*Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden

�Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden

�Department of Physiology, Institute of Neuroscience and Physiology, Goteborg University, Goteborg, Sweden

§Institute of Biomedicine, Goteborg University, Goteborg, Sweden

¶Howard Florey Institute, Melbourne, Australia

Adult neurogenesis occurs in the dentate gyrus of thehippocampus and in the olfactory bulb. The new neuronsarise from adult neural stem/progenitor cells (NSPCs) whichreside in the subgranular zone of the hippocampus and thesubventricular zone of the lateral ventricles (Taupin and Gage2002). These neurons can integrate into pre-existing circuitryand form active synapses, suggesting a role in basal neuronalreplacement (van Praag et al. 2002). Endogenous or graftedNSPCs are associated with reductions in damage or impair-ment following various pathological events including stroke(Zhang et al. 2003; Ishibashi et al. 2004). Although neuronalreplacement may be a factor in these instances, the presenteddata suggest that this may be a minor contribution.

It has been previously recognized that NSPCs mayinfluence the outcomes of pathological events by othermeans such as the secretion of various growth factors,including glial-derived neurotrophic factor (GDNF) and

nerve growth factor (Llado et al. 2004; Yasuhara et al.2006). Hence, it is likely that factors derived from endo-genous (i.e., recruited) or exogenous (i.e., grafted) NSPCspositively modulate the lesion environment and improveoutcomes. However, in areas where NSPCs reside in closeproximity to neurons, such as the dentate gyrus, theseendogenous factors could contribute to local neuroprotection.

Received November 4, 2008; revised manuscript received January 22,2009; accepted February 18, 2009.Address correspondence and reprint requests to Rogan Tinsley,

Howard Florey Institute, University of Melbourne, Parkville, Victoria3010, Australia. E-mail: rogan.tinsley@florey.edu.auAbbreviations used: CM, conditioned medium; EPSC, excitatory post-

synaptic current; GDNF, glial-derived neurotrophic factor; IDE, insulin-degrading enzyme; NMDAR, NMDA-type ionotropic glutamate receptor;NSPC, neural stem/progenitor cell; OHSC, organotypic hippocampal sliceculture; PBS, phosphate-buffered saline; PI, propidium iodide.

Abstract

Although the potential of adult neural stem cells to repair

damage via cell replacement has been widely reported, the

ability of endogenous stem cells to positively modulate dam-

age is less well studied. We investigated whether medium

conditioned by adult hippocampal stem/progenitor cells al-

tered the extent of excitotoxic cell death in hippocampal slice

cultures. Conditioned medium significantly reduced cell death

following 24 h of exposure to 10 lM NMDA. Neuroprotection

was greater in the dentate gyrus, a region neighboring the

subgranular zone where stem/progenitor cells reside com-

pared with pyramidal cells of the cornis ammonis. Using mass

spectrometric analysis of the conditioned medium, we identi-

fied a pentameric peptide fragment that corresponded to

residues 26–30 of the insulin B chain which we termed ‘pen-

tinin’. The peptide is a putative breakdown product of insulin, a

constituent of the culture medium, and may be produced by

insulin-degrading enzyme, an enzyme expressed by the stem/

progenitor cells. In the presence of 100 pM of synthetic

pentinin, the number of mature and immature neurons killed

by NMDA-induced toxicity was significantly reduced in the

dentate gyrus. These data suggest that progenitors in the

subgranular zone may convert exogenous insulin into a

peptide capable of protecting neighboring neurons from

excitotoxic injury.

Keywords: excitotoxicity, hippocampus, insulin, neuropro-

tection, NMDA, stem cells.

J. Neurochem. (2009) 109, 858–866.

JOURNAL OF NEUROCHEMISTRY | 2009 | 109 | 858–866 doi: 10.1111/j.1471-4159.2009.06016.x

858 Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866� 2009 The Authors

We chose to study this hypothesis using organotypichippocampal slice cultures (OHSCs). In such cultures, thearchitecture of the hippocampal formation remains largelyintact, while allowing various in vitro manipulations andvisualization of effects on groups of cells within thestructure. OHSCs have been used to model various brainpathologies, including stroke and epilepsy. Specifically, theapplication of glutamate receptor agonists, such as NMDAand kainate, has been shown to cause reproducible excito-toxic injury in OHSCs, which recapitulates importantfeatures of the pathophysiology of these disorders. Themodel therefore provides a valuable platform for analyzingthe interactions between NSPCs and the processes ofneurotoxicity and neuroprotection.

