Differentiation Of Human SH-SY5Y Cells Research Articles

RBM45 is an m6A-binding protein that affects neuronal differentiation and the splicing of a subset of mRNAs.

SUMMARYN6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are incompletely understood. RBM45 acts as an m6A-binding protein that is highly expressed during neurodevelopment. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings show a role for RBM45 in controlling mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Cytoplasmic long noncoding RNAs are differentially regulated and translated during human neuronal differentiation.

The expression of long noncoding RNAs is highly enriched in the human nervous system. However, the function of neuronal lncRNAs in the cytoplasm and their potential translation remains poorly understood. Here we performed Poly-Ribo-Seq to understand the interaction of lncRNAs with the translation machinery and the functional consequences during neuronal differentiation of human SH-SY5Y cells. We discovered 237 cytoplasmic lncRNAs up-regulated during early neuronal differentiation, 58%–70% of which are associated with polysome translation complexes. Among these polysome-associated lncRNAs, we find 45 small ORFs to be actively translated, 17 specifically upon differentiation. Fifteen of 45 of the translated lncRNA-smORFs exhibit sequence conservation within Hominidea, suggesting they are under strong selective constraint in this clade. The profiling of publicly available data sets revealed that 8/45 of the translated lncRNAs are dynamically expressed during human brain development, and 22/45 are associated with cancers of the central nervous system. One translated lncRNA we discovered is LINC01116, which is induced upon differentiation and contains an 87 codon smORF exhibiting increased ribosome profiling signal upon differentiation. The resulting LINC01116 peptide localizes to neurites. Knockdown of LINC01116 results in a significant reduction of neurite length in differentiated cells, indicating it contributes to neuronal differentiation. Our findings indicate cytoplasmic lncRNAs interact with translation complexes, are a noncanonical source of novel peptides, and contribute to neuronal function and disease. Specifically, we demonstrate a novel functional role for LINC01116 during human neuronal differentiation.

The role of E2F1-topoIIβ signaling in regulation of cell cycle exit and neuronal differentiation of human SH-SY5Y cells

This study aims to test the role of E2F1-topoIIβ signaling in neuronal differentiation of SH-SY5Y cells. With retinoic acid (RA) induction, a high percentage of cells were found to be arrested at the G0/G1 phase, with decreased levels of cyclinD1, CDK4, phosphorylation status of pRb and E2F1, in addition to an elevated level of p27. The cells were shown to differentiate into neuronal phenotypes characterized by highly expressed neuronal markers, MAP2 and enriched topoIIβ, and remarkable neurite outgrowth. Exogenously forced E2F1 expression with a specific E2F1 plasmid led to suppression of topoIIβ expression and disruption of the neuronal differentiation of SH-SY5Y cells. On further examination using the ChIP assay, we found that E2F1 bound directly to the promoter region of topoIIβ, and its binding ability was inversely correlated with topoIIβ expression in response to RA induction. Thus, our findings suggest that E2F1-topoIIβ signaling may play a role in regulation of cell cycle exit and neuronal differentiation.

Characterization of alginate/chitosan-based nanoparticles and mathematical modeling of their SpBMP-9 release inducing neuronal differentiation of human SH-SY5Y cells

The incidence of brain degenerative disease such as Alzheimer’s disease (AD) will increase as the world population is ageing. While current AD treatments have only a transient effect, there are many evidences indicating that some growth factors, such as BMP-9, may be used to treat AD. However, growth factors cannot readily access the brain because of their size and the presence of the blood brain barrier. We have therefore developed a small peptide derived from BMP-9, SpBMP-9, which can promote the differentiation of cholinergic neurons and inactivate GSK3beta, a Tau kinase. Here, we investigated the potential of a nanoparticle-based delivery system of SpBMP-9, made of alginate and chitosan (Alg/Chit NPs), as a new therapeutic strategy against AD. The Alg/Chit NPs size distribution revealed NPs with an average diameter of ∼240nm. The encapsulation efficiency of SpBMP-9 was ∼70% of the initial peptide mass loading. Release kinetics of SpBMP-9 were performed in physiological conditions and modelled with a mechanistic framework that took into account the size distribution of Alg/Chit NPs. The release of SpBMP-9 revealed to be mostly diffusive, but there were interactions between the peptide and the alginate chains. The Alg/Chit NPs could also increase the viability of SH-SY5Y cells in comparison to the control. Finally, the SpBMP-9 released from Alg/Chit NPs promoted the SH-SY5Y differentiation into mature neurons as demonstrated by a higher neurite outgrowth and an increased expression of the neuronal markers NSE and VAchT. In conclusion, the nano-scale SpBMP-9 delivery system made of Alg/Chit may be a promising therapeutic strategy against AD.

