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Quantitative proteomics and in-cell cross-linking reveal cellular reorganisation during early neuronal differentiation of SH-SY5Y cells

ABSTRACT: The proteomes of undifferentiated and differentiated SH-SY5Y cells are characterised and compared. For this, neuronal differentiation using retinoic acid (RA) or a combination of RA and phorbol-12-myristat-13-acetate (RA/PMA) was explored. An MS-based label-free quantification approach is applied to identify changes in the protein expression, the as proteins’ subcellular localisation abundance as well as their association with enriched KEGG pathways. By employing formaldehyde cross-linking insights into protein interaction networks of undifferentiated as well as RA- and RA/PMA-differentiated neurons are obtained. The analyses provided insights into the proteomes of undifferentiated and differentiated SH-SY5Y cells and suggest structural rearrangements, for instance, of the actin network during neuronal differentiation.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Neuroblastoma

SUBMITTER: Marie Alfes

LAB HEAD: Carla Schmidt

PROVIDER: PXD031054 | Pride | 2022-04-08

REPOSITORIES: Pride

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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 proteomics and in-cell cross-linking reveal cellular reorganisation during early neuronal differentiation of SH-SY5Y cells

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