Experimental procedures

Neural stem/progenitor cell culturesThe NSPCs used in this study were adult rat hippocampal progenitor

cells, the isolation of which has been previously described (Gage

et al. 1995; Palmer et al. 1997). Clonally derived cells were receivedat passage 4 as a gift from F. Gage (Laboratory of Genetics, The Salk

Institute, La Jolla, CA, USA). The cells were cultured in N2 medium

[Dulbecco’s modified Eagle’s medium/Nut Mix F12 (1 : 1), 2 mM

L-glutamine, and 1% N2 supplement; (Life Technologies, Taby,

Sweden)], supplemented with 20 ng/mL human recombinant basic

fibroblast growth factor (PeproTech, London, England). This

medium was also used as unconditioned control medium.

Adult rat hippocampal progenitor cells retained the potential to

differentiate into the three neural lineages [neuronal, astrocytic, and

oligodendrocytic (Song et al. 2002)] and had a stable phenotype in

long-term culture, retaining identical immunocytological character-

istics for more than 30 passages (Gage et al. 1995). In this study

cells were used between passages 5 and 20 post-cloning.

Conditioned medium (CM) was produced by seeding adult rat

hippocampal progenitor cells (5 · 104 cells/cm2) on to poly-

ornithine/laminin-coated 24-well plates. Cells were grown for

2 days before medium was collected and filtered (0.22 lm).

Penicillin–streptomycin (25 U/mL) and propidium iodide (PI,

2 lM) were added immediately before the CM was added to the

slice cultures. For studies involving GDNF, recombinant human

protein (Promega, Madison, WI, USA) was added to control

medium or CM at a final concentration of 1 ng/mL.

All experiments of neuroprotection in hippocampal slices were

performed with N2 medium containing human insulin. However, the

growth media for the mass spectrometric analysis contained N2

medium with either human or bovine insulin. The CM was collected

after 2 days of culturing, centrifuged to remove cellular material,

and stored at )20�C until the analysis was performed.

Hippocampal slice culturesRat OHSCs (400 lm thick) were prepared from P9 Sprague–

Dawley rats, using the method of Stoppini et al. (1991). OHSCswere cultured in slice medium [50% Eagle’s basal medium, 25%

Earle’s balanced salt solution, 23% horse serum, 7.5 mg/mL D-

glucose, 1 mM L-glutamine, and 25 U/mL penicillin–streptomycin

(Sigma-Aldrich Sweden AB, Stockholm, Sweden)] for 12–14 days

before experiments commenced.

NMDA-induced excitotoxicity and neuroprotectionOrganotypic hippocampal slice cultures were transferred to test

media 1 h before exposure to 10 lM NMDA for 24 h. The degree

of NMDA-induced excitotoxicity was determined by comparing PI

uptake prior to exposure with that following exposure. Pictures were

captured using a digital camera (Olympus DP50, Solna, Sweden)

coupled to an inverted fluorescence microscope (Olympus IX70),

equipped with a red long-pass WG fluorescence filter. Uptake of PI

was quantified as the mean pixel intensity of epifluorescence over

the whole slice, or in defined subregions (ImageJ v1.29x, National

Institutes of Health, Bethesda, MD, USA).

