Protein Phosphatase 2A Facilitates Axonogenesis by Dephosphorylating CRMP2

Abstract

Protein phosphatase 2A (PP2A) is indispensable in development, and deficits of PP2A and deterioration of neuronal axons have been observed in several neurodegenerative disorders, but the direct link between PP2A and the neuronal axon development is still missing. Here, we show that PP2A is essential for axon development in transfected rat brain and the dissociated hippocampal neurons. Upregulation of PP2A catalytic subunit (PP2Ac) not only promotes formation and elongation of the functional axons but also rescues axon retardation induced by PP2A inhibition. PP2A can dephosphorylate collapsin response mediator protein-2 (CRMP2) that implements the axon polarization, whereas constitutive expression of phosphomimic-CRMP2 abrogates the effect of PP2A upregulation. We also demonstrate that PP2Ac is enriched in the distal axon of the hippocampal neurons. Our results reveal a mechanistic link between PP2A and axonogenesis/axonopathy, suggesting that upregulation of PP2A may be a promising therapeutic for some neurodegenerative disorders.

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

“…42,43 Our study here, along with the previous reports, provides a possible novel strategy to generate new axons on polarity-established neurons, which might help generate new axons to promote functional recovery in patients with neural injuries. 33,44 Both loss of the MT2 receptor and a decrease in melatonin are highly correlated with Alzheimer's disease, 12,14,15 and the cellular protection of melatonin or MT2 receptor activation has been well clarified. 45,46 Moreover, dopamine, 47 estrogen, 48 thyroid hormone, 49 acetylcholine, 50 glutamate, 51 GABA, 52 or their receptors 53 can affect or crosstalk with the melatonin/MT2 receptor, and most of these molecules have an important role in axonogenesis or synapse formation.…”

“…Cultures of dissociated hippocampal neurons were prepared from embryonic E18 rats as previously described. 29,44 The hippocampi of embryos were dissected and trypsinized by TrypLE Express (Invitrogen, Grand Island, NY, USA) for 15 min at 37°C. Dissociated neurons were plated onto poly-D-lysine (Sigma) coated Figure 6 MT2 receptor is linked with Akt/GSK-3β/CRMP2 pathway.…”

“…Cultured neurons or brain sections were immunostained by previously described methods. 29,44 Neurons and brain sections were incubated in primary antibodies for 12-36 h at 4°C and subsequently with AlexaFluro 488-and AlexaFluro 546-conjugated secondary antibodies. Images were examined by using a Zeiss LSM510 laser confocal microscope (Zeiss, Jena, Germany) or an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan).…”

“…The western blotting was carried out by a well-established procedure in our laboratory. 29,44 Briefly, the cell extracts prepared from HEK 293 or cultured neurons were separated by SDS-PAGE (10%) and the proteins were transferred onto nitrocellulose membrane (Amersham, Piscataway, NJ, USA) for 1 h using the transfer apparatus (Bio-Rad, Berkeley, CA, USA). After block with 3% milk for 30 min at room temperature, the membranes were then incubated at 4°C with primary antibodies overnight.…”

“…The shRNA constructs were introduced into the lateral ventricle at E15.5-16.5 and specifically targeted into forebrain pyramidal neurons by using in utero electroporation as previously described. 44 Briefly, after anesthetization, the abdominal cavity was opened, and the uterine horns were exposed. Constructs with mU6-MT2-shRNA-pUbi-EGFP or the scrambled shRNA (Genechem Inc., Shanghai, China, 3 μg in 2 μl) were injected through a glass micropipette into the lateral ventricle of the embryos.…”