Enzyme replacement for craniofacial skeletal defects and craniosynostosis in murine hypophosphatasia

Abstract

Hypophosphatasia (HPP) is an inborn-error-of-metabolism disorder characterized by deficient bone and tooth mineralization due to loss-of function mutations in the gene (Alpl) encoding tissue-nonspecific alkaline phosphatase (TNAP). Alpl−/− mice exhibit many characteristics seen in infantile HPP including long bone and tooth defects, vitamin B6 responsive seizures and craniosynostosis. Previous reports demonstrated that a mineral-targeted form of TNAP rescues long bone, verterbral and tooth mineralization defects in Alpl−/− mice. Here we report that enzyme replacement with mineral-targeted TNAP (asfotase-alfa) also prevents craniosynostosis (the premature fusion of cranial bones) and additional craniofacial skeletal abnormalities in Alpl−/− mice. Craniosynostosis, cranial bone volume and density, and craniofacial shape abnormalities were assessed by microsocopy, histology, digital caliper measurements and micro CT. We found that craniofacial shape defects, cranial bone mineralization and craniosynostosis were corrected in Alpl−/− mice injected daily subcutaneously starting at birth with recombinant enzyme. Analysis of Alpl−/− calvarial cells indicates that TNAP deficiency leads to aberrant osteoblastic gene expression and diminished proliferation. Some but not all of these cellular abnormalities were rescued by treatment with inorganic phosphate. These results confirm an essential role for TNAP in craniofacial skeletal development and demonstrate the efficacy of early postnatal mineral-targeted enzyme replacement for preventing craniofacial abnormalities including craniosynostosis in murine infantile HPP.

Bone mineralization‐dependent craniosynostosis and craniofacial shape abnormalities in the mouse model of infantile hypophosphatasia

“…Of greater significance is the finding that craniosynostosis only occurs in ∼3‐week‐old Alpl −/− mice with severe cranial bone hypomineralization, as assessed quantitatively by micro‐CT. This finding corresponds well with our previous qualitative assessment that craniosynostosis occurs only in Alpl −/− mice with more severely deficient craniofacial bone mineralization (Liu et al, ). This result could indicate that craniosynostosis occurs indirectly, as a result of cellular changes that occur only in response to severely deficient cranial bone mineralization.…”

Postnatal Craniofacial Skeletal Development of Female C57BL/6NCrl Mice

“…All μCT analysis was performed using the Microview (version ABA 2.2) analysis software. Similar to our previous work (Liu et al, 2013 , 2015 ), a region of interest (ROI) was selected for each of the bones to be analyzed (frontal, parietal, pre-sphenoid, basi-sphenoid, basi-occipital and mandible) (Figures S1B–D). For the frontal bone, a 0.5 mm × 0.5 mm area was located 1.5 mm anterior to the intersection point of coronal suture and sagittal suture and 1 mm lateral to the posterior frontal suture.…”

“…Neural crest-derived calvarial osteoblasts from the frontal bone have superior intrinsic osteogenic potential and tissue regeneration ability compared to mesoderm-derived calvarial osteoblasts from parietal bone (Quarto et al, 2010 ). Our previous studies suggested that there is increased bone volume and density in mesoderm derived parietal bone compared to neural crest derived frontal bone at early postnatal stage in both BALB/c congenic mice at 1 month of age (Liu et al, 2013 ) and 50% C57BL/6-50%129SF2/J mice at 2 weeks of age (Liu et al, 2015 ). However, a thorough knowledge of quantitative differences in bone parameters during postnatal development between neural crest-derived skull bone and mesoderm-derived skull bone is lacking.…”

Alkaline Phosphatase and Hypophosphatasia

“…Patients with severe HPP receiving asfotase alfa still often require craniotomies [ 101 ] to relieve intracranial pressure. However, asfotase alfa [ 102 ] and ChimAP [ 103 ] (a soluble chimeric form of human ALP), both can prevent craniosynostosis, at least in mice (vide infra), if treatment starts at birth. So, prevention of craniosynostosis may depend on the time of initiation of the treatment, although there are data to indicate that in humans craniosynostosis may begin during embryonic life [ 104 ].…”

“…In untreated KO mice, multiple cranial vault and facial bones lacked adequate mineralization on μCT that was not evident in the treated mice. Digital caliper linear measurements demonstrated that treatment improved nose length, nasal bone length, and frontal bone length [ 102 ].…”