Metabolic Enhancement of Glycolysis and Mitochondrial Respiration Are Essential for Neuronal Differentiation

Abstract

Metabolic reactions provide energy and metabolic substance for cell function. It was recently shown that metabolic reprogramming is a key regulator of cell pluripotency and differentiation. Although many evidences point to a metabolic “switch” toward mitochondrial respiration, the importance of glycolysis and mitochondrial respiration is still controversial. In this study, we differentiated two different neuronal cells and compared the glycolytic and metabolic profile before and after differentiation. The results showed a significant increase in glycolysis (includes basal glycolysis and glycolytic capacity) and mitochondrial respiration (includes mitochondrial basal respiration, adenosine triphosphate production, and mitochondrial respiration capacity) of both SY5Y and neural stem cells (NSCs) during neuronal differentiation, whereas their mitochondrial DNA copies remain unchanged. Antimycin, a mitochondrial inhibitor, reduced cell density of differentiated SY5Y cells. However, for differentiated NSCs, antimycin dedifferentiated the cells, resulted in a significant increase in cell density, and lowered oxidative stress. In conclusion, this study demonstrated that metabolic enhancement of glycolysis and mitochondrial respiration (rather than a “switch”) are both important for neuronal differentiation, although only the blocking of mitochondrial respiration reverses the differentiation process.

Introduction

Neuronal development is represented as neuronal differentiation from neural stem cells (NSCs) to neurons, which occurs during infant nervous system development and nervous system repair/renewal, involving cell proliferation, cell cycle arrest, differentiation, migration, and apoptosis. The importance of this process for brain growth and neurocognitive ability has been well recognized (Candel Pau et al., 2015; Fuglestad et al., 2015). This process, also termed neurogenesis, shapes infant neurocognitive ability that affects their psychological adaptation for a lifetime and protects adults and elders from neurodegenerative diseases. Although one study found that hippocampal neurogenesis drops to undetectable levels in adults (Sorrells et al., 2018), other studies demonstrated that hippocampal neurogenesis is a seasons-crossing phenomenon.

Neurogenesis occurs not only in infants but persists in adults and aging population (Boldrini et al., 2018); it is reported that a third of the human hippocampal cell population is subject to turnover at a rate of 1.75% annually during adulthood (Kheirbek and Hen, 2013; Spalding et al., 2013), and shows variable degrees of maturation along differentiation stages in healthy adults (Moreno-Jimenez et al., 2019). However, the neurogenesis process declines as Alzheimer's disease progresses, showing the importance of neurogenesis to brain health.

Neuronal cells are highly energetic to maintain their signal transmission. Thus, cellular metabolism, in particular, how cellular bioenergetics may modulate cellular functionality, has gained intense interest recently. Increasing evidence has 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). It was reported that glycolysis plays a predominant role at early phase of differentiation, whereas mitochondrial bioenergetics is required for later stages of differentiation, including dendritic outgrowth and maturation. The splicing regulator RBM4 may play an important role in this glycolysis to mitochondrial respiration switch (Su et al., 2017).

However, on the contrary, it is reported that increased glycolytic rate, decreased mitochondrial DNA (mtDNA) copies, and mitochondrial activity are essential during the differentiation of human pluripotent stem cells to neural precursor cells (Lees et al., 2018). The importance of glycolysis is also well recognized in mature glial cells, which provides substances, like alanine and lactate, to fuel neuronal mitochondria (Volkenhoff et al., 2015). The confusing information on metabolic change during neuronal differentiation may be caused by the type of neuronal cells or the stage of differentiation. In this study, two neuronal cell lines, namely SH-SY5Y cells and human NSCs, were deployed and differentiated into mature neurons. The metabolic profiles of these two cell lines were investigated to explore our understanding of the metabolic variation during neuronal differentiation.

Materials and Methods

Materials

SH-SY5Y cells were purchased from American Type Culture Collection (Manassas, VA). Retinoic acid (RA); metabolic inhibitors, 2-deoxyglucose (2DG) and antimycin; and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO). Human neural stem cells (H9-derived) kit, neurobasal medium, B27 serum-free supplement, glutaMAX, glucose, and reactive oxidative species (ROS) fluorescence indicator H₂DCFDA were purchased from Thermo Fisher Scientific (Waltham, MA).

