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mTOR-dependent proliferation defect in humanES-derived neural stem cells affected by myotonicdystrophy type 1

Jerome Alexandre Denis1,*, Morgane Gauthier1,`, Latif Rachdi2,`, Sophie Aubert3,`, Karine Giraud-Triboult3,Pauline Poydenot3, Alexandra Benchoua3, Benoite Champon3, Yves Maury3, Christine Baldeschi1,Raphael Scharfmann2, Genevieve Pietu1, Marc Peschanski1 and Cecile Martinat1,§

1INSERM/UEVE U-861, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 5 rue Henri Desbrueres, 91030 Evrycedex, France2INSERM U-845 Research Center ‘Growth and Signaling’, Faculty of Medicine Paris Necker, 156 rue de Vaugirard, 75730 Paris cedex 15, France3CECS, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, 5 rue Henri Desbrueres, 91030 Evry cedex, France

*Present address: APHP, Saint Louis-Lariboisiere-Fernand Widal University Hospitals, Lariboisiere Hospital, Department of Medical Biochemistry and Molecular Biology, BiologicalResource Center, UMRS/INSERM U-942, 2 rue Ambroise Pare 75475 Paris cedex, 10, France`These authors contributed equally to this work§Author for correspondence (cmartinat@istem.fr)

Accepted 4 January 2013Journal of Cell Science 126, 1763–1772� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.116285

SummaryPatients with myotonic dystrophy type 1 exhibit a diversity of symptoms that affect many different organs. Among these are cognitive

dysfunctions, the origin of which has remained elusive, partly because of the difficulty in accessing neural cells. Here, we have takenadvantage of pluripotent stem cell lines derived from embryos identified during a pre-implantation genetic diagnosis for mutant-genecarriers, to produce early neuronal cells. Functional characterization of these cells revealed reduced proliferative capacity and increased

autophagy linked to mTOR signaling pathway alterations. Interestingly, loss of function of MBNL1, an RNA-binding protein whosefunction is defective in DM1 patients, resulted in alteration of mTOR signaling, whereas gain-of-function experiments rescued thephenotype. Collectively, these results provide a mechanism by which DM1 mutation might affect a major signaling pathway and

highlight the pertinence of using pluripotent stem cells to study neuronal defects.

Key words: Neural stem cells, Human embryonic stem cells, Pathological modeling, Myotonic dystrophy type 1, mTOR signaling pathways

IntroductionOwing to their ability to differentiate into a large spectrum of

cell types, human disease-specific pluripotent stem cells of

embryonic origin (hESCs), or more recently cells derived from

the reprogramming of somatic cells (hiPSCs), appear to be a

unique material in which to correlate molecular and functional

pathological mechanisms within difficult-to-obtain cell

populations (Maury et al., 2011). In the context of human

neurological disorders, most of our current knowledge about

molecular and cellular phenotypes has been gathered from

studies in postmortem brain tissues, which are typically the end-

stage of the disease and consequently may not be a fair

illustration of how the disease developed. Human disease-

specific pluripotent stem cells provide a unique opportunity to

complement findings in human biopsies and animal models by

analyzing the first stages of disease progression and deciphering

molecular mechanisms that could be at the origin of a disease-

related functional phenotype.

Over the past few years, a growing number of studies have

demonstrated the usefulness of this approach to study both

neurodevelopmental and neurodegenerative disorders. For

example, pre-implantation genetically diagnosed embryos have

been used to analyze molecular and functional defects in hESCs-

derived neurons carrying a mutant gene responsible for fragile X

(Eiges et al., 2007) and in motoneurons with myotonic dystrophy

type 1 (DM1) (Marteyn et al., 2011). Other early childhood-onset

monogenic disorders, such as Rett syndrome, familial

dysautonomia disorder and spinal muscular atrophy, have been

studied following the generation of hiPSCs from patients, and a

defective neuronal phenotype associated with the expression of the

mutant gene (Ebert et al., 2009; Kim et al., 2011; Lee et al., 2009).

Human iPSCs have also been instrumental in deciphering a

familial case of the late-onset neurodegenerative Parkinson’s

disease, associating the expression of a mutant gene with an

increased sensitivity of hiPSCs-derived dopamine neurons to

oxidative stress (Seibler et al., 2011).

Myotonic dystrophy type 1 is an autosomal dominant

neuromuscular disorder with late clinical onset, caused by the

expansion of trinucleotide CTG repeats in the 39-untranslated

region of the DMPK gene (Mahadevan et al., 1992). At the

skeletal muscle level, three main molecular events can be

described: (1) formation of nuclear foci that are composed at least

of mutant DMPK mRNA and recruited RNA-binding proteins,

such as splicing regulators and transcription factors (Ebralidze

et al., 2004; Mankodi et al., 2001; Timchenko and Caskey, 1996);

(2) disturbance of specific gene expression (Mankodi et al., 2002;

Savkur et al., 2001; Yadava et al., 2008); and (3) impairment of

cell proliferation and differentiation (Bigot et al., 2009; Furling

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et al., 2001; Timchenko et al., 2001a). Although DM1 has long

been considered mainly as a muscle disorder, there is extensive

evidence for the involvement of the central nervous system.

