Abstract
Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by pathogenic variants in the ABCC6 gene. Though complications of the disease can be treated, PXE itself remains currently intractable. A strategy for rapid and cost-effective discovery of therapeutic drugs would be to perform chemical compound screening using zebrafish, but this approach remains to be validated for PXE. In this paper, we validate a stable CRISPR/Cas9 abcc6a knockout zebrafish model–which has spinal column hypermineralization as its primary phenotypic feature–as a model system for compound screening in ectopic mineralization. We evaluated the anti-mineralization potential of five compounds, which had (anecdotal) positive effects reported in Abcc6 knockout mice and/or PXE patients. Abcc6a knockout zebrafish larvae were treated from 3 to 10 days post-fertilization with vitamin K1, sodium thiosulfate, etidronate, alendronate or magnesium citrate and compared to matching controls. Following alizarin red S staining, alterations in notochord sheath mineralization were semiquantified and found to largely congrue with the originally reported outcomes. Our results demonstrate that the use of this abcc6a knockout zebrafish model is a validated and promising strategy for drug discovery against ectopic mineralization.
Highlights
Pseudoxanthoma elasticum (PXE) is an ectopic calcification disorder caused by biallelic mutations in the ABCC6 (ATP-Binding Cassette, subfamily C member 6)–and in rare cases the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1)–gene (Nitschke et al, 2012; Nitschke and Rutsch 2012)
Upon genotyping only abcc6acmg52/cmg52 larvae had developed mineralized segments other than the notochord tip (C: n = 15, VK1: n = 10; Table 1) with vitamin K1-treated knockouts showing a significant 42% reduction in mineralization compared to their control counterparts (t-Test: p < 0.05, Figures 1A,B, Figure 2A)
We hypothesized that treatment with compounds implicated in PXE (VK1, sodium thiosulfate (STS), bisphosphonates and magnesium) would have a similar effect on the spinal hypermineralization phenotype as what has been reported in PXE murine models and/or patients
Summary
Pseudoxanthoma elasticum (PXE) is an ectopic calcification disorder caused by biallelic mutations in the ABCC6 (ATP-Binding Cassette, subfamily C member 6)–and in rare cases the ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1)–gene (Nitschke et al, 2012; Nitschke and Rutsch 2012). In PXE aberrant hydroxyapatite crystals are progressively deposited onto elastic fibers, which lose their elastic properties and fragment, leading to a myriad of symptoms. Compound Screening in Abcc6a Zebrafish haemorrhaging and vision loss) and cardiovascular manifestations (e.g., peripheral artery disease, increased risk of stroke). The phenotypic manifestations of PXE show a high degree of inter- and intrafamilial variability without solid genotype-phenotype correlations, making it difficult for health care professionals to accurately manage patients. Existing treatment options for PXE are limited and currently focus on slowing down disease progression in patients through lifestyle changes or by treating complications via e.g., cosmetic surgery or intravitreal injection of anti-VEGF antibodies (Vascular Endothelial Growth Factor; Shimada et al, 2021)
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