Cell culture and differentiation

SH-SY5Y cells were cultured in Dulbecco's modified eagle medium:F12 (DMEM/F12), with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL) in a humidified 5% carbon dioxide (CO₂) incubator at 37°C. Neuronal differentiation was induced by RA by following the method developed by Schneider et al. (2011). In brief, cells (<10 passages) were seeded at 3 × 10⁵ cells/mL in Seahorse culture plates or six-well plates, after a 24-hour attaching, 10 μM RA was added to induce cell differentiation for 5 days and differentiation medium was renewed every 2 days.

NSCs were cultured in complete NSC culture medium, which contains basic fibroblast growth factor, epidermal growth factor, and neural supplement by following manufacturer's protocol. For differentiation, cells (<3 passages) were seeded at 3 × 10⁵ cells/mL on polyornithine and laminin-precoated Seahorse culture plates or six-well plates; 48 hours after seeding, neural differentiation medium (neurobasal medium with B27 and glutaMAX) was used to induce neuronal differentiation for 7 days and medium was renewed every 3–4 days.

Cellular metabolic flux assay

Cells were seeded in precoated XF eight-well cell culture Microplates (Seahorse Bioscience, Copenhagen, Denmark) in triplicate at 3 × 10⁴ cells per well in 200 μL growth medium and then cultured at 37°C in a 5% CO₂ incubator. After treatment, assays were initiated by removing the growth medium from each well and replacing with 175 μL prewarmed assay medium [pH 7.4, containing XF Base medium (Seahorse Bioscience), 10 mM glucose, and 1 mM sodium pyruvate]. Then cells were incubated at 37°C in a non-CO₂ incubator for 1 hour to allow media temperature and pH to reach equilibrium before measurement.

MitoStress Assay Kit (Seahorse Bioscience) was used to measure extracellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) according to manufacturer's protocol. After initial baseline measurements, the following compounds were added as follows: adenosine triphosphate (ATP) synthase inhibitor oligomycin (2 μM), electron transport chain accelerator p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) at 2 μM, and finally, mitochondrial complex I/III inhibitors rotenone/antimycin mix (1 μM each). Three measurements were taken following the addition of each compound. Metabolic data of cells were normalized to total cellular protein content, which is determined using Pierce™ BCA Protein Assay (Thermo Fisher Scientific).

Measurement of cell density and viability

In brief, cells were seeded in a 24-well plate at a density of 3 × 10⁴ cells per well and treated with 50 mM 2DG or 1 μM antimycin. Cell viability was then determined by trypan blue exclusion: Floating cells were discarded with the culture medium, then cells were treated with 50 μL 0.25% trypsin ethylenediaminetetraacetic acid (EDTA) for 2 minutes, and then suspended in 150 μL fresh cell culture medium. Cell density and viability were determined by trypan blue exclusion. Briefly, 20 μL cells were mixed with 20 μL 0.4% trypan blue solution, and then cells were counted in a hemocytometer. Cell density was calculated as a percentage of (untreated) control cells.

Measurement of cellular ROS

Cellular ROS was measured as previously (Lee et al., 2011). Cells were cultured in 48-well plates. After inhibitor (2DG or antimycin) treatment, cells were incubated with 5 μM H₂DCFDA in DMEM/F12 (phenol red-free) at 37°C for 30 minutes. Fluorescence was then read with microplate reader Synergy 2 (BioTek Instruments, Inc., Winooski, VT) at excitation/emission 485/530 nm.

Mitochondrial DNA extraction and measurement

Cells in six-well plates were washed by prewarmed phosphate-buffered saline and then harvested by 200 μL 0.25% trypsin-EDTA. Cell pellet was suspended with 600 μL of extraction buffer (NaCl 75 mM, 50 mM Tris-HCl pH 8.5, EDTA 5 mM), 1% sodium dodecyl sulfate solution, proteinase K (100 μg/mL), and incubated at 55°C for 2 hours. Then samples were incubated for 10 minutes at 95°C to inhibit proteinase K and centrifuged at 8000 rpm for 15 minutes at 4°C. Supernatant was recovered and the same volume of phenol/chloroform/isoamyl alcohol (25:4:1) was added to separate nucleotides and proteins.