Psychological dysfunction, mental retardation, excessive daytime

sleepiness, and neuropathological abnormalities have been

described in DM1 patients (Abe et al., 1994; Delaporte, 1998;

Modoni et al., 2004; Modoni et al., 2008; Perini et al., 1999;

Turnpenny et al., 1994). Nevertheless, in contrast to the

substantial advances in understanding DM1 muscle pathology,

the molecular and functional bases of DM1 in the central nervous

system are still largely unknown.

In looking for molecular and functional mechanisms that could

be implicated in neural development in DM1, we based our

approach upon DM1-specific human pluripotent stem cells and

their ability to differentiate into neural cells. We recently

demonstrated that human embryonic stem cells (hESCs)

obtained during pre-implantation genetic diagnosis for DM1

(Mateizel et al., 2006) represent a relevant cellular model for

DM1. In particular, these human DM1-specific pluripotent stem

cells have allowed us to discover new molecular mechanisms

impeding the connectivity between DM1 hESCs-derived motor

neurons and their muscle targets (Marteyn et al., 2011). To more

broadly analyze the molecular and functional effects of the DM1

mutation in neural cells, we have taken advantage of progress

made in neural differentiation protocols to obtain robust and

homogenous neural precursor cell populations (Chambers et al.,

2009). We show here that these neural cells exhibit a

proliferation deficiency linked to a DM1-related impairment in

mTOR signaling pathway activity, associated with increased

autophagy.

ResultsTwo human embryonic stem cell (hES) lines carrying large CTG

expansions (VUB03_DM and VUB24_DM) obtained from

independent couples, and one wild-type control cell line

(VUB01_WT) were successfully differentiated toward a

homogenous population of neural stem cells (NSCs). No

differences in morphology and in the expression of NSC-

specific markers such as Nestin and Sox2 were observed during

the differentiation process (Fig. 1A–C). Moreover, no difference

in neuronal differentiation was observed between control and

mutant cells at day 20 for most of the analyzed gene markers,

Fig. 1. Characterization of DM1-NSCs.

(A) Morphology and expression of specific neural

markers (SOX2 and Nestin) in NSCs derived from

WT-hESC and two DM1-hESC lines. Nuclei were

counterstained with DAPI (blue). Scale bars: 20 mm.

(B) Quantification of Nestin expression by FACS

analysis. (C) Quantitative real-time RT-PCR analysis

of hESC-derived NSCs. RT-PCR levels are presented

as the fold change relative to undifferentiated hESC

lines after normalization with 18S rRNA levels.

(D) Detection of mutant RNA with a (CAG)10-Cy3

probe staining and MBNL1 (Muscleblind) protein by

immunofluorescence. Nuclei were counterstained

using DAPI (blue). The number of foci per nucleus

was quantified using ArrayScan, and is presented for

each cell line as a pie chart. (E) Expression of

MBNL1 and CUGBP1 proteins measured by western

blot analysis after nucleus (N) and cytoplasm

(C) subcellular fractionation. Lamin and b-actin were

used as loading controls.

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including SNAP25, DLG4 and MAPT, although we cannot

completely exclude a difference between DM1 and WT over

the time course of neuronal differentiation (supplementary

material Fig. S1). To validate their pathological relevance, we

performed in situ hybridization combined with immunostaining

which showed that DM1-NSCs and derived neurons harbored

intranuclear ribonucleoprotein aggregates (known as ‘foci’),

formed by the mutant RNA and the splice factor MBNL1,

which are typical of the disease (Fig. 1D and supplementary

material Fig. S1). The nuclear retention of MBNL1 was also

confirmed by western blotting (Fig. 1E).

Functional consequences of DM1 in neural stem cells

Quantitative analysis of doubling time by measurement of the ATP

concentration in cell cultures indicated that the proliferation rate of

DM1-NSCs was reduced compared to controls (Fig. 2A,B).

Consistent with this observation, a decreased number of Ki67-

positive cells was observed in DM1-NSC cultures (Fig. 2C), in

association with decreased PCNA gene expression and decreased

phosphorylation of the Rb protein (Fig. 2D,E). In addition,

expression of other cell cycle regulators was deregulated in DM1

cells in comparison with control, with increased p27kip1 (Fig. 2F)

and P15 proteins and decreased cyclin D1 and P21 (Fig. 2G).

Measurement of necrosis or apoptosis by annexinV and

propidium iodide staining, PARP cleavage and caspase-3

expression revealed no differences between DM1- and WT-

NSCs. Similarly, staining for b-galactosidase did not reveal any

differences in the proportion of senescent cells in the DM1- and

WT-NSC cultures (supplementary material Fig. S2). Altogether,

these results indicated that the reduced proliferative capacity of

DM1-NSCs was not associated with an increase in either cell

mortality or senescence.