ImmunohistochemistryTo characterize cell death, OHSCs were cultured in N2 medium

with different concentrations of NMDA and pentinin (in the

presence of PI). After 24 h, OHSCs were washed in phosphate-

buffered saline (PBS) and fixed in 4% paraformaldehyde (over-

night, 4�C). OHSCs were blocked and permeabilized by incubation

for 2 h in phosphate/Triton/serum buffer (0.1 M sodium phosphate

buffer, 0.3% Triton X-100, and 1% donkey serum; Jackson

Immunoresearch Laboratories Inc., West Grove, PA, USA) at

23�C, then incubated overnight (rocking, 4�C) with mouse anti-

NeuN antibody (1 : 500, Chemicon, Temecula, CA, USA), rabbit

anti-caspase 3A antibody (1 : 250, Cell Signaling Technology,

Danvers, MA, USA), and goat anti doublecortin antibody (Dcx,

1 : 400, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After

thorough washing (3 · 30 min in phosphate/Triton/serum buffer,

rocking), OHSCs were incubated overnight (rocking, 4�C) with

donkey anti-mouse Alexa 647-conjugated antibody (1 : 800,

Molecular Probes, Leiden, The Netherlands), donkey anti-rabbit

Alexa 488-conjugated antibody (1 : 800, Molecular Probes), and

donkey anti-goat Alexa 488-conjugated antibody (1 : 800, Molec-

ular Probes). OHSCs were washed thoroughly and mounted in

Prolong Gold mounting medium (Molecular Probes). Co-localiza-

tion of PI and/or caspase 3A staining with NeuN and Dcx

immunofluorescence was determined by confocal microscopy

(Leica TCS SP2, Leica Microsystems AG, Wetzlar, Germany).

Cells in four fields of the dentate gyrus were counted and averaged

for every slice (n = 8–12).

To determine whether insulin-degrading enzyme (IDE; EC

3.4.24.56) is expressed in NSPCs, cells were seeded in N2

medium onto polyornithine/laminin-coated glass coverslips, at a

density of 5.0 · 104 cells/cm2. After fixation (4% paraformalde-

hyde in PBS, 4�C, 10 min), cells were pre-incubated for 30 min

with PBS containing 3% bovine serum albumin and 0.05%

saponin (Sigma-Aldrich) at 23�C. Subsequently, cells were

incubated with mouse anti-IDE antibody (1 : 250, Covance

Research Products, Berkeley, CA, USA) and rabbit anti-musashi

antibody (1 : 250, Chemicon) for 1 h at 23�C in PBS containing

1% bovine serum albumin and 0.05% saponin. Following three

washes in PBS, cells were incubated for 1 h at 23�C with

secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse

(1 : 2000, Molecular Probes) and Alexa Fluor 555-conjugated

goat anti-rabbit (1 : 2000, Molecular Probes) and the nuclear dye

TO-PRO-3 (1 : 1000, Molecular Probes).

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Neuroprotection by the novel peptide pentinin | 859

Mass spectrometric analysisSamples of CM (50 lL) were desalted and concentrated using

ZipTipTM C18 (Millipore, Bedford, MA, USA) according to the

supplier’s instructions. The samples were eluted with 3 lL of matrix

solution (50 mg/mL 2,5-dihydroxybenzonic acid; Sigma-Aldrich, St.

Louis, MO, USA) in acetone and 0.1% trifluoric acid in water (4 : 1,

v/v) directly onto thehighly polished, stainless steel, sample probe, and

left to dry at ambient conditions. The matrix-assisted laser desorption/

ionization analyses were performed using an upgraded Bruker Reflex

II instrument (Bruker-FranzenAnalytik, Bremen,Germany) equipped

with a two-stage electrostatic reflectron, a delayed extraction ion

source, a high-resolution detector, and a 2 GHz digitizer. The spectra

were acquired in reflectron mode. Calibration was performed

externally by using a mixture of peptides with known masses.

ElectrophysiologyElectrophysiological experiments were performed on acutely

prepared hippocampal slices from 8- to 16-day-old Wistar rats.

The rats were anesthetized with isoflurane (Abbott Scandinavia AB,

Solna, Sweden) prior to decapitation, in accordance with the

guidelines of the local ethics committee for animal research. The

brain was removed and placed in an ice-cold solution containing (in

mM): 124 NaCl, 3 KCl, 0.5 CaCl2, 6 MgCl2, 26 NaHCO3, 1.25

NaH2PO4, and 10 D-glucose. Transverse hippocampal slices

(300 lm thick) were cut with a vibratome (HM 650 V, Microm,

Walldorf, Germany) in the same ice-cold solution and they were

subsequently stored in artificial CSF containing (in mM): 124 NaCl,

3 KCl, 2 CaCl2, 4 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 0.5 ascorbic

acid, 3 myo-inositol, 4 D,L-lactic acid, and 10 D-glucose. A surgical

cut was made between the CA3 and CA1 of the hippocampus. After

at least 1 h of storage at 25�C, a single slice was transferred to a

recording chamber where it was kept submerged in a constant flow

(�2 mL/min) at 30–32�C. The perfusion solution contained (in

mM): 124 NaCl, 3 KCl, 4 CaCl2, 4 MgCl2, 26 NaHCO3, 1.25

NaH2PO4, and 10 D-glucose. All solutions were continuously

bubbled with 95% O2 and 5% CO2 (pH �7.4). The perfusion systemconsisted of Teflon tubing and containers to prevent the pentinin