After a brief vortex and centrifugation at 13,000 rpm for 15 minutes at 4°C, the aqueous phase was recovered and DNA was precipitated with 1/10 NaAc (3M, pH 5.2) and 2 volume of isopropanol (or ethanol) at −20°C for 20 minutes. Then DNA pellet was air-dried after 70% ethanol washing. Finally, DNA sediment was dissolved in 20 μL sterile water at room temperature. DNA concentrations were measured using a Nanodrop 1000 spectrometer (Thermo Scientific). The copies of mitochondrial DNA (MT-DNA) were measured by quantitative polymerase chain reaction (qPCR) with Roche Master MIX and assays on Light Cycler480 (Roche). The primers are as follows:

MT-ND2: for 5′-CTACTCCACCTCAATCACACTAC-3′; rev 5′-AGGTAGGAGTAGCGTGGTAAG-3′

MT-ND5: for 5′-CTACCTAAAACTCACAGCCCTC-3′; rev 5′-GGGTAGAATCCGAGTATGTTGG-3′

MT-ATP6: for 5′-ACACCCCTTATCCCCATACTAG-3′; rev 5′-ATGGTTGATATTGCTAGGGTGG-3′

Statistical analysis

Data are presented as mean ± standard error of the mean from at least three independent experiments unless otherwise indicated in Figure 5. To assess statistical significance, one-way analysis of variance was performed in Prism version 6.0. Value of p < 0.05 was considered statistically significant.

Results

Neurite outgrowth of SH-SY5Y and human NSCs after differentiation

To compare the metabolic characteristics of SH-SY5Y and human NSCs during differentiation, SH-SY5Y cells were differentiated by RA for 5 days, whereas human NSCs were differentiated by B27-contained Neurobasal™ culture medium for 7 days, according to standard protocols. After 5 days of differentiation, SH-SY5Y cells displayed extensive neurite outgrowth and branching compared with the undifferentiated control cells (Fig. 1, upper panel).

FIG. 1.

Differentiation effects morphology of SH-SY5Y and human NSC. Cells were cultured on a PDL-precoated chamber slide; the figure represents typical morphology of eight independent experiments. The top panel compares the morphology of SH-SY5Y cells before and after 5 days differentiation. Before differentiation, cells showed a higher density but shorter neurites, whereas differentiated SY5Y cells showed less cell density but increased neurite outgrowth and branching. The bottom panel compares the morphology of NSCs before and after 7-day differentiation. Before differentiation, the cells showed a higher density and no neurite can be seen, whereas differentiated NSCs showed less cell density but increased neurite outgrowth and branching (cells are shown in 200 μm for better neurite view). DIFF, differentiated; NSC, neural stem cell; PDL, poly-d-lysine; UNDIFF, undifferentiated.

As expected, cell density appeared to be lower postdifferentiation compared with that of undifferentiated cells. This is consistent with previous studies, which showed that differentiated neurons are postmitotic and do not divide (in contrast to the highly proliferative neuroblasts before differentiation) (Ruijtenberg and van den Heuvel, 2016). After 7 days of differentiation, 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 7 days in culture, in contrast to the differentiated NSCs that have lower cell density but exhibited extensive neurite outgrowth and branching (Fig. 1, lower panel).

Glycolytic function of neuronal cells increased during differentiation

Glycolysis of SH-SY5Y and NSCs were measured by a Seahorse XF extracellular flux analyzer as described in subsection Cellular metabolic flux assay. Glycolytic profiles of SH-SY5Y and NSCs are given in Figure 2a and d. Both cell lines showed marked increase in ECAR (i.e., glycolytic activity) during differentiation (compared with the respective undifferentiated control). In particular, basal glycolysis of differentiated SH-SY5Y cells dramatically increased by 69% (p < 0.01; Fig. 2b), whereas maximal glycolytic capacity significantly increased by 93.7% (p < 0.01; Fig. 2c), relative to the undifferentiated control. Basal glycolysis of differentiated NSCs similarly increased by 67% (p < 0.01; Fig. 2e), whereas maximal glycolytic capacity significantly increased by 31% (p < 0.05; Fig. 2f), relative to the undifferentiated control.

