1

mTOR-dependent proliferation defect in human ES-derived neural stem cells affected by Myotonic Dystrophy Type1

Jérôme Alexandre Denisa,d, Morgane Gauthiera*, Latif Rachdib*, Sophie Aubertc*,

Karine Giraud-Triboultc, Pauline Poydenotc, Alexandra Benchouac, Benoite Champonc,

Yves Mauryc, Christine Baldeschia, Raphael Scharfmannb, Geneviève Piétua, Marc

Peschanskia, and Cécile Martinata+

a INSERM/UEVE U-861, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of

Monogenic Diseases, 5 rue Henri Desbruères, 91030 Evry cedex, France b INSERM U-845 Research Center “Growth and Signaling”, Faculty of Medicine Paris

Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France c CECS, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic

Diseases, 5 rue Henri Desbruères, 91030 Evry cedex, France d Present address: APHP, Saint Louis-Lariboisière-Fernand Widal University Hospitals,

Lariboisière Hospital, Dept. of Medical Biochemistry and Molecular Biology, Biological

Resource Center, UMRS/INSERM U-942, 2 rue Ambroise Paré 75475 Paris cedex 10,

France.

* Participated equally to the work

+ To whom correspondence should be addressed. Email: cmartinat@istem.fr

Running title: Defect of mTOR signaling pathway in DM1

Keys words: Neural stem cells; human embryonic stem cells; pathological modeling;

Myotonic Dystrophy type 1; mTOR signaling pathways

© 2012. Published by The Company of Biologists Ltd.Jo

urna

l of C

ell S

cien

ceA

ccep

ted

man

uscr

ipt

JCS Advance Online Article. Posted on 26 February 2013

2

ABSTRACT

Patients with Myotonic Dystrophy type 1 exhibit a diversity of symptoms that affect

many different organs. Among those are cognitive dysfunctions, the origin of which has

remained elusive due in part to the difficulty in accessing neural cells. Here, we have

taken advantage of pluripotent stem cell lines derived from embryos identified during a

pre-implantation genetic diagnosis as mutant gene-carriers, in order to differentiate cells

along the neural lineage. Functional characterization of these cells revealed reduced

proliferative capacity and increased autophagy linked to mTOR signaling pathway

alterations. Interestingly, loss of function of MBNL1, a RNA-binding protein whose

function is defective in DM1 patients, resulted in the mTOR signaling alteration whereas

gain-of-function experiments rescued the phenotype. 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.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

3

INTRODUCTION

Due 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 derived from

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

molecular and functional pathological mechanisms within difficult-to-obtain cell populations

(Gauthier et al., 2012; 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 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 these data by

analyzing the first stages of disease progression and to decipher molecular mechanisms that

could be at the origin of a disease-related functional phenotype.

Over the past years, a growing number of studies have demonstrated this application

both for neurodevelopmental and neurodegenerative disorders. Pre-implantation genetically

diagnosed embryos have, for instance, allowed 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) to be analyzed.

Other young childhood-onset monogenic disorders, such as the 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 3’-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.,

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

4

2009; Furling et al., 2001; Timchenko et al., 2001a). Although DM1 has long been mainly

considered as a muscle disorder, there is extensive evidence for 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 with 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 proliferative

deficiency linked to a DM1-related impairment in mTOR signaling pathway activity,

associated with increased autophagy.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

5

RESULTS

Two human embryonic stem cell lines (hES) 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 differences in neuronal differentiation were observed between control and

mutant cells at day 20 for most of the analyzed gene markers, such as SNAP25, DLG4 and

MAPT, although we cannot completely exclude a difference between DM1 and WT over the

time course of neuronal differentiation (Supp. Fig. S1). Validating their pathological

relevance, in situ hybridization combined with immunostaining indicated that DM1-NSCs and

derived-neurons harbored intranuclear ribonucleoprotein aggregates (named “foci”) formed

by the mutant RNA and the splice factor MBNL1, which are typical of the disease (Fig. 1d).

The nuclear retention of MBNL1 was also confirmed by western blot (Fig. 1e).

