Role Of MT2 Receptor, GSK-3 And PP-2A In Synaptic Plasticity And The Underlying Mechanisms

Backgroud:Synaptogenesis is the biological basement of learning and memory. During the neuronal development, the development and maturation of axon that maintains the axon-dendrites polarity is an essential and crucial step for the functional spine. Melatonin 2 receptor (MT2 receptor), Glycogen synthase kinase-3 (GSK-3), and protein phosphatase-2A (PP-2A) are widely expressed in the brain, play diverse functions in physiological and neurodegenerative conditions. MT2 receptor belongs to G protein coupled receptor (GPCR) superfamily, can couple to multiple and distinct signal transduction cascades whose activation can lead to unique cellular responses. Downregulation of MT2 receptors inhibits the differentiation of neural stem cells (NSCs) in adult hippocampus, long-term potentiation (LTP) and learning/memory. Activation of MT2 receptors disrupted the inhibitory neuronal function by reducing the GABAA receptor mediated currents in CA1 pyramidal cells. GSK-3 and PP-2A are the central of signal transduction cascades, they were recognized as the major tau protein kinase and phosphatase and play important role in neuronal polarity and synaptic plasticity. Previous reports from our lab revealed that melatonin (endogenous ligand for MT2 receptor) reverse AD-like tau hyperphosphorylation by GSK-3 inhibition and PP-2A activation. However, it is not known that the links between MT2 receptor, GSK-3 and PP-2A, and how they affect axonogenesis and synaptic plasticity.Objects:The current study is to explore the role of MT2 receptor, GSK-3 and PP-2A in axonogenesis and synaptic plasticity and the possible links among them both in vivo and in vitro; GSK-3 abnormality induces the synaptic disorder and the underlying mechanisms.Methods:On the model of cultured hippocampal neurons, the release of presynaptic neurotransmitters and the function of axon were measured by the uptake and release of FM4-64; Ca2+ influx and postsynaptic miniature excitatory postsynaptic currents (mEPSCs) were recorded by the whole cell patch clamp; vesicle exocytosis were examined by total internal reflection fluorescence microscopy (TIRFM); fluorescence resonance energy transfer (FRET) imaging and immunoprecipitation were employed to investigate the association between MT2 and Akt, and several synaptic proteins, which play a crucial role in exocytosis in synaptosome fraction. The development of axon and distributions of proteins were observed by immuofluorescence; immunoblot were applied to detect the levels of total and phosphorylated proteins.Results:(1) Role of MT2 receptor on the development and function of axon and the mechanisms:Activation of MT2 promotes functional axonogenesis with an enhanced synaptic transmission in hippocampal neurons, whereas inhibition of MT2 arrests axon differentiation; the signaling components downstream of MT2 involves Akt/GSK-3β/CRMP-2 cascade; MT2 directly binds to Akt to inhibit GSK-3; disruption of MT2-Akt binding by application of a MT2 intracellular C terminal peptide fragment abolishes the MT2-stimulated GSK-3 inhibition, axonogenesis and synaptic transmission; knock-out of MT2 receptor induced tau hyperphosphorylation by PP-2A inhibition.(2) Effect of GSK-3 on presynaptic release of neurotransmitters and the mechanisms:Upregulation of GSK-3βretards the presynaptic vesicle exocytosis; GSK-3βphosphorylates the LⅡ-Ⅲ(synprint site) of P/Q-type Ca2+ channel and upregulation of GSK-3 decreases Ca2+ influx from P/Q-type VDCC; GSK-3βdecreases the association of LⅡ-Ⅲwith synaptotagmin, SNAP25 and syntaxin; the association of synaptobrevin with SNAP25 and syntaxin, and the dissociation of synaptobrevin from synaptophysin I, all of which are required for Ca2+-dependent SNARE complex formation and thus for an efficient exocytosis.(3) Effect of PP-2A on neurite outgrowth in neuronal cells:PP-2A is essential for the initiation of neurite, and upregulation of PP-2A could promote the outgrowth of neurites, especially axons.Conclusions:Our data in the present study show that the activation of MT2 receptor promotes the axonogenesis and synaptogenesis by GSK-3 inhibition; knock-out of MT2 receptor induces AD-like tau hyperphosphorylation by PP-2A downregulation in vivo; Activation of GSK-3 retards presynaptic vesicle releasing by phosphorylating P/Q calcium channel; Upregulation of PP-2A promote the neurite outgrowth, especially for axon.