Project Topics
Get Complete Project Material File(s) Now! »
Chapter 3 Metabolic comparison of SY5Y with human neural stem cells
Introduction
Nowadays, more than six million American people are suffering from neurodevelopmental dysfunction and neurodegenerative diseases (Center), mainly as a result of compromised functioning of neuronal cells. Thus, many studies have focused on understanding the mechanisms of dysregulation in these cells. Because of the ethical problems of obtaining human neuronal cells and mature neurons do not divide, a stable immortal human neuronal cell line is critical for the understanding of nervous system biology (Gordon et al., 2013). A Neuroblastoma SH-SY5Y cell line is the most widely used neuronal model for in vitro studies of neuronal differentiation and cytotoxicity. The genetic characteristics of this cell line before and after retinoic acid (RA) induced differentiation have long been reported (Lopes et al., 2010; Schneider et al., 2011). The topic of cellular metabolism, in particular how cellular bioenergetics may modulate cellular functionality, has gained intense interest recently as increasing lines of evidence have shown that metabolism is strongly associated with neuronal differentiation, maturation, aging and oxidative response (Agathocleous et al., 2012; Agostini et al., 2016; Zheng et al., 2016). After exposure of RA, SY5Y cells exhibited greater stimulation of mitochondrial respiration with uncoupling and increased maximum respiration capacity while the mitochondrial number was not changed in terms of both mitochondrial DNA copy and citrate synthase (Schneider et al., 2011). RA-induced differentiation also confers SY5Y cells higher tolerance to neurotoxicity and ROS, potentially by up-regulating surviving signals and antioxidative ability (Cheung et al., 2009; Schneider et al., 2011). However, controversy surrounding the use of SH-SY5Y cells exists largely due to their origin, in that they were originally derived from bone tumour biopsy (Gordon et al., 2013). Little has been done on the comparison between SH-SY5Y cells and NSCs, let alone comparison of their metabolic profiles. In this chapter, we aim to compare metabolic characteristics of SH-SY5Y cells with those of human NSCs before and after differentiation and provide evidence in support of using SH-SY5Y as an appropriate neuronal model in the context of metabolic studies.
Results
Morphological changes of SH-SY5Y and human NSCs after differentiation
To compare the characteristics of differentiated SH-SY5Y and human NSCs, SH-SY5Y cells were differentiated by addition of RA into culture medium for five days while human NSCs were induced by using B27-contained Neurobasal™ medium for seven days. After five days of differentiation, mature SH-SY5Y neurons displayed extensive neurite outgrowth and branching compared to the undifferentiated control cells (Fig 3-1). As expected, cell density appeared to be lower post differentiation when compared to that of undifferentiated cells. This is consistent with previous studies which showed that differentiated neurons are post-mitotic and do not divide (in contrast to the highly proliferative undifferentiated neuroblasts) (Ruijtenberg and van den Heuvel, 2016). After seven days of differentiation, the human NSCs showed a similar decrease in cell density and an increase in neurite outgrowth and branching. Similar to undifferentiated SH-SY5Y neuroblasts, undifferentiated NSCs were highly proliferate and grew denser after seven days in culture, in contrast to the differentiated NSCs which have lower cell density in culture but exhibited extensive neurite outgrowth and branching (Fig 3-1).
Cells were cultured in a PDL pre-coated chamber slide; the figure represents typical morphology of eight independent experiments. The top panel compares the morphology of SH-SY5Y cells before and after five days differentiation. Before differentiation, cells showed a higher density but shorter neurites, while differentiated SY5Y cells showed less cell density but increased neurite outgrowth and branching. The bottom panel compares the morphology of NSCs before and after seven days differentiation. Before differentiation, the cells showed a higher density and no neurite can be seen; while differentiated NSCs showed less cell density but increased neurite outgrowth and branching. UNDIFF: undifferentiated; DIFF: differentiated.
Mitochondrial DNA copies (mtDNA) of SH-SY5Y and human NSCs didn’t change by differentiation
It is reported that mitochondrial DNA levels increase during neuronal differentiation (Agostini et al., 2016). To investigate whether mitochondrial DNA levels were changed during the differentiation of these two neural cell lines, three mitochondrial genes (encoded within the mitochondrial genome), MT-ND2, MT-ND5 and MT-ATP6, were measured by real time qPCR. As shown in Fig 3-2, expression of the three mitochondrial genes remained unchanged both in differentiated SH-SY5Y cells and human NSCs compared to their undifferentiated control cells, indicating that mitochondrial DNA copies were unchanged during the differentiation process of these two cell lines. This result is consistent with the previous report that mitochondrial DNA is not changed during the differentiation of human NSCs to motor neurons (O’Brien et al., 2015).
