maurer et al 2007 jpr gsk3 nsc differentiation pr0605825

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Glycogen Synthase Kinase 3 (GSK3) Regulates Differentiation and

Proliferation in Neural Stem Cells from the Rat Subventricular Zone

Martin H. Maurer,*, Jens O. Bro1mme,, Robert E. Feldmann, Jr.,, Anne Ja1rve,

Fatemeh Sabouri, Heinrich F. Bu1rgers, Dominik W. Schelshorn, Carola Kru1ger,

Armin Schneider, and Wolfgang Kuschinsky

Department of Physiology and Pathophysiology, Division of Systems Physiology, University of Heidelberg,Im Neuenheimer Feld 326, 69120 Heidelberg, Germany, and SYGNIS Pharma AG, Im Neuenheimer Feld 515,

Heidelberg, Germany

Received November 3, 2006

On the basis of its inhibition by SB216763, we identified the multifunctional enzyme Glycogen Synthase

Kinase 3 (GSK3) as a central regulator for differentiation and cell survival of adult neural stem cells.

Detected by proteomic approaches, members of the Wnt/-catenin signaling pathway appear to

participate in enhanced neuronal differentiation and activated transcription of -catenin target genes

during GSK3 inhibition, associated with decreased apoptosis.

Keywords: Neural stem cell Neurosphere Glycogen Synthase Kinase 3 Two-dimensional gel electrophoresis Rat Subventricular zone

Introduction

Glycogen Synthase Kinase 3 (GSK3) was originally named

for its role in glycogen metabolism. Nowadays, GSK3 is

considered a multifunctional enzyme participating in cell

survival, formation of the cytoskeleton, cell growth and polarity,

metabolism, and transcriptional control.1-3 In mammals, GSK3

also takes part in oncogenesis and neurological disorders.4

During development, GSK3 coordinates cell growth and

polarity.5-7

GSK3 is located both in the cytoplasm and the nucleus. Two

functionally different protein isoforms have been identified: the

unbound form is regulated by protein kinases, whereas the

second form is associated with two others proteins, axin and

the adenomateous polyposis coli protein (APC), and is regu-

lated by the developmental Wnt signaling pathway.1 Of note,

phosphorylation of GSK3 at its serine residues results in

inactivation of the enzyme, not in signaling activation. The

inactivation of GSK3 results in the stabilization of its down-

stream target -catenin, which is otherwise ubiquitinated and

degraded in the proteasome. The stabilized -catenin is

transferred to the nucleus and binds to the TCF/Lef-1 tran-

scription factor, activating transcription for specific targetgenes.8-10

In the present study, we analyzed the molecular basis of

GSK3 for proliferation and differentiation of adult neural stem

cells. First, we measured changes in protein levels during

inhibition of GSK3 to detect proteins involved in this process.

After verifying the transcriptional activation by-catenin

stabilization and nuclear transfer, we focused on cell viability

and found an increase in neurosphere differentiation.

Materials and Methods

Neurosphere Cultures. Neural stem cells were isolated fromadult rat brains as described earlier.11,12 Protocols are concor-

dant with the policy on the use of animals, as endorsed by the

National Institutes of Health, and fulfill the local legal require-

ments. The subventricular zones of 6 rat brains were dissected,

washed in 10 mL ice-cold Dulbeccos Phosphate Buffered Saline

(DPBS) supplemented with 4.5 g/L glucose (DPBS/Glc) and

centrifuged for 5 min at 1600g at 4 C. The pellet was

mechanically homogenized, resuspended in 20 mL DPBS/Glc,

and centrifuged for 5 min at 1600g at 4 C. The pellet was

enzymatically digested in 10 mL of 0.01% (w/v) papain, 0.1%

(w/v) Dispase II (neutral protease), 0.01% (w/v) DNase I, and

12.4 mM MgSO4 in Hanks Balanced Salt Solution (HBSS),

triturated by a plastic pipet tip, and incubated at room

temperature for 40 min. In three washing steps, the homoge-nate was centrifuged for 5 min at 1600gat 4 C, and the pellet

was resuspended in 10 mL Dulbeccos Modified Eagles Me-

dium (DMEM)-Hams F12 medium supplemented with 100

units/mL penicillin, 100 units/mL streptomycin, and 2 mM

L-glutamine. Cells were resuspended in 1 mL of neurobasal-

B27 medium, and the cell number was counted. Cells were

plated in 2 mL dishes at 200 000 cells in B27-neurobasal

medium supplemented with 100 units/mL penicillin, 100 units/

mL streptomycin, 20 ng/mL EGF, 20 ng/mL FGF-2, and 2 g/

mL heparin. About 4/5 of the medium was replaced weekly,

* To whom correspondence should be addressed. Dr. Martin H. Maurer,Dept. of Physiology and Pathophysiology, University of Heidelberg, ImNeuenheimer Feld 326, 69120 Heidelberg, Germany. Phone: +49-6221-544075. Fax: +49-6221-544561. E-mail: [email protected].

University of Heidelberg. SYGNIS Pharma AG. Equal cotntributions.

