Cholesterol supports the retinoic acid‐induced synaptic vesicle formation in differentiating human SH‐SY5Y neuroblastoma cells

Abstract

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

Constitutive activity of the A2A adenosine receptor and compartmentalised cyclic AMP signalling fine-tune noradrenaline release

“…Neurites and their boutons responded to receptor activation ( Fig. 1e) and have been proposed to provide release sites [39,40]. We therefore probed the effect of A 2A -receptor activation on noradrenaline release.…”

An In Vitro Model for Neuroscience: Differentiation of SH-SY5Y Cells into Cells with Morphological and Biochemical Characteristics of Mature Neurons

“…Commonly used differentiation agents include phorbol esters and retinoic acid (RA) [11], growth factors such as brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neuregulins [12][13][14]. Other less frequently used differentiation factors include the Vitamin D metabolite 1,25-dihydroxycholecalciferol (VitD 3 ), and cholesterol [15][16][17]. Another way of improving neuronal differentiation is to culture SH-SY5Y cells in a three dimensional (3D) environment [18] such as an extracellular matrix (ECM) gel, which provides cell support and trace amounts of substances that promote differentiation [19,20].…”

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells

“…All trans retinoic acid (RA), a vitamin A metabolite involved in growth and development, is known to induce neuronal differentiation of SH-SY5Y cells as well as to inhibit the cellular proliferation (Borriello et al 2006;Cheung et al 2009;Constantinescu et al 2007;Cuende et al 2008;Nakamura et al 2003;Pahlman et al 1984). A more mature neuronal phenotype can be reached using growth medium supplemented with specific chemical factors, such as recombinant human brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), dibutyryl-cyclic AMP (db-cAMP), phorbol esters and others (Agholme et al 2010;Dwane et al 2013;Encinas et al 2000;Gimenez-Cassina et al 2006;Jamsa et al 2004;Kou et al 2008;Kume et al 2008;Sarkanen et al 2007). Thereafter, SH-SY5Y cells have been largely utilized to investigate the disease mechanisms associated with various neurodegenerative disorders (Agholme et al 2014;Ferreira et al 2013;Hadzhieva et al 2013;Jamsa et al 2004;Ke et al 2012;Krishna et al 2014;Lim et al 2015;Lopes et al 2010Lopes et al , 2012Palomo et al 2011;Scifo et al 2013Scifo et al , 2015.…”