Functional consequences of DM1 in neural stem cells

Quantitative analysis of doubling time through measurement of the ATP concentration in

cell cultures indicated that the proliferation rate of DM1-NSCs was reduced when compared

to controls (Fig. 2a and 2b). 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 and 2e). 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, β−galactosidase staining did not reveal any differences in the proportion of

senescent cells in the DM1 and WT NSCs cultures (Supp. Fig. S2). Altogether, these results

indicated that the reduced proliferative capacity of DM1-NSCs was not associated to 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 (also named SQSTM1) and ATG12 protein was observed

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

6

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 (Supp. 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, showed in an increased number of

DM1-NSCs harboring LC3-GFP aggregates, relative to controls (Fig. 3c and 3d). As a

positive control, treatment of WT-NSC with either EBSS medium or chloroquine resulted in

an increased number of cells containing LC3-GFP aggregates. No effect of chloroquine

treatment was 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, GSK3α/β and rpS6

(Ser235/236) (for 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 and Supp. FigS4). In contrast, analysis of the post-

translational activation of these proteins through phosphorylation revealed major differences

between DM1 and controls. Whereas phosphorylation of the upstream components Akt

(Ser473) and AMPK (Thr172) appeared unaffected, the downstream GSK3α/β (Ser21/9) and

rpS6 (Ser235/236 and Ser240/245) were decreased in DM1-NSCs (Fig. 4a and 4b) through a

mechanism independent of a stress-induced activation of P53 (Supp. Fig S4).

To eliminate the possibility of a non-specific effect of the growth factors present in the

medium (i.e. EGF, FGF2 and BDNF) on mTOR signaling pathway activity, NSCs were

starved for forty eight hours and then treated for one hour with each factor alone or in

combination. No effect of EGF and/or BDNF was observed on phosphorylated Ser235/236-

rpS6 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 and 4e). The effect of FGF2-mediated ERK1/2 activation on the phosphorylation of P-

rpS6 was transient, as it disappeared forty eight hours after treatment. As all our experiments

were performed forty eight hours after treatment with growth factors, these results suggest

that a differential impact of growth factors present in the medium on mTOR signaling

pathways in DM1- and WT- NSCs can be ruled out.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

7

Molecular correlates of functional defects in DM1 neural cells

A potential link between the functional defects observed in DM1-NSCs for proliferation

and autophagy 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 (Ser235/236)-rpS6 without substantial modulation of

either phosphorylated (Ser473)-Akt, (Ser21)-GSK3α or (Ser9)-GSK3β (Fig. 5a and 5b and

Supp. 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 and 5d and Supp. Fig S4) and induction of

autophagy, as shown by LC3-GFP aggregation after transfection with the fusion protein

plasmid (Fig. 5e and 5f ).

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 960 CTG repeats. Treated cells exhibited a profound decrease in P-rpS6 as well as

P-GSK3β, which was consistent with our working hypothesis (Fig. 6a and 6b and Supp. 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-GSK3α/β (Ser21/9)

(Fig. 6b). Conversely, overexpression of MBNL1 in DM1-NSCs elicited a partial restoration

of the phosphorylation of Ser232/236-rpS6 (Fig. 6c). These changes in MBNL1 expression in

DM1 and WT-NSCs led to altered proliferation and autophagy (Fig. 6d and 6e).

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

8

DISCUSSION

The primary finding of the current study was the identification of an altered mTOR

signaling pathway, which results in reduced proliferative capacity and induction of autophagy

in neural cells derived from DM1 gene-carrying human embryonic stem cells. This alteration

in the mTOR signaling pathway could be reproduced by reducing the bioavailability of the

RNA-binding protein MBNL1 in WT cells, mimicking the defect associated to 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 exhibited 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 where 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

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

9

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 associated with the disease. GSK3 activity is inhibited through

phosphorylation of serine 21 in GSK-3α and serine 9 in GSK-3β, 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 to 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 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 DM1 mutation can modulate the phosphorylation of specific

molecular targets of the mTOR 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). Whereas various evidences suggests that the toxic gain-of-function of the

mutant RNA corresponds to the main mechanism by which the DM1 mutation 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 both to DMPK haploinsufficiency and to aberrant regulation of alternate splicing of

the insulin receptor due to 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 loss of MBNL1 bioavailability. Further studies will be required to

determine the mechanism by which MBNL1 may modulate the GSK3/mTOR signaling

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

10

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

implication of other modulators in the proliferation 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 of cell cycle inhibitor p21 expression in

DM1.

