BDNF and DYRK1A Are Variable and Inversely Correlated in Lymphoblastoid Cell Lines from Down Syndrome Patients

Down syndrome or trisomy 21 is the most common genetic disorder leading to mental retardation. One feature is impaired short- and long-term spatial memory, which has been linked to altered brain-derived neurotrophic factor (BDNF) levels. Mouse models of Down syndrome have been used to assess neurotrophin levels, and reduced BDNF has been demonstrated in brains of adult transgenic mice overexpressing Dyrk1a, a candidate gene for Down syndrome phenotypes. Given the link between DYRK1A overexpression and BDNF reduction in mice, we sought to assess a similar association in humans with Down syndrome. To determine the effect of DYRK1A overexpression on BDNF in the genomic context of both complete trisomy 21 and partial trisomy 21, we used lymphoblastoid cell lines from patients with complete aneuploidy of human chromosome 21 (three copies of DYRK1A) and from patients with partial aneuploidy having either two or three copies of DYRK1A. Decreased BDNF levels were found in lymphoblastoid cell lines from individuals with complete aneuploidy as well as those with partial aneuploidies conferring three DYRK1A alleles. In contrast, lymphoblastoid cell lines from individuals with partial trisomy 21 having only two DYRK1A copies displayed increased BDNF levels. A negative correlation was also detected between BDNF and DYRK1A levels in lymphoblastoid cell lines with complete aneuploidy of human chromosome 21. This finding indicates an upward regulatory role of DYRK1A expression on BDNF levels in lymphoblastoid cell lines and emphasizes the role of genetic variants associated with psychiatric disorders.     

Kirenol inhibits RANKL-induced osteoclastogenesis and prevents ovariectomized-induced osteoporosis via suppressing the Ca2+-NFATc1 and Cav-1 signaling pathways

BackgroundOsteoporosis is a threat to aged people who have excessive osteoclast activation and bone resorption, subsequently causing fracture and even disability. Inhibiting osteoclast differentiation and absorptive functions has become an efficient approach to treat osteoporosis, but osteoclast-targeting inhibitors available clinically remain rare. Kirenol (Kir), a bioactive diterpenoid derived from an antirheumatic Chinese herbal medicine Herba Siegesbeckiae, can treat collagen-induced arthritis in vivo and promote osteoblast differentiation in vitro, while the effects of Kir on osteoclasts are still unclear.PurposeWe explore the role of Kir on RANKL-induced osteoclastogenesis in vitro and bone loss in vivo.MethodsThe in vitro effects of Kir on osteoclast differentiation, bone resorption and the underlying mechanisms were evaluated with bone marrow-derived macrophages (BMMs). In vivo experiments were performed using an ovariectomy (OVX)-induced osteoporosis model.ResultsWe found that Kir remarkably inhibited osteoclast generation and bone resorption in vitro. Mechanistically, Kir significantly inhibited F-actinring formation and repressed RANKL-induced NF-κB p65 activation and p-p38, p-ERK and c-Fos expression. Moreover, Kir inhibited both the expression and nuclear translocation of NFATc1. Ca²⁺ oscillation and caveolin-1 (Cav-1) were also reduced by Kir during osteoclastogenesis in vitro. Consistent with these findings, 2–10 mg/kg Kir attenuated OVX-induced osteoporosis in vivo as evidenced by decreased osteoclast numbers and downregulated Cav-1 and NFATc1 expression.ConclusionsKir suppresses osteoclastogenesis and the Cav-1/NFATc1 signaling pathway both in vitro and in vivo and protects against OVX-induced osteoporosis. Our findings reveal Kir as a potential safe oral treatment for osteoporosis.     