Large cytoplasmic vacuoles were observed in DM1-NSCs,

suggesting the induction of autophagy (Fig. 3A). Confirming this

functional phenotype, increased expression of the autophagic

markers LC3B-II, p62 and ATG12 protein was observed in DM1-

NSCs (Fig. 3B–F), as well as significantly increased expression

of the lysosomal cathepsin B marker both at the mRNA and the

protein level (supplementary material Fig. S3). In addition, the

nucleofection of a plasmid encoding the LC3–GFP fusion

protein, used as a marker for autophagic vesicles and pre-

autophagic compartments, resulted in more LC3–GFP aggregates

in DM1-NSCs relative to controls (Fig. 3C,D). As a positive

control, treatment of WT-NSCs with either EBSS medium or

chloroquine resulted in an increased number of cells containing

LC3–GFP aggregates. No effect of chloroquine treatment was

Fig. 2. DM-NSCs exhibit lower proliferative capacity.

(A) Proliferation rate of WT-NSCs and DM1-NSCs

detected by quantification of intracytoplasmic ATP during

6 days in culture. (B) Analysis of the doubling time for

WT-NSCs and DM1-NSCs. (C) Decrease in the number of

Ki67-positive DM1-NSCs (VUB03_DM) after 48 hours in

culture in comparison with WT-NSCs. The number of

Sox2-positive cells was used as a control for the NSCs

phenotype. Ki67-positive cells were detected and

quantified using the automated ArrayScan Imager.

(D) Decreased expression of the proliferative marker PCNA

in DM1-NSCs in comparison with WT-NSCs, as revealed

by real-time RT-PCR analysis. RT-PCR levels are

presented as a fold change over control WT-NSCs after

normalization with 18S rRNA levels. (E) Decreased

expression level of phosphorylated Rb (S807–811) in

DM1-NSCs (VUB03_DM) compared with WT-NSCs, as

determined by western blotting. (F) Increased expression

level of P27 in DM1-NSCs compared with control NSCs as

revealed by western blotting. (G) Altered expression of

p15ink4B, p21waf1 and Cyclin D1 proteins in DM1-NSCs

determined by western blot analysis. All western blot

analyses were performed using protein extracted from

NSCs after 48 hours in culture. Band densities were

quantified using ImageJ software. Data are means 6 s.e.m.

of least three independent samples. One-way ANOVA with

Dunnett’s post-hoc test was performed; ns, not significant,

*P,0.05, **P,0.01, ***P,0.001.

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observed on DM1-NSCs suggesting that these cells could not

accumulate additional autophagic vacuoles.

Alteration of mTOR signaling pathways in DM1 neural cells

To assess the central role of AKT/mTOR signaling pathways in

both cell-cycle control and autophagy, expression of their main

components, namely Akt, AMPK, GSK3a/b and rpS6 (Ser235/

236) (ribosomal protein S6) was systematically analyzed. Gene

expression and protein levels were unaltered for all of these

components, when analyzed by quantitative PCR and western

blotting, respectively (Fig. 4; supplementary material Fig. S4). In

contrast, analysis of the post-translational activation of these

proteins through phosphorylation revealed major differences

between DM1-NSCs and controls. Whereas phosphorylation of

the upstream components Akt (Ser473) and AMPK (Thr172)

appeared unaffected, the downstream GSK3a/b (Ser21/9) and

rpS6 (Ser235/236 and Ser240/245) were decreased in DM1-NSCs

(Fig. 4A,B) through a mechanism independent of a stress-

induced activation of P53 (supplementary material Fig. S4).

To eliminate the possibility of a non-specific effect of the

growth factors present in the medium [i.e. epidermal growth

factor (EGF), fibroblast growth factor (FGF2) and brain-derived

neurotrophic factor (BDNF)] on mTOR signaling pathway

activity, NSCs were starved for 48 hours and then treated for 1

hour with each factor alone or in combination. No effect of EGF

and/or BDNF was observed on phosphorylated rpS6 (Ser235/

236) levels. Treatment with FGF2 resulted in a dramatic increase

in phosphorylated rpS6 (Ser235/236) through the activation of

phospho-Erk1/2, equally in DM1- and WT-NSCs (Fig. 4C,E). The

effect of FGF2-mediated ERK1/2 activation on the phosphorylation

of rpS6 was transient, as it disappeared 48 hours after treatment. As

all our experiments were performed 48 hours after treatment with

growth factors, these results suggest that there might be a differential

impact of growth factors in the medium on mTOR signaling

pathways in DM1- and WT-NSCs.

