Aggregation susceptibility of mutant GFAPs can be assessed using zebrafish. a Schematic representation of an expression plasmid encoding human GFAP C-terminally fused to a FLAG epitope and EGFP driven by a zebrafish gfap promoter. EGFP: enhanced green fluorescent protein; F: 3× FLAG epitope tag; hGFAP: human GFAP; pA: polyadenylation sequence; and pGFAP: zebrafish gfap promoter. b Regions of zebrafish embryos at 30 h post-fertilization (hpf) imaged in (c). c One-cell stage zebrafish embryos were microinjected with expression plasmids encoding WT or indicated alleles of GFAP and imaged with a confocal laser microscope at 30 hpf. Images represent stacking of Z-series of images. Insets represent magnifications of the boxed areas. R79C: p.Arg79Cys; R79H: p.Arg79His; R239C: p.Arg239Cys; R239H: p.Arg239His; and D128N: p.Asp128Asn. Scale bar = 150 μm. d and e GFP aggregates, indicated by green dots, were counted in the brain (d) and trunk (e) regions of each group in (c). n = WT: 10; R79C: 9; R79H: 12; R239C: 15; R239H: 8; and D128N: 11. *: P < 0.05; **: P < 0.01; ***: P < 0.001. f Aggregation assays were performed with WT or D157N allele of GFAP as described in (c). Insets represent magnifications of the boxed areas. D157N: p. Asp157Asn. g and h GFP aggregates were counted as described in (d and e). NS, not significant. Scale bar = 150 μm

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