Competitive blocking of LRP4-sclerostin binding interface strongly promotes bone anabolic functions.

Induction of bone formation by Wnt ligands is inhibited when sclerostin (Scl), an osteocyte-produced antagonist, binds to its receptors, the low-density lipoprotein receptor-related proteins 5 or 6 (LRP5/6). Recently, it was shown that enhanced inhibition is achieved by Scl binding to the co-receptor LRP4. However, it is not clear if the binding of Scl to LRP4 facilitates Scl binding to LRP5/6 or inhibits the Wnt pathway in an LRP5/6-independent manner. Here, using the yeast display system, we demonstrate that Scl exhibits a stronger binding affinity for LRP4 than for LRP6. Moreover, we found stronger Scl binding to LRP6 in the presence of LRP4. We further show that a Scl mutant (SclN93A), which tightly binds LRP4 but not LRP6, does not inhibit the Wnt pathway on its own. We demonstrate that SclN93A competes with Scl for a common binding site on LRP4 and antagonizes Scl inhibition of the Wnt signaling pathway in osteoblasts in vitro. Finally, we demonstrate that 2 weeks of bi-weekly subcutaneous injections of SclN93A fused to the fragment crystallizable (Fc) domain of immunoglobulin (SclN93AFc), which retains the antagonistic activity of the mutant, significantly increases bone formation rate and enhances trabecular volumetric bone fraction, trabecular number, and bone length in developing mice. Our data show that LRP4 serves as an anchor that facilitates Scl-LRP6 binding and that inhibition of the Wnt pathway by Scl depends on its prior binding to LRP4. We further provide evidence that compounds that inhibit Scl-LRP4 interactions offer a potential strategy to promote anabolic bone functions.

Positive Outcomes of Denosumab Treatment in 2 Patients With Cherubism.

Cherubism is a rare autosomal dominant disease whose severity ranges widely, from asymptomatic to life-threatening. Bilateral symmetrical painless expansion of the mandible and maxilla resulting in a typical appearance of the face resembling a cherub, are the highlighted features of the condition. In most cases, cherubism-induced lesions in the jaws appear around the age of 3 years and tend to expand and increase in numbers until puberty. Treatment options for cherubism range from observation to surgical correction and various pharmacologic therapies. Given the excess sensitivity of cherubism osteoclasts to RANKL (receptor activator of nuclear factor κB ligand) and the positive effects of denosumab (XGEVA; Amgen, Thousand Oaks, CA) treatment in patients with giant cell granuloma, we have designed a treatment based on denosumab for 2 cherubism patients that achieves what we consider promising results.

Amyloid ß attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca2+ , ERK1/2 and Clusterin signaling in neurons.

A hallmark of Alzheimer's disease is accumulation of amyloid beta (Aß) deposits, which are associated with neuronal dysfunction, spine loss, and impaired Ca2+ homeostasis. Amyloid beta (Aß) binds to and is aggregated by Zn2+ , a metal released from synaptic glutamatergic vesicles during neuronal activity. Synaptically released Zn2+ activates a metabotropic Gq-coupled Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling in post-synaptic neurons. We examined if Aß, as a Zn2+ binding protein, regulates neuronal Zn2+ -signaling mediated by mZnR/GPR39 using SHSY-5Y cells and cortical neurons from GPR39 wild-type and knockout mice. Following acute or chronic treatment with Aß neuronal Zn2+ -dependent Ca2+ release via mZnR/GPR39 is significantly reduced. This impairment is overcome when excess Zn2+ is applied, suggesting that impaired Ca2+ -signaling results from Aß binding of Zn2+ . The Zn2+ -dependent mZnR/GPR39 activation triggers phosphorylation of extracellular regulated kinase and up-regulates expression of the chaperone protein clusterin (Clu). Importantly, neuronal Zn2+ -dependent extracellular regulated kinase1/2 phosphorylation and up-regulation of Clu are attenuated by silencing mZnR/GPR39 as well as by Aß treatment. In contrast, Zn2+ -dependent AKT phosphorylation is not mediated by mZnR/GPR39 and is not attenuated by Aß treatment. Thus, Zn2+ signaling via mZnR/GPR39 is distinctively disrupted by a critical pathological component of Alzheimer's disease. Synaptically released Zn2+ activates a Zn2+ -sensing receptor, mZnR/GPR39, and induces Ca2+ -signaling, followed by ERK1/2 MAPK activation and up-regulation of clusterin. Amyloid beta (Aß) binds to Zn2+ thus forming oligomers that are a hallmark of Alzheimer's disease. We show that Aß attenuates Zn2+ -dependent Ca2+ -responses, abolishes ERK1/2 activation and down-regulates clusterin expression. Thus, Zn2+ signaling via mZnR/GPR39 is disrupted by Aß, a critical pathological component of Alzheimer's disease.

Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation.

Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, beta-alanyl-L-histidine. Carnosine is believed to be involved in cellular defense such as free radical detoxification and inhibition of protein cross-linking. The more stable N-acetyl derivative of carnosine has also been identified in the brain. The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. In this study, BV2 microglial cells were stimulated with bacterial LPS, a potent inflammatory stimulus. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively. By competitive spectrophotometric measurement and electrospray mass spectrometry analysis, we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFalpha secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.

Intracellular zinc inhibits KCC2 transporter activity.

We found that K(+)/Cl(-) co-transporter 2 (KCC2) activity, monitored with wide-field fluorescence, was inhibited by intracellular Zn(2+), a major component of neuronal injury. Zn(2+)-mediated KCC2 inhibition produced a depolarizing shift of GABA(A) reversal potentials in rat cortical neurons. Moreover, oxygen-glucose deprivation attenuated KCC2 activity in a Zn(2+)-dependent manner. The link between Zn(2+) and KCC2 activity provides a previously unknown target for neuroprotection and may be important in activity-dependent regulation of inhibitory synaptic transmission.