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
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
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.
