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

## TL;DR

This study creates a zebrafish model of Hypophosphatasia, a rare metabolic disorder, and finds vitamin B6-related metabolic issues and bone problems similar to those in humans.

## Contribution

The first zebrafish model of HPP is developed, showing multiple disease features and metabolic disturbances linked to vitamin B6.

## Key findings

- Alpl-/- zebrafish show reduced alkaline phosphatase activity and impaired bone mineralization.
- Vitamin B6 metabolism is disrupted, with pyridoxal depletion and d3-PLP accumulation in alpl-/- embryos.
- Pyridoxine treatment reduces seizures, and metabolic profiling reveals polyamine and neurotransmitter abnormalities.

## Abstract

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, 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 alpl-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl-/- compared to alpl+/+ embryos. The survival of alpl-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl-/- embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl-/- embryos. Metabolic profiling of alpl-/- 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.