peptide from binding to the surface of the perfusion system.

Picrotoxin (100 lM, Sigma-Aldrich) was always present in the

perfusion solution to block GABAA receptor-mediated activity.

D-AP5 (50 lM, Ascent Scientific, Weston-Super-Mare, UK) was

used to block NMDA receptors when indicated.

Excitatory post-synaptic currents (EPSCs) were recorded from

visually identified CA1 pyramidal neurons using whole-cell patch-

clamp recordings, holding the cell at +40 mV. The pipette solution

contained (in mM): 130 Cs methanesulfonate, 2 NaCl, 10 HEPES,

0.6 EGTA, 5 QX-314, 4 Mg-ATP, and 0.4 GTP (pH �7.3 and

osmolality 270–300 mOsm). Patch pipette resistances were

2.5–6 MW. EPSCs were recorded at a sampling frequency of

10 kHz and filtered at 1 kHz, using an EPC-9 amplifier (HEKA

Elektronik, Lambrecht, Germany). Series resistance was monitored

using a 5-ms, 10-mV hyperpolarizing pulse and it was not allowed

to change more than 10%, otherwise the recording was not included

in the analysis. Schaffer collateral afferents were activated in stratum

radiatum at 0.2 Hz using biphasic constant current pulses

(200 ls + 200 ls, 30–50 lA) delivered through tungsten wires

(resistance �0.5 MW, STG 1004, Multi Channel Systems MCS

GmbH, Reutlingen, Germany). NMDA EPSCs were measured as

the mean amplitude at 30–50 ms after the stimulation artifact. The

EPSCs were analyzed off-line using custom made IGOR Pro

(WaveMetrics, Lake Oswego, OR, USA) software.

Statistical analysisTreatment groups were compared by using ANOVA. Where groups

differed, Tukey’s post hoc analysis was applied. All analyses were

performed in SPSS 12.1 (SPSS, Chicago, IL, USA). Data are

expressed as mean and SE.

Results

NMDA-induced excitotoxicity in hippocampal sliceculturesAs subsequent experiments exploited the uptake of thenuclear dye PI as a marker of cell death, we performedimmunofluorescent analyses to confirm the identity of PI-labeled cells. OHSCs were maintained in N2 medium orexposed to 5 or 10 lM NMDA in N2 medium for 24 h, inthe presence of PI. Slices were fixed and stained for NeuN, amarker of mature neurons (Fig. 1). In addition, caspase 3Aimmunoreactivity was used as a marker of caspase-dependent apoptosis (data not shown).

There was a low level of PI staining in control cultures,which was most pronounced in the dentate gyrus. NMDAincreased PI staining in a concentration-dependent manner.The vast majority of PI-labeled cells was co-labeled withNeuN, except in the dentate gyrus, where a small proportion ofPI+ cells was NeuN). On the basis of latter experiments (see‘‘Pentinin protects both immature andmature neuronal cells’’),these are likely to be Dcx+ immature neuronal precursors.Hence, NMDA-induced cell death was primarily mediated byexcitatory neurotoxicity. Although caspase 3A immunoreac-tivity was detected, this was not co-localized with NeuN.Furthermore, caspase3A immunoreactivity was not NMDA-dependent andwasmostly found on the surface of the slice. It islikely that these cells are dying through other mechanismswhich may be related to the organotypic culture such as tissueloss at the air interface. These cellswere also not PI-labeled andhence did not affect the level of PI staining. The PI+ cell deathinduced by NMDA is likely to be mediated by the influx ofCa2+ and Na+ which causes cellular swelling and eventuallynecrosis (Lee et al. 1999). The influx of Ca2+ also activatednumerous pathways, including those which produced nitricoxide, which in turn perturbed normal cellular functions andcould lead to cell death (Aarts and Tymianski 2004).

