Several papers document a variety of differentiation protocols for SH-SY5Y cells, and these protocols have been characterised to differing extents. As our work is principally focussed on neurodegenerative diseases, we were interested in protocols reporting to induce cholinergic or dopaminergic neuronal phenotypes, as these are relevant to Alzheimer’s Disease and Parkinson’s Disease, respectively. For this reason, we focussed on a differentiation protocol using both retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), with a 10-day timecourse (De Medeiros et al. 2019) (Fig. 1). Importantly, this protocol is easy to perform without complex or expensive media preparations; requires no extra cell passaging (in comparison to some published protocols (Shipley et al. 2016)); and results in lysates with sufficient protein concentration for Western blotting-based quantification of major neuronal proteins (see Figs. 3, 5 and 6).

Using this protocol, we observed profound and robust morphological changes over the 10-day timecourse. During this time, the SH-SY5Y cells went from a largely epithelial morphology (Fig. 2A) to an increasingly neurone-like morphology with extensive neurite outgrowth and branching by Day 10 (Fig. 2B,C and D). Additionally, these neurites stained strongly positive for the neurone-specific β-III tubulin (Fig. 2E–H). Therefore, the differentiation protocol we have employed results in neuronal morphology in 10 d, driven by β-III tubulin polymerisation along neurites.

The differentiation protocol leads to reliable and robust outgrowth of neurites expressing β-III tubulin within 10 d. Transmitted light (left) and immunocytochemistry (ICC, right) images of SH-SY5Y cells at different timepoints during differentiation ((A): Day 0, (B): Day 3, (C): Day 7 and (D): Day 10) showing morphological changes during the differentiation. ICC images are stained for β-III tubulin (green) and DAPI (blue). Images are the composite of 33 confocal slices. Note that the transmitted light and ICC images are taken from different batches of cells. Scale bar = 50 µM.

To further characterize this differentiation protocol, we investigated and quantified the expression of key neuronal and synaptic markers during this timecourse using Western blotting (Fig. 3A-C). As expected from the ICC results in Fig. 2, we observed a significant and robust induction of β-III tubulin expression across the timecourse of differentiation, reflecting neurite formation. Additionally, we observed a strong induction of the expression of both the pre-synaptic marker, synaptophysin (Fig. 3B) and the post-synaptic marker, PSD-95 (Fig. 3C). Whilst both markers showed a significant increase over the timecourse of differentiation, it is interesting to note that PSD-95 expression increased most strongly between days 0–3, whereas synaptophysin expression increased most strongly between days 3–7. These results therefore indicate that, as well as neuronal morphology, differentiated SH-SY5Y cells may be forming synaptic structures.

Time-dependent expression of key neuronal and synaptic markers during differentiation of SH-SY5Y cells. All experiments involved SDS-PAGE and Western blotting of whole cell lysates at the designated timepoints. (A): Expression of β-III tubulin. Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. (B): Expression of synaptophysin. Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. (C): Expression of PSD-95. Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. For all graphs, values are mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 1-way ANOVA with Tukey’s post-hoc test.

Having established that this differentiation protocol results in the expression of synaptic markers, we next used immunocytochemistry to characterise the localisation of these proteins. Our reasoning was that if SH-SY5Y cells differentiated using this protocol are capable of synapse formation, we would observe punctate distribution of both the post-synaptic marker PSD-95 and the pre-synaptic marker synaptophysin. As shown in Fig. 4A-C, synaptophysin showed a neurite-localised, punctate distribution by Day 10. This is what we would expect if presynaptic structures were being formed in these cells, and is broadly similar to synaptophysin staining observed in primary neurones, e.g. (Swarnkar et al. 2021). We observed a similar distribution of PSD-95 along neurite processes (Fig. 4D-F), which was possibly even more punctate than that of synaptophysin in places. Whilst it is notable that both synaptic markers are visible throughout the cell bodies at Day 10, these results nevertheless suggest that this differentiation protocol leads to the formation of both pre- and post-synaptic structures. We note, however, that we should be cautious in over-interpreting these data, and that the puncta we show here may simply represent transport cargo. However, costaining for both PSD-95 and synaptophysin (Fig. 4G-I) revealed several sites where these pre- and post-synaptic markers were in close apposition. Whilst we acknowledge that this does not provide definitive evidence, it is however consistent with the formation of synaptic structures.

