Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially availabl...Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.展开更多
Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibi...Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.展开更多
Abstract Nogo-66 plays a central role in the myelin- mediated inhibition of neurite outgrowth. Tau is a micro- tubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whethe...Abstract Nogo-66 plays a central role in the myelin- mediated inhibition of neurite outgrowth. Tau is a micro- tubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau inter- acts directly with growth factor receptors, or engages in cross-talk with regeneration inhibitors like Nogo-66. Here, we report that plasmid overexpression of tau significantly elevated the protein levels of total tau, phosphorylated tau, and microtubule-affinity regulating kinase （MARK）. Nogo- 66 transiently elevated the total tau protein level and per- sistently reduced the level of p-s262 tau （tau phosphory- lated at serine 262）, whereas it had little influence on the level of p-T205 tau （tau phosphorylated at threonine 205）. Nogo-66 significantly decreased the protein level of MARK. Hymenialdisine, an inhibitor of MARK, signifi- cantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma cortical neurons. However, 2a （N2a） cells and primary concomitant inhibition ofMARK abolished the rescue of neurite outgrowth by tan in N2a cells. We conclude that dephosphorylation of tau at S262 is able to regulate Nogo-66 signaling, and that overexpression of tau can rescue the Nogo-66-induced inhibition of neurite outgrowth in vitro.展开更多
目的通过转染小干扰RNA(siRNA)沉默RAW264.7细胞源性泡沫细胞神经轴突生长抑制因子B受体(Ng BR)表达,研究Ng BR对泡沫细胞胆固醇逆转运(RCT)的影响,探索从RCT途径抗动脉粥样硬化(As)的新方法,为冠心病的临床防治提供新思路。方法利用氧...目的通过转染小干扰RNA(siRNA)沉默RAW264.7细胞源性泡沫细胞神经轴突生长抑制因子B受体(Ng BR)表达,研究Ng BR对泡沫细胞胆固醇逆转运(RCT)的影响,探索从RCT途径抗动脉粥样硬化(As)的新方法,为冠心病的临床防治提供新思路。方法利用氧化型低密度脂蛋白(ox-LDL)诱导RAW264.7细胞形成泡沫细胞,油红O染色进行鉴定。将泡沫细胞分为四组:空白对照组、siRNA阴性对照组、Ng BR-siRNA1转染组(si Ng BR-1组)、Ng BR-siRNA2转染组(si Ng BR-2组)。利用siRNA沉默泡沫细胞Ng BR基因表达,并利用real-time PCR和Western blot对其进行干扰效率鉴定。随后采用real-time PCR检测各组细胞肝X受体α(LXRα)、三磷酸腺苷结合盒转运体A1(ABCA1)及三磷酸腺苷结合盒转运体G1(ABCG1)mRNA表达,Western blot检测各组细胞相应蛋白含量,液闪计数仪检测胆固醇流出率。结果 ox-LDL成功诱导泡沫细胞形成;si Ng BR-1组和si Ng BR-2组Ng BR mRNA及其蛋白明显下调(P<0.05);si Ng BR-1组和si Ng BR-2组LXRα、ABCA1和ABCG1的mRNA及其蛋白表达显著降低(P<0.05),胆固醇流出显著减少(P<0.05)。结论 Ng BR可以增加巨噬细胞源性泡沫细胞RCT的调控基因LXRα及其下游基因ABCA1、ABCG1的表达,从而减弱或者避免As的发生和发展,为冠心病的临床防治提供理论依据。展开更多
基金supported by the National Natural Science Foundation of China,No.81171178
文摘Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.
基金a Ph D fellowship by FCT-Fundacao para a Ciência Tecnologia (SFRH/BD/135868/2018)(to SSC)。
文摘Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.
基金supported by the National Natural Science Foundation of China(81371380 and 31171028)
文摘Abstract Nogo-66 plays a central role in the myelin- mediated inhibition of neurite outgrowth. Tau is a micro- tubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau inter- acts directly with growth factor receptors, or engages in cross-talk with regeneration inhibitors like Nogo-66. Here, we report that plasmid overexpression of tau significantly elevated the protein levels of total tau, phosphorylated tau, and microtubule-affinity regulating kinase （MARK）. Nogo- 66 transiently elevated the total tau protein level and per- sistently reduced the level of p-s262 tau （tau phosphory- lated at serine 262）, whereas it had little influence on the level of p-T205 tau （tau phosphorylated at threonine 205）. Nogo-66 significantly decreased the protein level of MARK. Hymenialdisine, an inhibitor of MARK, signifi- cantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma cortical neurons. However, 2a （N2a） cells and primary concomitant inhibition ofMARK abolished the rescue of neurite outgrowth by tan in N2a cells. We conclude that dephosphorylation of tau at S262 is able to regulate Nogo-66 signaling, and that overexpression of tau can rescue the Nogo-66-induced inhibition of neurite outgrowth in vitro.
文摘目的通过转染小干扰RNA(siRNA)沉默RAW264.7细胞源性泡沫细胞神经轴突生长抑制因子B受体(Ng BR)表达,研究Ng BR对泡沫细胞胆固醇逆转运(RCT)的影响,探索从RCT途径抗动脉粥样硬化(As)的新方法,为冠心病的临床防治提供新思路。方法利用氧化型低密度脂蛋白(ox-LDL)诱导RAW264.7细胞形成泡沫细胞,油红O染色进行鉴定。将泡沫细胞分为四组:空白对照组、siRNA阴性对照组、Ng BR-siRNA1转染组(si Ng BR-1组)、Ng BR-siRNA2转染组(si Ng BR-2组)。利用siRNA沉默泡沫细胞Ng BR基因表达,并利用real-time PCR和Western blot对其进行干扰效率鉴定。随后采用real-time PCR检测各组细胞肝X受体α(LXRα)、三磷酸腺苷结合盒转运体A1(ABCA1)及三磷酸腺苷结合盒转运体G1(ABCG1)mRNA表达,Western blot检测各组细胞相应蛋白含量,液闪计数仪检测胆固醇流出率。结果 ox-LDL成功诱导泡沫细胞形成;si Ng BR-1组和si Ng BR-2组Ng BR mRNA及其蛋白明显下调(P<0.05);si Ng BR-1组和si Ng BR-2组LXRα、ABCA1和ABCG1的mRNA及其蛋白表达显著降低(P<0.05),胆固醇流出显著减少(P<0.05)。结论 Ng BR可以增加巨噬细胞源性泡沫细胞RCT的调控基因LXRα及其下游基因ABCA1、ABCG1的表达,从而减弱或者避免As的发生和发展,为冠心病的临床防治提供理论依据。