Neural stem/progenitor cells secrete neuroprotectivefactorsTo test the neuroprotective qualities of test media, OHSCswere first pre-incubated with PI for 24 h. Fluorescent photo-micrographs were taken and used to determine the level ofbackground staining. OHSCs were then transferred to test

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866� 2009 The Authors

860 | J. Faijerson et al.

media 1 h before 10 lM NMDA was added. Photomicro-graphswere taken again after 24 h ofNMDAexposure, and thechange in PI staining intensity was quantified.

Test media were N2 medium and medium conditioned byNSPCs, with or without GDNF added. GDNF had previouslybeen shown to be neuroprotective in OHSCs when given atrelatively high doses (50–100 ng/mL; Dahl et al. 2003).

Figure 2 shows representative photomicrographs afterNMDA exposure, in the presence of different test media aswell as quantitative data pooled from several experiments(Fig. 2). Exposure to 10 lM NMDA caused a greater thanthree-fold increase in PI staining. Incubation in CM led to a33% reduction in NMDA-induced PI staining. The additionof a low dose of GDNF (1 ng/mL) to N2 medium (controlmedium) did not reduce excitotoxicity. However, when thisdose was added to CM, PI staining was significantly reducedto control levels. This dose was found to be the lowestrequired to achieve full neuroprotection in the presence ofCM (data not shown).

Neuroprotection is region dependentInterestingly, the different hippocampal regions showedselective vulnerability for NMDA excitotoxicity as well as

preferential neuroprotection by test media. Regional analysisof NMDA-induced toxicity is shown in Fig. 2. The dentategyrus showed a low relative increase in PI staining afterNMDA exposure. This increase was abolished in thepresence of CM, even without the addition of GDNF. Thecornus ammonis regions (CA1 and CA3) exhibited highvulnerability to NMDA, which was partly ameliorated in thepresence of CM. However, addition of GDNF was requiredto restore control levels of PI staining in both these regions.

Neural stem/progenitor cells produce a peptide derivedfrom insulin – ‘pentinin’Our laboratory had previously developed a preparative two-dimensional proteomic approach involving mass spectro-metry for the analysis of CM from cultured NSPCs (Dahlet al. 2003). This approach was demonstrated to be suitablefor identification of relatively small secreted proteins in thewell-defined CM. A number of proteins in the molecularweight range of 10–25 kDa were identified in the CM whichcould potentially mediate neuroprotection. The neuroprotec-tive effects of some of these candidates have been tested,however, no evidence of protection against excitotoxicitywas observed (data not shown).

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Fig. 1 NMDA induces excitotoxicity in hippocampal slices. Control

hippocampal slices (a, d, and g) and OHSCs cultured in the presence

of 5 (b, e, and h) or 10 lM NMDA (c, f, and i) were investigated.

Photomicrographs of NeuN immunoreactivity (blue) and PI incorpo-

ration (red) in representative fields in the dentate gyrus (a, b, and c),

CA3 (d, e, and f), and CA1 (g, h, and i). Scale bar = 50 lm.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866

Neuroprotection by the novel peptide pentinin | 861

In the present study, we analyzed the peptide pattern of CMdirectly with mass spectrometry. We focused on peptides withmolecular masses ranging from approximately 700 to8000 Da. The mass spectrometric analysis revealed changesin the peptide pattern evolving during culturing of the NSPCs,and a truncated form with a mass identical to the loss ofresidues 26–30 in the COOH-terminal of the insulin B chainwas identified (Fig. 3). The cells were cultured in growthmedia containing either human or bovine insulin. Culturing ingrowth media containing human insulin resulted in a massshift of the intact protein, truncated form, and the lostfragment compared with culturing in growth media contain-ing bovine insulin. The molecular masses of the lost fragmentfrom human and bovine insulin were 590.6 and 560.3 Dawhich was equivalent to the masses of the amino acidsequences corresponding to residues 26–30 (Fig 3a, b). Thus,

the mass shift between the intact insulin and its peptide wascontingent on the origin of the protein, and the consequentdifferences in amino acid sequences. These peptides were notpresent in the control media (data not shown).

