Neuroscience Letters 585 (2015) 166–170

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Neuroscience Letters

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Short communication

Vitamin E isomer �-tocopherol enhances the efficiency of neural stemcell differentiation via L-type calcium channel

Sihao Denga, Guoqiang Houb, Zhiqin Xuea, Longmei Zhangb, Yuye Zhoub, Chao Liua,Yanqing Liuc, Zhiyuan Lia,b,∗

a Department of Anatomy and Neurobiology, School of Basic Medical Sciences, Central South University, Changsha, Hunan 410013, Chinab Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine andHealth, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, Chinac Xiang-Ya Boai Hospital, Hunan, Changsha 410013, China

h i g h l i g h t s

• �-Tocopherol enhances efficiency of neural stem cell differentiation.• �-Tocopherol promotes morphological maturation of the differentiated neurons.• L-type Ca2+ channels are involved in �-tocopherol induced neuronal differentiation.

a r t i c l e i n f o

Article history:Received 12 September 2014Received in revised form14 November 2014Accepted 19 November 2014Available online 20 November 2014

Keywords:�-TocopherolVitamin ENeural stem cellDifferentiationCalcium channel

a b s t r a c t

The effects of the vitamin E isomer �-tocopherol on neural stem cell (NSC) differentiation have notbeen investigated until now. Here we investigated the effects of �-tocopherol on NSC neural differentia-tion, maturation and its possible mechanisms. Neonatal rat NSCs were grown in suspended neurospherecultures, and were identified by their expression of nestin protein and their capacity for self-renewal.Treatment with a low concentration of �-tocopherol induced a significant increase in the percentage of�-III-tubulin-positive cells. �-Tocopherol also stimulated morphological maturation of neurons in cul-ture. We further observed that �-tocopherol stimulation increased the expression of voltage-dependentCa2+ channels. Moreover, a L-type specific Ca2+ channel blocker verapamil reduced the percentage ofdifferentiated neurons after �-tocopherol treatment, and blocked the effects of �-tocopherol on NSC dif-ferentiation into neurons. Together, our study demonstrates that �-tocopherol may act through elevationof L-type calcium channel activity to increase neuronal differentiation.

© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Natural forms of vitamin E consist of (�-, �-, �-, �-) tocopheroland (�-, �-, �-, �-) tocotrienol isomers, which are hydrophobicfat-soluble compounds found in various food sources [1,2]. Recentstudies have reported that vitamin E has many beneficial healtheffects such as antioxidant and anti-inflammatory properties [3,4].Vitamin E can delay or prevent a clinical diagnosis of Alzheimer’sdisease in elderly persons with mild cognitive impairment. In

∗ Correspondence author at: Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Sciences, Kaiyuan Road 190, Guangzhou, Guangdong 510530,China. Tel.: +86 02032015241.

E-mail addresses: deng sihao2011@csu.edu.cn (S. Deng), li zhiyuan@gibh.ac.cn(Z. Li).

particular, vitamin E supplementation protects cultured hippocam-pal neurons against the neurotoxic effects of oxidative damage[5] and also reverses melamine-induced deficits in spatial cogni-tion and hippocampal synaptic plasticity in rats [6]. Dunn-Thomasreported that vitamin E could influence retinal precursor cell dif-ferentiation by reducing stress-related function [7]. These studiesof vitamin E have primarily utilized �-tocopherol, as it exerts thehighest biological activity [8]. However, recent studies have sug-gested that �-tocopherol may be more effective. �-tocopherol, butnot �-tocopherol, showed promise as a chemopreventive agentdue to its higher cancer preventive activity in animal models[9,10]. �-Tocopherol can reportedly reduce cholesterol accumula-tion potentially through enhancement of lysosomal exocytosis, andit is a novel lead compound for drug development to treat lysoso-mal storage diseases [11]. However, the effects of �-tocopherol onthe differentiation of NSCs are less clear.

http://dx.doi.org/10.1016/j.neulet.2014.11.0310304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

S. Deng et al. / Neuroscience Letters 585 (2015) 166–170 167

Neural stem cells (NSCs) cultured as neurospheres are capableof differentiating into neurons, astrocytes, and oligodendrocytes[12]. The grafting of NSCs improves neurological deficits in neu-rodegenerative diseases such as ischemic stroke and spinal cordinjury [13]. However, the induction of new neuronal cells in thedamaged brain is relatively low and might be nonfunctional [13,14].An alternative is the transplantation of neural stem cells that coulddifferentiate into neurons for tissue repair [15]. However, trans-planted cells face the same challenge of neuronal differentiation,maturation, and integration into host neural networks [16]. Thus,determining how to promote NSC differentiation and maturation isa critical problem for establishing safe and practical cell therapies.

