Phosphatases are recognised to have important functions in the initiation of skeletal mineralization. Tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 are indispensable for bone and cartilage mineralization but their functional relationship in the mineralization process remains unclear. In this study, we have used osteoblast and ex-vivo metatarsal cultures to obtain biochemical evidence for co-operativity and cross-talk between PHOSPHO1 and TNAP in the initiation of mineralization. Clones 14 and 24 of the MC3T3-E1 cell line were used in the initial studies. Clone 14 cells expressed high levels of PHOSPHO1 and low levels of TNAP and in the presence of β-glycerol phosphate (BGP) or phosphocholine (P-Cho) as substrates and they mineralized their matrix strongly. In contrast clone 24 cells expressed high levels of TNAP and low levels of PHOSPHO1 and mineralized their matrix poorly. Lentiviral Phospho1 overexpression in clone 24 cells resulted in higher PHOSPHO1 and TNAP protein expression and increased levels of matrix mineralization. To uncouple the roles of PHOSPHO1 and TNAP in promoting matrix mineralization we used PHOSPHO1 (MLS-0263839) and TNAP (MLS-0038949) specific inhibitors, which individually reduced mineralization levels of Phospho1 overexpressing C24 cells, whereas the simultaneous addition of both inhibitors essentially abolished matrix mineralization (85 %; P<0.001). Using metatarsals from E15 mice as a physiological ex vivo model of mineralization, the response to both TNAP and PHOSPHO1 inhibitors appeared to be substrate dependent. Nevertheless, in the presence of BGP, mineralization was reduced by the TNAP inhibitor alone and almost completely eliminated by the co-incubation of both inhibitors. These data suggest critical non-redundant roles for PHOSPHO1 and TNAP during the initiation of osteoblast and chondrocyte mineralization.

Hypophosphatasia (HPP), caused by loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNAP), is characterized by skeletal and dental hypomineralization that can vary in severity from life-threatening to milder manifestations only in adulthood. PHOSPHO1 deficiency leads to early-onset scoliosis, osteomalacia, and fractures that mimic pseudo-HPP. Asfotase alfa, a life-saving enzyme replacement therapy approved for pediatric-onset HPP, requires subcutaneous injections 3 to 6 times per week. We recently showed that a single injection of an adeno-associated virus vector serotype 8 harboring TNAP-D10 (AAV8-TNAP-D10) effectively prevented skeletal disease and prolonged life in Alpl -/- mice phenocopying infantile HPP. Here, we aimed to determine the efficacy of AAV8-TNAP-D10 in improving the skeletal and dental phenotype in the Alpl Prx1/Prx1 and Phospho1 -/- mouse models of late-onset (adult) HPP and pseudo-HPP, respectively. A single dose of 3 × 1011 vector genomes per body (vg/b) was injected intramuscularly into 8-week-old Alpl Prx1/Prx1 and wild-type (WT) littermates, or into 3-day-old Phospho1 -/- and WT mice, and treatment efficacy was evaluated after 60 days for late-onset HPP mice and after 90 days for Phospho1 -/- mice. Biochemical analysis showed sustained serum alkaline phosphatase activity and reduced plasma PPi levels, and radiographic images, micro-computed tomography (micro-CT) analysis, and hematoxylin and eosin (H&E) staining showed improvements in the long bones in the late-onset HPP mice and corrected scoliosis in the Phospho1 -/- mice. Micro-CT analysis of the dentoalveolar complex did not reveal significant changes in the phenotype of late-onset HPP and pseudo-HPP models. Moreover, alizarin red staining analysis showed that AAV8-TNAP-D10 treatment did not promote ectopic calcification of soft organs in adult HPP mice after 60 days of treatment, even after inducing chronic kidney disease. Overall, the AAV8-TNAP-D10 treatment improved the skeletal phenotype in both the adult HPP and pseudo-HPP mouse models. This preclinical study will contribute to the advancement of gene therapy for the improvement of skeletal disease in patients with heritable forms of osteomalacia. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Hitherto, membralin has been a protein of unknown function. Here, we show that membralin mutant mice manifest a severe and early-onset motor neuron disease in an autosomal recessive manner, dying by postnatal day 5-6. Selective death of lower motor neurons, including those innervating the limbs, intercostal muscles, and diaphragm, is predominantly responsible for this fatal phenotype. Neural expression of a membralin transgene completely rescues membralin mutant mice. Mechanistically, we show that membralin interacts with Erlin2, an endoplasmic reticulum (ER) membrane protein that is located in lipid rafts and known to be important in ER-associated protein degradation (ERAD). Accordingly, the degradation rate of ERAD substrates is attenuated in cells lacking membralin. Membralin mutations or deficiency in mouse models induces ER stress, rendering neurons more vulnerable to cell death. Our study reveals a critical role of membralin in motor neuron survival and suggests a novel mechanism for early-onset motor neuron disease.

The Hyp mouse is a commonly used model for the study of the phosphate wasting disease X-linked hypophosphataemia. The defect in this mouse line is a deletion that includes exons 16 to 22 of Phex, although the exact extent of this X chromosome deletion remains unknown. This complicates genotyping which increases costs, time and difficulty of working with this important model. We aimed to determine the molecular breakpoints of this deletion in order develop a robust assay for its detection. We designed short mapping PCRs around the Phex locus to refine the putative breakpoint locations, then used gap PCR to amplify a product containing the breakpoint junction. DNA sequencing showed the deleted region was approximately 297 kb, significantly larger than previous reports, but did not contain any genes other than Phex. DNA sequence analysis revealed that this deletion may be the result of microhomology-mediated end joining. Finally, we designed a multiplex PCR assay for genotyping Hyp colonies and validated it using a panel of Hyp colony mice. This study provides confirmation of the Hyp phenotype as a single gene defect, a potential mechanism for its formation and an improved method for genotyping that will make working with this strain significantly easier.

Background Homoarginine ( hA rg) has been shown to be cardioprotective in a model of ischemic heart failure; however, the mechanism remains unknown. hA rg can inhibit tissue-nonspecific alkaline phosphatase ( TNAP ), an enzyme that promotes vascular calcification. We hypothesized that hA rg will exert beneficial effects by reducing calcification in a mouse model of coronary artery disease associated with TNAP overexpression and hypercholesterolemia. Methods and Results TNAP was overexpressed in the endothelium in mice homozygous for a low-density lipoprotein receptor mutation (wicked high cholesterol [ WHC ] allele). WHC and WHC -endothelial TNAP mice received placebo or hA rg supplementation (14 mg/L in drinking water) starting at 6 weeks of age simultaneously with an atherogenic diet. Outcomes were compared between the groups after 4 to 5 weeks on treatment. Experiments were performed in males, which presented a study limitation. As expected, WHC -endothelial TNAP mice on the placebo had increased mortality (median survival 27 days, P<0.0001), increased coronary calcium and lipids ( P<0.01), increased left ventricular end-diastolic diameter ( P<0.0001), reduced ejection fraction ( P<0.05), and increased myocardial fibrosis ( P<0.0001) compared with WHC mice. Contrary to our hypothesis, hA rg neither inhibited TNAP activity in vivo nor reduced coronary artery calcification and atherosclerosis in WHC -endothelial TNAP mice; however, compared with the placebo, hA rg prevented left ventricular dilatation ( P<0.01), preserved ejection fraction ( P<0.05), and reduced myocardial fibrosis ( P<0.001). Conclusions The beneficial effect of hA rg supplementation in the setting of calcified coronary artery disease is likely due to its direct protective actions on the myocardial response to the ischemic injury and not to the inhibition of TNAP activity and calcification.