Role of autophagy and mTOR signaling in neural differentiation of bone marrow mesenchymal stem cells

Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10320

SHORT COMMUNICATION

Role of autophagy and mTOR signaling in neural differentiation ofbone marrow mesenchymal stem cellsYanfei Li, Cuiqin Wang, Guangyu Zhang, Xiaohan Wang, Ranran Duan, Huili Gao, Tao Peng,Junfang Teng and Yanjie Jia*

Department of Neurology, First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China

Abstract

Autophagy is involved in cell differentiation. We present evidence that autophagy is activated during b-mercaptoethanol (b-ME)-induced neuronal differentiation of bone marrow mesenchymal stem cells (MSCs), in which mammalian target ofrapamycin (mTOR) signaling is important. mTOR activity declined after being transported from the nucleus to the cytoplasm.Using 3-methyladenine (3-MA) and rapamycin to regulate the activity of mTOR, it was found that the efficiency of neuronaldifferentiation was affected.

Keywords: autophagy; MSCs; mTOR; neuronal differentiation

Introduction

Autophagy is an evolutionarily conserved catabolic programcontrolled by the autophagy-related family (ATG family) ofgenes that recycles proteins and organelles. As a conse-quence of autophagy, cells generate metabolic precursors formacromolecular biosynthesis or ATP generation (He andKlionsky, 2009). Recent studies have shown that autophagyparticipates in differentiation, a cell remodeling mechanismthat promotes morphological and structural changes.

Target of rapamycin (TOR) is an evolutionarily conservedprotein kinase family and exists widely in all types ofbiological cells. Mammalian TOR (mTOR) is a member ofthe phosphatidylinositol kinase-related family of serine-threonine kinases which forms two different complexes:mTOR complex 1 (mTORC1), which is sensitive torapamycin, and mTOR complex 2 (mTORC2), which isinsensitive to rapamycin. mTORC1 contains mTOR,regulatory-associated protein of mTOR (raptor), mammali-an ortholog of LST8 (mLST8), and proline-rich Aktsubstrate of 40 kDa (PRAS40). mTORC2 contains mTOR,mLST8, rapamycin-insensitive companion of mTOR (ric-tor), and mitogen-activated protein kinase-associated pro-tein 1 (mSIN1). mTORC1 has been shown to regulate cellgrowth, proliferation and some intracellular activities, suchas mRNA transcription, translation and autophagy, whereas

mTORC2 functions in cytoskeleton structure reassemblyand cell survival (Loewith et al., 2002). 3-MA is commonlyused as a specific inhibitor of autophagic sequestration.Rapamycin, an immunosuppressant drug, can interact withmTORC1 and suppress its kinase activity (Lei et al., 2012).

Autophagy is negatively controlled by mTOR, andinhibitors of mTOR act as autophagy inducers. mTORregulates protein synthesis by phosphorylating two charac-terized substrates, 70 kDa S6 kinase (p70S6K) and eukaryoticinitiation factor 4E-binding protein 1 (4E-BP1) (Yonezawaet al., 2004). Microtubule-associated protein 1 light chain 3(LC3), a homologue ofApg8pessential for autophagy in yeast,is associated to autophagosome membranes after processing.Two forms of LC3, called LC3 I and II post-translationally invarious cells. The amount of LC3 II is correlated with theextent of autophagosome formation (Kabeya et al., 2000).

Bone marrow mesenchymal stem cells (MSCs) haveextensive capacity for multilineage differentiation and pro-liferation. Adult MSCs are able to differentiate into a numberof mesenchymal phenotypes, including those that form bone,cartilage, muscle, fat, and other connective tissues (Caplan,2009). MSCs can differentiate into neurons under particularconditions (Jing et al., 2011). Tau is a type of neuronmicrotubule-associatedprotein (MAP),whichhelpsmaintainstructure of the axon. Microtubule-associated protein-2(MAP-2) is a kind of protein found specifically in dendritic

�Corresponding author: email: [email protected]

1337Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology

branching of neurons. Tau and MAP-2 are commonbiomarkers of neurons. A cell-based therapy for replacingdopaminergic neurons in subjects with Parkinson's disease isthe transplantation of differentiated autologousMSCs, whichmight be a safe and effective approach (Hayashi et al., 2013).Wethereforeexplored thepotential rolesofautophagyand themTORsignalingpathwayinneuronaldifferentiationofMSCs.

Materials and methods

Cell culture

MSCswereobtained from the femurs and tibias ofWistar rats,aged 6–8 weeks. The cells were extracted and cultured in acomplete medium consisting of Dulbecco's modified Eagle'smedium (DMEM, Invitrogen, USA), 10% fetal bovine serum(FBS, Gibco, USA), and 0.3mg/mL geneticin. Cells werecultured in a 5%CO2 in air atmosphere at 37�C for 4 days. Atconfluency, the cells were harvested with 0.25% Trypsin-EDTA solution and counted for viable cells using trypan blue,and then plated into 24-well plates at 5� 104/well.