Molecular correlates of functional defects in DM1 neural

cells

A potential link between the functional defects in proliferation and

autophagy observed in DM1-NSCs and the decreased activation of

mTOR signaling was explored by analyzing the functional

consequences of a partial pharmacological blockade of the

pathway in WT-NSCs. Cells were treated with rapamycin, a

specific mTOR inhibitor. Rapamycin provoked a dramatic decrease

in phosphorylated rpS6 (Ser235/236) (P-rpS6) without substantial

modulation of either phosphorylated Akt (Ser473), GSK3a(Ser21)

or GSK3b (Ser9) (Fig. 5A,B; supplementary material Fig. S4).

Functional defects associated with this molecular alteration were

reminiscent of the phenotype described above for DM1-NSCs, as

rapamycin-treated WT-NSCs exhibited impaired proliferative

capacity (Fig. 5C,D; supplementary material Fig. S4) and

induction of autophagy, as shown by LC3–GFP aggregation after

transfection with the fusion protein plasmid (Fig. 5E,F).

Association of mTOR signaling alterations with expression of

the mutant DMPK gene was controlled by transient transfection

of WT-NSCs with a plasmid containing an extended stretch of

Fig. 3. Induction of autophagy in DM-NSCs. (A) Phase-

contrast microscopy of cytoplasmic vacuoles in DM1-NSCs

compared with control cells after 24 hours in culture.

(B) Increased expression level of LC3B-II in DM1-NSCs

(VUB03_DM) as determined by western blotting after

48 hours in culture. (C) Nucleofection of DM1- and WT-

NSCs with a plasmid expressing a LC3–GFP fusion for

48 hours (VUB03_DM and VUB01_WT, respectively).

(D) Increased number of cells containing LC3–GFP dots in

the cytoplasm of DM1-NSCs, as quantified using the

automated ArrayScan Imager. EBSS and chloroquine at

50 mM were used as positive controls. Data are presented as

the percentages of positive cells. (E) Detection by

immunostaining of two autophagic markers p62 and ATG12

in WT and DM1-NSCs (VUB03_DM and VUB01_WT,

respectively). Data are expressed as means 6 s.e.m. of at

least three independent samples. One-way ANOVA with

Dunnett’s post-hoc test was performed; ns, not significant,

*P,0.05, **P,0.01, ***P,0.001.

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960 CTG repeats. Treated cells exhibited a profound decrease in

P-rpS6 as well as P-GSK3b, which was consistent with our

working hypothesis (Fig. 6A,B; supplementary material Fig. S5).

The main consequence of the presence of an extended stretch of

CUG repeats in DM1-NSCs is the decreased bioavailability of the

MBNL1 protein due to its sequestration in intranuclear foci. To

reproduce this phenomenon in WT-NSCs, MBNL1 gene

expression was knocked down using specific siRNAs. MBNL1-

depleted cells exhibited significantly decreased levels of P-rpS6

and, to a lesser extent, decreased levels of P-GSK3a/b (Ser21/9)

(Fig. 6C). Conversely, overexpression of MBNL1 in DM1-NSCs

elicited a partial restoration of the phosphorylation of rpS6

(Ser232/236) (Fig. 6D). These changes in MBNL1 expression inDM1- and WT-NSCs led to altered proliferation and autophagy(Fig. 6E,F).

DiscussionThe primary finding of the current study was the identification of

an altered mTOR signaling pathway, which results in reducedproliferative capacity and induction of autophagy in neural cellsderived from DM1 gene-carrying human embryonic stem cells.

This alteration in the mTOR signaling pathway could bereproduced by reducing the bioavailability of the RNA-bindingprotein MBNL1 in WT cells, mimicking the defect associated

Fig. 4. mTOR signaling pathway is defective in DM1-NSCs. (A) Expression level of key modulators of Akt/mTOR signaling in DM1-NSCs and WT-NSCs

including: phospho(Ser473)-Akt, phospho(Thr172)-AMPKa, phospho(Ser21/9)-GSK3a/b and phospho(Ser235–236)-ribosomal protein S6 (rpS6) and the

corresponding total forms. Cell lysates were obtained after 48 hours in culture. (B) Decreased phosphorylation of S6 ribosomal protein at serine 235–236 and

serine 240–245 in DM1-NSCs compared with control NSCs after 48 hours in culture. (C) Effect of the different growth factors required for NSC culture (i.e. EGF,

FGF2, BDNF) on the phosphorylation of P-rpS6 (Ser235–236). DM1-NSCs and WT-NSCs were growth-factor starved for 48 hours and subsequently treated for 1

hour with or without each of the growth factors. (D) FGF2 dose–response analysis of DM1- and WT-NSCs after growth factor starvation and treatment with 1–100

nM FGF2 for 1 hour. The expression level of rpS6 and ERK1/2 was analyzed. (E) Kinetics of FGF2 treatment on DM1-and WT-NSCs as determined by the

expression of rpS6 and ERK1. Cells were starved for 48 hours and then treated for various durations with 10 nM FGF2. Western blots were quantified using

ImageJ software and quantitative data are presented as the phosphorylated form normalized to the total form. VUB03_DM cells were used as a source for DM1-

NSCs. Data are expressed as means 6 s.e.m. of three independent samples. One-way ANOVA with Dunnett’s post-hoc test was performed; ns, not significant,

*P,0.05, **P,0.01, ***P,0.001.