A large number of neurological abnormalities have been reported in patients both at

clinical and histological levels (de Leon and Cisneros, 2008). Central nervous system

involvement in DM1 reveals itself through 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 the thalamus 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 DM1 patients post-mortem (Jiang et al., 2004).

Alternative splicing of NMDAR1 (N-methyl-D-aspartate receptor1), APP (amyloid beta

precursor protein) and microtubule-associated protein tau (MAPT) is abnormally 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 neuropathological contributions of these molecular abnormalities are still

unclear. MBNL1 knock-out mice exhibit cognitive impairments, suggesting that MBNL1

loss-of-function might be implicated in the neuropathology 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 to clinical symptoms, yet. Last, a very recent study using a MBNL2 knock-out

mouse model suggests an essential role for this gene in the DM1 developing brain (Charizanis

et al., 2012). However, this conclusion 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 a MBNL1-dependent mTOR signaling defect, leading to functional abnormalities,

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, one may

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

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

11

neuropathology, as a similar process has been suggested to play a role in other neurologic

disorders involving abnormal protein aggregations, such as Parkinson, Alzheimer and

Huntington diseases (Garelick and Kennedy, 2010).

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

12

MATERIALS AND METHODS

Cell cultures

hES cells were propagated as previously described (Marteyn et al., 2011). The differentiation

of hES into NSC was performed by using a SMAD inhibitor protocol (Chambers et al., 2009).

Briefly, hES colonies were mechanically dissociated and incubated in suspension with N2B27

medium containing: 1:2 of DMEM/F12 medium with Glutamax I; 1:2 of Neurobasal medium;

N2 supplement; B27 supplement without vitamin A; β-Mercaptoethanol (all from Invitrogen).

The following were also added: human recombinant Noggin, a BMP pathway inhibitor, used

at 300 ng/mL (PeproTech); SB-431542, a TGFβ pathway inhibitor, used at 20 µM (Tocris);

Y-27632, a ROCK inhibitor, used at 20 µM (Calbiochem). After 6 hours, aggregates were

transferred in 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 of differentiation, the medium was replaced by the N2B27

medium supplemented with EGF (Epidermal Growth Factor) at 10 ng/mL (R&D Systems);

FGF2 (Fibroblast Growth Factor) at 10 ng/mL (PeproTech); hBDNF (human Brain-Derived

Neurotrophic Factor) at 20 ng/mL (R&D Systems). For differentiation into neurons, NSC

cells 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 chemical compounds such as rapamycin (Cell

Signaling), chloroquine and bafylomycin (Sigma) was performed following the

manufacturer’s instructions.

Senescence, apoptosis and proliferation assay

Senescence assay was performed using a senescence detection kit based on β -galactosidase

activity as per the manufacturer’s instructions (Abcam). Apoptosis assay was performed using

Vybrant Apoptosis Assay Kit (Molecular Probes) according to manufacturer’s

recommendations and proliferation was measured using CTG Cell Titer Glo® (Promega).

Briefly, at Day 1 NSCs were plated in 96-well plates in 100µL media at 2,000 cells/cm2.

CellTiter-Glo® reagent (Promega) was added to the cells and the bioluminescence signal was

read 40 min later on an AnalystGT microplate reader (Molecular devices).

Mean Doubling time (Tmean) was calculated for each cell line as followed:

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

13

Number of cells at a defined time of the experiment was calculated using calibration curve

obtained by serial dilution of a defined number of cells and correlated to luminescence signal

(proportional to [ATP] intracellular concentration).

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 reverse transcriptase (Invitrogen) and real-

time PCR were performed with SyberGreen PCR Master Mix (Applied Biosystems) on a

Chromo4 Real-Time system (Bio-Rad) as previously described (Marteyn et al., 2011).

Primers are listed in Supp. Table S1.

Nucleofection experiment with p960-CTG, siMBNL1 and expression plasmid MBNL1

The sequence encoding the 43kDa isoform of MBNL1 inserted in the pCDNA3.1 vector and

the plasmid (CTG)960 were kindly provided by Dr Nicolas Charlet (CERBM-GIE, Illkirch,

France). The plasmid expressing the LC3-GFP fusion protein was kindly provided by Prof.