Cell grouping

MSCs were divided into groups as follows: 3-MA group,rapamycin group and a control group. The concentrations of3-MA used were 0, 1, 5, and 10mM and were set as groups 1,2, 3, 4. The concentrations of rapamycin used were 0, 10, 20and 30mM and were set as groups 1, 2, 3, 4. The controlgroup was MSCs with no 3-MA or rapamycin treatment.

Neuronal differentiation of MSCs in vitro

To induce neuronal differentiation, subconfluent cultureswere treated with pre-induction medium, which consisted ofDMEM, 10% FBS and 1mM b-mercaptoethanol (b-ME) for24 h.After pre-induction, cellswerewashedwith PBS, and themedium for the 3-MA group was replaced with 3-MA andserum-freemediumwith 10mMb-ME for 24 h. Themediumfor the rapamycin group was replaced with rapamycin andserum-freemediumwith10mMb-ME for 24h. In the controlgroup, the induction medium was replaced with serum-freemedium with 10mM b-ME for 24h. The morphology of theMSCs was observed before and after induction using aninvertedmicroscope (Woodbury et al., 2000). Tomeasure theefficiency of neuronal differentiation, 2 neuronal markers,microtubule-associated protein 2 (MAP-2) and tau 24 h wereanalyzed after induction.

Immunofluorescence

After washing with PBS, cells were fixed at 4�C in 4%paraformaldehyde for 20min, permeabilized with 0.1%

Triton X-100 for 10min and blocked with 10% bovine serumalbumin (BSA) for 1 h. The cells were incubated with thefollowing antibodies: LC3B (LC3 I and II, 1:200, CellSignaling Technology, USA), LC3 I is cytosolic, whereas LC3II is membrane bound. Mutational analyses suggest that LC3I is formed by the removal of the C-terminal 22 amino acidsfrom newly synthesized LC3, followed by the conversion of afraction of LC3 I into LC3 II. MAP-2 (1:200, Santa Cruz,USA), Tau (1:200, Santa Cruz), mTOR (1:200, Cell SignalingTechnology) at 4�C overnight. After being washed with PBS,cells were incubated with a secondary antibody (anti-Ig-G-Cy3 goat anti-rabbit, 1:500, Santa Cruz) at room tempera-ture for 1 h. Cells were observed with an invertedfluorescence microscope.

Western blot analysis

Cell lysates (100mL) were collected from each group.Approximately 20mg of total protein from each lysate wassubjected to SDS–PAGE and transferred to a polyvinylidenefluoride (PVDF) membrane (Millipore, USA). The mem-branes were blocked in 5%non-fat milk for 2 h and incubatedat 4�C overnight with one of the following antibodies: LC3B(1:1000, Cell Signaling Technology), MAP-2 (1:1000, SantaCruz), Tau (1:1000, Santa Cruz), mTOR (1:1000, Cell Signal-ing Technology), p-mTOR (1:1000, Cell Signaling Technolo-gy), p-p70s6k (1:1,000, Cell Signaling Technology), p-4EBP1(1:1000, Cell Signaling Technology), orb-actin (1:1000, SantaCruz). Themembranes were then incubated with a secondaryantibody (1:3000) for 2 h at room temperature and developedusing a color reagent. Values were expressed as percentagesrelative to the loading control, b-actin.

mTOR kinase activity assay

We used a K-LISATM (Recombinant) Activity Kit (Millipore,USA) tomeasure the kinase activity ofmTOR.TheK-LISATM

mTOR (Recombinant) Activity Kit is an ELISA-based activityassay that utilizes a p70S6K-GST fusion protein as a specificmTOR substrate. ThemTOR substratewas bound to thewellsof a glutathione-coated 96-well plate and then incubated withrecombinant mTOR-containing samples. Active mTORphosphorylates p70S6K at Thr389 in the presence of ATP.The phosphorylated substrate is detected with an anti-p70S6K-pT389 antibody, followed by detection with anHRP-antibody conjugate and TMB substrate. Sensitivity isincreased by the addition of ELISA Stop Solution, and relativeactivity is determined by absorbance at 450 and 540nm.

Statistical analysis

Each experiment was repeated at least three times. Data aremeans� standard deviation. Statistical differences in

Role of autophagy and mTOR signaling Y. Li et al.

