Gene Expression Analysis in gla-Mutant Zebrafish Reveals Enhanced Ca2+ Signaling Similar to Fabry Disease

Abstract

Fabry disease (FD) is an X-linked inborn metabolic disorder due to partial or complete lysosomal α-galactosidase A deficiency. FD is characterized by progressive renal insufficiency and cardio- and cerebrovascular involvement. Restricted access on Gb3-independent tissue injury experimental models has limited the understanding of FD pathophysiology and delayed the development of new therapies. Accumulating glycosphingolipids, mainly Gb3 and lysoGb3, are Fabry specific markers used in clinical follow up. However, recent studies suggest there is a need for additional markers to monitor FD clinical course or response to treatment. We used a gla-knockout zebrafish (ZF) to investigate alternative biomarkers in Gb3-free-conditions. RNA sequencing was used to identify transcriptomic signatures in kidney tissues discriminating gla-mutant (M) from wild type (WT) ZF. Gene Ontology (GO) and KEGG pathways analysis showed upregulation of immune system activation and downregulation of oxidative phosphorylation pathways in kidneys from M ZF. In addition, upregulation of the Ca²⁺ signaling pathway was also detectable in M ZF kidneys. Importantly, disruption of mitochondrial and lysosome-related pathways observed in M ZF was validated by immunohistochemistry. Thus, this ZF model expands the pathophysiological understanding of FD, the Gb3-independent effects of gla mutations could be used to explore new therapeutic targets for FD.

Keywords: Fabry disease; alpha-galactosidase A; calcium signaling; cardiac involvement; gla; oxidative stress; zebrafish.

Figure 1

Quality Control and Fold Change. Total samples (n = 16), with sex-matched groups: MtM: Mutant Male (n = 4), MtF: Mutant Female (n = 4), WtM: Wild type Male (n = 4), WtF: Wild type Female (n = 4). (A) Pearson Correlation for pairwise comparison of mRNA FPKM between samples, rounded to 2 decimals; (B) Heatmap clustering of variance stabilized counts for all genes binned into five hundred clusters, showing that both sex and condition are clustering together; (C) PCA analysis of variance stabilized counts for all genes, with batch correction for both Sex and Replicates; (D) Volcano plot of Log2 fold change (LFC) for all genes using DESeq2 differential expression analysis, with resulting −log10 adjusted p-values. (Negative LFC means LFC (Mutant Mt) < LFC (Wild type Wt). A high confidence set of genes are displayed with gene symbols (adjusted p-value < 0.000001 and absolute value of LFC > 1.7).

Figure 2

Gene ontology (GO) enrichment analysis of pathways upregulated and downregulated in renal tissues from the mutant, compared to wildtype ZF. Data refers to GO Biological Process (BP: (A) and (B), respectively), Cellular component (CC, (C) and (D), respectively) and Molecular function (MF: (E) and (F), respectively). In all cases, the twenty most enriched pathways are reported. FDR ≤ 0.05.

Figure 3

KEGG pathway enrichment analysis and heatmap of m6pr and atf6 genes in mutant compared to wildtype ZF. (A) KEGG pathways associated with the upregulated genes; (B) KEGG pathways associated with the downregulated genes; (C) heatmap (percentage column normalized, where dark blue is the maximum per column) showing the expression of the selected downregulated genes m6pr and atf6.

Figure 4

Immunohistochemical and Western blot detection of selected proteins in kidneys from WT and M ZF reveals protein expression disturbances in mitochondria and lysosomes. (A) Representative IHC staining specific for mitochondrial marker isocitrate dehydrogenase (NAD⁽⁺⁾)3 alpha (Idh3a) and lysosomal marker cathepsin B (Ctsb) in kidney tissue sections from WT and M ZF (upper right and upper left panels, and lower right and lower left panels, respectively; (B) Quantification of immunohistochemical staining of sections from WT and M ZF kidneys. Signal intensity is significantly higher in WT than in M for both proteins. (C) Representative immunoblots of Ctsb and Idh3a in kidney tissue section from WT and M ZF; (D) Quantification of immunoblots from WT and M ZF kidneys. (Mann–Whitney test U * p < 0.05). Wildtype (WT), mutant (M). ns: not significant. Scale bar (bottom left corner, in black) = 100 µm.