An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP^(+)mononuclear cells and PDGF-BB secretion

Osteoporosis(OP)is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures.Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed.We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation.We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment.The secretion of platelet-derived growth factor-BB(PDGF-BB),the number of tartrate-resistant acid phosphatase-positive(TRAP+)mononuclear cells,and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice.The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP-1"cells.These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP4 mononuclear cells.To test the therapeutic potential of the Siglec-15 neutralizing antibody,we injected the antibody in an ovariectomy-induced osteoporotic mouse model,which mimics postmenopausal osteoporosis in women,and in two fracture healing models because fracture is the most serious health consequence of osteoporosis.The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis.Of note,the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models.Thus,the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

Sialylation of TLR2 initiates osteoclast fusion

The molecular control of osteoclast formation is still not clearly elucidated. Here, we show that a process of cell recognition mediated by Siglec15-TLR2 binding is indispensable and occurs prior to cell fusion in RANKL-mediated osteoclastogenesis. Siglec15 has been shown to regulate osteoclastic bone resorption. However, the receptor for Siglec15 has not been identified, and the signaling mechanism involving Siglec15 in osteoclast function remains unclear. We found that Siglec15 bound sialylated TLR2 as its receptor and that the binding of sialylated TLR2 to Siglec15 in macrophages committed to the osteoclast-lineage initiated cell fusion for osteoclast formation, in which sialic acid was transferred by the sialyltransferase ST3 Gal1. Interestingly, the expression of Siglec15 in macrophages was activated by M-CSF, whereas ST3 Gal1 expression was induced by RANKL. Both Siglec15-specific deletion in macrophages and intrafemoral injection of sialidase abrogated cell recognition and reduced subsequent cell fusion for the formation of osteoclasts, resulting in increased bone formation in mice. Thus, our results reveal that cell recognition mediated by the binding of sialylated TLR2 to Siglec15 initiates cell fusion for osteoclast formation.

PGE2 activates EP4 in subchondral bone osteoclasts to regulate osteoarthritis

Prostaglandin E2(PGE2), a major cyclooxygenase-2(COX-2) product, is highly secreted by the osteoblast lineage in the subchondral bone tissue of osteoarthritis(OA) patients. However, NSAIDs, including COX-2 inhibitors, have severe side effects during OA treatment. Therefore, the identification of novel drug targets of PGE2 signaling in OA progression is urgently needed. Osteoclasts play a critical role in subchondral bone homeostasis and OA-related pain. However, the mechanisms by which PGE2 regulates osteoclast function and subsequently subchondral bone homeostasis are largely unknown. Here, we show that PGE2 acts via EP4 receptors on osteoclasts during the progression of OA and OA-related pain. Our data show that while PGE2 mediates migration and osteoclastogenesis via its EP2 and EP4 receptors, tissue-specific knockout of only the EP4 receptor in osteoclasts(EP4 Lys M) reduced disease progression and osteophyte formation in a murine model of OA. Furthermore, OA-related pain was alleviated in the EP4 Lys M mice, with reduced Netrin-1 secretion and CGRP-positive sensory innervation of the subchondral bone. The expression of plateletderived growth factor-BB(PDGF-BB) was also lower in the EP4 Lys Mmice, which resulted in reduced type H blood vessel formation in subchondral bone. Importantly, we identified a novel potent EP4 antagonist, HL-43, which showed in vitro and in vivo effects consistent with those observed in the EP4 Lys Mmice. Finally, we showed that the Gαs/PI3 K/AKT/MAPK signaling pathway is downstream of EP4 activation via PGE2 in osteoclasts. Together, our data demonstrate that PGE2/EP4 signaling in osteoclasts mediates angiogenesis and sensory neuron innervation in subchondral bone, promoting OA progression and pain, and that inhibition of EP4 with HL-43 has therapeutic potential in OA.