1338 Cell Biol Int 38 (2014) 1337–1343 © 2014 International Federation for Cell Biology

multiple groups were determined by one-way ANOVA.Statistical significance between two groups was determinedby Student's t-test using the SPSS software. Differencesbetween groups were considered significant if P< 0.05.

Results

Autophagy was activated during the neuronaldifferentiation of MSCs

The abundance of LC3-II or the ratio of LC3-II/LC3-I is agood indicator of autophagosome formation (Levine andKlionsky, 2004). We did not detect LC3-II expression inMSCs before induction, and only a few fluorescent LC3 dots(autophagosomes) were seen by immunofluorescencestaining. Both the expression of LC3-II and the ratio ofLC3-II/LC3-I were higher after induction (P< 0.05, Figures1b and 1c). The number of fluorescent LC3 dots alsoincreased after induction (Figure 1a), indicating upregula-tion of autophagy during differentiation.

Downregulation of mTOR signaling during the neuronaldifferentiation of MSCs

To explore the possible mechanism by which autophagywas activated during neuronal differentiation of MSCs,we examined mTOR signaling activity 24 h after b-mercaptoethanol (b-ME) induction. The K-LISATM

mTOR (Recombinant) Activity Kit was used to detect

mTOR and Western blots to analyze the expressionof mTOR, phosphorylated mTOR (p-mTOR) and 2 subs-trates of mTOR, p-70S6K, and 4E-BP1. Before induction,increasing concentrations of 3-MA (1–10mM) graduallyenhanced mTOR activity. Increasing concentrations ofrapamycin (10–30mM) resulted in a gradual decline in theactivity of mTOR. After induction, similar results wereobtained. The activity of mTOR was also lower after thanbefore induction with no rapamycin or 3-MA treatment.Overall, mTOR activity declined after induction comparedto pre-induction (P< 0.05, Figure 2h). Before induction,the expression of p-mTOR, p-p70S6K, and p-4E-BP1 wasincreased with increasing concentrations of 3-MA, butrapamycin had the opposite effect. Total expression ofmTOR was unchanged. Using 3-MA or rapamycin,similar expression patterns of associated phosphorylationproteins after induction were observed. It can be concludedthat the expression of p-mTOR, p-p70S6K, and p-4E-BP1after induction, declined (P< 0.05, Figures 2a–2g, 2iand 2j).

An appropriate level of mTOR activity is important forneuronal differentiation of MSCs

Considering that mTOR is critical in neuronal differentia-tion, we tested whether changes in mTOR activity affecteddifferentiation. To examine the efficiency of differentiation,2 neuronal markers, microtubule-associated protein 2(MAP-2) and tau, were analyzed 24 h after induction.

Figure 1 Autophagy activity during the neuronal differentiation of MSCs. Autophagy activity increased during the process of neuronaldifferentiation of MSCs: (a) LC3-positive dots (in red) in MSCs (scale bar: 20mm). The number of fluorescent LC3 dots increased after 24 h induction. (b)Autophagy-dependent expression of LC3-II inMSCs. (c) Protein levels of LC3were analyzed byWestern blotting. The expression of LC3-II and the ratio ofLC3-II/LC3-I was higher after induction of the neuronal differentiation of MSCs (n¼ 3, P< 0.05).

Y. Li et al. Role of autophagy and mTOR signaling


One day after induction, rising concentrations ofrapamycin (10–20mM) gradually enhanced the fluorescencesignal of neurite elongation (Figures 3b and 3c, 3i and 3j).When the concentration reached 30mM, the fluorescencesignal was weakened (Figures 3d and 3k). 3-MA (1–10mM)caused a gradual weakening in fluorescence signal of neuriteelongation (Figures 3e–3g, 3l–3n). The expression ofMAP-2and tau in the rapamycin 20mM group was the highestcompared to the other groups (P< 0.05, Figures 3o–3q).

Cytoplasmic nuclear shuttling of mTOR during theneuronal differentiation of MSCs

Before induction, the fluorescence signal was mainlydistributed in the nucleus; it was enhanced in the 3-MAgroup (Figures 4d–4f), but weakened in the rapamycingroup (Figures 4a–4c) compared to the control group. Afterinduction, the fluorescence signal in the nucleus weakenedin the rapamycin group compared (Figures 4g–4i). In the

Figure 2 Downregulation of mTOR signaling. (a) The expression levels of mTOR (b), p-mTOR (b), p-p70S6K (b), and p-4E-BP1 (b) were analyzed byWestern blotting techniques prior to induction with b-ME. The expression levels of mTOR (a), p-mTOR (a), pp70S6K (a), and p-4E-BP1 (a) were analyzedbyWestern blotting techniques 24 h after induction with b-ME. The expression of b-actin was used as a loading control. (b–g and i and j): Quantificationof the expression levels of mTOR, p-mTOR, p-p70S6K, and p-4E-BP1 (mean� SD). Before induction, the expression levels of p-mTOR, p-p70S6K, andp-4E-BP1 were elevated with increasing concentrations of 3-MA and were decreased with increasing concentrations of rapamycin. The expression ofp-mTOR, p-p70S6K, and p-4E-BP1 declined after induction compared to before induction (n¼ 3, P< 0.05). (h): The activity ofmTORwas analyzed by theK-LISA assay before and after induction. mTOR activity declined after induction compared to preinduction (n¼ 3, P< 0.05).