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with the expression of the DM1 mutation. The functional

abnormalities associated with the altered mTOR pathway might

contribute to the neurological aspects of this disease. This study

further highlights the value of mutant gene-carrying human stem

cell lines obtained from pre-implantation genetically diagnosed

embryos in helping to decipher the molecular mechanisms and

the functional consequences related to these mutations.

Molecular mechanisms involved in DM1 pathophysiology

have been mainly identified by using animal models. Few human

cell culture models have been developed; most are based on the

use of muscle precursor cells derived from congenital forms of

DM1, which are characterized by very large stretches of CTG

repeats (.2000). Myoblasts derived from congenital forms of

DM1 have alteration in various cell-cycle modulators, including

p21 and cyclin D1 (Timchenko et al., 2001b; Timchenko et al.,

2004). They also exhibit premature senescence through a p16-

dependent mechanism and defect in p38MAPK and ERK MEK

(Bigot et al., 2009), leading to defective proliferation and

differentiation (Beffy et al., 2010; Bigot et al., 2009; Timchenko

et al., 2001b; Timchenko et al., 2004). In addition, the induction

of autophagy has been recently observed in myoblasts derived

from congenital DM1 patients; a link to a p53-dependent

inhibition of mTOR pathway in response to metabolic stress

has been hypothesized (Beffy et al., 2010).

Our results have in part confirmed these data, by extending them

to DM1 neural precursor cells, in which altered proliferation and

induction of autophagy were observed. However, molecular

mechanisms responsible for those effects were not similar as

neither persistent activation of the FGF2-dependent MEK–ERK

pathway nor abnormal p53 activation was observed in DM1-NSCs.

Therefore, the current results do not support the hypothesis of a

cellular stress response in DM1 cells, which would be secondarily

responsible for the mTOR signaling pathway alterations.

Nevertheless, we cannot exclude the possibility that these

discrepancies are due to the differences between either the cell

types analyzed or the number of CTG repeats in the two cellular

systems. Indeed, mTOR signaling was altered in the current study

in the presence of only 1000 CTG repeats in DM1-NSCs or after

transfection of a plasmid with 960 repeats in WT-NSCs, i.e. in

conditions that are unlikely to be linked with congenital forms of

the disease.

Conversely, our data point to a specific defect in the activity of

the glycogen synthase kinase 3 (GSK3) being involved in the

disease. GSK3 activity is inhibited through phosphorylation of

serine 21 in GSK-3a and serine 9 in GSK-3b, which have been

identified as targets of Akt and AMPK (Fang et al., 2000). The

inhibition of GSK3 may participate in the activation of mTOR,

which results in the increased phosphorylation of rpS6

(Jastrzebski et al., 2007). According to this cascade of events

and in the absence of any alteration in the activation of Akt or

AMPK in DM1 neural cells, the decreased mTOR-dependent

phosphorylation of rpS6 can be directly associated with this

decreased inhibition of GSK3. Alternatively, as GSK3 acts as an

integrator of Akt and Wnt signals, the observed alteration of the

GSK3/mTOR pathway is related to a Wnt defect (Ma et al., 2010;

Vigneron et al., 2011). Consistent with the results of the current

study, alteration of GSK3 phosphorylation was also observed in a

rat PC12 cell line expressing 90 CUG repeats, which resulted in

Fig. 5. The mTOR defect is correlated with

decreased proliferation and induction of autophagy

in DM-NSCs. (A) Effect of rapamycin treatment on the

expression of key players of the Akt/mTOR signaling

pathway in DM1-and WT-NSCs, determined by

western blot analysis. (B) Detection, by

immunofluorescence, of P-rpS6 (Ser235–236) in DM1-

and WT-NSCs after treatment with rapamycin (10 nM)

for 48 hours. (C) Effect of rapamycin treatment (10 nM

for 4 days) on proliferation rate of WT-NSCs as

determined by measuring intracytoplasmic ATP.

(D) Quantification of the proliferation index of the WT-

NSCs with or without rapamycin treatment (10 nM) for

4 days. Data are presented as the mean signal [ATP] at

day x+1/x compared with the control condition

(arbitrarily 100%). (E) Detection of autophagy using

anti-LC3B antibody in WT-NSCs treated with

rapamycin (10 nM) or treated with chloroquine

(50 mM) for 6 hours. (F) Quantification of

autophagosomes after transfection of WT-NSCs with a

plasmid expressing LC3–GFP fusion protein for

48 hours and treatment with rapamycin and/or

chloroquine for 6 hours. Data are presented as a

percentage of positive cells quantified using the

automated ArrayScan Imager. Statistical data are

expressed as means 6 s.e.m. of at least three

independent samples. One-way ANOVA with

Dunnett’s post-hoc test was performed; **P,0.01,

***P,0.001.

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altered expression and phosphorylation of Tau (Hernandez-

Hernandez et al., 2006). Given the crucial role of Tau in

neurodegenerative diseases, further investigations into whether

the decreased GSK3 phosphorylation observed in DM1-NSCs

could lead to defects in Tau expression would be of interest.

