The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer’s disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices, or primary neurons treated with an MT2 receptor-selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons.
Synaptic circuits are established at the sites of axon–dendritic, axon–somatic, or axon–axonal contact, in which functional axonogenesis is a critical step. Axonogenesis can be regulated by many intracellular signals that involve cytoskeletal rearrangements, local protein degradation, as well as diffusional barriers. Additionally, several extracellular neurotrophic factors and hormones have also been shown to have a role in axon guidance and synaptic formation in central neurons. To date, the role of melatonin and its receptors in axonogenesis remains unclear. Most of the biological functions of melatonin are mediated by its two receptors, MT1 and MT2 receptors, both of them belong to the G protein-coupled receptor (GPCR) subfamily and are widely expressed throughout the central nervous system (CNS). Activation of the MT2 receptor in response to melatonin is critical for controlling circadian rhythms and regulation of slow-wave sleep. Early studies have shown that activation of the MT2 receptor in the retina reduces the release of dopamine, while dopamine inhibits growth cone motility and neurite outgrowth during embryonic development, suggesting the involvement of the MT2 receptor in functional axonogenesis. In mutant mice with deficient expression of the MT2 gene, the induction of long-term potentiation (LTP) of excitatory synaptic transmission is impaired, and this impairment is closely related to deficits in learning. In the hippocampus, the MT2 receptor inhibits GABAA receptor-mediated current, which is implicated in synaptic transmission. In Alzheimer’s disease, expression of the MT2 receptor is significantly reduced, especially in the hippocampus. A partial agonist of the MT2 receptor, UCM765, exhibits anxiolytic-like properties by increasing the time spent in the open arm of an elevated plus-maze test, and by reducing the latency to eat in a novel environment in the novelty suppressed feeding test, suggesting its role in anxiety. Together, these findings suggest that the MT2 receptor links the signaling cascades that mediate learning and memory formation, one of the important biological functions of melatonin; however, the cellular and molecular events underlying this linkage are yet to be established.
Dissociated hippocampal neurons have been commonly used as an excellent in vitro model in the study of axon development and synaptic transmission because they maintain morphological, functional, and molecular characteristics of the hippocampal neurons in vivo. In dissociated hippocampal neurons, the transition for axon formation and maturation involves the following five stages: stage 1 neurons (~2 to 4 h after plating) display abundant lamellipodia and filopodia that develop into several immature short neurites at stage 2 (~12 to 24 h); polarization occurs at stage 3 (~24 to 48 h), in which a single neurite initiates a rapid elongation to become the axon while others acquire dendritic identity; stage 4 (~3–4 days) is characterized by rapid outgrowth of axon and dendrites; and at stage 5 (7 days onwards), the maturation of axon and dendrites is essential for functional synapse formation. In the present study, we have identified a novel role for the MT2 receptor in functional axonogenesis and show that activation of the MT2 receptor is crucial for functional axonogenesis and synaptic transmission in central neurons. Using fluorescence resonance energy transfer (FRET) imaging combined with peptide blocking assays, we have identified Akt as an interacting partner and a substrate of the MT2 receptor. Activation of the MT2 receptor-Akt signaling cascade promotes the formation of functional synapses in the hippocampus, whereas inhibition of the MT2 receptor arrests axonogenesis and synaptic transmission. Given the implications of the MT2 receptor in learning and memory, we propose that targeting MT2 receptor-Akt signaling may be a feasible strategy for stimulating functional synaptic circuit assembly.
Accumulation of the MT2 receptor in polarized axons
To explore the role of MT1 and MT2 receptors in axon development, we first measured their cellular localization in dissociated rat hippocampal neurons by co-immunostaining for the MT1 receptor or MT2 receptor and Tuj1, a neuron-specific class III β-tubulin. We found that the MT2 receptor was uniformly distributed on all neurites with tip enrichment in stage 2 neurons, while a strong fluorescence signal was only detected at the polarized axon tip but not in the dendrites in stage 3 neurons. Quantitative analysis showed that the MT2 receptor was differentially enriched at the neurite tips in stage 2 neurons, whereas more exclusive axon tip enrichment of the MT2 receptor was observed in stage 3 neurons. The MT1 receptor had a similar distribution to the MT2 receptor at stage 2, but no polarized distribution of the MT1 receptor was detected at stage 3. The specificity of the MT2 receptor antibody was verified by the peptide blocking experiment. These results suggest that the MT2 receptor might have a potential role in axon differentiation, an early stage of synapse development.