Oxygen and differentiation status modulate the effect of X-ray irradiation on physiology and mitochondrial proteome of human neuroblastoma cells

Cytotoxic effects, including oxidative stress, of low linear energy transfer (LET)-ionizing radiation are often underestimated and studies of their mechanisms using cell culture models are widely conducted with cells cultivated at atmospheric oxygen that does not match its physiological levels in body tissues. Also, cell differentiation status plays a role in the outcome of experiments. We compared effects of 2 Gy X-ray irradiation on the physiology and mitochondrial proteome of nondifferentiated and human neuroblastoma (SH-SY5Y) cells treated with retinoic acid cultivated at 21% and 5% O2. Irradiation did not affect the amount of subunits of OxPhos complexes and other non-OxPhos mitochondrial proteins, except for heat shock protein 70, which was increased depending on oxygen level and differentiation status. These two factors were proven to modulate mitochondrial membrane potential and the bioenergetic status of cells. We suggest, moreover, that oxygen plays a role in the differentiation of human SH-SY5Y cells.

Chronic ethanol exposure changes dopamine D2 receptor splicing during retinoic acid-induced differentiation of human SH-SY5Y cells

There is evidence for ethanol-induced impairment of the dopaminergic system in the brain during development. The dopamine D2 receptor (DRD2) and the dopamine transporter (DAT) are decisively involved in dopaminergic signaling. Two splice variants of DRD2 are known, with the short one (DRD2s) representing the autoreceptor and the long one (DRD2l) the postsynaptic receptor. We searched for a model to investigate the impact of chronic ethanol exposure and withdrawal on the expression of these proteins during neuronal differentiation. RA-induced differentiation of human neuroblastoma SH-SY5Y cells seems to represent such a model. Our real-time RT-PCR, Western blot, and immunocytochemistry analyses of undifferentiated and RA-differentiated cells have demonstrated the enhanced expression of both splice variants of DRD2, with the short one being stronger enhanced than the long one under RA-treatment, and the DRD2 distribution on cell bodies and neurites under both conditions. In contrast, DAT was down-regulated by RA. The DAT is functional both in undifferentiated and RA-differentiated cells as demonstrated by [3H]dopamine uptake. Chronic ethanol exposure during differentiation for up to 4 weeks resulted in a delayed up-regulation of DRD2s. Ethanol withdrawal caused an increased expression of DRD2l and a normalization of DRD2s. Thus the DRD2s/DRD2l ratio was still disturbed. The dopamine level was increased by RA-differentiation compared to controls and was diminished under RA/ethanol treatment and ethanol withdrawal compared to RA-only treated cells. In conclusion, chronic ethanol exposure impairs differentiation-dependent adaptation of dopaminergic proteins, specifically of DRD2s. RA-differentiating SH-SY5Y cells are suited to study the impact of chronic ethanol exposure and withdrawal on expression of dopaminergic proteins during neuronal differentiation.

Accumulation of the amyloid precursor-like protein APLP2 and reduction of APLP1 in retinoic acid-differentiated human neuroblastoma cells upon curcumin-induced neurite retraction

Amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. The function of these three proteins is not yet fully understood. One of the proposed roles of APP is to promote neurite outgrowth. The aim of this study was to investigate the regulation of the expression levels of APP family members during neurite outgrowth. We observed that retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y cells resulted in increased expression of APP, APLP1 and APLP2. We also examined the effect of the NFκB, AP-1 and c-Jun N-terminal kinase inhibitor curcumin (diferuloylmethane) on the RA-induced expression levels of these proteins. We found that treatment with curcumin counteracted the RA-induced mRNA expression of all APP family members. In addition, we observed that curcumin treatment resulted in neurite retraction without any effect on cell viability. Surprisingly, curcumin had differential effects on the APLP protein levels in RA-differentiated cells. RA-induced APLP1 protein expression was blocked by curcumin, while the APLP2 protein levels were further increased. APP protein levels were not affected by curcumin treatment. We propose that the sustained levels of APP and the elevated levels of APLP2, in spite of the reduced mRNA expression, are due to altered proteolytic processing of these proteins. Furthermore, our results suggest that APLP1 does not undergo the same type of regulated processing as APP and APLP2.