Mechanisms by which the mutation causing DM1 could

modulate the phosphorylation of specific molecular targets of themTOR signaling pathways remain to be determined. Three main

hypotheses may be proposed: (1) haploinsufficiency of the

DMPK gene (Reddy et al., 1996); (2) alteration of the expression

of neighboring genes such as SIX5 and DMDW (Klesert et al.,

2000; Sarkar et al., 2000); (3) toxic gain-of-function of the

mutant mRNA because of the sequestration of proteins involved

in RNA processing, such as the MBNL1 protein (Ranum and

Cooper, 2006). Although several lines of evidence suggest that

the toxic gain-of-function of the mutant RNA corresponds to the

main mechanism by which the mutation resulting in DM1 causes

the complex pattern of the disease, the contributions of each of

these mechanisms are not mutually exclusive. As an example,

insulin-resistance associated with DM1 has been correlated with

both DMPK haploinsufficiency and aberrant regulation of

alternate splicing of the insulin receptor because of the loss of

bioavailability of MBNL1 (Llagostera et al., 2007; Savkur et al.,

2001). The current results following gain- or loss-of-function of

MBNL1 support the hypothesis of a potential role for a toxic

RNA mechanism in mTOR signaling alterations that involves

Fig. 6. mTOR signaling defect in DM1-

NSCs is related to MBNL1. (A) Decreased

expression level of P-rpS6 after transfection of

WT-NSCs with a plasmid expressing 960 CTG

for 24 hours. (B) Western blots were analyzed

using ImageJ software and quantitative data are

presented as the phosphorylated form

normalized to the total form. (C) Expression

level of the key modulators of the Akt/mTOR

signaling pathway in WT-NSCs nucleofected

with a SiRNA targeting MBNL1 or with a

siScramble RNA for 48 hours. Data are

presented as the phosphorylated form

normalized to the total form. (D) Effect of the

overexpression of MBNL1 in DM1-NSCs on

the expression level of the key modulators of

the Akt/mTOR signaling pathway. Cells were

nucleofected with a plasmid encoding the

43 kDa isoform of the MBNL1 protein

(pMBNL1) or mock nucleofected for 48 hours.

Quantitative data are presented as

phosphorylated form normalized to their total

form. Western blots were quantified using

ImageJ software. (E) Effect of the

downregulation of MBNL1 in WT-NSCs and

the overexpression of MBNL1 in DM1-NSCs

on proliferation. Proliferation was measured by

immunostaining for Ki67 48 hours after

nucleofection. (F) Effect of the downregulation

of MBNL1 in WT-NSCs and the

overexpression of MBNL1 in DM1-NSCs on

the induction of autophagy. Cells were co-

nucleofected with a plasmid expressing the

LC3–GFP protein fusion and autophagy was

quantified 48 hours after nucleofection by

measuring the number of cells containing

LC3–GFP dots. Data are expressed as means 6

s.e.m. of at least three independent samples.

One-way ANOVA with Dunnett’s post-hoc test

was performed, *P,0.05, **P,0.01.

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loss of MBNL1 bioavailability. Further studies will be required to

determine the mechanism by which MBNL1 may modulate theGSK3/mTOR signaling pathway. However, it is worth

mentioning that the effect of gain- and loss-of-function of

MBNL1 is less pronounced on proliferation than on autophagy,

which suggests the involvement of other modulators in theproliferation phenotype. Accordingly, CUGBP1, another RNA-

binding protein whose expression is affected by the presence of

the DM1 mutation, has been shown to affect the expression ofcell-cycle inhibitor p21 expression in DM1.

A large number of neurological abnormalities have beenreported in patients both at the clinical and histological levels (de

Leon and Cisneros, 2008). Central nervous system involvement

in DM1 implicates cognitive impairment, hypersomnolence, and

personality and behavioral disturbances (Abe et al., 1994;Delaporte, 1998; Meola et al., 2003; Modoni et al., 2008;

Turnpenny et al., 1994). Grey matter reduction has also been

described in various cortical regions, the hippocampus and thethalamus of DM1 patients (Minnerop et al., 2008; Minnerop et al.,

2011; Weber et al., 2010). At the molecular level, nuclear

aggregation of mutant mRNAs in combination with MBNL1 has

been described in cortical and subcortical neuronal cells of DM1patients post-mortem (Jiang et al., 2004). Alternative splicing of N-

methyl-D-aspartate receptor1 (NMDAR1), amyloid beta precursor

protein (APP) and microtubule-associated protein tau (MAPT) isabnormally regulated in DM1 brain tissue samples, even though

direct involvement of MBNL1 in these splice defects has not been

demonstrated (Jiang et al., 2004). However, the functional

consequences as well as the precise neuropathologicalcontributions of these molecular abnormalities are still unclear.

