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 em vitroena Vivo. Descobrimos que a formação de axônios é retardada em camundongos knockout para o receptor MT2, fatias de cérebro eletroporadas MT2-shRNA ou neurônios primários tratados com um antagonista seletivo do receptor MT2. A ativação do receptor MT2 promove axonogênese que está associada a um aumento na transmissão sináptica excitatória em neurônios centrais. Os componentes de sinalização a jusante do receptor MT2 consistem no Akt/GSK-3 /CRMP-2 cascata. O motivo terminal C-do receptor MT2 liga-se diretamente à Akt. A inibição do receptor MT2 ou a interrupção da ligação do receptor MT2-Akt reduz a axonogênese e a transmissão sináptica. Nossos dados sugerem que o receptor MT2 ativa Akt/GSK-3 /CRMP-2 e é necessário e suficiente para mediar a axonogênese funcional e a formação sináptica em neurônios centrais.
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.
Neurônios do hipocampo dissociados têm sido comumente usados como um excelenteem vitromodelo no estudo do desenvolvimento do axônio e transmissão sináptica porque eles mantêm as características morfológicas, funcionais e moleculares dos neurônios do hipocampona 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.
Acúmulo do receptor MT2 em axônios polarizados
Para explorar o papel dos receptores MT1 e MT2 no desenvolvimento do axônio, primeiro medimos sua localização celular em neurônios dissociados do hipocampo de ratos por co-imunocoloração para o receptor MT1 ou receptor MT2 e Tuj1, um neurônio-específico classe III -tubulina. Descobrimos que o receptor MT2 foi distribuído uniformemente em todos os neuritos com enriquecimento da ponta nos neurônios do estágio 2, enquanto um forte sinal de fluorescência foi detectado apenas na ponta do axônio polarizado, mas não nos dendritos nos neurônios do estágio 3. A análise quantitativa mostrou que o receptor MT2 foi enriquecido diferencialmente nas pontas dos neuritos nos neurônios do estágio 2, enquanto o enriquecimento mais exclusivo da ponta do axônio do receptor MT2 foi observado nos neurônios do estágio 3. O receptor MT1 teve uma distribuição semelhante ao receptor MT2 no estágio 2, mas nenhuma distribuição polarizada do receptor MT1 foi detectada no estágio 3. A especificidade do anticorpo do receptor MT2 foi verificada pelo experimento de bloqueio de peptídeo. Esses resultados sugerem que o receptor MT2 pode ter um papel potencial na diferenciação do axônio, um estágio inicial do desenvolvimento da sinapse.