Codogno (INSERM U984, Chatenay-Malabry, France). The siRNA sequence for MBNL1

knock-down was previously described (Dansithong et al., 2005).

These expression vectors and siRNAs were nucleofected into NSCs using the “Rat Neural

Stem Cell Nucleofector Kit” (Lonza) according to the manufacturer's instructions.

Immunocytochemistry and Fluorescent in Situ Hybridization (FISH)

Immunocytochemisty and fluorescent in situ hybridization were performed as previously

described (Marteyn et al., 2011). Primary antibodies are listed in Supp. Table S2.

Immunostaining was analyzed by epifluorescence microscopy with the Zeiss Imager Z1 and

the Zeiss Axiovert 40CFL, and images were captured with the Zeiss Axiocam mRM.

ArrayScan analysis

Number of nuclei with foci, number of foci per nucleus and number of cells with at least two

LC3-eGFP positive autophagosomes were counted using the ArrayScan VTI HCS Reader

(Cellomics). Data were examined using the Cell Selecting software (Cellomics) as previously

described (Marteyn et al., 2011).

SDS Poly-Acrylamide Gel Electrophoresis and Western Blotting

Cells were lyzed in RIPA lysis buffer (Sigma) supplemented with antiprotease cocktail

(Sigma) and antiphosphatase (Roche). Nuclear and cytoplasmic fractions were extracted using

the NE-PER Kit (Thermoscientific). Protein concentration of the cell extracts was determined

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

14

at 562 nm using the Pierce® BCA Protein Assay Kit (Perbio Thermo Scientific) according to

the manufacturer's instructions and using a plate analyzer (Biotek). SDS Poly-Acrylamide Gel

Electrophoresis (SDS-PAGE) was performed using NuPAGE® Novex 4-12% Bis-Tris Gels

(Invitrogen); 10 to 30 µg of total protein was loaded per lane. iBlot® Gel Transfer Stack

(Invitrogen) was used for the transfer onto nitrocellulose membranes. Membranes were

blocked with 5% non-fat milk in Phosphate Buffered Saline containing 0.1% Tween 20

(PBST) for 1 hour (except for MBNL1 antibody which requires SVF 2% in PBST), then

incubated overnight at 4°C in 5% non-fat milk in PBST (except for MBNL1 : PBST only)

with primary antibodies at the appropriate dilution. The antibodies used are listed in Supp.

Table S2. Immunoreactive bands were revealed by using Amersham ECL Plus™ Western

Blotting Detection Reagents (GE Healthcare). Equal protein loading was verified by the

detection of Actin. Quantification was performed using the Image J software (NIH).

Statistical analysis

All the data are given as mean ± standard error of the mean (SEM) and were analyzed by

GraphPad Prism 6.0 software. All the statistical tests were performed at least 3 times and one-

way ANOVA with Dunnett's post hoc test was used.

ACKNOWLEDGEMENTS

This work was supported by the Association Française contre les Myopathies (AFM),

MediCen (IngeCell program), the European Commission (FP6, STEM-HD) and Genopole.

We thank Dr. Genevieve Gourdon (INSERM U781, Paris, France) for performing CTG

repeats analysis; Karen Sermon (AZ-VUB Brussels, Belgium) for providing DM1 and control

hES cell lines; Dr. Nicolas Charlet (INSERM, Strasbourg, France) and Dr. Glen Morris for

providing resources to us. Prof. Patrice Codogno and Isabelle Beau (INSERM U984) for

providing us LC3BeGFP construction and their 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 their helpful discussions and their expertise

on the field of Myotonic Dystrophy disease and Dr. Delphine Laustriat, Dr. Fabrice

Casagrande, Julien Come, Pauline Georges, Jacqueline Gide and Rémi Vernet (UMR861,

CES, I-Stem, Evry, France) for technical assistance.