Punctate distribution of pre- and post-synaptic markers along neurites of D10 differentiated SH-SY5Y cells. (A-C): Immunocytochemistry for DAPI (blue, A), synaptophysin (green, B) and merged (C) in SH-SH5Y cells after 10 d of differentiation, showing diffuse somatic and more punctate neurite localisation. Images are the composite of 33 confocal slices. (D-F): Immunocytochemistry for DAPI (blue, D), PSD-95 (green, E) and merged (F) in SH-SH5Y cells after 10 d of differentiation, showing diffuse somatic and more punctate neurite localisation. (G-I): Immunocytochemistry for PSD-95 (green, G), synaptophysin (red, H) and merged (I). Arrows in I indicate areas of close apposition of PSD-95 and synaptophysin staining. Images are the composite of 32 confocal slices. Scale bar = 50 µM.

There are differing reports as to the neuronal subtype best represented by differentiated SH-SY5Y cells, with different protocols reporting either a dopaminergic (Niaz et al. 2021) or cholinergic phenotype (De Medeiros et al. 2019). For this reason, we used Western blotting to examine the presence and relative levels of markers of different neuronal subtypes. Consistent with our differentiated SH-SY5Y cells having a cholinergic phenotype, we observed a robust increase in the expression of choline acetyltransferase (ChAT) over the timecourse of differentiation (Fig. 5A). In contrast, expression of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis, was expressed at high levels on Day 0, however this level rapidly decreased as the cells differentiated, becoming almost undetectable by Day 10 (Fig. 5B). Interestingly, expression of glutamate-aspartate transporter (GLAST), a key protein in cellular glutamate uptake and often used as a marker of glutamatergic neurones (Rodríguez-Campuzano and Ortega 2021), was also increased during differentiation (Fig. 5C), implying that the fully differentiated cells have either a mixed cholinergic and glutamatergic phenotype or that there is a mixed population. Interestingly, glutamine synthetase (GLUL) levels did not change significantly during our differentiation protocol (Fig. 5D), implying that either this is not a reliable marker of glutamatergic neurones, or that our protocol results in an incomplete glutamatergic phenotype. It should be noted that there is scant evidence in the literature of differentiated SH-SY5Y cells expressing AMPA or NMDA receptors, although some studies have demonstrated the expression of mRNA (Anchesi et al. 2023) and protein (Yang et al. 2020) of both under certain conditions (often a combination of RA and other factors, e.g. GLP-1 or THC).

Differentiated cells display a mixed cholinergic/glutamatergic phenotype. All experiments involved SDS-PAGE and Western blotting of whole cell lysates at the designated timepoints. (A): Expression of choline acetyltransferase (ChAT). Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. (B): Expression of tyrosine hydroxylase (TH). Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. (C): Expression of glutamate-aspartate transporter (GLAST). Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. (D): Expression of glutamine synthetase (GLUL). Top: quantification of the relative expression levels normalised to D10. Bottom: representative Western blot. No significant differences observed. For all graphs, values are mean ± SEM (n = 3). 1-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ****, p < 0.0001.