Voltage-gated Ca2+ channels (VGCCs) are classified as two maintypes, including high voltage-activated (HVA; L-, N-, and P/Q-type)and low voltage-activated (LVA; T-type) channels [17]. Recentstudies showed that calcium influx through L-type Ca2+ channelsmight modulate activity-dependent processes involved in neuronaldifferentiation. L-type Ca2+ channel currents play a key role in ori-enting the in vitro differentiation of NSCs toward the neuronalphenotypes [18].

Thus, in the current study, we investigated the effects of �-tocopherol on NSC differentiation and maturation, and found thatL-type Ca2+ channels could play a role in �-tocopherol-induced NSCdifferentiation.

2. Materials and methods

2.1. Cell culture and treatment

Primary NSCs were derived from the brains of postnatal day1 wistar rats (Hunan slack scene of laboratory animal co.) andwere prepared as neurospheres according to methods previouslydescribed [19]. Animal used was in accordance with the NationalInstitutes of Health Guide for the care and use of laboratory animals.All procedures were approved by the ethics committee of Cen-tral South University for experimental usage of laboratory animals.Briefly, rats were deeply anesthetized with sodium pentobarbital(50 mg/kg, i.p.) and the tissue surrounding the lateral ventricleswas carefully removed and digested enzymatically with 2 mg/mlpapain solution at 37 ◦C for 30 min. The digested tissue was sub-sequently triturated gently and passed through a mesh to makea single-cell suspension. Cells were centrifuged and resuspendedin neurobasal-A medium (Gibco) containing 2% B27 supplements(Gibco), 20 ng/ml basic fibroblast growth factor (bFGF; Sigma),20 ng/ml epidermal growth factor (EGF; Sigma), 2 mM l-glutamine(Gibco) and 5 �g/ml heparin (Sigma). Cells were then seeded at aninitial cell density of 1 × 105 cells/ml and maintained in a humidi-fied atmosphere of 5% CO2 at 37 ◦C.

For the differentiation assay, neurospheres were dissociated andplated at 50,000 cells/ml on Matrigel (BD Biosciences)-coated glasscoverslips. The cells were maintained for 7 days in neurobasal-Amedium supplemented with 2% B27, 1 �M retinoic acid (RA, Sigma)and 10 ng/ml recombinant Human brain derived neurotrophicfactor (BDNF, PeproTech). These NSCs were assigned to 6 treat-ment groups: control, �-tocopherol (10 �M), verapamil (10 �M,Sigma), �-conotoxin (10 �M, Alomone labs), �-tocopherol (10 �M)plus verapamil (10 �M), �-tocopherol (10 �M) plus �-conotoxin(10 �M). The duration of the treatment was 6 days. The mediumwas changed every 3 days.

2.2. Immunofluorescence

Cells were fixed in 4% paraformaldehyde (PF) for 20 min andpre-incubated in PBS containing 5% donkey serum for 2 h. Sub-sequently, cells were incubated in PBS containing appropriate

mixture of primary antibodies overnight at 4 ◦C. We used rabbitanti-nestin (1:500, Sigma), mouse anti-�-III-tubulin (1:500, Mil-lipore) and rabbit anti-glial fibrillary acidic protein (GFAP, 1:500,Sigma). This was followed by a 2 h reaction with Alexa Fluor 488and/or Alexa Fluor 594 conjugated donkey anti-mouse, rabbit, orgoat IgGs (1:400, Jackson ImmunoResearch) in the dark. Cellswere then counterstained with bisbenzimide (Hoechst 33,258,1:50,000), washed and mounted before microscopic examination.Samples were then viewed and photographed by using Olympus(BX60) fluorescent microscopes equipped with image analysis sys-tems (cellSens Standard, Olympus). For quantitative analysis, thenumber of �-III-tubulin-positive or GFAP-positive cells on threecoverslips was quantified in twenty randomly selected micro-scopic visual fields per coverslip, and results were expressedas the percentages of bisbenzimide-positive cells that were also�-III-tubulin-positive or GFAP-positive cells. Cell counting was per-formed under a fluorescence microscope with a 10× objective.