MBNL1 knockout mice exhibit cognitive impairments, suggesting

that MBNL1 loss-of-function might be implicated in theneuropathology of DM1 (Matynia et al., 2010). The MBNL1

knockout mouse model has also identified novel splicing defects in

the brain, including Sorbs1, Dclk1 and Camk2d (Suenaga et al.,

2012), but these genes have not been associated with clinicalsymptoms, yet. Lastly, a very recent study using a MBNL2

knockout mouse model suggests an essential role for this gene in

the DM1 developing brain (Charizanis et al., 2012). However, thisconclusion needs to be qualified in the light of redundant functions

of the different types of MBNL1 and MBNL2 proteins (Wang

et al., 2012).

The results of the current study shed some light on the

neuropathology of DM1, as we have identified an MBNL1-

dependent mTOR signaling defect, leading to functionalabnormalities, in neural precursors that have been shown by

previous authors to exhibit a telencephalic phenotype (Chambers

et al., 2009). Tentatively, and subject to further validation, onemay relate the proliferation defects observed in DM1-NSCs to

the reduced volume of the grey matter in DM1 patients. mTOR-

dependent induction of autophagy may also participate in the

neuropathology, as a similar process has been suggested to play arole in other neurological disorders involving abnormal protein

aggregations, such as Parkinson’s, Alzheimer’s and Huntington’s

diseases (Garelick and Kennedy, 2011).

Materials and MethodsCell cultures

hES cells were propagated as previously described (Marteyn et al., 2011). Thedifferentiation of hES into NSCs was performed using a SMAD inhibitor protocol(Chambers et al., 2009). Briefly, hES colonies were mechanically dissociated andincubated in suspension with N2B27 medium containing DMEM/F12 medium

with Glutamax I mixed with neurobasal medium; N2 supplement; B27 supplementwithout vitamin A; b-mercaptoethanol (all from Invitrogen) (1:1 vol). Thefollowing were also added: human recombinant noggin, a BMP pathway inhibitor,used at 300 ng/ml (PeproTech); SB-431542, a TGFb pathway inhibitor, used at20 mM (Tocris); and Y-27632, a ROCK inhibitor, used at 20 mM (Calbiochem).After 6 hours, aggregates were transferred to a dish pre-coated with 0.01%polyornithine (Sigma) and laminin 1 ng/ml (Sigma) and maintained with medium(without Y-27632). After the appearance of neural rosettes following 8–10 days ofdifferentiation, the medium was replaced with the N2B27 medium supplementedwith EGF at 10 ng/ml (R&D Systems), FGF2 at 10 ng/ml (PeproTech) and humanBDNF (hBDNF) at 20 ng/ml (R&D Systems). For differentiation into neurons,NSCs were seeded at a density of 50,000 cells per cm2 on polyornithine/laminin-coated dishes in N2B27 medium containing hBDNF but without EGF and FGF2.Medium was changed every other day for 20 days. Treatment with chemicalcompounds such as rapamycin (Cell Signaling), chloroquine and bafilomycin(Sigma) was performed according to the manufacturer’s instructions.

Senescence, apoptosis and proliferation assay

A senescence assay was performed using a senescence detection kit based on b-galactosidase activity, as per the manufacturer’s instructions (Abcam). Apoptosisassay was performed using Vybrant Apoptosis Assay Kit (Molecular Probes)according to manufacturer’s recommendations and proliferation was measuredusing CTG Cell Titer GloH (Promega). Briefly, at day 1 NSCs were plated in 96-well plates in 100 ml medium at 2000 cells/cm2. CellTiter-GloH reagent (Promega)was added to the cells and the bioluminescence signal was read 40 minutes later onan AnalystGT microplate reader (Molecular devices).

Mean doubling time (Tmean) was calculated for each cell line as follows:

Tmean~Xday6

day~0

Ln(2)day(xz1){day(x)

Ln ncellsday(xz1)ð Þ{ ncellsday(x)ð Þ =6,

The number of cells (ncells) at a defined time of the experiment was calculatedusing a calibration curve obtained by serial dilution of a defined number of cellsand correlated to the luminescence signal (proportional to the intracellularconcentration of ATP).

RNA extraction and real-time RT-PCR

Total RNA from cells was extracted using the ‘RNeasy Mini Kit Protocol’(Qiagen). Reverse transcription was performed with SuperScript III reversetranscriptase (Invitrogen) and real-time PCR were performed with SyberGreenPCR Master Mix (Applied Biosystems) on a Chromo4 real-time system (Bio-Rad)as previously described (Marteyn et al., 2011). Primers are listed in supplementarymaterial Table S1.

