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 vitroinin vivo. Ugotavljamo, da je tvorba aksonov upočasnjena pri miših z izločanjem receptorjev MT2, MT2-shRNA elektroporiranih možganskih rezinah ali primarnih nevronih, zdravljenih z antagonistom, selektivnim na receptor MT2. Aktivacija receptorja MT2 spodbuja aksonogenezo, ki je povezana z izboljšanjem ekscitatornega sinaptičnega prenosa v osrednjih nevronih. Signalne komponente navzdol od receptorja MT2 so sestavljene iz Akt/GSK-3 /CRMP-2 kaskada. Motiv terminala C- receptorja MT2 se neposredno veže na Akt. Inhibicija receptorja MT2 ali prekinitev vezave MT2 receptor-Akt zmanjša aksonogenezo in sinaptični prenos. Naši podatki kažejo, da receptor MT2 aktivira Akt/GSK-3 /CRMP-2 signalizacija in je potrebna in zadostna za posredovanje funkcionalne aksonogeneze in sinaptične tvorbe v osrednjih nevronih.
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.
Disociirani hipokampalni nevroni so se pogosto uporabljali kot odličnoin vitromodel pri preučevanju razvoja aksonov in sinaptičnega prenosa, ker ohranjajo morfološke, funkcionalne in molekularne značilnosti nevronov hipokampusain 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.
Kopičenje receptorja MT2 v polariziranih aksonih
Da bi raziskali vlogo receptorjev MT1 in MT2 pri razvoju aksonov, smo najprej izmerili njihovo celično lokalizacijo v disociiranih nevronih hipokampusa podgan s so-imunskim obarvanjem za MT1 receptor ali MT2 receptor in Tuj1, nevron-specifičen razred III -tubulin. Ugotovili smo, da je bil receptor MT2 enakomerno porazdeljen na vse nevrite z obogatitvijo konice v nevronih stopnje 2, medtem ko je bil močan fluorescenčni signal zaznan le na polarizirani konici aksona, ne pa tudi v dendritih v nevronih stopnje 3. Kvantitativna analiza je pokazala, da je bil receptor MT2 različno obogaten na konicah nevritov v nevronih 2. stopnje, medtem ko so bolj ekskluzivno obogatitev aksonskega vrha receptorja MT2 opazili pri nevronih 3. stopnje. Receptor MT1 je imel podobno porazdelitev kot MT2 receptor na 2. stopnji, vendar v 3. stopnji niso zaznali polarizirane porazdelitve receptorja MT1. Specifičnost protitelesa receptorja MT2 smo preverili s poskusom blokiranja peptidov. Ti rezultati kažejo, da bi lahko imel receptor MT2 potencialno vlogo pri diferenciaciji aksonov, zgodnji fazi razvoja sinaps.