AUTHOR INFORMATION

The authors declare having no competing financial interests.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

15

REFERENCES Abe, K., Fujimura, H., Toyooka, K., Yorifuji, S., Nishikawa, Y., Hazama, T. and Yanagihara, T. (1994). Involvement of the central nervous system in myotonic dystrophy. J Neurol Sci 127, 179-85. Beffy, P., Del Carratore, R., Masini, M., Furling, D., Puymirat, J., Masiello, P. and Simili, M. (2010). Altered signal transduction pathways and induction of autophagy in human myotonic dystrophy type 1 myoblasts. Int J Biochem Cell Biol 42, 1973-83. Bigot, A., Klein, A. F., Gasnier, E., Jacquemin, V., Ravassard, P., Butler-Browne, G., Mouly, V. and Furling, D. (2009). Large CTG Repeats Trigger p16-Dependent Premature Senescence in Myotonic Dystrophy Type 1 Muscle Precursor Cells. Am J Pathol. Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M. and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-80. Charizanis, K., Lee, K. Y., Batra, R., Goodwin, M., Zhang, C., Yuan, Y., Shiue, L., Cline, M., Scotti, M. M., Xia, G. et al. (2012). Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy. Neuron 75, 437-50. Dansithong, W., Paul, S., Comai, L. and Reddy, S. (2005). MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1. J Biol Chem 280, 5773-80. de Leon, M. B. and Cisneros, B. (2008). Myotonic dystrophy 1 in the nervous system: from the clinic to molecular mechanisms. J Neurosci Res 86, 18-26. Delaporte, C. (1998). Personality patterns in patients with myotonic dystrophy. Arch Neurol 55, 635-40. Ebert, A. D., Yu, J., Rose, F. F., Jr., Mattis, V. B., Lorson, C. L., Thomson, J. A. and Svendsen, C. N. (2009). Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277-80. Ebralidze, A., Wang, Y., Petkova, V., Ebralidse, K. and Junghans, R. P. (2004). RNA leaching of transcription factors disrupts transcription in myotonic dystrophy. Science 303, 383-7. Eiges, R., Urbach, A., Malcov, M., Frumkin, T., Schwartz, T., Amit, A., Yaron, Y., Eden, A., Yanuka, O., Benvenisty, N. et al. (2007). Developmental Study of Fragile X Syndrome Using Human Embryonic Stem Cells Derived from Preimplantation Genetically Diagnosed Embryos. Cell Stem Cell 1, 568-577. Fang, X., Yu, S. X., Lu, Y., Bast, R. C., Jr., Woodgett, J. R. and Mills, G. B. (2000). Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A. Proc Natl Acad Sci U S A 97, 11960-5. Furling, D., Coiffier, L., Mouly, V., Barbet, J. P., St Guily, J. L., Taneja, K., Gourdon, G., Junien, C. and Butler-Browne, G. S. (2001). Defective satellite cells in congenital myotonic dystrophy. Hum Mol Genet 10, 2079-87. Garelick, M. G. and Kennedy, B. K. (2010). TOR on the brain. Exp Gerontol 46, 155-63. Gauthier, M., Marteyn, A., Denis, J., Furling, D., Charlet, N., Marie, J., Sermon, K., Peschanski, M., Pietu, G. and Martinat, C. (2012). Mutant gene-carrying human embryonic stem cells reveal a defective Krab protein in myotonic dystrophy type 1. submitted. Hernandez-Hernandez, O., Bermudez-de-Leon, M., Gomez, P., Velazquez-Bernardino, P., Garcia-Sierra, F. and Cisneros, B. (2006). Myotonic dystrophy expanded CUG repeats disturb the expression and phosphorylation of tau in PC12 cells. J Neurosci Res 84, 841-51.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