1198 Journal of Proteome Research 2007, 6, 1198-1208 10.1021/pr0605825 CCC: $37.00 2007 American Chemical Society

Published on Web 01/18/2007


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and cells were passaged every 10-14 days. The neurospheres

were cultured for 6-10 weeks in 5% CO2 at 37 C before use.

For inhibition of GSK3, cell cultures were incubated for 3

days in the presence of 10 M (final concentration) 3-(2,4-

dichlorphenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-di-

one (SB216763; Tocris, Ellisville, MO), a specific low-molecular

weight inhibitor of GSK3.13-15 Five cell cultures were used in

each group for comparative replicates, run in individual gels.

Two-Dimensional Gel Electrophoresis (2-DE). 2-DE was

performed using standard protocols as previously described.11,12,16Cells were harvested, and protein extraction for 2-DE was

performed for 60 min at room temperature in a lysis buffer

containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/

v) Triton X-100, 100 mM DTT, 0.05% IPG buffer, pH 3-10

(Amersham Biosciences, Uppsala, Sweden), and 0.156% (w/v)

Complete protease inhibitor tablets (Roche, Mannheim, Ger-

many). Sample protein amounts were determined by the

modified Bradford method.17,18 A total of 250 g (5-10 L) of

the protein solution was suspended in rehydration solution

consisting of 6 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5%

(v/v) IPG buffer, pH 3-10, and a few grains of bromophenol

blue to give a final volume of 350 L. The samples were applied

to pH 3-10 nonlinear gradient IEF gel strips for isoelectric

focusing in the IPGphor apparatus (Amersham Biosciences,Uppsala, Sweden). The IEF gel strips reswelled for 12 h at 30 V

to remove high salt concentrations and to improve protein

entry into the gel. Then 200, 500, and 1000 V were applied for

1 h each. Voltage was increased to 8000 V in 30 min and kept

constant at 8000 V for 12 h, resulting in a total of 100 300 Vh.

Gel strips were equilibrated for 20 min each in an SDS

equilibration buffer consisting of 50 mM Tris-HCl, pH 8.8, 6 M

urea, 30% (v/v) glycerol, 2% (w/v) SDS, a few grains of

bromophenol blue, and 1% (w/v) dithiothreitol or 2.5% (w/v)

iodoacetamide, respectively. The second-dimension separation

was performed using 12.5% polyacrylamide gels in the presence

of 0.1% (w/v) sodium dodecylsulfate. The gels were run at 30

mA for 30 min and 100 mA for about 4 h in a 20 cm 20 cm

water-cooled vertical electrophoresis apparatus (OWL, Woburn,MA). For image analysis, gels were stained by the Blue silver

method, a sensitive Coomassie-based quantitative stain.19

Briefly, gels were soaked overnight in 0.12% Coomassie Blue

G-250, 10% phosphoric acid, 10% ammonium sulfate, and 20%

methanol and destained the next day for 5-6 h.

Gel Image Analysis and Mass Spectrometry. Gels were

scanned, and images were analyzed using the Phoretix 2D

Expression software (Nonlinear Dynamics, Newcastle-upon-

Tyne, U.K.). Image analysis was performed as described.20

Normalized spot volumes defined as integral of spot area

multiplied by optical densities were compared to normalized

means ( standard deviations from 3 gels of each group by

Students t test for unpaired data.21 Spots of interest were

excised and digested by trypsin for mass spectrometry (Centre

for Molecular Medicine, ZMMK, University of Cologne, Ger-

many), and mass spectra were identified by searching the NCBI

nonredundant protein database with Mascot22 (http://

www.matrixscience.com) as described elsewhere in detail.23

Briefly, trypsinized protein samples were loaded onto prespot-

ted AnchorChip targets which are stainless steel supports

coated with hydrophobic material equipped with an array of

384 circular interruptions (anchors) of 600 mm diameter

(Bruker-Daltonics, Bremen, Germany). They were prepared

using R-cyano-4-hydroxycinnamic acid (HCCA) as matrix,

whereby 0.3 mL of analyte solution and 1.2 mL of matrix

solution (0.3 g/L HCCA in ethanol/acetone ) 2:1) were applied

onto the anchors using an Investigator ProMS MALDI Spotting

Robot (Genomic Solutions, Ann Arbor, MI). Samples were

allowed to air-dry at room temperature. Peptide mass spectra

were obtained using an Ultraflex TOF/TOF (Bruker-Daltonics)

in the fully automated reflectron TOF mode operated by the

flexControl software. The mass spectrometer was equipped with

a SCOUT-MALDI source for multisample handling, a pulsed

UV laser, a two-stage gridless reflector, a 2 GHz digitizer, a LIFT-

TOF/TOF unit to analyze fragment ions of selected peptide ions(see below), and multichannel-plate detectors for linear and

reflector mode measurements. All measurements were carried

out in positive ionization mode using a reflector voltage of 20

kV. The external instrument calibration was achieved using

signals from [M 1 H]1 ions of the following reference standards

(m/z): Bradykinin clip (1-7) mono 757.39916; Angiotensin II

mono 1046.5418; Angiotensin I mono 1296.6848; Substance P

mono 1347.7354; Bombesin mono 1619.8223; Renin substrate

mono 1758.93261; adrenocorticotropic hormone (ACTH) clip

(1-17) mono 2093.0862; ACTH clip (18-39) mono 2465.1983;