After differentiation, total RNA was extracted from SH-SY5Y cells and human NSC and levels of MT-ND2, MT-ND5 and MT-ATP6 were assessed by real time pPCR and normalized to nucleus PPIA. UNDIFF: undifferentiated; DDIFF: differentiated. Values are mean (± s.e.m) from three independent experiments, performed in triplicate. Statistically significant difference: one-way ANOVA, * P<0.05.
Glycolytic function of SH-SY5Y and human NSCs before and after differentiation
Glycolysis of SH-SY5Y and NSCs were measured by a Seahorse XF extracellular flux analyser as described in section 2.10. Glycolytic profiles of SH-SY5Y and NSCs are shown in Fig 3-3a and Fig 3-3d. Both cell lines showed marked increase in extracellular acidification rate (i.e., glycolytic activity) and post differentiation (compared to the respective undifferentiated control). Particularly, basal glycolysis of differentiated SH-SY5Y cells dramatically increased by 69% (p<0.01; Fig 3-3b), while maximal glycolytic capacity significantly increased by 93.7% (p<0.01; Fig 3-3c), relative to the undifferentiated control. Basal glycolysis of differentiated NSCs similarly increased by 67% (p<0.01, Fig 3-3e) while maximal glycolytic capacity significantly increased by 31% (p<0.05; Fig 3-3f), relative to the undifferentiated control.
SH-SY5Y and human NSCs were differentiated (or not) as described in Section 2.2.2 followed by glycolysis test as described in Section 2.10. (a, d) Glycolysis profiles of SH-SY5Y and human NSCs respectively. (b, e) Basal glycolysis of SH-SY5Y and human NSCs respectively. (c, f) Maximum glycolytic capacity of SH-SY5Y and human NSCs respectively. ECAR: extracellular acidification rate; DIFF: differentiated; UNDIFF: Undifferentiated; Oligo: oligomycin; 2DG: 2-deoxyglucose. Values are mean (± s.e.m) from three independent experiments, and each measuring point was repeated three times, OCR and ECAR values were normalised by collecting protein from each well after experimental completion. Statistical Significance: one-way ANOVA, **p<0.01
Mitochondrial respiration of SH-SY5Y and human NSCs before and after differentiation
An increase in mitochondrial respiration has been reported during the maturation of neural stem cells (Agostini et al., 2016). To compare the characteristics of mitochondrial respiration between SH-SY5Y and human NSC cells, mitochondrial respiration of both cell lines was measured by using the Seahorse metabolic flux analyser and the profiles (measured in the form of oxygen consumption rate, OCR) are shown in Fig 3-4a and Fig 3-4f. Both cell lines showed an overall increase in activity after differentiation (compared to their undifferentiated control,). Differentiated SH-SY5Y cells showed a dramatic 298% increase (p<0.01) in basal mitochondrial respiration (i.e., mitochondrial complex I/III-linked respiration) when comparing to their undifferentiated control (Fig 3-4b). In addition, ATP production and maximal mitochondrial respiration capacity increased by 304% (p<0.01; Fig 3-4c) and 326% (p<0.01; Fig 3-4d) respectively, while proton leak was modestly increased by 176% albeit not statistically significant (Fig 3.4e) in differentiated cells. In contrast, for differentiated NSCs, maximal mitochondrial capacity was significantly increased by 194% (p<0.01, Fig 3.4i), while basal respiration (Fig 3.4g, 27.6% increase), ATP production (Fig 3.4h, 23.2% increase) and proton leak (Fig 3.4j, 72%% increase) showed no statistically significant difference.
Collectively, these results indicated a similar energetic enhancement of glycolysis and mitochondrial respiration during neuronal differentiation of SH-SY5Y and NSCs. Moreover, the increase of mitochondrial function of SH-SY5Y cells was more significant than that of NSCs, which provides more ATP to support neuronal functions.