2.3. Morphometric measurements of neurites

Neurite branching was analyzed by counting the number ofneurites for each neuron. Only neurons whose soma and neuriteswere completely contained inside the image border were ana-lyzed. Neurite length measurements were taken for the longestneurite present on 150 �-III-tubulin-positive cells from controland �-tocopherol treated groups, using the measurement tool inImageJ. Fields were sampled randomly, and the person execut-ing the measurements was blinded as to the treatment condition.Briefly, images of �-III-tubulin-positive cells were captured at 20×magnification and neurites were measured for each neuron, how-ever, only one longest neurite per cell was used for comparativepurposes. The longest neurite on each cell was drawn using thepencil tool in the ImageJ program. Lengths were determined as thedistance between the edge of the cell body and the tip of the growthcone. Lengths of outlined neurites were then computed using theassociated macro.

2.4. Immunocytochemistry

For immunocytochemistry, cells were fixed with 4% PF in PBS for20 min and permeabilized with PBS/tween20 (0.1%) for 15 min. Per-meabilization was followed by 30 min incubation in 3% H2O2. Cellswere then blocked with 10% normal horse serum in PBS for 1 h.Subsequently, cells were incubated in PBS containing polyclonalrabbit anti-Cav�3 antibody (1:100, Alomone Labs) overnight. Inthe following day, the cells were reacted with a pan-specific sec-ondary antibody (biotinylated goat anti-rabbit IgG) at 1:400 for2 h, and then with the ABC reagents (1:400, Vector Laboratories)for another hour. Immunoreactivity (IR) was visualized with 0.05%diaminobenzidine (DAB) and 0.003% H2O2. Three 10-min washeswith PBS were used between incubations.

2.5. Electrophysiological recording

Matrigel coated coverslips with differentiated NSCs at day 14were used for electrophysiological recordings. The coverslip wastransferred to the recording chamber of an upright microscope(DMLFS, Leica). Cells were perfused with an extracellular solutioncontaining (in mM): 127 NaCl, 3 KCl, 1 MgSO4, 26 NaHCO3, 1.25NaH2PO4, 1 d-glucose, and 2 CaCl2 (pH 7.4). The pipette solutioncontained (in mM): 5 NaCl, 1 CaCl2, 10 HEPES, 0.2 EGTA, 3 ATP,and 0.4 GTP (pH 7.4). Whole-cell patch-clamp recordings werecarried out with patch-clamp amplifiers (Axopatch 200B, Axoninstruments) and Clampfit 10.2 for data acquisition and analyses.To examine the excitability of neurons, transient membrane cur-rents were induced by stepping the holding membrane potential

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from −80 mV to +80 mV (500 ms) with activities recorded. Thenthe cell was switched to current-clamp and currents (−0.02 nA to0.05 nA, 300 ms) were injected through the patch pipette to exam-ine whether action potentials could be induced. Spontaneous actionpotentials were collected during a 10 min period of continuousrecording in current-clamp (0 nA) and analyzed.

2.6. Statistical analysis

The data are expressed as mean ± SD values of three or moreindependent experiments (each with two replicates) on graphsplotting pooled data. Statistical analysis was calculated using prismsoftware. Two-tailed Student’s t tests were used for two-groupcomparisons. One-way ANOVA with post hoc Tukey’s test wereused for comparisons of more than two groups. Values of P < 0.05were considered significant.

