Effects of CB1R activation on STAT3 and ERK1/2 pathways, and on the neuritogenesis mediated by APP and its phosphorylation

• Autor(a) principal: Ribeiro, Nuno Fernando Lobo Oliveira
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/35959
• topic: Neuritogenesis; CB1R; Cannabinoid; STAT3 signaling; ERK1/2 signaling; APP; Gαo; SH-SY5Y cells

Neuritogenesis is a very important event in neuronal differentiation, and its study could pave the way for the discovery of new therapies related to the regeneration of the nervous system. Among the several proteins and signaling pathways involved in neuritogenesis, are Alzheimer's Amyloid Precursor Protein (APP), the Gαo protein, and the STAT3 and ERK1/2 signaling pathways, which can be activated by various receptors. Previous studies by our group showed that APP has effects on neuritogenesis, and that part of these effects may be due to its interaction with the Gαo protein, the most abundant Gα heterotrimeric subunit in the brain. In APP and Gαo-mediated neuritogenesis, both the STAT3 and ERK1/2 pathways are involved. However, it is not known whether physiological activators of Gαo, such as the neuritogenic GPCR cannabinoid receptor 1 (CB1R), affect APP-mediated neuritogenesis. To study this possible functional interaction, this work began by characterizing several synthetic cannabinoids that are known to activate CB1R, such as HU-210, WIN 55,212-2 and ACEA. Another cannabinoid with a very low affinity for CB1R, CBDV, was also studied as a possible control. The activation of STAT3 and ERK1/2 signaling was monitored after administration of the 4 cannabinoids to SH-SY5Y neuroblastoma cells, for 3 different time points: 15, 30 and 60 min. All were found to lead to a decrease in phosphoSTAT3 and a significant increase in STAT3, resulting in an apparent decrease in STAT3 activation, potentially due to earlier (5-10 min) robust activation, followed by retroinhibition. Relative to ERK1/2, all cannabinoids increased phosphoERK1/2 and total ERK1/2 levels. HU-210 and CBDV were able to significantly increase the pERK1/2/ERK1/2 ratio. Following, HU-210 was chosen to study its effects on APP-mediated neuritogenesis, since it is a known activator of CB1R and Gαo, with reported neuritogenic effects. Specifically, HU-210 effects were studied in conditions of overexpression of three forms of APP: wild-type (Wt); serine 655 mutated to alanine (SA– 'dephospho') or glutamate (SE– 'phospho'), since APP phosphorylation at S655 increases ERK1/2 activation and APP-Gαo binding. In the experimental conditions here used, in cells unstimulated with HU-210, transfection with any of the APP-GFPs resulted in increased average length of the branches, in relation to cells transfected with the GFP vector. A 15 min pulse of HU-210 was able to increase the number of primary cell projections, in all conditions except SA APP, and also increased the number of branches, although it generally decreased their lengths. Of the tested APP forms, the SE APP showed the longest total length of primary and secondary projections (branches), either under basal conditions or after treatment with HU-210. SA was the APP form that increased the most the number of branches with HU-210 treatment but did not have the number of primary projections increased. Under these conditions, HU-210 was not able to produce any synergistic effect with the APPs’ expression, and even reduced the branches’ length and increased their number. In the future, new experimental conditions will have to be tested, to verify if APP and CB1R activation could have an earlier synergic effect in the promotion of the number or length of new neuritic projections.