SH-SY5Y and human NSCs were differentiated (or not) as described in Section 2.2.2 followed by mitochondrial stress test as described in Section 2.10. (a, f) Mitochondrial respiration profiles of SH-SY5Y and human NSCs respectively. (b, g) Basal mitochondrial respiration of SH-SY5Y and human NSCs respectively; (c,h) ATP related respiration of SH-SY5Y and human NSCs respectively; (d,i) Maximum mitochondrial respiration capacity of SH-SY5Y and human NSCs respectively; (e, j) Proton leak related mitochondrial respiration of SH-SY5Y cells and NSCs. ECAR: extracellular acidification rate; DIFF: differentiated; UNDIFF: Undifferentiated; Oligo: oligomycin; 2DG: 2-deoxyglucose. Values are mean (± s.e.m) from three independent experiments, and each measuring point was repeated 3 times, OCR and ECAR values were normalized by collecting protein from each well after experimental completion. Statistical Significance: one-way ANOVA, **p<0.01
Differential cellular bioenergetics between that of differentiated SH-SY5Y and human NSCs
Emerging evidence suggests a shift in metabolic utilisation away from the exhaustive glycolysis to favour oxidative phosphorylation is important for the initiation of neural differentiation (Agostini et al., 2016). Therefore, we compared similarities (or differences) in the bioenergetics of energy production between differentiated SH-SY5Y and human NSCs. Following acquisition of mitochondrial basal respiration (OCR) and basal glycolysis (ECAR) profiles, an energy map of SH-SY5Y cells and NSCs pre- and post-differentiation was then generated. Fig 3-5 illustrates the association between OCR and ECAR in the neuronal cells. SH-SY5Y cells (outline-symbolled data) showed a steep increase in OCR and a mild increase in ECAR. In contrast, the solid black data for NSC cells, showed a mild increase in ORC but a steep increase in ECAR. These results suggest that both SH-SY5Y cells and NSCs increased not only mitochondrial function, but also glycolysis for energy generation following differentiation, although the reliance on glycolytic flux is greater in the differentiated SH-SY5Y cells.
Basal glycolysis (ECAR) from section 3.2.2 and basal mitochondrial respiration (OCR) from section 3.2.3 were used to generate this energy map, which showed the energetic source for SH-SY5Y and NSCs before and after differentiation. The dashed arrow indicates the bioenergetics changes of SH-SY5Y cells while the black arrow indicates that of NSCs.
Metabolic shift towards glycolysis following glucose starvation in SY5Y neuroblasts
It is reported that undifferentiated SY5Y cells are more dependent on glycolysis, and a transition from glycolysis to oxidative phosphorylation is tightly coupled to neuronal differentiation (Agathocleous et al., 2012). However, we saw increased glycolysis as well as mitochondrial oxidative phosphorylation in the differentiated cells (compared to their undifferentiated state). To further investigate the dynamics of cellular bioenergetics of neuronal cells, we assessed the glycolytic flux and mitochondrial respiration following glucose starvation in SY5Y neuroblasts. In the glucose-fed (control) cells, both mitochondrial respiration and glycolytic flux were unchanged during the assay (Fig 3-6a). In the glucose-starved cells, baseline mitochondrial respiration was 1.5-fold higher than that of the glucose-fed (control) cells. However, upon acute exposure to injected glucose in the test media, mitochondrial respiration in the glucose-starved cells was reduced to a level like that of glucose-fed cells at baseline (Fig 3-6a). Interestingly, this decrease was concomitant with a dramatic rise in glycolytic flux (in the glucose-starved cells) (Fig 3-6b). Glycolytic flux was 3-fold higher in the glucose-starved cells compared to the control cells upon acute exposure to injected glucose in the test media (Fig 3-6b).
Collectively, these differential bioenergetic responses to acute glucose exposure suggest that SY5Y neuroblasts utilise both glycolysis and mitochondrial respiration as energy sources when glucose is present. However, as shown in Figure 3-6c, the cells significantly increased mitochondrial respiration when glucose was deprived. Furthermore, upon re-introduction of glucose, the energy map (OCR/ECAR) of cells was markedly shifted from mitochondrial respiration to glycolysis in the glucose-starved cells. In contrast, the shift in the glucose-fed (control) cells was marginal upon re-introduction of glucose (Fig 3-6c). The apparent shift in the OCR/ECAR ratio indicated an over-compensation of glycolysis in the glucose-starved cells after the re-introduction of glucose.