3. Results

3.1. Characterization of NSCs isolated from SVZ of the postnatalrat brain

We cultured primary NSCs as mentioned in the methods section.Cells maintained the ability to grow clonally and neurosphere-likecell clusters appeared on day 7 of the incubation (Fig. 1A). Almost allof the cells in the neurospheres expressed nestin (Fig. 1B). Further-

Fig. 1. Identification of NSCs derived from adult rat SVZ. (A) Phase-contrastphotographs of neurospheres-like cell clusters. (B) The vast majority of cells in neu-rospheres express the NSC-specific marker Nestin (red). (C) Immunofluorescenceanalysis of NSCs revealed differentiation into �-III-tubulin-positive neuronal cells(red) and GFAP-positive astrocytes (green); nuclei were counterstained with bis-benzimide (blue). (D) Representative traces of spontaneous postsynaptic currentsin neurons clamped at −80 mV. (E) Current-clamp recording shows a representativetrain of action potentials elicited in a presumptive neuron differentiated from NSCs.The action potentials were evoked with current steps ranging from −20 to 50 pA. (F)Current trace elicited by a 50 pA injected current. (G and H) Voltage-clamp recordingof K+ currents and Na+ currents induced by stepping holding potential from –80 mVto +80 mV. Scale bars: 100 �m in A, 50 �m in B and C. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

more, after withdraw of growth factors, cells that were plated ontomatrigel differentiated into neurons (�-III-tubulin-positive cells),and astrocytes (GFAP-positive cells) (Fig. 1C). Using whole-cellpatch-clamp recordings we examined the functional characteristicsof NSC-derived neurons. The neuron-like cells exhibited sponta-neous postsynaptic currents (Fig. 1D) and also generated repetitivetrains of action potentials in response to depolarizing current steps(Fig. 1E–F). Consistent with this, voltage-clamp recording revealedrapidly inactivating inward Na+ currents and persistent outward K+

currents that are essential for normal neuronal firing (Fig. 1G–H).Taken together, these results indicate that the cells isolated fromneonatal rat brains display properties of NSCs and can be grownsuccessfully as suspended neurospheres for further studies.

3.2. ı-Tocopherol treatment promotes neuronal differentiation ofNSCs

To study the role of �-tocopherol in NSC differentiation, we cul-tured NSCs on matrigel-coated coverslips in 24-well culture plateswith differentiation culture medium for 7 days. NSC differentiationwas evaluated by immunofluorescent staining using �-III-tubulinfor neurons and GFAP for astrocytes (Fig. 2A–B). �-tocopheroltreatment promoted neuronal differentiation of NSCs in a dosedependent manner (dose–response curve Fig. 2C–D). Treatmentwith 10 �M �-tocopherol under differentiation conditions exerteda significant increase in the percentage of neurons (27.51 ± 3.91%in �-tocopherol-treated cells versus 18.29 ± 3.78% in controlcells), whereas astrocytes were decreased from 57.85 ± 3.64%to 47.92 ± 4.47% (P < 0.05, n = 3). This suggests that in vitro �-tocopherol tends to enhance neuronal differentiation.

3.3. ı-Tocopherol promotes morphological maturation of neurons

We observed that the neurons obtained in the pres-ence of �-tocopherol displayed a higher degree of maturation(Fig. 2E–F), as indicated by the increased number (6.84 ± 1.89 vs3.24 ± 1.01 in control; Fig. 2G) and length (218.2 ± 46.31 �m vs110.43 ± 15.89 �m in control; Fig. 2H) of their neurites. Theseresults indicate that �-tocopherol administration promotes thematuration of neurons during NSC differentiation.

3.4. Role of L-type calcium channels in the effects of ı-tocopherolon NSC differentiation

To examine whether L-type calcium channel activity contributesto the effect of �-tocopherol on neuronal differentiation, immuno-histochemistry was performed to examine immunoreactivity forvoltage-dependent Ca2+ channels (Cav channels) in stimulatedcultures (Fig. 3A–B). As seen in Fig. 3, control cells exhibitedweak Cav channel antibody staining (�3 subunit), while the �-tocopherol treated cells showed more intense and diffuse Cav

channel immunoreactivity. These results suggest that �-tocopherolstimulation may increase the expression of Cav channels.