16

Jastrzebski, K., Hannan, K. M., Tchoubrieva, E. B., Hannan, R. D. and Pearson, R. B. (2007). Coordinate regulation of ribosome biogenesis and function by the ribosomal protein S6 kinase, a key mediator of mTOR function. Growth Factors 25, 209-26. Jiang, H., Mankodi, A., Swanson, M. S., Moxley, R. T. and Thornton, C. A. (2004). Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 13, 3079-88. Kim, J. E., O'Sullivan, M. L., Sanchez, C. A., Hwang, M., Israel, M. A., Brennand, K., Deerinck, T. J., Goldstein, L. S., Gage, F. H., Ellisman, M. H. et al. (2011). Investigating synapse formation and function using human pluripotent stem cell-derived neurons. Proc Natl Acad Sci U S A 108, 3005-10. Klesert, T. R., Cho, D. H., Clark, J. I., Maylie, J., Adelman, J., Snider, L., Yuen, E. C., Soriano, P. and Tapscott, S. J. (2000). Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat Genet 25, 105-9. Lee, G., Papapetrou, E. P., Kim, H., Chambers, S. M., Tomishima, M. J., Fasano, C. A., Ganat, Y. M., Menon, J., Shimizu, F., Viale, A. et al. (2009). Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402-6. Llagostera, E., Catalucci, D., Marti, L., Liesa, M., Camps, M., Ciaraldi, T. P., Kondo, R., Reddy, S., Dillmann, W. H., Palacin, M. et al. (2007). Role of myotonic dystrophy protein kinase (DMPK) in glucose homeostasis and muscle insulin action. PLoS One 2, e1134. Ma, Y., Jin, J., Dong, C., Cheng, E. C., Lin, H., Huang, Y. and Qiu, C. (2010). High-efficiency siRNA-based gene knockdown in human embryonic stem cells. Rna 16, 2564-9. Mahadevan, M., Tsilfidis, C., Sabourin, L., Shutler, G., Amemiya, C., Jansen, G., Neville, C., Narang, M., Barcelo, J., O'Hoy, K. et al. (1992). Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 255, 1253-5. Mankodi, A., Takahashi, M. P., Jiang, H., Beck, C. L., Bowers, W. J., Moxley, R. T., Cannon, S. C. and Thornton, C. A. (2002). Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol Cell 10, 35-44. Mankodi, A., Urbinati, C. R., Yuan, Q. P., Moxley, R. T., Sansone, V., Krym, M., Henderson, D., Schalling, M., Swanson, M. S. and Thornton, C. A. (2001). Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet 10, 2165-70. Marteyn, A., Maury, Y., Gauthier, M. M., Lecuyer, C., Vernet, R., Denis, J. A., Pietu, G., Peschanski, M. and Martinat, C. (2011). Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy. Cell Stem Cell 8, 434-44. Mateizel, I., De Temmerman, N., Ullmann, U., Cauffman, G., Sermon, K., Van de Velde, H., De Rycke, M., Degreef, E., Devroey, P., Liebaers, I. et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21, 503-11. Matynia, A., Ng, C. H., Dansithong, W., Chiang, A., Silva, A. J. and Reddy, S. (2010). Muscleblind1, but not Dmpk or Six5, contributes to a complex phenotype of muscular and motivational deficits in mouse models of myotonic dystrophy. PLoS One 5, e9857. Maury, Y., Gauthier, M., Peschanski, M. and Martinat, C. (2011). [Human pluripotent stem cells: opening key for pathological modeling]. Med Sci (Paris) 27, 443-6.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

