PMC:5590178 / 20135-21687 JSONTXT

{"project":"AxD_symptoms","denotations":[{"id":"T50","span":{"begin":853,"end":864},"obj":"Phenotype"}],"attributes":[{"id":"A50","pred":"hp_id","subj":"T50","obj":"http://purl.obolibrary.org/obo/HP_0009592"}],"text":"To check if GFP aggregations in zebrafish embryos are akin to GFAP aggregations in AxD patients, we performed transmission electron microscopy (TEM) on zebrafish embryos microinjected with expression plasmids encoding WT or p.Arg79Cys allele of GFAP, and indeed found electron dense inclusions in the cells of both groups of embryos (Fig. 4a-c), which is reminiscent of TEM findings of RFs in the astrocytes of the AxD brain [35]. Of note, more inclusions were observed in the TEM images of p.Arg79Cys embryos than WT embryos, consistent with CLM images. Intriguingly, found in the p.Arg79Cys embryos were the spherical structures with double layer membranes containing electron dense inclusions (Fig. 4c). These were reminiscent of autophagosome, which were previously reported in the AxD patient’s brain, mouse brain expressing p.Arg236His, and human astrocytoma U251 cells expressing p.Arg239Cys [35, 36]. Taken together, this outcome indicates that the GFAP aggregation assay in zebrafish embryos can be employed to assess the pathogenicity of GFAP mutations identified in patients tentatively diagnosed with AxD.\nFig. 4 Aggregation susceptibility of mutant GFAPs can be assessed using zebrafish. a-d Zebrafish embryos at one-cell stage were microinjected with expression plasmids encoding WT (a), p.Arg79Cys GFAP (b and c), or p.Asp128Asn (d), and imaged at 30 hpf with transmission electron microscopy. Arrows and arrowhead indicate electron dense inclusions and a spherical structure with double-layered membranes, respectively. Scale bar = 2 μm"}

{"project":"2_test","denotations":[{"id":"28882119-18276609-12765650","span":{"begin":426,"end":428},"obj":"18276609"},{"id":"28882119-18276609-12765651","span":{"begin":900,"end":902},"obj":"18276609"},{"id":"28882119-24165736-12765652","span":{"begin":904,"end":906},"obj":"24165736"}],"text":"To check if GFP aggregations in zebrafish embryos are akin to GFAP aggregations in AxD patients, we performed transmission electron microscopy (TEM) on zebrafish embryos microinjected with expression plasmids encoding WT or p.Arg79Cys allele of GFAP, and indeed found electron dense inclusions in the cells of both groups of embryos (Fig. 4a-c), which is reminiscent of TEM findings of RFs in the astrocytes of the AxD brain [35]. Of note, more inclusions were observed in the TEM images of p.Arg79Cys embryos than WT embryos, consistent with CLM images. Intriguingly, found in the p.Arg79Cys embryos were the spherical structures with double layer membranes containing electron dense inclusions (Fig. 4c). These were reminiscent of autophagosome, which were previously reported in the AxD patient’s brain, mouse brain expressing p.Arg236His, and human astrocytoma U251 cells expressing p.Arg239Cys [35, 36]. Taken together, this outcome indicates that the GFAP aggregation assay in zebrafish embryos can be employed to assess the pathogenicity of GFAP mutations identified in patients tentatively diagnosed with AxD.\nFig. 4 Aggregation susceptibility of mutant GFAPs can be assessed using zebrafish. a-d Zebrafish embryos at one-cell stage were microinjected with expression plasmids encoding WT (a), p.Arg79Cys GFAP (b and c), or p.Asp128Asn (d), and imaged at 30 hpf with transmission electron microscopy. Arrows and arrowhead indicate electron dense inclusions and a spherical structure with double-layered membranes, respectively. Scale bar = 2 μm"}