Zebrafish can be used to assess the formation of mutant GFAP aggregates
Of mutant GFAPs reported in AxD patients, the most common are p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His [9]. To test if these mutant GFAPs aggregate in zebrafish embryos, we first generated expression plasmids individually encoding WT or one of the four GFAP mutants C-terminally fused to a FLAG epitope, and compared their expression levels in human embryonic kidney HEK293T cells by Western blotting. Expression levels of all of the mutants were comparable to that of WT GFAP (Fig. 2a and b), indicating that the four mutation alleles do not affect GFAP stability. We next individually cloned the WT or mutant alleles of GFAP C-terminally fused to a FLAG epitope and enhance green fluorescent protein (EGFP) into the 3′ end of the zebrafish gfap promoter [25], and the resulting constructs (Fig. 3a) were microinjected into one-cell stage zebrafish embryos. Subsequently, brain and trunk regions of the embryos expressing comparable levels of GFP at 30 hpf were imaged with a confocal laser microscope (CLM; Fig. 3b). Embryos microinjected with WT GFAP plasmids showed GFP aggregations. This was not surprising as supplementation of human GFAP to zebrafish that have their own GFAP proteins expressed, could lead to GFAP aggregation zebrafish. This is supported by the previous report that expression of WT human GFAP in mouse triggered aggregation of GFAP [34]. Nevertheless, the number of aggregations was significantly higher in both the head and trunk regions of the embryos microinjected with plasmids encoding common GFAP mutants (Fig. 3c, d, and e). To further validate this method as a tool to determine pathogenicity of GFAP mutations, we repeated the experiment with p.Asp157Asn GFAP that was previously reported to be a non-disease causing variant [13]. As expected, so significant difference in aggregation was noted between WT and p.Asp157Asn GFAP (Fig. 3f-h).
Fig. 3 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
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.
Fig. 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