To determine whether the increase in Cav channel expres-sion was responsible for the effects exerted by �-tocopherol onNSC differentiation, NSC fate was investigated in control and�-tocopherol cultures treated with or without the L-type calciumchannels inhibitor verapamil. Verapamil, by itself, resulted in alower percentage of �-III-tubulin-positive and a higher percentageof GFAP-positive cells. The neuronal yield of NSC differentiationin the presence of verapamil was decreased (15.04 ± 3.53% in ver-apamil treated cells versus 18.29 ± 3.78% in control cells) (Fig. 3Cand E). Moreover, verapamil treatment also blocked the effects of�-tocopherol on NSC differentiation into neurons (20.56 ± 3.29%in �-tocopherol plus verapamil treated cells versus 27.51 ± 3.91%in �-tocopherol treated cells) (Fig. 3D–E). However, this

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Fig. 2. �-Tocopherol enhances efficiency of neural stem cell differentiation and maturation of the in vitro differentiated neurons. �-III-tubulin (red) and GFAP (green)immunofluorescent images of control cells (A) and �-tocopherol treated cells (B). Scale bar: 50 �m. Dose–response of �-tocopherol (C and D) revealed that �-tocopherolsignificantly increased �-III-tubulin-positive cells compared to control condition. In contrast, astrocytes were decreased (D). *P < 0.05 compared with control; n ≥ 3 assays. (Eand F) Representative images of immunofluorescent analysis of �-III-tubulin in cells differentiated from NSCs after 7 days of culture under differentiation conditions in theabsence or presence of 10 �M �-tocopherol. Scale bar: 50 �m. (G and H) Bar graph representing the quantitative data of morphometric analyses of the number of neuritesand neuritic lengths in control and �-tocopherol treated cells. *P < 0.05 compared with control; n ≥ 3 assays. (For interpretation of the references to color in this figure legend,the reader is referred to the web version of this article.)

Fig. 3. �-Tocopherol promotes neuronal differentiation of NSCs primarily by increasing Ca2+ influx through L-type calcium channels. Control cells exhibited weak Cav channelantibody staining (�3 subunit) (A), while Cav channel immunoreactivity was much more intense and diffuse in �-tocopherol treated cells (B). Scale bar: 50 �m. Representativeimages of verapamil treated (C) and �-tocopherol plus verapamil-treated (D) cells, showing the percentage of �-III-tubulin-positive neuronal cells (red) and GFAP-positiveastrocytes (green). Blue color showed the nuclei. Scale bar: 50 �m. The neuronal yield of NSC differentiation decreased in the presence of the L-type calcium channel blockerverapamil, but not N-type calcium antagonist (�-conotoxin; 10 �M). Moreover, verapamil, but not �-conotoxin, also blocked the effects of �-tocopherol on NSC differentiationinto neurons (E). In comparison, astrocytes showed the opposite changes in the verapamil treated group versus control. In the presence of �-tocopherol and verapamil, butnot �-conotoxin, an increase in the percentage of astrocytes was seen (F). Ctrl: control; �-T: �-tocopherol; *P < 0.05 compared with ctrl; #P < 0.05 compared with �-T; n ≥ 3assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

differentiation did not change in the presence of the N-typecalcium channels inhibitor �-conotoxin (Fig. 3E). In contrast,GFAP-positive astroglial cells, showed the opposite change in theverapamil treatment group compared to the control. Further, inthe presence of �-tocopherol, verapamil increased the percentageof GFAP-positive astroglial cells relative to just �-tocopherol.The percentage of GFAP-positive cells was not changed by theN-type calcium channel inhibitor �-conotoxin (Fig. 3F). The elec-trophysiological function of these NSC-derived neurons culturedin the presence of drugs was also examined by whole-cell patch-clamp recording. Spontaneous postsynaptic currents, evokedaction potentials, inward Na+, and outward K+ currents from�-tocopherol, verapamil, and �-tocopherol plus verapamil groupsshowed normal neuronal function (data not show). Collectively,these data indicate an important role of verapamil-sensitivecalcium channels in the response of NSC to �-tocopherol, and�-tocopherol treatment appeared to promote neuronal differenti-ation of NSCs primarily by increasing Ca2+ influx through L-typecalcium channels.