17

Meola, G., Sansone, V., Perani, D., Scarone, S., Cappa, S., Dragoni, C., Cattaneo, E., Cotelli, M., Gobbo, C., Fazio, F. et al. (2003). Executive dysfunction and avoidant personality trait in myotonic dystrophy type 1 (DM-1) and in proximal myotonic myopathy (PROMM/DM-2). Neuromuscul Disord 13, 813-21. Minnerop, M., Luders, E., Specht, K., Ruhlmann, J., Schneider-Gold, C., Schroder, R., Thompson, P. M., Toga, A. W., Klockgether, T. and Kornblum, C. (2008). Grey and white matter loss along cerebral midline structures in myotonic dystrophy type 2. J Neurol 255, 1904-9. Minnerop, M., Weber, B., Schoene-Bake, J. C., Roeske, S., Mirbach, S., Anspach, C., Schneider-Gold, C., Betz, R. C., Helmstaedter, C., Tittgemeyer, M. et al. (2011). The brain in myotonic dystrophy 1 and 2: evidence for a predominant white matter disease. Brain 134, 3530-46. Modoni, A., Silvestri, G., Pomponi, M. G., Mangiola, F., Tonali, P. A. and Marra, C. (2004). Characterization of the pattern of cognitive impairment in myotonic dystrophy type 1. Arch Neurol 61, 1943-7. Modoni, A., Silvestri, G., Vita, M. G., Quaranta, D., Tonali, P. A. and Marra, C. (2008). Cognitive impairment in myotonic dystrophy type 1 (DM1): a longitudinal follow-up study. J Neurol 255, 1737-42. Perini, G. I., Menegazzo, E., Ermani, M., Zara, M., Gemma, A., Ferruzza, E., Gennarelli, M. and Angelini, C. (1999). Cognitive impairment and (CTG)n expansion in myotonic dystrophy patients. Biol Psychiatry 46, 425-31. Ranum, L. P. and Cooper, T. A. (2006). RNA-Mediated Neuromuscular Disorders. Annu Rev Neurosci. Reddy, S., Smith, D. B., Rich, M. M., Leferovich, J. M., Reilly, P., Davis, B. M., Tran, K., Rayburn, H., Bronson, R., Cros, D. et al. (1996). Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat Genet 13, 325-35. Sarkar, P. S., Appukuttan, B., Han, J., Ito, Y., Ai, C., Tsai, W., Chai, Y., Stout, J. T. and Reddy, S. (2000). Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nat Genet 25, 110-4. Savkur, R. S., Philips, A. V. and Cooper, T. A. (2001). Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 29, 40-7. Seibler, P., Graziotto, J., Jeong, H., Simunovic, F., Klein, C. and Krainc, D. (2011). Mitochondrial Parkin Recruitment Is Impaired in Neurons Derived from Mutant PINK1 Induced Pluripotent Stem Cells. J Neurosci 31, 5970-6. Suenaga, K., Lee, K. Y., Nakamori, M., Tatsumi, Y., Takahashi, M. P., Fujimura, H., Jinnai, K., Yoshikawa, H., Du, H., Ares, M., Jr. et al. (2012). Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain. PLoS One 7, e33218. Timchenko, L. T. and Caskey, C. T. (1996). Trinucleotide repeat disorders in humans: discussions of mechanisms and medical issues. Faseb J 10, 1589-97. Timchenko, N. A., Cai, Z. J., Welm, A. L., Reddy, S., Ashizawa, T. and Timchenko, L. T. (2001a). RNA CUG repeats sequester CUGBP1 and alter protein levels and activity of CUGBP1. J Biol Chem 276, 7820-6. Timchenko, N. A., Iakova, P., Cai, Z. J., Smith, J. R. and Timchenko, L. T. (2001b). Molecular basis for impaired muscle differentiation in myotonic dystrophy. Mol Cell Biol 21, 6927-38. Timchenko, N. A., Patel, R., Iakova, P., Cai, Z. J., Quan, L. and Timchenko, L. T. (2004). Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis. J Biol Chem 279, 13129-39.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

18

Turnpenny, P., Clark, C. and Kelly, K. (1994). Intelligence quotient profile in myotonic dystrophy, intergenerational deficit, and correlation with CTG amplification. J Med Genet 31, 300-5. Vigneron, F., Dos Santos, P., Lemoine, S., Bonnet, M., Tariosse, L., Couffinhal, T., Duplaa, C. and Jaspard-Vinassa, B. (2011). GSK-3beta at the crossroads in the signalling of heart preconditioning: implication of mTOR and Wnt pathways. Cardiovasc Res 90, 49-56. Wang, E. T., Cody, N. A., Jog, S., Biancolella, M., Wang, T. T., Treacy, D. J., Luo, S., Schroth, G. P., Housman, D. E., Reddy, S. et al. (2012). Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins. Cell 150, 710-24. Weber, Y. G., Roebling, R., Kassubek, J., Hoffmann, S., Rosenbohm, A., Wolf, M., Steinbach, P., Jurkat-Rott, K., Walter, H., Reske, S. N. et al. (2010). Comparative analysis of brain structure, metabolism, and cognition in myotonic dystrophy 1 and 2. Neurology 74, 1108-17. Yadava, R. S., Frenzel-McCardell, C. D., Yu, Q., Srinivasan, V., Tucker, A. L., Puymirat, J., Thornton, C. A., Prall, O. W., Harvey, R. P. and Mahadevan, M. S. (2008). RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression. Nat Genet 40, 61-8.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

