Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

The extracellular Ca2+-sensing receptor (CaR), a G protein-coupled receptor responsible for maintenance of calcium homeostasis, is implicated in regulation of skeletal metabolism. To discern the role of the osteoblast CaR in regulation of bone development and remodeling, we generated mice in which the CaR is excised in a broad population of osteoblasts expressing the 3.6 kb a1(I) collagen promoter. Conditional knockouts had abnormal skeletal histology at birth and developed progressively reduced mineralization secondary to retarded osteoblast differentiation, evident by significantly reduced numbers of osteoblasts and decreased expression of collagen I, osteocalcin and sclerostin mRNAs. Elevated expression of ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1, and osteopontin mRNAs in the conditional knockout indicate altered regulation of genes important in mineralization. Knockout of the osteoblast CaR also resulted in increased expression of the receptor activator of nuclear factor kappa B ligand (RANK-L), the major stimulator of osteoclast differentiation and function, consistent with elevated osteoclast numbers in vivo. Osteoblasts from the conditional knockouts exhibited delayed differentiation, reduced mineralizing capacity, altered expression of regulators of mineralization and increased ability to promote osteoclastogenesis in co-culture experiments. We conclude that CaR signaling in a broad population of osteoblasts is essential for bone development and remodeling and plays an important role in the regulation of differentiation and expression of regulators of bone resorption and mineralization.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is a lysosomal storage disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase (GALNS), an enzyme that degrades keratan sulfate (KS). Currently no therapy for MPS IVA is available. We produced recombinant human (rh)GALNS as a potential enzyme replacement therapy for MPS IVA. Chinese hamster ovary cells stably overexpressing GALNS and sulfatase modifying factor-1 were used to produce active (∼2 U/mg) and pure (≥97%) rhGALNS. The recombinant enzyme was phosphorylated and was dose-dependently taken up by mannose-6-phosphate receptor (Kuptake = 2.5 nM), thereby restoring enzyme activity in MPS IVA fibroblasts. In the absence of an animal model with a skeletal phenotype, we established chondrocytes isolated from two MPS IVA patients as a disease model in vitro. MPS IVA chondrocyte GALNS activity was not detectable and the cells exhibited KS storage up to 11-fold higher than unaffected chondrocytes. MPS IVA chondrocytes internalized rhGALNS into lysosomes, resulting in normalization of enzyme activity and decrease in KS storage. rhGALNS treatment also modulated gene expression, increasing expression of chondrogenic genes Collagen II, Collagen X, Aggrecan and Sox9 and decreasing abnormal expression of Collagen I. Intravenous administration of rhGALNS resulted in biodistribution throughout all layers of the heart valve and the entire thickness of the growth plate in wild-type mice. We show that enzyme replacement therapy with recombinant human GALNS results in clearance of keratan sulfate accumulation, and that such treatment ameliorates aberrant gene expression in human chondrocytes in vitro. Penetration of the therapeutic enzyme throughout poorly vascularized, but clinically relevant tissues, including growth plate cartilage and heart valve, as well as macrophages and hepatocytes in wild-type mouse, further supports development of rhGALNS as enzyme replacement therapy for MPS IVA.

Recent studies have shown that inoculation of susceptible mice with amyloid-β (Aβ) peptides accelerates Aβ deposition in the brain, supporting the idea that Aβ may be self-amplifying; however, the exact mechanism is not understood. Here we provide evidence that Aβ may self-amplify, in part, by inhibiting α-secretase ADAM10 (a disintegrin and metalloprotease) cleavage of full-length Aβ precursor protein (FL AβPP) and therefore allow greater β-secretase processing, and that Aβ itself is a substrate for ADAM10. Exposure of primary neuronal cultures from PDAβPP mice to exogenous rat Aβ1- 40 resulted in increased de novo human Aβ1-42 production and exposure of cells to Aβ decreased production of ADAM10 cleavage product soluble AβPPα (sAβPPα). In a cell-free assay, Aβ decreased ADAM10 cleavage of the chimeric substrate MBP-AβPPC125 and Aβ itself was apparently cleaved by the enzyme. The axonal guidance and trophic factor netrin-1, however, reduced the Aβ1- 40-induced Aβ1-42 increase, increased sAβPPα, and reversed the Aβ-induced sAβPPα decrease in vitro. In vivo, induction of netrin-1 expression in PDAβPPSwe/Ind transgenic mice resulted in reductions in both Aβ1-42 and Aβ1- 40, and ICV delivery of netrin-1 to PDAβPPSwe/Ind mice increased sAβPPα, decreased Aβ, and improved working memory. Finally, to support further study of netrin-1's potential as a therapeutic for Alzheimer's disease, pilot gene therapy studies were performed and a netrin mimetic peptide synthesized and tested that, like netrin, can increase sAβPPα and decrease Aβ1-42in vitro. Taken together, these data provide mechanistic insights into Aβ self-amplification and the ability of netrin-1 to disrupt it.

Significance Our work identifies a therapeutically tractable yet previously unrecognized repressive epigenetic feature of primary myelofibrosis that controls a key cytokine circuit in the bone marrow microenvironment. Though lacking previous clinical utility, we show that an orally active retinoic acid receptor antagonist can normalize thrombopoietin production, restore bone integrity, and dramatically reduce fibrosis. This identification of a pathway-specific drug for primary myelofibrosis opens up an avenue of treatment options and provides insight into the complex mechanisms underlying myelofibrosis.

Objective: Loss-of-function calcium-sensing receptor (CAR) mutations cause elevated parathyroid hormone (PTH) secretion and hypercalcaemia. Although full Car deletion is possible in mice, most human CAR mutations result from a single amino acid substitution that maintains partial function. However, here, we report a case of neonatal severe hyperparathyroidism (NSHPT) in which the truncated CaR lacks any transmembrane domain (CaR R392X ), in effect a full CAR 'knockout'. Case report: The infant (daughter of distant cousins) presented with hypercalcaemia (5.5-6 mmol/l corrected calcium (2.15-2.65)) and elevated PTH concentrations (650-950 pmol/l (12-81)) together with skeletal demineralisation. NSHPT was confirmed by CAR gene sequencing (homozygous c.1174C-to-T mutation) requiring total parathyroidectomy during which only two glands were located and removed, resulting in normalisation of her serum PTH/calcium levels. Design and methods: The R392X stop codon was inserted into human CAR and the resulting mutant (CaR R392X ) expressed transiently in HEK-293 cells. Results: CaR R392X expressed as a 54 kDa dimeric glycoprotein that was undetectable in conditioned medium or in the patient's urine. The membrane localisation observed for wild-type CaR in parathyroid gland and transfected HEK-293 cells was absent from the proband's parathyroid gland and from CaR R392X -transfected cells. Expression of the mutant was localised to endoplasmic reticulum consistent with its lack of functional activity. Conclusions: Intriguingly, the patient remained normocalcaemic throughout childhood (2.5 mM corrected calcium, 11 pg/ml PTH (10-71), age 8 years) but exhibited mild asymptomatic hypocalcaemia at age 10 years, now treated with 1-hydroxycholecalciferol and Ca 2C supplementation. Despite representing a virtual CAR knockout, the patient displays no obvious pathologies beyond her calcium homeostatic dysfunction.

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