A literature survey revealed that this truncation of insulinmay be produced as a result of the action of IDE. This enzymecleaved insulin at various sites, including residue 26 of the Bchain (Stentz et al. 1989), and this produced a truncated Bchain as well as a pentameric fragment (Fig. 3c). A tripeptide(amino acid sequence: GPE) cleaved from insulin-like growthfactor 1 has previously been shown to have neuroprotectiveproperties (Saura et al. 1999; Guan et al. 2000, 2004). Hence,we were interested in testing the effects of the identifiedpentameric peptide which we termed ‘pentinin’.

Furthermore, we confirmed that NSPCs expressed IDEusing immunofluorescent staining (Fig. 3d). Immunoreactiv-

Fig. 2 Medium conditioned by NSPCs reduce excitotoxicity. Photo-

micrographs of PI staining were taken to assess toxicity in the different

test media (top panels show representative photomicrographs), and

these were quantified in the whole slice or in subregions (lower panel).

When the whole slice was analyzed, exposure to 10 lM NMDA

caused greater than three-fold increase in PI staining. Incubation in

conditioned medium (CM) led to a 33% reduction in NMDA-induced PI

staining. A low dose of GDNF (1 ng/mL) added to N2 medium (control

medium) did not reduce excitotoxicity. However, when this dose was

added to CM, PI staining was reduced to control levels. In the dentate

gyrus, a relatively low increase in PI staining was observed after

NMDA exposure. This increase was abolished in the presence of CM,

even without addition of GDNF. The CA1 and CA3 regions exhibited

high vulnerability to NMDA, which was partly ameliorated in the

presence of CM. However, addition of GDNF was required to restore

control levels of PI staining in both regions. *p < 0.05, **p < 0.01,

***p < 0.001; ns, not significant; Ctrl, control.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866� 2009 The Authors

862 | J. Faijerson et al.

ity was seen in all cells and had a perinuclear localization.This was consistent with reports of IDE being a cytosolicenzyme (Akiyama et al. 1988). The properties of pentininwere further investigated using a stable synthetic peptide(YTPKT, Sigma GENOSYS, The Woodlands, TX, USA),which was applied to the OHSC model.

Pentinin reduces excitotoxic cell deathWe tested the neuroprotective properties of pentinin byadding the synthetic peptide to unconditioned medium in theNMDA-induced excitotoxicity model. A dose–responseassay showed that 100 pM provided an effective dose andthis concentration was chosen for subsequent experiments(data not shown). At 100 pM, pentinin potently reducedexcitotoxicity induced by 10 lM NMDA (Fig. 4a).

Pentinin protects both immature and mature neuronal cellsTo determine the cell types which were protected bypentinin, we fixed OHSCs and performed immunofluores-cence for markers of mature neurons (NeuN) and neuronallycommitted progenitors (Dcx). Cells in the dentate gyrus werecounted and the percentage of cells double-labeled with PIand each marker was determined after NMDA-inducedexcitotoxicity. In the presence of 100 pM pentinin, thepercentage of cells immunoreactive for mature and immatureneuronal markers, co-labeled with PI, were significantlyreduced (64% and 86%, respectively, Fig. 4b).

Pentinin does not modulate NMDA-receptor activationWe hypothesized that if pentinin acts in a similar way toGPE, it may antagonize the NMDA-type ionotropic gluta-mate receptor (NMDAR). This would reduce calcium influxand hence cause neuroprotection (Sara et al. 1989). To testthis we measured NMDAR-mediated EPSCs using whole-cell patch-clamp recordings from CA1 pyramidal cells inacute hippocampal slices. After establishing a 5-minutebaseline, the pentinin peptide (10 nM) was added to theextracellular solution. The peptide did not have any effect onthe NMDAR-EPSC magnitude (Fig. 5a). Fifteen minutesafter the peptide was added the average NMDAR-EPSCmagnitude was 89 ± 5% (n = 7) of the baseline (p > 0.05,Fig. 5b). To compare this to the effect of a well-characterizedNMDAR antagonist, we added D-AP5 (50 lM) to theextracellular solution in the presence of pentinin. Figure 5cshows that D-AP5 rapidly abolished the NMDAR-EPSC.These results demonstrated that the pentinin peptide has norapid antagonistic effect on NMDAR-mediated synapticresponses.