SH-SY5Y neuroblasts were cultured as described in Section 2.10. Prior to initiation of glycolytic flux assay, cells were incubated with Seahorse Assay medium (pyruvate included) with (or without) 10 mM glucose for 1 h at 37oC in a CO2-free incubator. Following three baseline readings during the assay, additional glucose (10 mM) was added to acutely stimulate glycolysis. Profiles of (a) mitochondrial respiration and (b) glycolysis in SH-SY5Y neuroblasts. (c) Relationship between OCR and ECAR (energy map) at baseline and after glucose injection. OCR: oxygen consumption rate; ECAR: extracellular acidification rate; ‘Glucose’ or ‘+glu’: 10 mM glucose injection into test media. Values are mean (± s.e.m) from three independent experiments. OCR and ECAR values were normalized by collecting protein from each well after experimental completion.
Effect of inhibitors 2DG and antimycin on cellular bioenergetics of undifferentiated and differentiated neural cells
To further investigate the extent of reliance of undifferentiated human neural cells on glycolytic and mitochondrial function, we treated undifferentiated SH-SY5Y and human NSCs cells with 2DG (glycolysis inhibitor) or antimycin (mitochondrial respiration complex III inhibitor) for 24 h and then assessed their bioenergetic response. We found that the morphology of undifferentiated SH-SY5Y cells remained unaltered following the treatment of 2DG or antimycin when compared to the control cells (Fig 3-7a). In contrast, cell density was significantly reduced by 44% (p<0.01) following 2DG treatment, and a lesser extent of 34% (p=0.07) following antimycin treatment (Fig 3-7a&b), while cell viability remained relatively unchanged (Fig 3-7b). Compared to the untreated control, cellular ROS production was significantly increased by 38.8% following 2DG treatment, and 144.9% after antimycin treatment (Fig 3-7c). Not surprisingly, SH-SY5Y neuroblasts treated with glycolysis inhibitor 2DG showed reduced lactate generation by 53% (Fig. 3-7d), and glucose consumption to a negligible level (Fig. 3-7e), compared to the untreated control. On the contrary, the antimycintreated SH-SY5Y neuroblasts showed an increased production of lactate and consumption of glucose, 40% for both (p<0.01) as compared to control cells (Fig 3-7d&e).
Although it was not statistically significant, cell density of undifferentiated NSCs was reduced following 2DG and antimycin treatment by 31% and 27%, respectively, when compared to the untreated NSC control (Fig 3-7g). Antimycin treatment significantly increased ROS production by 128% (p<0.01) while 2DG treatment only yielded a not significant 14% increase (Fig 3-7h). The effect of 2DG and antimycin on lactate generation and glucose consumption were similar to those seen in SH-SY5Y cells. 2DG significantly decreased lactate generation by 65% (p<0.01; Fig. 3-7i), and glucose consumption by 81% (p<0.0.1; Fig. 3-7h) compared to the (untreated) control. Also, similar to the responses of SH-SY5Y neuroblasts, antimycin-treated NSCs significantly increased both lactate generation and glucose consumption by about 26.9% and 46.2%, compared to the control, respectively (p<0.01), (Fig 3-7i&j). In summary, the undifferentiated NSCs responded similarly to the metabolic inhibitors when compared to those of the SH-SY5Y neuroblasts.
Undifferentiated SH-SY5Y and NSCs were treated (or not) with 10 mM 2DG or 1 uM antimycin for 24h in culture prior to measurements of glycolytic and mitochondrial function. (a, f) Morphology of undifferentiated (a) SH-SY5Y cells and (f) NSCs visualized under light microscopy. Images were evenly taken at 10x magnification. (b, g) Cell density (column) and viability (#) of undifferentiated SH-SY5Y and NSCs measured by trypan blue exclusion, respectively. (c, h) Intracellular ROS levels of undifferentiated SH-SY5Y and NSCs respectively. (d, i) Lactate production of undifferentiated SH-SY5Y and NSCs respectively. (e, j) Glucose consumption of undifferentiated SH-SY5Y and NSC respectively. Values of images are from four independent experiments. Values of cell density, ROS, glucose and lactate are mean (± s.e.m) from three independent experiments. Statistical significance: one-way ANOVA, **p<0.01 relative to untreated control. 2DG: 2-deoxyglucose; ANTI: antimycin.
