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Quantitative proteomic analysis identifies proteins and pathways related to neuronal development in differentiated SH-SY5Y neuroblastoma cells

ABSTRACT: SH-SY5Y neuroblastoma cells are widely used as in vitro neuronal model. They can be induced to a differentiated phenotype, presenting neurites and synaptical-like structures in response to retinoic (RA) acid and brain-derived neurotrophic factor (BDNF), providing a model to analyze neuronal differentiation. We report a large scale MS quantification of SH-SY5Y cells proteome during its differentiation process after treatment with RA/BDNF. Using isobaric tags for relative and absolute quantification (iTRAQ) approach and phosphopeptide enrichment protocols, we identified a total of 5587 proteins, 366 of them showed differential abundance between both conditions of culture. Differentiated SH-SY5Y cells showed regulation of proteins and phosphosites strongly related to neuronal development, in contrast, undifferentiated cells expressed proteins more related to cell proliferation and control of cell cycle. Interactive network analysis covered processes as focal adhesion, cytoskeleton dynamics and neurodegenerative diseases and pathway analysis displayed regulation of mitogen-activated protein kinase and phosphoinositide 3-kinase/Akt signaling pathways mainly; the proteins involved in those processes might be considered as markers for neuronal differentiation. Overall the data collection presented here can be explored for any studies which intent to use SH-SY5Y as neuronal model.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Magno Junqueira

PROVIDER: MSV000080133 | MassIVE |

REPOSITORIES: MassIVE

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Project description:Background: SH-SY5Y cells exhibit a neuronal phenotype when treated with all-trans retinoic acid (RA), but the molecular mechanism of activation in the signaling pathway mediated by phosphatidylinositol 3-kinase (PI3K) is not sufficiently understood. To shed new light on the mechanism, we comprehensively compared the gene expression profiles between SK-N-SH cells and two subtypes of SH-SY5Y cells (SH-SY5Y-A and SH-SY5Y-E), each of which showed a different phenotype during RA-mediated differentiation. Results: SH-SY5Y-A cells differentiated in the presence of RA, whereas RA-treated SH-SY5Y-E cells required additional treatment with brain-derived neurotrophic factor (BDNF) for full differentiation. In combination with perturbation using a PI3K inhibitor, LY294002, we identified 386 genes and categorized them into two clusters dependent on the PI3K signaling pathway during RA-mediated differentiation in SH-SY5Y-A cells. Transcriptional regulation of the gene cluster was greatly reduced in SK-N-SH cells or partially impaired in SH-SY5Y-E cells in coincidence with a defect in the neuronal phenotype of these cell lines. Additional stimulation with BDNF induced a set of neural genes which were down-regulated in RA-treated SH-SY5Y-E cells but were abundant in the differentiated SH-SY5Y-A cells. Conclusions: We identified the gene clusters controlled by PI3K- and TRKB-mediated signaling pathways during differentiation in two subtypes of SH-SY5Y cells. TRKB-mediated bypass pathway compensates for the impaired neural functions generated by defects in several signaling pathways including PI3K in SH-SY5Y-E cells. The expression profiling data are useful for further studies to elucidate the signal transduction-transcriptional network including PI3K and/or TRKB. Experiment Overall Design: Human neuroblastomas, SK-N-SH (HTB-11) and SH-SY5Y-A cells (CRL-2266) were obtained from the American Type Culture Collection (ATCC). We also obtained SH-SY5Y-E cells (EC94030304) from the European Collection of Cell Cultures (ECACC). Tissue culture cells were maintained in D-MEM/F12 1:1 mixture supplemented with 15% FBS (Fetal Bovine Serum) and 1% NEAA (Non-essential amino acid) in a 5% CO2 humidified incubator at 37oC. The culture medium was changed twice a week. For the RA-inducible experiment, random culture cells from two clone subtypes of SH-SY5Y and SK-N-SH were seeded in laminin coated culture dishes (BioCoat Laminin Cellware; BD Biosciences, Billerica, MA, USA) for 1 day and then transferred to a medium containing 10 μM of RA in the presence or the absence of LY294002 (10μM) for five days. For BDNF-induced sequential differentiation of the SH-SY5Y-E strain, cells were washed with D-MEM/F12 twice after five days in the presence of RA and then incubated with 50 ng/ml of BDNF in D-MEM/F12 without serum for three days.

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Quantitative proteomic analysis identifies proteins and pathways related to neuronal development in differentiated SH-SY5Y neuroblastoma cells

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