Nucleofection experiments with p960-CTG, siMBNL1 and the expressionplasmid MBNL1

The sequence encoding the 43 kDa isoform of MBNL1 inserted in the pCDNA3.1vector and the plasmid (CTG)960 were kindly provided by Dr Nicolas Charlet(CERBM-GIE, Illkirch, France). The plasmid expressing the LC3–GFP fusionprotein was kindly provided by Prof. Codogno (INSERM U984, Chatenay-Malabry, France). The siRNA sequence for MBNL1 knock-down was previouslydescribed (Dansithong et al., 2005).

These expression vectors and siRNAs were nucleofected into NSCs using theRat Neural Stem Cell Nucleofector Kit (Lonza) according to the manufacturer’sinstructions.

Immunocytochemistry and fluorescent in situ hybridization

Immunocytochemisty and fluorescent in situ hybridization were performed aspreviously described (Marteyn et al., 2011). Primary antibodies are listed insupplementary material Table S2. Immunostaining was analyzed byepifluorescence microscopy with the Zeiss Imager Z1 and the Zeiss Axiovert40CFL, and images were captured with the Zeiss Axiocam mRM.

ArrayScan analysis

The number of nuclei with foci, the number of foci per nucleus and the number ofcells with at least two LC3–eGFP-positive autophagosomes were counted using theArrayScan VTI HCS Reader (Cellomics). Data were examined using the CellSelecting software (Cellomics) as previously described (Marteyn et al., 2011).

SDS poly-acrylamide gel electrophoresis and western blotting

Cells were lysed in RIPA lysis buffer (Sigma) supplemented with anti-proteasecocktail (Sigma) and anti-phosphatase (Roche). Nuclear and cytoplasmic fractionswere extracted using the NE-PER Kit (Thermoscientific). The proteinconcentration of the cell extracts was measured at 562 nm using the PierceHBCA Protein Assay Kit (Perbio Thermo Scientific) according to themanufacturer’s instructions and using a plate analyzer (Biotek). SDS-PAGE was

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performed using NuPAGEH Novex 4–12% Bis-Tris gels (Invitrogen); 10–30 mg oftotal protein was loaded per lane. iBlotH Gel Transfer Stack (Invitrogen) was usedfor the transfer onto nitrocellulose membranes. Membranes were blocked with 5%non-fat milk in phosphate-buffered saline containing 0.1% Tween 20 for 1 hour(except for MBNL1 antibody which requires 2% fetal calf Serum in PBST), thenincubated overnight at 4 C in 5% non-fat milk in PBST (except for MBNL1 inPBST only) with primary antibodies at the appropriate dilution. The antibodiesused are listed in supplementary material Table S2. Immunoreactive bands wererevealed using the Amersham ECL PlusTM Western Blotting Detection Reagents(GE Healthcare). Equal protein loading was verified by the detection of actin.Quantification was performed using ImageJ software (NIH).

Statistical analysis

All the data are given as means 6 standard error of the mean (s.e.m.) and wereanalyzed using GraphPad Prism 6.0 software. All the statistical tests wereperformed at least three times and one-way ANOVA with Dunnett’s post-hoc testwas used.

AcknowledgementsWe thank Dr Genevieve Gourdon (INSERM U781, Paris, France) forperforming CTG repeats analysis; Karen Sermon (AZ-VUBBrussels, Belgium) for providing DM1 and control hES cell lines;Dr Nicolas Charlet (INSERM, Strasbourg, France) and Dr GlenMorris for providing us with resources; Prof. Patrice Codogno andIsabelle Beau (INSERM U984) for the LC3BeGFP construction andtheir very helpful advice concerning autophagy; Dr Denis Furling(INSERM U787, Paris, France), Dr Nicolas Sergeant and Dr Marie-Laure Caillet-Boudin (INSERM U815, Lille, France) for theirhelpful discussions and their expertise on the field of myotonicdystrophy disease; and Dr Delphine Laustriat, Dr FabriceCasagrande, Julien Come, Pauline Georges, Jacqueline Gide andRemi Vernet (UMR861, CES, I-Stem, Evry, France) for technicalassistance.

Author contributionsJ.A.D. designed and performed experiments and analyzed data;M.G., L.R. and S.A. contributed equally to this work; M.G.performed gain and loss of function experiments and L.R. and S.A.performed western blot analyses for mTOR signaling pathway;K.G.T, P.P., B.C., Y.M. and C.B. participated differentially todifferent technical aspects of this project; A.B. helped with thedifferentiation of neural stem cells from hESCs; R.S. and G.P.contributed to the design of the project; M.P. and C.M. supervisedthe project.

FundingThis work was supported by the Association Francaise contre lesMyopathies, MediCen (IngeCell program); the EuropeanCommission (FP6, STEM-HD) [grant number LSHB-CT-2006-037349]; and Genopole.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.116285/-/DC1

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