Tau is a microtubule-binding protein which plays important roles in neuronal growth and development including microtubule polymerisation and stabilisation e.g. (Kadavath et al. 2015), however, it is best known as the major component of neurofibrillary tangles (NFTs) in Alzheimer’s disease (Masters et al. 2015). Indeed several studies have indicated that cognitive decline correlates more closely with the extent of Tau pathology (termed Braak staging) than any other pathological hallmark of Alzheimer’s Disease (Lowe et al. 2018). In NFTs, Tau is typically found in a hyperphosphorylated state, leading to the general view that all Tau phosphorylation is neurotoxic. However, this is not always the case, and Tau phosphorylation (even at residues associated with AD-pathology) has been demonstrated to occur during development of neurones and the nervous system (Fuster-Matanzo et al. 2009, 2012; Hefti et al. 2019). We therefore examined differentiation-dependent regulation of phosphorylation of Tau at three specific residues, S202/T205 (collectively known as the AT8 epitope) and pS396, which are all associated with AD pathology. The pS396 antibody detects bands of 4 different molecular weights – at ~ 50 kDa (presumably representing pS396 monomeric Tau), at > 100 kDa band (presumably oligomers or dimers of pS396 Tau) and two bands between, at approximately 55 and 70 kDa, which we presume represent either multiple phosphorylated forms of Tau or different isoforms. Interestingly, we noticed a reciprocal relationship between the 100 kDa band and the lower molecular weight bands during differentiation, with the 50, 55 and 70 kDa bands increasing and the 100 kDa band decreasing in abundance between Day 0 and Day 10 (Fig. 6A-D). These results suggest that during neuronal differentiation, whilst phosphorylation of Tau at pS396 increases, the formation of higher-order structures decreases. In contrast, for the S202/T205 epitope, we noted a consistent increase in Tau phosphorylation across the timecourse. In our hands, this antibody also revealed multiple bands between 75 kDa and > 200 kDa, however it was the doublet at around 78 kDa (the predicted molecular weight according to the manufacturer, see Table 1 for details) which showed a strong developmental trend (Fig. 6E and F). It should also be noted that total Tau expression is strongly induced by our differentiation protocol (Fig. 6G), indicating that this is potentially behind the increase in abundance of phosphorylated species of Tau seen in this study. Importantly, however this increase in total Tau expression is in contrast to the decrease in pS396 Tau oligomers. Whilst we cannot ascribe a specific function to these phosphorylated Tau species in differentiation, the fact that they are differentially developmentally regulated implies that dysregulation of neurogenesis, known to occur in AD, might be an instigator of aberrant Tau phosphorylation. Interestingly, both the S396 and S202/T205 epitopes have been reported to be phosphorylated by CDK5/p35 and GSK3β (Johnson and Stoothoff 2004; Li et al. 2006; Gao et al. 2020), implying that these kinases are activated during the process of differentiation in SH-SY5Y cells. Indeed, this observation is in line with studies demonstrating a critical role for CDK5 in adult hippocampal neurogenesis (Lagace et al. 2008). Thus, the SH-SY5Y differentiation protocol outlined in this study represents a viable and robust model to investigate this.

Tau phosphorylation at residues associated with AD pathology displays differentiation-dependent regulation. All experiments involved SDS-PAGE and Western blotting of whole cell lysates at the designated timepoints. (A-C): Quantification of 3 separate pS396 bands detected on Western blot. These bands are ~ 100 kDa ((A), presumably dimers of phosphorylated Tau (pTau)), ~ 75 kDa ((B), presumably either a multiply phosphorylated monomeric pTau or a monomer of a longer isoform also detected by this antibody) and ~ 50 kDa ((C), presumably monomeric pTau). Bands quantified are indicated in representative Western blot (D). Quantification of the relative signal is normalised to D10. Values are mean ± SEM (n = 3). Bottom: representative Western blot. (E): Quantification of S202/T205 phosphorylation epitope (also referred to as AT8), relative signal levels normalised to D10. (F): representative Western blot, with the 78 kDa band quantified indicated with an arrow. (G): Above: Total tau quantification, relative signals normalised to D10. Below: Representative Western blot of total tau and GAPDH. For all graphs, values are mean ± SEM (n = 3). 1-way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, ****, p < 0.0001.