4. Discussion

Neurons are considered as the most functional cells in humanbrain, the generation of neurons from neural stem cells makesit a promising potential therapeutic tool for the treatment ofmany nervous system disorders [20]. These cells must be able togenerate functional neural cells that integrate appropriately andconsequently mediate functional repair [21]. As a consequence, itbecomes important to search for the ways to enhance neuronaldifferentiation of NSCs in the research for NSC-based therapies. Inthe present study, we investigated the effects of a vitamin E iso-mer �-tocopherol on the differentiation and maturation of NSCs invitro. We found that �-tocopherol supplementation enhanced neu-ronal differentiation efficiency of neural stem cells at the expense ofastrocytic differentiation. When the neurospheres were dissociatedand maintained in differentiation medium, �-tocopherol increasedthe percentage of neurons and decreased the number of astrocytes.Moreover, �-tocopherol also promoted morphological maturationof neurons. In our report, �-tocopherol facilitated precursor cell

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differentiation, providing new perspectives for raising the possi-bility of cell therapies. Thus, the present work provides insight intopotential therapies for brain and spinal cord diseases.

There are a number of studies demonstrating that L-type Ca2+

channels are needed for orienting NSC differentiation toward theneuronal phenotype [17,18,22]. Ca2+ entry through L-type Ca2+

channels is more efficient than through other types of Ca2+ chan-nels in coupling excitation and transcription of neurons. Anothermain finding of the present study is that L-type calcium channelsmediate the stimulation by �-tocopherol to neuronal differentia-tion of NSCs. The evidences are that, firstly, �-tocopherol treatmentof NSC cultures enhances L-type Ca2+ channel immunoreactivity.Second, our data suggest that �-tocopherol may act as a L-typeCa2+ channel activator for neuron-like cell fate determination, witha decrease in neuronal phenotypes when grown in the presence ofverapamil. When NSCs are maintained in the presence of verapamil,the percentage of neuronal-like cells is decreased. Moreover, partialinhibition of L-type Ca2+ channels activity by verapamil (L-type cal-cium channel blocker of the phenylalkylamine class) and nifedipine(dihydropyridine L-type calcium channel blocker, data not show)attenuates the effects of �-tocopherol on NSC differentiation intoneurons. Nevertheless, the percentages of �-III-tubulin-positiveand GFAP-positive cells are unchanged by N-type calcium chan-nels blocker �-conotoxin treatment. This observation is in line withprevious findings on neural stem cells derived from the brain cor-tex of postnatal mice, in which differentiation is closely related tothe expression L-type Ca2+ channels pharmacologically character-ized by using Cav channel blocker and activator [17,18]. Thus theactivated L-type Ca2+ channels initiated by �-tocopherol may be acritical factor for regulating NSC differentiation.

In summary, the present study demonstrates that �-tocopherolacts through elevation of L-type calcium channel activity toincrease neuronal differentiation and decrease astrocytic differ-entiation in NSCs, and it might become a useful tool to increasedifferentiation toward the neuronal lineage when NSCs are manip-ulated and expanded in vitro prior to transplantation in vivo.

Acknowledgments

This work was supported by the National Natural Sci-ence Foundation of China (81171037/H0903) and 973program(2012CB966400).

References

[1] N. Hussain, F. Irshad, Z. Jabeen, I.H. Shamsi, Z. Li, L. Jiang, Biosynthesis,structural, and functional attributes of tocopherols in planta; past present,and future perspectives, J. Agric. Food Chem. 61 (2013) 6137–6149.

[2] E. Cardenas, R. Ghosh, Vitamin E: a dark horse at the crossroad of cancermanagement, Biochem. Pharmacol. 86 (2013) 845–852.

[3] S. Salinthone, A.R. Kerns, V. Tsang, D.W. Carr, Alpha-tocopherol (vitamin E)stimulates cyclic AMP production in human peripheral mononuclear cells andalters immune function, Mol. Immunol. 53 (2013) 173–178.

[4] D. Pekmezci, Vitamin E and immunity, Vitam. Horm. 86 (2011) 179–215.[5] R. Pazdro, J.R. Burgess, Differential effects of �-tocopherol and

N-acetyl-cysteine on advanced glycation end product-induced oxidativedamage and neurite degeneration in SH-SY5Y cells, Biochim. Biophys. Acta1822 (2012) 550–556.

[6] L. An, T. Zhang, Vitamins C and E reverse melamine-induced deficits in spatialcognition and hippocampal synaptic plasticity in rats, Neurotoxicology 44C(2014) 132–139.

[7] T.E. Dunn-Thomas, D.L. Dobbs, D.S. Sakaguchi, M.J. Young, V.G. Honovar, M.H.Greenlee, Proteomic differentiation between murine retinal andbrain-derived progenitor cells, Stem Cells Dev. 17 (2008) 119–131.