The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis

Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis.     

p53 downregulates Down syndrome-associated DYRK1A through miR-1246

Several microRNAs mediate the functions of p53 family members. Here we characterize miR-1246 as a new target of this family. In response to DNA damage, p53 induces the expression of miR-1246 which, in turn, reduces the level of DYRK1A, a Down syndrome-associated protein kinase. Knockdown of p53 has the opposite effect. Overexpression of miR-1246 reduces DYRK1A levels and leads to the nuclear retention of NFATc1, a protein substrate of DYRK1A, and the induction of apoptosis, whereas a miR-1246-specific inhibitor prevented the nuclear import of NFATc1. Together, these results indicate that p53 inhibits DYRK1A expression through the induction of miR-1246.     

DYRK1A enhances the mitogen-activated protein kinase cascade in PC12 cells by forming a complex with Ras, B-Raf, and MEK1

Dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) is the human homologue of the Drosophila mnb (minibrain) gene. In Drosophila, mnb is involved in postembryonic neurogenesis. In human, DYRK1A maps within the Down syndrome critical region of chromosome 21 and is overexpressed in Down syndrome embryonic brain. Despite its potential involvement in the neurobiological alterations observed in Down syndrome patients, the biological functions of the serine/threonine kinase DYRK1A have not been identified yet. Here, we report that DYRK1A overexpression potentiates nerve growth factor (NGF)-mediated PC12 neuronal differentiation by up-regulating the Ras/MAP kinase signaling pathway independently of its kinase activity. Furthermore, we show that DYRK1A prolongs the kinetics of ERK activation by interacting with Ras, B-Raf, and MEK1 to facilitate the formation of a Ras/B-Raf/MEK1 multiprotein complex. These data indicate that DYRK1A may play a critical role in Ras-dependent transducing signals that are required for promoting or maintaining neuronal differentiation and suggest that overexpression of DYRK1A may contribute to the neurological abnormalities observed in Down syndrome patients.     

The role of Mac-1 (CD11b/CD18) in osteoclast differentiation induced by receptor activator of nuclear factor-kappaB ligand

Multinuclear osteoclasts are derived from CD11b-positive mononuclear cells in bone marrow and in circulation. FACS sorting experiments showed impaired osteoclastogenesis in RAW264.7 cells with low CD11b expression. Neutralizing antibodies and siRNA against CD11b inhibited osteoclastogenesis induced by RANKL. Although primary cultured mouse bone marrow macrophages expressed CD11a and CD11b, osteoclastogenesis induced by M-CSF and RANKL was inhibited in the presence of anti-CD11b or anti-CD18 but not anti-CD11a antibodies. Furthermore, anti-CD11b antibodies inhibited NFATc1 expression induced by M-CSF and RANKL in BMMs. These findings suggest, at least partly, an important role of CD11b in osteoclastogenesis.     

Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade

SHIP is an SH2-containing inositol-5-phosphatase expressed in hematopoietic cells. It hydrolyzes the PI3K product PI(3,4,5)P(3) and blunts the PI3K-initiated signaling pathway. Although the PI3K/Akt pathway has been shown to be important for osteoclastogenesis, the molecular events involved in osteoclast differentiation have not been revealed. We demonstrate that Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. Inhibition of the PI3K by LY294002 reduces formation of osteoclasts and attenuates the expression of NFATc1, but not that of c-Fos. Conversely, overexpression of Akt in bone marrow-derived macrophages (BMMs) strongly induced NFATc1 expression without affecting c-Fos expression, suggesting that PI3K/Akt-mediated NFATc1 induction is independent of c-Fos during RANKL-induced osteoclastogenesis. In addition, we found that overexpression of Akt enhances formation of an inactive form of GSK3β (phospho-GSK3β) and nuclear localization of NFATc1, and that overexpression of a constitutively active form of GSK3β attenuates osteoclast formation through downregulation of NFATc1. Furthermore, BMMs from SHIP knockout mice show the increased expression levels of phospho-Akt and phospho-GSK3β, as well as the enhanced osteoclastogenesis, compared with wild type. However, overexpression of a constitutively active form of GSK3β attenuates RANKL-induced osteoclast differentiation from SHIP-deficient BMMs. Our data suggest that the PI3K/Akt/GSK3β/NFATc1 signaling axis plays an important role in RANKL-induced osteoclastogenesis.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.