19

Figure Legends Figure 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 are counterstained with DAPI (blue) (Scale bar=20µm). b. Quantification of Nestin expression by FACS c. Quantitative real-time RT-PCR analysis of hESC-derived NSC. RT-PCR levels are presented as the fold change relative to undifferentiated hESC lines after normalization with 18S ribosomal RNA levels. d. Co-detection of mutant RNA with a (CAG)10-Cy3 probe staining and MBNL1 (Muscleblind) protein by immunofluorescence. Nuclei are counterstained using DAPI (blue). The number of foci per nuclei 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 fractioning. Lamin and β-actin were used as loading controls. Figure 2: DM-NSCs exhibit lower proliferative capacity a. Proliferation rate for 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. Decreased number of Ki67 positive DM1-NSCs (VUB03_DM) after 48 hours of culture in comparison with WT-NSCs. The number of Sox2 positive cells was used as a control for NSCs phenotype. Detection and quantification of Ki67 positive cells was done by 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 ribosomal RNA levels. e. Decreased expression level of Phosphorylated Rb (S807-811) in DM1-NSCs (VUB03_DM) when compared with WT-NSCs as determined by western blotting. f. Increased expression level of P27 in DM1-NSCs as compared with control NSCs as revealed with western blotting. g. Altered expression of p15ink4B, p21waf1 cell and Cyclin D1 proteins in DM1-NSCs determined by western blot analysis. All western blot analyses were performed using protein extracted from NSCs cells after 48 hours of culture. Band quantification was performed using NIH Image J software. Data are shown as the mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01. *** p-value < 0.001. Figure 3: Induction of autophagy in DM-NSCs a. Detection by phase contrast microscopy of cytoplasmic vacuoles in DM1-NSCs as compared with control after 24 hours of culture. b. Increased expression level of LC3B-II in DM1-NSCs (VUB03_DM) as determined by western blotting after 48 hours of 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. EBSS and chloroquine at 50µM were used as a positive control. Data are presented as a percentage of positive cells quantified by the automated ArrayScan Imager. e. Detection by immunostaining of two autophagic markers p62

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

20

and ATG12 in WT and DM1-NSCs (VUB03_DM and VUB01_WT, respectively). Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01. *** p-value < 0.001. Figure 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)-AMPKα, Phospho(Ser21/9)-GSK3α/β, and Phospho(Ser235-236)-Ribosomal protein S6 (rpS6) and the corresponding total forms. Cell lysates were obtained after 48 hours of culture. b. Decreased phosphorylation of S6 Ribosomal protein at Serine 235-236 and Serine 240-245 in DM1-NSCs when compared to control NSCs after 48 hours of culture. c. Effect of the different growth factors required for NSC culture (i.e. EGF (epidermal growth factor), FGF2 (fibroblast growth factor), BDNF (Brain derived neurotrophic factors)) on the phosphorylation of P-rpS6 (Ser235-236). DM1-NSCs and WT-NSCs were growth factor starved for 48 hours and subsequently treated for one hour with or without each of the growth factors. d. FGF2 dose-response analysis after growth factor starvation and treatment with FGF2 from 1 to 100 nM for 1 hour of DM1- and WT-NSCs. The expression level of rpS6 and ERK1/2 was analyzed. e. Kinetic of FGF2 treatment on DM1-and WT-NSCs for the expression of rpS6 and ERK1. Cells were starved for 48 hours and then treated for various durations with FGF2 (10 nM). Western blot were quantified using NIH Image J software and quantitative data are presented as phosphorylated form normalized to their total form. VUB03_DM cells were used as source for DM1-NSCs. Statistical data are expressed as mean ± SEM using three independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01 *** p-value < 0.001. Figure 5: mTOR defect is correlated to 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 (10nM) 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 four days. Data are presented as the mean of signal [ATP] at day x+1/x compared to the control condition (arbitrary 100%) e. Detection of autophagy using anti-LC3B antibody in WT-NSCs treated with rapamycin (10 nM) or treated with chloroquine (50 µM) 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 by the automated ArrayScan Imager. Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ** p-value < 0.01. *** p-value < 0.001.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

21

Figure 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. Western blots were analyzed by NIH Image J software and quantitative data are presented as phosphorylated form normalized to their total form. b. 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 phosphorylated form normalized to their total form. c. 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 NIH Image J software d. Effect of the down-expression of MBNL1 in WT-NSCs and the over-expression of MBNL1 in DM1-NSCs on the proliferation rate. Proliferation was measured by immunostaining for Ki67 48 hours after nucleofection. e. Effect of the down-expression of MBNL1 in WT-NSCs and the over-expression 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. Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, *: p-value < 0.05, ** p-value < 0.01.

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t

Jour

nal o

f Cel

l Sci

ence

Acc

epte

d m

anus

crip

t