The role of soluble adenylyl cyclase in the BDNF-dependent block of MAG/myelin-mediated inhibition.

摘要

In the adult mammalian central nervous system axons do not spontaneously regenerate following injury. This lack of axonal regeneration is partly due to the presence of inhibitory proteins, such as myelin-associated glycoprotein (MAG). Previously, we showed that elevating cyclic AMP (cAMP) by pretreating (priming) neurons with neurotrophins, such as brain-derived neurotrophic factor (BDNF), is sufficient to overcome the block of axonal outgrowth by MAG. Additionally, we demonstrated this BDNF-mediated effect to be PKA-, ERK-, calcium- and CREB-dependent. However, increasing cAMP levels in response to BDNF could be dependent on several factors. A balance between the production of cAMP by adenylyl cyclases and its degradation by PDEs will determine intracellular cAMP levels. Given that the source of the cAMP produced in response to BDNF is unknown, we sought to investigate which adenylyl cyclase is activated, transmembrane adenylyl cyclase (tmAC) or soluble adenylyl cyclase (sAC). tmACs and sAC differ in their spatial localization within the cell, structure and regulation. Our hypothesis is that the rise in cAMP in response to BDNF priming is partially dependent on sAC activation.In this study, we have detected an isoform of sAC, somatic sAC, expressed in various postnatal rat primary neurons and demonstrated that specifically blocking sAC with the pharmacological inhibitors, KH7 and OH-E or by knocking down sAC expression with siRNA, abolishes the ability of BDNF to overcome inhibition by MAG. Additionally, infection of primary neurons with a lentivirus that expresses sAC is sufficient to overcome the block of axonal growth by MAG and myelin in vitro and promotes optic nerve regeneration in vivo. As previously mentioned, priming with BDNF leads to ERK activation, which results in overcoming MAG-induced inhibition of neurite growth. We found that blocking sAC with pharmacological inhibitors blocked the BDNF-dependent phosphorylation of ERK whereas blocking tmAC had no effect on ERK activation by BDNF. Lastly, we sought to determine if alternative modes of sAC regulation exist, such as interactions with TrkB. Our data demonstrated that sAC does not associate with inactive or active TrkB receptors, yet does not rule out that other potential modes of regulation may exist. Taken together, our data suggest that sAC plays an integral role in BDNF signaling to overcome inhibition of axonal growth by MAG.