Ox-A get involved in several physiological processes that are important for neuronal homeostasis. It also exerts neuroprotective effects on dopaminergic [34, 35], cortical [36, 37], and hippocampal [38] neurons, in SH-SY5Y cells and other cell lines [39, 40]. It has been proposed that Ox-A could remodel neuronal activity and promote morphological changes that reinforce synaptic plasticity. However, studies related to neurite growth and its association with proteins involved in these mechanisms are limited. Here we investigated the possibility that Ox-A can induce neurite outgrowth in vitro in SH-SY5Y human neuroblastoma cells, a model commonly used for neurodegenerative diseases research. We also evaluated the effect of Ox-A on Activity-regulated cytoskeleton associated protein (Arc). Finally, we assessed the effects of selective OX1R and OX2R receptor antagonists on Ox-A-induced Arc enhancement.
Ours findings showed that Ox-A (100 nM for 24 h) increased the number of SH-SY5Y cells with longer neurites by 79.6%, 215 cells with neurites of 21 to 80 µm in lengths. While many control cells (237 cells, 87.8%) had neurites with lengths from 0 to 40 µm. Furthermore, Ox-A significantly augmented Arc protein, whose translation is associated with activity-dependent synaptic plasticity. This effect was inhibited by specific antagonists of both receptors, OX1R and OX2R. Ours data coincide with those reported by Forte et al. [22], who found that Ox-A (200 nM by 3 days) promoted branching of neuronal processes of human induced pluripotent stem cells (h-iPSCs), effect that was prevented by adding SB-334867 (10 µM). Likewise, Stonayova et al. [36] demonstrated that Ox-A stimulates synaptogenesis and thus neuronal interactions in dissociated postnatal cortical neurons of rat. Stonayova et al. [36] proposed the hypothesis that Ox-A acts as a synaptogenic factor which was supported by an early and sustained increase in synaptophysin expression, an integral membrane protein localized to synaptic vesicles. On the other hand, Björnström et al. [41] demostrated that Ox-A (10 nM) inhibits propofol-induced neurite retraction PLD/PKCƐ pathway in cortical neuronal cells. These data together suggest that Ox-A, in addition to inducing proliferation and neurogenesis, also stimulates cellular communication and the establishment of neuronal networks by increasing the number of neurites and their branches and by protecting the neurite retraction. Therefore, Ox-A may play a key role in mediating short- and long-term events related to cellular communication.
In this study, we showed that Ox-A, in addition to increasing the number of cells with longer neurites, also increased the amount of Arc protein. Arc is highly expressed in several situations of synaptic plasticity, including acute administration of addictive drugs, e.g., morphine [42], cocaine [43], and methamphetamine [44] alcohol [45], stress [46] seizure [47], and LTP [48]. These findings are relevant considering that Ox-A modulates reward-processing, promotes drug-seeking behavior, improves memory and learning [49]. We propose that the effects induced by Ox-A on a greater number of cells with longer neurites and greater expression of Arc protein may be part of the molecular mechanisms involved in reinforcing synaptic strength in response to highly motivational stimuli, such as abuse consumption of drugs. Furthermore, the results of this study become critical in differentiated and undifferentiated SH-SY5Y cells of dopaminergic origin since they are a popular cell model for neurodegenerative diseases such as Parkinson's (PD) research. Disease characterized by the loss of dopaminergic cells and their projections, thus affecting neuronal connectivity and synaptic transmission.