and Somatostatin clip (28) mono 3147.4710. Fragment m/z

spectra were obtained by integration over up to 2000 successive

laser pulses (f ) 50 Hz). The spectra were calibrated using

autodigestion peptide signals for trypsin (m/z 842.5094,1045.5637, 2211.1040, and 2283.1802) as reference values and

were the basis of mining the NCBI nonredundant database for

protein identification via Mascot query (Matrix Science, Lon-

don, U.K.) with the following parameters: enzyme, trypsin;

missed cleavages, 1; allowed modifications, carbamidomethyl

(fixed) and methionine oxidation (variable); tolerance, 75 ppm,

that is, mass measurement accuracies were typically( 75 ppm.

The Mascot-delivered probability based score was regarded as

a quality parameter for the correct identification.22 Protein spots

that could not successfully be identified with the previous

method were additionally analyzed by peptide sequencing with

the same device, Ultraflex MALDI LIFT-TOF/TOF. Therefore,

a high-resolution timed ion selector to separate selected

peptide ions, a LIFT device for raising the potential energy offragment ions, a velocity focusing stage with subsequent

postacceleration, and a postLIFT metastable suppressor device

were used. For the analysis, up to five precursor ions were

selected. The ions were subjected initially to acceleration with

8 kV in mass spectrometer step one, then selected using a timed

ion-gate, and finally energy-lifted to a voltage of 19 kV.

Fragmentized ion species were then accelerated in the second

ion source and analyzed in mass spectrometer step two,

running in reflector mode. Mascot analysis of the obtained

spectra was performed with 0.8 Da tolerance, one missed

cleavage, and carbamidomethyl and methionine oxidation as

allowed modifications.

Reverse Transcription-Polymerase Chain Reaction (RT-

PCR). Neurospheres were harvested for total RNA extraction

using the RNeasy kit (Qiagen, Hilden, Germany). RNA was

reversely transcribed using the Omniscript RT kit (Qiagen,

Hilden, Germany). Specific cDNA target sequences for Glycogen

Synthase Kinase 3 (GSK3; NM_032080), forward, 5-

GGATCTGCCATCGAGACATT-3; reverse, 5-CCAACTGATCCA-

CACCACTG-3; Wnt5a (NM_022631), forward, 5-TGGAGTGG-

TAAATGCCATGA-3; reverse, 5-ATACTGTCCTGCGACCTGCT-

3; Wnt7a (XM_342723), forward, 5-CCCGAACCCTCATGAACTTA-

3; reverse, 5-TAGCCTGAGGGGCTGTCTTA-3; and Bone Mor-

phogenic Protein 4 (BMP4; NM_012827), forward, 5-CC-

TGGTAACCGAATGCTGAT-3; reverse, 5-TCCTCACAGTGTTG-

GSK3 Regulates Neural Stem Cell Differentiation research articles

Journal of Proteome Research Vol. 6, No. 3, 2007 1199


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GCTCTG-3 were amplified by quantitative RT-PCR in the

LightCycler 2.0 system (Roche, Mannheim, Germany) using the

DNA Master SYBR Green I kit.24 The amplification protocol was

5 min initial denaturation at 94 C, 50 cycles of 5 s denaturation

Figure 1. (A) Comparison of two-dimensional electropherograms of neurospheres in the presence of the GSK3 inhibitor SB216763

(green spots) compared to neurospheres differentiated in vitro (red spots). Overlaid spot images result in yellow spot color. Some

distinct protein isoforms are solely expressed in either of the groups, others are differentially expressed. (B) Three-dimensional close-

up of spots identified as isoforms of the Collapsin response mediator protein-2 (CRMP-2). Whereas the main spot decreases, there is

an isoform shift for other isoforms of CRMP-2: spot no. 76 decreases and spot no. 1392 appears at a new pI, indicating changes in

phosphorylation.

Table 1. Differentially Expressed Proteins during in Vitro Differentiation of Neurospheres Which Are Related to GSK3 in the Wnt

Signaling Pathwaya

GenBank annotation

protein

abbrev.

UniProt

accession

number remarks

theoretical

pI

theoretical

MW (Da)

fold-change

(inhibited

vs control)

RuvB-like protein 1; Pontin 52 Ruvbl1 P60123 binds -catenin 6.02 50524 1.47Proteasome subunit, alpha type 1 Psa1 P18420 degradates -catenin 6.14 29784 1.6Rho GDP-dissociation inhibitor 1 Gdir Q99PT1 binds rho 5.12 23450 1.5Proteasome subunit, alpha type 6 Psa6 P34062 degradates -catenin 6.35 27838 1.3Microtubule-associated proteinRP/EB family member 1

Mare1 Q66HR2 binds APC 5.02 30168 -2.3

a Proteins were identified by two-dimensional gel electrophoresis and mass spectrometry (Figure 1). Spot volumes were compared by statistical tests ( P

maurer et al 2007 jpr gsk3 nsc differentiation pr0605825

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