FIG. 2.

Differentiation affects cellular glycolytic function of SY5Y and NSCs. SH-SY5Y and human NSCs were differentiated (or not) as described in subsection Cell culture and differentiation followed by glycolysis test as described in subsection Cellular metabolic flux assay. (a, d) Glycolytic 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. Values are mean (±SEM) from three independent experiments, and each measuring point was repeated three times, OCR and ECAR values were normalized by collecting protein from each well after experimental completion. Statistical significance: one-way ANOVA, **p < 0.01. 2DG, 2-deoxyglucose; ANOVA, analysis of variance; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; Oligo, oligomycin; SEM, standard error of the mean.

Unchanged mtDNA of SH-SY5Y and human NSCs after differentiation

It was reported that mtDNA levels increase during neuronal differentiation (Agostini et al., 2016). To investigate whether mtDNA 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 given in Figure 3, expression of the three mitochondrial genes remained unchanged both in differentiated SH-SY5Y cells and human NSCs compared with their undifferentiated control cells, indicating that the number of mtDNA copies were unchanged during the differentiation process of these two cell lines. This result is consistent with the previous report that mtDNA is not changed during the differentiation of human NSCs to motor neurons (O'Brien et al., 2015).

FIG. 3.

Differentiation has no effect on the mitochondrial content of SY5Y and NSCs. After differentiation, total RNA was extracted from SH-SY5Y cells and human NSC and levels of MT-ND2, MTND5 and MT-ATP6 were assessed by real time qPCR and normalized to nucleus PPIA. Values are mean (±SEM) from three independent experiments, performed in triplicate. Statistically significant difference: one-way ANOVA, *p < 0.05. PPIA, peptidylprolyl isomerase A; qPCR, quantitative polymerase chain reaction.

Mitochondrial respiration of neuronal cells during 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 NSCs, mitochondrial respiration of both cell lines were measured by Seahorse metabolic flux analyzer and the profiles (measured in the form of OCR) are given in Figure 4a and f. Both cell lines showed an overall increase in activity after differentiation (compared with 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) compared with their undifferentiated control (Fig. 4b).

FIG. 4.

Differentiation affects cellular mitochondrial function of SY5Y and NSC. SH-SY5Y and human NSCs were differentiated (or not) as described in subsection Cell culture and differentiation followed by mitochondrial stress test as described in subsection Cellular metabolic flux assay. (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. Values are mean (±SEM) from three independent experiments, and each measuring point was repeated three times, OCR and ECAR values were normalized by collecting protein from each well after experimental completion. Statistical significance: one-way ANOVA, **p < 0.01. ATP, adenosine triphosphate; R/A, rotonone antimycin.

In addition, ATP production and maximal mitochondrial respiration capacity increased by 304% (p < 0.01; Fig. 4c) and 326% (p < 0.01; Fig. 4d), respectively, whereas proton leak was modestly increased by 176% although not statistically significant (Fig. 4e) in differentiated cells. In contrast, for differentiated NSCs, maximal mitochondrial capacity was significantly increased by 194% (p < 0.01; Fig. 4i), whereas basal respiration (Fig. 4g, 27.6% increase), ATP production (Fig. 4h, 23.2% increase), and proton leak (Fig. 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.

Effect of glycolytic inhibitor 2DG and mitochondrial inhibitor antimycin on cellular bioenergetics of differentiated neural cells

To further investigate the extent of reliance of differentiated neural cells on glycolysis and mitochondrial respiration, differentiated SH-SY5Y and human NSCs were treated with 2DG (glycolytic inhibitor) or antimycin (mitochondrial respiration complex III inhibitor) for 24 hours and then assessed their bioenergetic response. Surprisingly, morphology and cell density of the differentiated SY5Y cells remained unaltered in the presence of either 2DG or antimycin (Fig. 5a, b), whereas cellular ROS production was significantly decreased by 2DG but remained at a similar level as the control cells after the addition of antimycin (Fig. 5c). 2DG reduced glucose consumption by 74% and lactate generation by 49%, whereas antimycin increased glucose consumption by 127% and lactate generation by 78% (Fig. 5d, e).

