ABSTRACT
Nogo-66 plays a central role in the myelin-mediated inhibition of neurite outgrowth. Tau is a microtubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau interacts 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 persistently reduced the level of p-S262 tau (tau phosphorylated 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, significantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma 2a (N2a) cells and primary cortical neurons. However, concomitant inhibition of MARK abolished the rescue of neurite outgrowth by tau 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.
Subject(s)
Neurites/drug effects , Neurons/cytology , Neurons/drug effects , Nogo Proteins/pharmacology , tau Proteins/metabolism , Analysis of Variance , Animals , Azepines/pharmacology , Cells, Cultured , Cerebral Cortex/cytology , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Neuroblastoma/pathology , Protein Serine-Threonine Kinases/metabolism , Pyrroles/pharmacology , Rats , Rats, Sprague-Dawley , Time Factors , Transfection , tau Proteins/geneticsABSTRACT
Evidence suggested that glycogen synthase kinase-3ß (GSK-3ß) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3ß-adeno associated virus (GSK-3ß-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3ß-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3ß promotes axonal regeneration after SCI.
Subject(s)
Glycogen Synthase Kinase 3 beta/genetics , Nerve Regeneration/genetics , Spinal Cord Injuries/genetics , Syringomyelia/genetics , Animals , Axons/drug effects , Axons/metabolism , Dependovirus/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Humans , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Rats , Sensorimotor Cortex/drug effects , Sensorimotor Cortex/pathology , Spinal Cord Injuries/pathology , Spinal Cord Injuries/therapy , Syringomyelia/pathology , Syringomyelia/therapyABSTRACT
The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.