致病性α-synuclein在帕金森病中的神经毒性与传播机制

帕金森病(Parkinson’s disease,PD)是继阿尔兹海默病(Alzheimer’s disease,AD)后的第二大慢性神经退行性疾病,其主要病理特征是由α-突触核蛋白(α-synuclein,α-syn)的错误折叠和聚集导致的黑质多巴胺神经元的损伤与死亡。近期研究表明,腺苷二磷酸核糖(ADP-ribose)聚合酶1(PARP-1)及其催化产物PAR可以加剧α-syn的病理改变,促进α-syn的聚集,同时由病理性α-syn错误折叠形成的聚集体能够通过与lymphocyte-activation gene 3(LAG3)相互作用在相邻神经元细胞之间传播,加速神经元细胞的损伤,导致PD的发生与进行性发展。本文就近年来在α-syn神经毒性与传播机制等方面的研究做一综述。

Extracellular RNAs-TLR3 signaling contributes to cognitive impairment after chronic neuropathic pain in mice

Chronic pain is often associated with cognitive decline,which could influence the quality of the patient’s life.Recent studies have suggested that Toll-like receptor 3(TLR3)is crucial for memory and learning.Nonetheless,the contribution of TLR3 to the pathogenesis of cognitive decline after chronic pain remains unclear.The level of TLR3 in hippocampal neurons increased in the chronic constriction injury(CCI)group than in the sham group in this study.Importantly,compared to the wild-type(WT)mice,TLR3 knockout(KO)mice and TLR3-specific neuronal knockdown mice both displayed improved cognitive function,reduced levels of inflammatory cytokines and neuronal apoptosis and attenuated injury to hippocampal neuroplasticity.Notably,extracellular RNAs(exRNAs),specifically double-stranded RNAs(dsRNAs),were increased in the sciatic nerve,serum,and hippocampus after CCI.The co-localization of dsRNA with TLR3 was also increased in hippocampal neurons.And the administration of poly(I:C),a dsRNA analog,elevated the levels of dsRNAs and TLR3 in the hippocampus,exacerbating hippocampus-dependent memory.In additon,the dsRNA/TLR3 inhibitor improved cognitive function after CCI.Together,our findings suggested that exRNAs,particularly dsRNAs,that were present in the condition of chronic neuropathic pain,activated TLR3,initiated downstream inflammatory and apoptotic signaling,caused damage to synaptic plasticity,and contributed to the etiology of cognitive impairment after chronic neuropathic pain.

Poly(I:C)preconditioning protects the heart against myocardial ischemia/reperfusion injury through TLR3/PI3K/Akt-dependent pathway

Emerging evidence suggests that Toll-like receptors(TLRs)ligands pretreatment may play a vital role in the progress of myocardial ischemia/reperfusion(I/R)injury.As the ligand of TLR3,polyinosinic-polycytidylic acid(poly(I:C)),a synthetic double-stranded RNA,whether its preconditioning can exhibit a cardioprotective phenotype remains unknown.Here,we report the protective effect of poly(I:C)pretreatment in acute myocardial I/R injury by activating TLR3/PI3K/Akt signaling pathway.Poly(I:C)pretreatment leads to a significant reduction of infarct size,improvement of cardiac function,and downregulation of inflammatory cytokines and apoptotic molecules compared with controls.Subsequently,our data demonstrate that phosphorylation of TLR3 tyrosine residue and its interaction with PI3K is enhanced,and protein levels of phospho-PI3K and phospho-Akt are both increased after poly(I:C)pretreatment,while knock out of TLR3 suppresses the cardioprotection of poly(I:C)preconditioning through a decreased activation of PI3K/Akt signaling.Moreover,inhibition of p85 PI3K by the administration of LY294002 in vivo and knockdown of Akt by siRNA in vitro significantly abolish poly(I:C)preconditioning-induced cardioprotective effect.In conclusion,our results reveal that poly(I:C)preconditioning exhibits essential protection in myocardial I/R injury via its modulation of TLR3,and the downstream PI3K/Akt signaling,which may provide a potential pharmacologic target for perioperative cardioprotection.