FIG. 5.

Effects of inhibitors on differentiated neural cells. Differentiated SH-SY5Y and NSCs were treated (or not) with 10 mM 2DG or 1 μM antimycin for 24 hours in culture before measurements of glycolytic and mitochondrial function. (a, f) Morphology of differentiated (a) SH-SY5Y cells and (f) NSCs visualized under light microscopy. Images were taken at 10 × magnification. (b, g) Cell density (column) and viability (#) of differentiated SH-SY5Y and NSCs, respectively. (c, h) Intracellular ROS levels of differentiated SH-SY5Y and NSCs, respectively. (d, i) Lactate production of differentiated SH-SY5Y and NSCs, respectively. (e, j) Glucose consumption of differentiated SH-SY5Y and NSCs, respectively. Values of images are from four independent experiments. Values of cell density, ROS, glucose and lactate are mean (±SEM) from three independent experiments. Statistical significance: one-way ANOVA, **p < 0.01, *p < 0.05 relative to untreated control. ANTI, antimycin; ROS, reactive oxidative species.

The differentiated NSCs 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 after 2DG treatment compared with the untreated control. However, antimycin reversed the neurite network and increased cell proliferation, resulting in a higher cell density (Fig. 5f, g). ROS production was decreased by the treatment of both 2DG (33.7%, p = 0.06) and antimycin (42.4%, p < 0.05), although the effect of 2DG was less significant than that of antimycin (Fig. 5h). 2DG tended to reduce glucose consumption and lactate generation of differentiated NSC, but not significantly, whereas antimycin significantly increased both the glucose consumption (586%, p < 0.01) and lactate generation (162.5%, p < 0.01) compared with the untreated control (Fig. 5j).

Discussion

Although the human brain accounts for only 2% of body weight, it consumes 20% of the energy derived from glucose; hence, making brain the main consumer of glucose (Mergenthaler et al., 2013). Extensive metabolic reprogramming occurs during cell differentiation (Esteban-Martinez and Boya, 2018). Previous studies reported that glycolysis is critical for energy generation in highly proliferative cells (Agostini et al., 2016), and high metabolic flux through exhaustive glycolysis is a common feature of undifferentiated neurons to facilitate both cellular bioenergetics and biosynthesis for cell proliferation (O'Brien et al., 2015). Therefore, a metabolic “switch” from cytoplasmic glycolysis to mitochondrial oxidative phosphorylation is needed to meet the high energy requirement of mature neuronal cells in the brain (Agathocleous et al., 2012; Zheng et al., 2016).

However, a metabolic shift toward glycolysis is also reported as essential for retinal ganglion cell (RGC) neurogenesis (Esteban-Martinez and Boya, 2018), indicating that our understanding on metabolic reprogramming during neuronal differentiation needs more investigation. In our study, both SH-SY5Y and NSCs lines experienced an increase in metabolic activities after differentiation. However, we did not observe a “switch” from glycolysis to mitochondrial respiration, but rather strengthen in both glycolysis and mitochondrial respiration.

Increased glycolysis may facilitate neuronal differentiation by supplying pyruvate in vitro (Suzuki et al., 2016). Pyruvate can be utilized by lactate dehydrogenase A, relieving the burden of mitochondria as an energy house and exporting out of mitochondria to generate acetyl-CoA and promote histone acetylation in selected gene loci (Peng et al., 2016), which are associated with neuronal differentiation (Moussaieff et al., 2015). Moreover, glycolysis fuels pentose-phosphate pathway, which modulates neuronal differentiation through regulating the protein expression and activity of glucose 6-phosphate dehydrogenase (Almeida et al., 2018). In contrast, pharmacological and genetic inhibition of glycolysis consistently inhibited RGC differentiation (Esteban-Martinez et al., 2017).

The human mitochondrial genome is a double-strand circular molecule of 16 569 bp and encodes 13 proteins that are core subunits for oxidative phosphorylation (Olson and Mello, 2010).