[8] K.M. Lebold, M.G. Traber, Interactions between alpha-tocopherol,polyunsaturated fatty acids, and lipoxygenases during embryogenesis, FreeRadical Biol. Med. 66 (2014) 13–19.

[9] G.X. Li, M.J. Lee, A.B. Liu, Z. Yang, Y. Lin, W.J. Shih, C.S. Yang, Delta-tocopherolis more active than alpha- or gamma-tocopherol in inhibiting lungtumorigenesis in vivo, Cancer Prev. Res. (Phila.) 4 (2011) 404–413.

[10] F. Guan, G. Li, A.B. Liu, M.J. Lee, Z. Yang, Y.K. Che, Y. n Lin, W. Shih, C.S. Yang,Delta- and gamma-tocopherols but not alpha-tocopherol, inhibit coloncarcinogenesis in azoxymethane-treated F344 rats, Cancer Prev. Res. (Phila.) 5(2012) 644–654.

[11] M. Xu, K. Liu, M. Swaroop, F.D. Porter, R. Sidhu, S. Firnkes, D.S. Ory, J.J.Marugan, J. Xiao, N. Southall, W.J. Pavan, C. Davidson, S.U. Walkley, A.T.Remaley, U. Baxa, W. Sun, J.C. McKew, C.P. Austin, W. Zheng, Delta-tocopherolreduces lipid accumulation in Niemann-Pick type C1 and Wolman cholesterolstorage disorders, J. Biol. Chem. 287 (2012)39349–39360.

[12] J.F. Bonner, C.J. Haas, I. Fischer, Preparation of neural stem cells andprogenitors: neuronal production and grafting applications, Methods Mol.Biol. 1078 (2013) 65–88.

[13] C.J. Taylor, D.J. Jhaveri, P.F. Bartlett, The therapeutic potential of endogenoushippocampal stem cells for the treatment of neurological disorders, Front.Cell. Neurosci. 7 (2013) 5.

[14] F. Daniela, A.L. Vescovi, D. Bottai, The stem cells as a potential treatment forneurodegeneration, Methods Mol. Biol. 399 (2007) 199–213.

[15] H. Sabelstrom, M. Stenudd, P. Reu, D.O. Dias, M. Elfineh, S. Zdunek, P. Damberg,C. Goritz, J. Frisen, Resident neural stem cells restrict tissue damage andneuronal loss after spinal cord injury in mice, Science 342 (2013) 637–640.

[16] C. Andressen, Neural stem cells: from neurobiology to clinical applications,Curr. Pharm. Biotechnol. 14 (2013) 20–28.

[17] L.M. Louhivuori, V. Louhivuori, H.K. Wigren, E. Hakala, L.C. Jansson, T.Nordstrom, M.L. Castren, K.E. Akerman, Role of low voltage activated calciumchannels in neuritogenesis and active migration of embryonic neuralprogenitor cells, Stem Cells Dev. 22 (2013)1206–1219.

[18] M. D’Ascenzo, R. Piacentini, P. Casalbore, M. Budoni, R. Pallini, G.B. Azzena, C.Grassi, Role of L-type Ca2+ channels in neural stem/progenitor celldifferentiation, Eur. J. Neurosci. 23 (2006) 935–944.

[19] G.N. Marshall, H.H. Ross, O. Suslov, T. Zheng, D.A. Steindler, E.D. Laywell,Production of neurospheres from CNS tissue, Methods Mol. Biol. 438 (2008)135–150.

[20] G. Gincberg, H. Arien-Zakay, P. Lazarovici, P.I. Lelkes, Neural stem cells:therapeutic potential for neurodegenerative diseases, Br. Med. Bull. 104(2012) 7–19.

[21] A. Bithell, B.P. Williams, Neural stem cells and cell replacement therapy:making the right cells, Clin. Sci. (Lond.) 108 (2005) 13–22.

[22] L. Wen, Y. Wang, H. Wang, L. Kong, L. Zhang, X. Chen, Y. Ding, L-type calciumchannels play a crucial role in the proliferation and osteogenic differentiationof bone marrow mesenchymal stem cells, Biochem. Biophys. Res. Commun.424 (2012) 439–445.