Broad Vitamin B6-Related Metabolic Disturbances in a Zebrafish Model of Hypophosphatasia (TNSALP-Deficiency)

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in <i>ALPL</i>, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated <i>alpl</i>^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no <i>alpl</i> mRNA and 89% lower total alkaline phosphatase activity was detected in <i>alpl</i>^-/- compared to <i>alpl</i>^+/+ embryos. The survival of <i>alpl</i>^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in <i>alpl</i>^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in <i>alpl</i>^-/- embryos. Accumulation of d3-pyridoxal 5'-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in <i>alpl</i>^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in <i>alpl</i>^-/- embryos. Metabolic profiling of <i>alpl</i>^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.