Investigation of alpl expression and Tnap-activity in zebrafish implies conserved functions during skeletal and neuronal development

Abstract

Different tissues express different alkaline phosphatase (AP) isoforms and HPP patients' phenotypes can greatly differ in the responsiveness between tissues 1. Remarkably, comparative studies have already shown that the predominant TNAP transcript variant in bones is the same as in brain tissues 6 , which implies a common mode of TNAP function in both tissues. TNAP's most important biochemical function in bones and teeth is providing the basis for mineralization processes. The enzyme enables hydroxyapatite crystallization in bone via catalyzing the dephosphorylation of mineralization inhibitors such as inorganic pyrophosphate (PP i) and phosphorylated osteopontin 7. In the nervous system, two predominant mechanisms, the availability of vitamin B6 and alterations in purinergic signaling 8,9 , significantly influence the outcome of the disease. In case of decreased TNAP-activity, pyridoxal-5-phosphate (PLP), which is the transportable form of vitamin B6, accumulates within the serum and cannot be redistributed into the brain without dephosphorylation via TNAP. PLP is an essential enzymatic co-factor within central neurotransmitter synthesis pathways in the brain and the lack of PLP results in neurological impairment due to limited biochemical conversion 8. Additionally, TNAP enzyme supports the development and maintenance of synapse functionality and is involved in the outgrowth and myelination of neurites 10-12. Localization of TNAP within primates' brains has been described as distinct patterns within layer 4 and 5 of the cortex 13,14 and has been detected in the retina across a number of different vertebrate species 15. In mice, TNAP localization was detected predominantly in endothelial cells, primordial germ cells, pioneer growth cones, and neural precursors 16. Akp2 (the murine version of the human ALPL gene) knockout mice display reduced serum levels of alkaline phosphatase, elevated substrate levels, impaired bone mineralization, and frequently die from epileptic seizures 17,18. Due to the early lethal phenotype in mice and the lack of supplementary in vivo studies within other vertebrates, our knowledge about TNAP's interconnected functions within bone and neuronal tissues is still scarce. Due to a number of biological properties, the zebrafish (Danio rerio) has become an important vertebrate model organism for bone 19,20 and brain research 21. Along with other advantages, including low cost and easy housing of the animals, the possibility to investigate transgenic reporter lines by non-invasive in vivo monitoring and perform large scale screenings, zebrafish offer a wide repertoire of different approaches for investigation of bone and brain development 22-24. Furthermore, basic molecular pathways for vertebrate development, like bone development, are evolutionarily conserved and share common properties with other species 25,26. Consequently, a rising number of in vivo models for musculoskeletal diseases have been established in recent years 27-29 and even the molecular background of complex diseases, such...

Tissue-Nonspecific Alkaline Phosphatase—A Gatekeeper of Physiological Conditions in Health and a Modulator of Biological Environments in Disease

“…Moreover, alpl expression has been described within the mantle and interneuromast cells of the lateral line system of Tg(alpl:mCherry) transgenic lines [162] and in the retina of developing zebrafish embryos [163]. The neuronal function of Tnap in the central nervous system of zebrafish is so far unknown, but our inhibitor experiments indicate an influence on axonal growth within the midbrain [7].…”

“…However, the zebrafish alpl gene is located on chromosome 11, encoding for only three transcript versions (ZFIN gene ID: ZDB-GENE-040420-1). The zebrafish ENSEMBL database reference alpl transcript (ENSDART00000146461.3) contains 12 exons that are all coding and has a transcript length of 1871 bp (genome assembly: GRCh38.p13, GRCm38.p6, GRCz11; summarized in [7] and [8]).…”

“…alpl expression has been reported in zebrafish as a readout of osteogenic defects, e.g., in Sp7/osterix mutants and after ferric iron ammonium citrate treatment, although studies exclusively focusing on Tnap function in zebrafish bone development are still rare [81,82]. An incubation experiment in zebrafish using the potent chemical inhibitors levamisole and MLS-0038949 that specifically inhibit the Tnap enzyme resulted in impaired craniofacial development and bone mineralization delay or even abolishment, depending on the respective inhibitor concentration that was used for the treatment of the zebrafish larvae [7]. Future experiments must establish more precise in vivo zebrafish models that are adapted to investigate Tnap function, either by tissue-specific knockout or by introducing disease-causing mutations.…”

“…The function of CYP26B1, i.e., inactivation of all-trans-retinoic acid and thereby potential regulation of concentration gradients [138], is conserved in vertebrates and is essential for osteoblast-osteocyte transition, subsequently resulting in the observed prominent malformations of extremities within in knockout mice [139]. Detailed genetic investigation of alpl function in zebrafish skull development and in suture formation are still missing, but the application of diverse Tnap inhibitors implies abnormal craniofacial development in zebrafish embryos [7].…”

“…Investigation in rats further imply that 17β-Estradiol promotes the outgrowth of dendritic spines in the hippocampus and its systemic application leads to downregulation of NTPDase1 and NTPDase2 as well as to upregulation of TNAP activity, which is indicating an interaction of the purinergic and estradiol signaling pathways during rearrangements of dendritic spines [159,160]. Analyses of alpl expression in neuronal tissues during zebrafish embryogenesis via in situ hybridization (ISH) are described in the zfin.org database (http://zfin.org/ZDB-GENE-040420-1) [161] and in Ohlebusch et al [7], and imply restricted expression patterns in distinct brain regions starting from segmentation stages. Moreover, alpl expression has been described within the mantle and interneuromast cells of the lateral line system of Tg(alpl:mCherry) transgenic lines [162] and in the retina of developing zebrafish A number of different mouse models have been reported, and the role of TNAP in murine brain development is extensively discussed in Zimmerman and Langer.…”