Discussion

This study examined the influence of factors produced byadult NSPCs on NMDA-induced excitotoxicity in thehippocampus. We found that medium conditioned by NSPCsprovided a significant degree of neuroprotection, and indeed

(a)

(c)

(d) (i) (ii) (iii)

(b)

Fig. 3 Identification of pentinin in medium

conditioned by NSPCs. (a): The measured

molecular mass of human insulin and its

truncated form are 5808.6 and 5218.0 Da.

(b): Mass spectra of CM containing bovine

insulin. The measured molecular mass of

bovine insulin and its truncated form are

5734.6 and 5174.3 Da. The mass differ-

ences of 590.6 and 560.3 Da are almost

identical with the mass of residues 26–30

in the B chain of the two insulins with the

amino acid sequence YTPKT and YTPKA,

respectively. (c) Amino acid sequences for

human, bovine, and rat insulin B chain.

Shaded areas indicate cleavage sites for

insulin degrading enzyme. (d) Insulin

degrading enzyme (green, i and iii) is ex-

pressed in NSPCs expressing the imma-

ture cell marker musashi (red, ii, iii). Cell

nuclei were visualized with ToPro-3 (blue, ii

and iii).

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Neuroprotection by the novel peptide pentinin | 863

completely abolished NMDA-dependent cell death in thedentate gyrus. In the CA1 and CA3 regions, abolition ofneurotoxicity could be achieved by supplementing the CMwith a very low dose of GDNF.

In order to determine the source of this neuroprotection wereanalyzed data from a previous proteomic investigationperformed in our laboratory. Although we hypothesizedthat the proteins identified in that study may have beenneuroprotective, further analyses of selected proteins, bothexperimental and in silico, did not demonstrate anyneuroprotective activities of the proteins.

In the present paper we focused on the analysis of peptidesevolving during culturing, in contrast to the previous strategythat was optimized for identification of relatively smallproteins in the CM (Dahl et al. 2003). These analysesdemonstrated that the NSPCs cleave insulin, resulting in atruncated form of the protein and a pentapeptide which wetermed pentinin.

We reasoned that pentinin may have neuroprotectiveproperties, through analogy with GPE, an N-terminalpeptide of insulin-like growth factor which is neuropro-tective in different paradigms (Saura et al. 1999; Guan

et al. 2000, 2004). In addition, a C-terminal peptide ofmechano-growth factor, a splice variant of insulin-likegrowth factor-1, has also been shown to be neuroprotectivein a NMDA/OHSC model as well as in vivo (Dluzniewskaet al. 2005).

Indeed, pentinin displayed neuroprotective properties andtreatment with 100 pM of the peptide resulted in a potentreduction of NMDA-induced excitotoxicity in both matureand immature neurons. We hypothesized that pentinin wasproduced in vitro by the cleavage of insulin. This wassupported by immunofluorescence of IDE in NSPCs, anenzyme which is known to produce this pentapeptide as abreakdown product (Stentz et al. 1989). IDE has beenidentified in several subcellular locations, but is primarilycytosolic. Insulin processing usually occurs in endosomesas part of insulin receptor recycling. Although a proportionwas fully degraded by lysosomes, both intact insulin andfragments were secreted by diacytosis (Duckworth et al.1998). Hence, we would predict that the NSPCs take upinsulin via insulin receptors, partially degrade the proteinby IDE in the endosomes and secrete the resultingfragments when the insulin receptor is returned to the

(a)

(c)

(b)

Fig. 4 Pentinin reduces NMDA-induced excitotoxicity in OHSCs. (a)

One hundred pM pentinin potently reduces the propidium iodide (PI)

incorporation induced by 10 lM NMDA in OHSCs (whole slice). (b)

Immunohistochemical analyses show that pentinin markedly reduced

the fraction of NeuN+ and doublecortin+ cells incorporating PI in the

dentate gyrus. *p < 0.05, **p < 0.01, ***p < 0.001. (c) Representative

photomicrographs showing neuroprotection at the level of the whole

slice (left) or at the cellular level (right). In the right hand panels, cells

were labeled with NeuN (blue), doublecortin+ (green), and PI (red).