Many studies have demonstrated that the number of mtDNA varies among different tissues during differentiation, aging, and pathological conditions (Rathod et al., 2016). O'Brien found a significant increase in mtDNA-encoded genes, ND2 and ND4, at 21 days after the differentiation of motor neurons from human embryonic stem cells (hESC), but no significant changes in mtDNA at 21 days after the differentiation of motor neurons from induced pluripotent stem cells (iPSC) (O'Brien et al., 2015). In an ex vivo study of cortical neuronal development, Agostini et al. (2016) isolated primary cortical neurons from E17.5 mouse embryos and differentiated them by B27 in a neurobasal medium, a significant increase in mtDNA copies was recorded as well as protein expression of electron transport chain (ETC) complexes at 7 days in vitro.

To further study the variations of mtDNA copies during differentiation, Augustyniak et al., 2017, and Zemach et al., 2010, studied the mtDNA copies during the differentiation of three iPSC origined cells, NSCs, early neural progenitor (eNP), and NP. Differentiated by idebenone for 5 days, mtDNA copies only increased in eNP cells, whereas the number of mtDNA copies of NSCs and NP remained unchanged. This result indicates that the mtDNA increase is a developmental stage-specific event (Lev Maor et al., 2015). It is also reported that mtDNA copies of SH-SY5Y cells and hESC were both unchanged after differentiation, and this is consistent with a previous report (Schneider et al., 2011), indicating that the increased mitochondrial function was mainly caused by increased activity of mitochondrial ETC complexes (Schneider et al., 2011).

Extensive literatures point to mitochondrial ROS impairing stemness, hence resulting in a preference of glycolysis, rather than mitochondrial oxidative phosphorylation, for energy generation in undifferentiated (proliferative) cells (Varum et al., 2011; Vega-Naredo et al., 2014). However, mitochondrial oxidative phosphorylation is essential for cell differentiation and function. Inducing NSC differentiation into oligodendrocytes and neurons, mitochondrial respiration, membrane potential, and ATP synthesis were all increased in NSCs (Mendivil-Perez et al., 2017). This mitochondrial biogenesis may relate to the epigenetic regulation by Pan-HDAC inhibitors and sodium butyrate (Uittenbogaard et al., 2018).

Antimycin, a mitochondrial complex III inhibitor, prevented embryonic stem cells to differentiate into dopaminergic neurons, maintaining undifferentiated with a high Oct4 levels and a reduction in cell proliferation (Pereira et al., 2013). Not only mitochondrial respiration, but also mitochondrial ROS may also play a role in cell differentiation. ROS can act as a signal transduction molecule, regulating stem cell proliferation and differentiation (Pashkovskaia et al., 2018). On the contrary, dysfunction of mitochondrion cause cell death, and are common pathological processes associated with neurodegenerative diseases, like Parkinson's disease (Iannielli et al., 2018).

Furthermore, differentiated SH-SY5Y cells were less susceptible to metabolic inhibitors of glycolysis and mitochondrial respiration, as evidenced by differences in cell density and cellular morphology outcomes after the exposure to glycolytic inhibitor-2DG and mitochondrial inhibitor-antimycin. This feature of differentiated SH-SY5Y neurons may be related to an upregulation of survival pathways mediated by RA in culture (Cheung et al., 2009). The differentiation process may also confer neuronal cells higher tolerance to neurotoxicity and ROS, potentially by upregulating surviving signals and antioxidative ability (Cheung et al., 2009; Schneider et al., 2011).

However, neurons differentiated from human NSCs were highly susceptible to perturbations in mitochondrial respiration rather than glycolysis. Inhibition of mitochondrial respiration not only abolished neurite outgrowth, but also dedifferentiated NSCs, promoting cellular proliferation that was evidenced by an increase in cell density and absence of neurite outgrowth. These findings highlight that intact mitochondrial respiration is essential for neurite outgrowth of differentiated human NSCs without the protection of RA.

Footnotes

Author Disclosure Statement

The authors declare they have no conflicting financial interests.

Funding Information

The authors received no funding for this research.

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