The Role Of Protein Phosphatase 2A In The Establishment Of Hippocampal Neuronal Polarity

Typically, Neurons contain one axon and multiple dendrites. These processes play their special roles, receiving information in the dendrites, and conveying it to other cells in the axon. Neuronal polarity is essential for the unidirectional signal flow from somata or dendrites to axons in neurons. Previous studies have demonstrated that collapsin response mediator protein-2 (CRMP-2) is enriched in the growing axon of hippocampal neurons, which can interact with tubulin heterodimers and promotes microtubule assembly in vitro. Overexpression of CRMP-2 induces multiple axons, whereas inhibition of CRMP-2 function impairs axon formation. GSK-3βcan phosphorylate CRMP-2 at Thr-514 and inactivate the protein. Down-regulation of GSK-3βdecreases the level of CRMP-2 phosphorylation at Thr-514 and promotes axon specification and elongation. Protein phosphatase-2A (PP2A) is the most important Ser/Thr protein phosphatase in the brain. Here, we found that axon-dendrite polarity of the hippocampal neurons was established after 48 h culture in vitro in normal and DMSO (vehicle control) treated groups. When treated with okadaic acid (OA), a PP2A inhibitor, the growth of neurites was significantly inhibited and the formation of axon was obviously retarded, the latter suggests an inhibited establishment of neuronal polarity. Based on these results, we chose 10 nmol/L of OA and 10 nmol/L of D-erythro-Sphingosine to treat the hippocampal neurons cultured for 12 h. OA significantly inhibited axon elongation and branching and deceased the percentage of neurons with monoaxon and multi-axons (P<0.001). However, D-erythro-Sphingosine promoted axon elongation (P<0.01) and increased the percentage of neurons with multiply neurites (P<0.001). Results from Western-blot showed that OA and D-erythro-Sphingosine had no effect on level of PP2Ac subunit. The level of the methylated PP2A deceased in OA treated group, whereas the results were reversed when treated wth D-erythro-Sphingosine. To explore the role of PP2A in the maintenance of the neuronal polarity, we cultured the neurons for 2 day in vitro (DIV) to reach stage 3 and then treated with OA or D-erythro-Sphingosine or DMSO for another 24 h. The majority of the neurons treated with DMSO at day 3 developed normal polarity. OA treatment did not affect the formation of axon and neuronal polarity (P>0.05) and D-erythro-Sphingosine treatment also did not cause the formation of multiple axons (P>0.05). These results suggest that PP2A may not affect the maintenance of neuronal polarity. To test the role of PP2A in the formation of neuronal polarity, we used a constitutively active PP2A(PP2Awt) and a dominant-negative mutant PP2A (PP2Adn). Neurons were co-transfected with RFP (or GFP)/pcDNA4.0, or RFP (or GFP)/PP2Awt or RFP (or GFP)/PP2Adn and the neurons were cultured for another 48 h, then fixed and immunostained with specific antibodies. Majority control hippocampal neurons cotransfected with RFP or GFP/pcDNA4.0 developed normal polarity showing one axon and multiple dendrites. Overexpression of PP2Awt significantly promoted the single axon elongation (P<0.01) and the formation of multiple axons (P<0.001), whereas overexpression of PP2Adn significantly inhibited the formation of axon and neuronal polarity (P<0.001). Furthermore, we examined the function of the multiple axons and found that PP2Awt caused the formation of multiple axons positive for FM4-64 uptake after stimulation by 45 mM K+ for 1 min. After the initial FM dye loading, treatment with 90 mM K+depleted the dye from two axons of a PP2Awt-transfected neuron, suggesting mutiple axons had normal vesicle recycling. We also investigated whether the expression of the nonphosphorylated form of CRMP-2 was downstream of PP2A. Hippocampal neurons were cotransfected with PP2Awt or pcDNA4.0 and RFP. Hippocampal neurons were fixed at 3 DIV and then immunostained with anti-pT514 antibodies. We found that the phosphorylation level of CRMP-2 at Thr514 decreased both in the cell body and neurites in the neurons overexpressing PP2Awt, indicating that PP2Awt dephosphorylated CRMP-2 at Thr514 and induced axon elongation and the formation of multiple axons-like neurites in hippocampal neurons. We conclude that upregulation of PP2A may promote axon polarity and formation of multiple axons possibly through dephosphorylating CRMP-2, and the palingenetic axons show normal function in uptake and release of vesicles.