udy, differentiated neural cells showed increased glycolysis and mitochondrial respiration (Sections 3.2.2 and 3.2.3). We then investigated whether inhibition of glycolytic flux and mitochondrial respiration would alter (i.e., inhibit) the characteristics of differentiated neuronal cells. Surprisingly, morphology and cell density of the differentiated SY5Y cells remained unaltered in the presence of either 2DG or antimycin (Fig 3-8a&b). Interestingly, cellular ROS production was significantly decreased by 2DG but remained at a similar level as the control cells after the addition of antimycin (Fig 3-8c). Similar to the effect on the undifferentiated SY5Y cells, 2DG reduced glucose consumption by 74% and lactate generation by 49%, while antimycin increased glucose consumption by 127% and lactate generation by 78% (Fig 3-8d&e). The differentiated NSCs cells showed a more complicated neurite connection and have fewer cells than their undifferentiated counterpart. The cell morphology and cell density of the differentiated NSCs remained unchanged following 2DG treatment when compared to the untreated control. However, antimycin reversed the neurite network and increased cell proliferation, resulting in a higher cell density (Fig 3-8f&g). ROS production was decreased by the treatment of both of 2DG (33.7%, p=0.06) and antimycin (42.4%, p<0.05), though the effect of 2DG was less significant than that of antimycin (Fig 3.8h). 2DG tended to reduce glucose consumption and lactate generation of differentiated NSC, but not significantly, while antimycin significantly increased both the glucose consumption (586%, p<0.01) and lactate generation (162.5%, p<0.01) compared to the untreated control (Fig 3-8j).
Taken together, the findings in this study showed that the bioenergetic characteristics of undifferentiated SH-SY5Y cells and human NSCs are largely similar: both glycolysis and mitochondrial respiration are needed as their energy resources and blocking glycolysis by 2DG or mitochondrial respiration by antimycin results in elevated ROS production and proliferation inhibiting. Intriguingly, the SH-SY5Y cells differentiated by RA show different profiles from the differentiated NSCs. Though these two cells showed a similar response of lactate generation and glucose consumption to 2DG and antimycin, the SH-SY5Y cells’ cell density and morphology remained unchanged following the treatment of these two inhibitors while antimycin reversed the differentiation of the NSCs, indicating that mitochondrial respiration is essential for differentiated NSCs.
Table of Contents
Chapter 1 Literature review
1.1 Neurodevelopment: from cell commitment to maturation
1.2 Micronutrients and neural development
1.3 Lutein
1.4 The antioxidant effect of lutein
1.5 Lutein is more than simply an antioxidant for neurons
1.6 DHA
1.7 Metabolic re-programming underpins neuronal differentiation
1.8 Changes in mitochondrial functionality and structure support cell differentiation
1.9 Metabolism regulates differentiation via epigenetic regulation
1.10 Modification of histones facilitates the expression of neural genes during differentiation
1.11 DNA methylation regulates the sequential neuronal gene expression during differentiation
1.12 The effects of micronutrients on epigenetics and gene expression
1.13 Micronutrients enhance neuronal metabolism during the differentiation process
1.14 Reactive oxidative stress
1.15 ROS and neurodegenerative diseases
1.16 ROS and neurodevelopment
1.17 Hypothesis and Aims
Chapter 2 Materials and methods
2.1. Materials
2.2. Cell culture of SH-SY5Y
2.3. Measurement of enzymatic activities
2.4. Measurement of metabolic products
2.5. RNA isolation, mRNA array, miRNA array, and quantitative real time PCR analysis
2.6. Mitochondrial DNA extraction and MT-DNA measurement
2.7. Measurement of ROS
2.8 Mitochondrial membrane potential assay
2.9 Measurement of glucose consumption and lactate generation
2.10 Extracellular mitochondrial respiration and Glycolysis
2.11 DPPH free radical scavenging assay
2.12 Western Blotting
2.13 Statistical analysis
Chapter 3 Metabolic comparison of SY5Y with human neural stem cells
3.1. Introduction
3.2. Results
3.3. Discussion
Chapter 4 Modulation of cellular metabolism underpins lutein and DHA enhanced neural differentiation
4.1 Introduction
4.2 Results
4.3 Discussion
Chapter 5 The role of ROS in micronutrients mediated neuronal differentiation
5.1 Introduction
5.2 Results
5.3 Discussion
Chapter 6 Micronutrient affect gene expression via epigenetics during neuronal differentiation
6.1 Introduction
6.2 Results
6.3 Discussion
Chapter 7 Conclusion
Reference
GET THE COMPLETE PROJECT
Dietary micronutrients enhance neural differentiation through the modulation of cellular metabolism