Arrows mark NeuN+ cells, arrowheads mark NeuN+/doublecortin+

cells, and asterisks mark PI+/NeuN+ cells.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866� 2009 The Authors

864 | J. Faijerson et al.

plasma membrane. Interestingly, it has also been reportedthat insulin lacking these five residues from the B chain isfully active in vitro (Fischer et al. 1985), suggesting thatboth fragments may be bioactive, although it should benoted that IDE can further cleave the B chain to makesmaller fragments.

The expression of IDE is not unique to NSPCs. IDE is infact expressed throughout the body, with its highest expres-sion in testes, tongue, and brain (Kuo et al. 1993). However,it remains unknown whether the processing of insulinfragments (lysosomal degradation vs. secretion), especiallypentinin, is differentially regulated.

Insulin is present in the CNS (Margolis and Altszuler1967; Havrankova et al. 1978). Local production and releaseof insulin in the CNS has been suggested (Coker et al. 1990),but it seems that the insulin found in the brain largely isproduced by beta cells in the pancreas and enters the brainacross the blood–brain barrier (Pardridge 1986; Schwartzet al. 1991; Schulingkamp et al. 2000).

In order to investigate the molecular mechanism of theobserved neuroprotection by pentinin, we performed elec-trophysiological recordings of excitatory neurons in thehippocampus. These recordings show that pentinin does nothave a rapid antagonistic effect on NMDA signaling,indicating that the actions of pentinin are either downstreamof the NMDAR or act via a parallel pathway to dampen thecalcium signal or counter its effects, such as nitric oxideproduction (Aarts and Tymianski 2004), thereby promotingcell survival. Furthermore, it has been shown that the NR2Aand NR2B subunits of NMDAR favour either survival orexcitotoxicity, respectively (Liu et al. 2007). Pentinin couldpotentially interfere with the intracellular activation ofproteins after NMDAR stimulation, either by potentiatingNR2A-mediated cell survival or by inhibiting NR2B-mediated cell death.

We have shown that medium conditioned by undifferen-tiated adult NSPCs protects hippocampal neurons fromNMDA-induced excitotoxicity. One component of thatmedium, a peptide which we termed pentinin, contains ahigh proportion of its neuroprotective activity. These data notonly imply the presence of a new neuroprotective factor inthe brain, but also suggest an important role for undifferen-tiated stem/progenitor cells as modulators of lesions in thebrain.

Acknowledgments

The authors would like to dedicate this manuscript to their revered

colleague and friend Peter S. Eriksson, M.D., Ph.D. (1959–2007).

This work was supported by the Swedish Medical Research

Council, the Swedish Stroke Society, the Edit Jacobson Foundation,

the Royal Society of Arts and Sciences in Goteborg, and the

Swedish Society of Medicine. The authors thank Barbro Jilderos

and Niklas Mattsson for excellent technical assistance.

1.5

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20151050Time (min)

Pentinin (10 nM)

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2015 10 5 0 Time (min)

Pentinin (10 nM)

400

300

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0NM

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121086420Time (min)

Pentinin (10 nM)D-AP5 (50 µM)

20 ms 100 pA

a b

a b

100 ms100 pA

a b

a b

(a)

(b)

(c)

Fig. 5 The effect of pentinin on NMDA receptor-mediated signaling.

(a) An experiment illustrating that application of pentinin (10 nM) does

not affect the magnitude of NMDA receptor-mediated EPSCs. Aver-

age NMDAR-EPSC taken at time points a and b are shown on top. (b)

Graph summarizing seven experiments such as that shown in a. Be-

fore averaging, each experiment was normalized to the baseline value

just preceding the application of pentinin. (c) An experiment illustrating

the effect of the competitive NMDA receptor antagonist D-AP5

(50 lM) on NMDAR-EPSCs. Average NMDAR-EPSC taken at time

points a and b are shown on top. Note that a small AMPA-receptor

EPSC persists after application of D-AP5.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 109, 858–866

Neuroprotection by the novel peptide pentinin | 865

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866 | J. Faijerson et al.