 in neurons. In pure astrocyte cultures we detected a strong APLP1 signal and a more limited signal for APP and APLP2 compared to neurons. These results are consistent with the detection of relatively higher levels of APLP1 mRNA compared to APP or APLP2 mRNA in astrocytes in the RNA-seq analysis. Second, we studied the impact of APP, APLP1 and APLP2 as a substrate on astrocyte morphology. APP, APLP1 and APLP2 were overexpressed in HEK cells and astrocytes were cultured on top of them. HEK cells overexpressing mGFP were used as a negative control and neurexin-1-alpha, known to increase astrocyte morphology complexity, were used as positive control. Preliminary Scholl analysis suggests that APLP1 but not APP or APLP2 as a substrate could increase the morphological complexity of astrocytes. Presently, conditions to effectively evaluate the knockdown of APP, APLP1 and APLP2 in astrocytes are being established. In parallel, we have begun a collaboration with the team of Dr. Akihiko Nakano at RIKEN to study the interaction between astrocyte processes and the synaptic terminal by super resolution imaging. So far, we have set the best conditions to visualize astrocyte processes and the synaptic terminal in rat hippocampal neuronal/astrocyte co-cultures.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Overall, the experiments are progressing as expected. However, we had a slight delay because of antibodies that were not working particularly well, which required several additional tests. The rest of the experiments are expected to proceed as planned. We also chose to study in addition of APLP1 and APLP2, the APP protein. All 3 proteins are homologues and seem to have redundant functions, which makes their joint study relevant.
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| 今後の研究の推進方策 |
In year 2, we will investigate the effects of APP, APLP1 and APLP2 knockdown on astrocyte morphology, synaptic plasticity, and synapse-astrocyte contact dynamics by fluorescence imaging. To this end, astrocytes specifically knocked down for APP, APLP1 or APLP2 will be cultured on top of wild type neurons. Their morphology will be assessed by Sholl analysis and the impact on the number of synapses will be determined using markers for the pre- and post- synaptic compartments. Synapse-astrocyte contact dynamics will be examined using high-resolution imaging and a fluorescence proximity indicator based on FRET called NAPA in which a signal is detected when synapse and astrocyte processes are in close contact (Octeau et al., Neuron 2018). Synaptic function will be assessed by monitoring presynaptic vesicle turnover using the VGLUT-pHluorin or neurotransmitter release using iGluSnFR, while postsynaptic glutamate receptor dynamics will be monitored with pHluorin-tagged GluA1 and GluA2. Imaging of pHluorin-based probes are established in the host laboratory. In parallel we will also assess the impact of overexpressing APLPs in astrocytes using the same functional assays. Finally, once the role of APLPs in astrocyte/neuron interactions is determined, we will investigate the underlying mechanisms. We will also assess the role of the homophilic and heterophilic APLP1/APLP2 interactions on astrocyte complexity and synaptic plasticity. To this end we will use mutants known to impede the inter-cellular interaction of APLPs.
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