3-MA groups, there was an obvious fluorescence signalaccumulating in the nucleus (Figures 4j–4l).

Discussion

Zhao et al. (2010) found that autophagy activity wasmarkedly increased when glioma stem/progenitor cells(GSPCs) were induced to differentiate using fetal calf serum(FCS), and that rapamycin, the activator of autophagy, couldpromote their differentiation. The mTOR signaling pathwaywas involved the regulation of autophagy, and mTOR couldturn into its activated form, phospho-mTOR, which canrespond directly to mTOR activity (Fielhaber et al., 2012).Our study demonstrated that the expression of p-mTOR, p-p70S6K, and p-4E-BP1 declined after induction compared,which indicates that mTOR activity decreased during theneuronal differentiation.

mTOR activity is modified in various pathological statesof the nervous system, including brain tumors, tuberoussclerosis and neurodegenerative disorders such as Alzheim-er's, Parkinson's, and Huntington's diseases (Swiech et al.,2008). mTOR is important in the regulation of neuraldifferentiation and synaptic plasticity (Magri et al., 2011). Insome treatments for neurodegenerative disorders, decreasein neuronal mTOR activity can alleviate disease progression.mTOR activity that is either too low or too high impairs celldifferentiation. High concentration rapamycin in neuronaldifferentiation can reduce nerve swelling, cell size and neuralmarkers of immune activity (Zeng and Zhou, 2008). We

found that rising concentrations of rapamycin (10–20mM)gradually increased the expression of MAP-2 and tau afterinduction; when it reached 30mM, MAP-2 and tauexpression declined, which indicates an appropriate levelof mTOR activity be needed in neuronal differentiation.

In human embryonic kidney (HEK) 293 cells, mTOR is acytoplasmic-nuclear shuttling protein, which may berelevant to the mitogen-stimulated rapamycin-sensitivemTOR signaling pathway and translation initiation (Kimand Chen, 2000). mTOR shuttled from its normalpredominantly mitochondrial location to the cell nucleusunder certain conditions (Tirado et al., 2003; Bachmannet al., 2006). Altering mTOR nuclear shuttling withexogenous nuclear import and export signals has shownthat they are consistent with a direct link between the nuclearshuttling ofmTOR and themitogenic stimulation of p70S6Kactivation and 4EBP1 phosphorylation. Before induction,the fluorescence signal is mainly distributed in the nucleusand this signal is enhanced in the 3-MA group, but weakenedin the rapamycin group. After induction, the fluorescencesignal in the nucleus weakened in the rapamycin group andthere was an obvious fluorescence signal accumulating in thenucleus in the 3-MA groups, which indicates nuclearshuttling of mTOR might occur in neuronal differentiation.

During the neuronal differentiation of MSCs, autophagyactivity increased. The expression of mTOR signaling wasdownregulated during differentiation. Neuronal differentia-tion required proper mTOR activity, elevated or down-regulated mTOR activity could perturb axon growth and

Figure 3 Expression of Tau and MAP-2. (a–g): The expression levels of MAP-2 by immunofluorescence staining (scale bar: 50mm). (h–n): Theexpression levels of Tau by immunofluorescence staining (scale bar: 50mm). (o): The expression levels of MAP-2 and Tau were analyzed by Westernblotting analysis 24 h after inductionwithb-ME. (p and q): The quantification of the expression levels ofMAP-2 and Tau (mean� SD, n¼ 6). rapamycin: 1(0mM), 2 (10mM), 3 (20mM), 4 (30mM). 3-MA: 1 (0mM), 2 (1mM), 3 (5mM), 4 (10mM).



navigation, dendritic arborization, and synaptogenesis. Inconclusion, autophagy is activated and might be importantin neuronal differentiation of MSCs. Neuronal differentia-tion appears to require normal mTOR activity.

Acknowledgment and funding

This work was supported by the National Natural ScienceFoundation of China (Grant Numbers 81071114).

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Received 20 January 2014; accepted 25 April 2014.Final version published online 18 August 2014.



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Role of autophagy and mTOR signaling in neural differentiation of bone marrow mesenchymal stem cells