The Regulatory Mechanism of Neurogenesis by IGF-1 in AdultMice

Honghua Yuan & Renjin Chen & Lianlian Wu &

Quangang Chen & Ankang Hu & Tengye Zhang &

Zhenzhen Wang & Xiaorong Zhu

Received: 24 February 2014 /Accepted: 14 April 2014# Springer Science+Business Media New York 2014

Abstract Growth factors like insulin-like growth factor 1(IGF-1) is reported to mediate neurogenesis in the subgranularzone (SGZ) and the subventricular zone (SVZ) of the adultmammalian brain, but its regulatory mechanism remains un-clear. We generated transgenic mice overexpressing IGF-1specifically in neural stem cells (NSCs) and assessed the effectof IGF-1 on neurogenesis in adult mice NSCs. Overexpres-sion of IGF-1 could stimulate the expression of phospho-Aktand phospho-ERK1/2 while inducing proliferation and differ-entiation of NSCs in the SGZ and SVZ. The MEK/ERKinhibitor U0126 could inhibit ERK1/2 phosphorylation, fur-ther inhibiting the proliferation of NSCs in the SGZ and SVZbut had no effect on the phosphorylation of Akt. By contrast,The PI3K/Akt inhibitor LY294002 inhibited phosphorylationof Akt and differentiation of NSCs in the SGZ and SVZ,resulting in no change in the proliferation of NSCs andERK1/2 phosphorylation. These results demonstrate thatIGF-1 upregulates the proliferation of NSCs by triggeringMEK/ERK pathway signaling in the adult mice SGZ andSVZ. Meanwhile, IGF-1 also induces differentiation of NSCsvia the PI3K/Akt pathway in adult mice. However, we foundno evidence of crosstalk between the PI3K/Akt and MEK/ERK pathways in adult mice NSCs. Our work provides newexperimental evidence of the involvement of the PI3K/Aktand MEK/ERK pathways in the proliferation and differentia-tion of the NSCs of adult mice.

Keywords IGF-1 . Transgenic mice . Neural stem cells .

Regulatorymechanism . Differentiation . Proliferation

Introduction

The discovery that adult mammalian brain neurogenesis isprevalent and that neural stem cells (NSCs) or neural progen-itor cells (NPCs) found in adult mammalian brain have theability to generate new neurons to replace lost and damagedones. This inherent cell replacement mechanism offers hopefor the treatment of various disorders of the nervous system [1,2]. Neurogenesis in the adult mammalian occurs mainly in thesubventricular zone (SVZ) of the lateral ventricles and thesubgranular zone (SGZ) of the dentate gyrus of the hippocam-pus. Neurogenesis is a dynamic process regulated by bothintrinsic and extrinsic factors [3–5]. Under normal conditions,neurogenesis is maintained through processes that includeproliferation and differentiation of NSCs, neuronal survivaland migration, and integration of young neurons into pre-existing circuit [6, 7]. Some endogenous growth factors likeinsulin-like growth factor 1 (IGF-1), vascular endothelialgrowth factor (VEGF), and fibroblast growth factor 2 (FGF-2) can directly or indirectly improve the proliferation, differ-entiation, and survival of NSCs in the SVZ and SGZ of theadult mammalian [8]. Several reports have confirmed thatIGF-1 can prevent apoptosis, facilitate their proliferation, aswell as induce neuronal differentiation of NSCs [9–11].

The biological functions of IGF-1 are regulated through itsbinding to the receptor IGF-1R, a heterotetramer with intrinsictyrosine kinase activity. Once activated, IGF-1R phosphory-lates downstream targets that include docking or adaptorproteins of the insulin receptor substrate family, IRS-1, Grab2,and Shc [12]. This in turn activates multiple downstreamsignal transduction pathways, including those mediated byphosphatidylinositol 3-kinase (PI3K/Akt) and mitogen-

Honghua Yuan, Renjin Chen and Lianlian Wu contributed equally to thiswork.

H. Yuan : L. Wu : T. Zhang : Z. WangResearch Center for Neurobiology, Xuzhou Medical College,Xuzhou, Jiangsu, China

R. Chen :Q. Chen :A. Hu :X. Zhu (*)Laboratory Animal Center, Xuzhou Medical College,Xuzhou 221004, Jiangsu, Chinae-mail: xzyxdwzx@163.com

Mol NeurobiolDOI 10.1007/s12035-014-8717-6

activated protein kinase/extracellular signal-regulated kinase(MEK/ERK) [13, 14]. These signaling mechanisms can regu-late cellular processes like survival, proliferation, and differ-entiation of NSCs. The Akt protein kinase is an importanttransducer of the PI3K/Akt signaling cascade [15, 16]. Acti-vation of PI3K causes it to phosphorylate Akt at Thr308 andSer473 and then activating pro-survival signaling pathways[17]. ERK1/2 is an important mediator of MEK/ERK signal-ing. Through signal transduction, it can propagate the extra-cellular stimulus to effector molecules in the cytoplasm andfurther into the nucleus.

IGF-1 regulates the proliferation and differentiation ofNSCs via PI3K/Akt and MEK/ERK pathways to promotethe neurogenesis [18–20]. However, these findings aboveemphasize that the role of signaling cascade in neuronaldifferentiation and proliferation is distinct in the source ofNSCs and is dependent on different cell types. The preciseregulatory mechanism of neurogenesis by PI3K/Akt andMEK/ERK pathways in the adult mammalian brain has notyet been fully demonstrated. In this study, we generatedtransgenic mice overexpressing IGF-1 and attempt to eluci-date the regulatory mechanism of IGF-1 in neurogenesis inadult mice.

Materials and Methods

Animals

C57BL/6 J mice (3 months old) were purchased from theAnimal Center of Xuzhou Medical College, Xuzhou, China.The C57BL/6 J transgenic mice of overexpression of IGF-1(3 months old) were generated by Cyagen Biosciences Inc.The complete mice IGF-1 cDNA (GenBank, AY878193.1)was subcloned into the pcDNA3.1 vector to generate thepcDNA3.1-IGF-1 construct. The CMV promoter sequenceof the recombinant vector was replaced by the sequence ofintone 2 (GenBank, AY438043.1) of nestin gene and the 5′flanking sequence (GenBank, AY331185) of pNX7 vector.All animals were reared in specific pathogen-free (SPF) con-dition. All animal experiments were manipulated in accor-dance with the ethical guidelines of Xuzhou Medical College.

5-Bromo-2-Deoxyuridine Incorporation Assay

To detect cell proliferation NSCs in vivo, 50 mg/kg of 5-bromo-2-deoxyuridine (BrdU) was cavities injected into theC57BL6/J mice and transgenic mice. BrdU solution wasprepared in sterile saline at 10 mg/ml. All the mice wereinjected with BrdU for 3 days (twice per day), and then killed24 h after last injection. For BrdU staining, the slides wereincubated in 2 N HCl at 37 °C for 30 min. After washing with0.05 M borate buffer (pH 8.5) for 10 min followed by

phosphate-buffered saline (PBS), the slides were blocked with10% normal goat serum in PBS and incubated with mice anti-BrdU antibody (1:400; Calbiochem) at 4 °C. Fluorescenceimages were captured using an OLYMPUS IX71 microscope.

Immunofluorescence Staining

The C57BL/6 J mice and transgenic mice were anesthe-tized and transcardially perfused with 4 % cold parafor-maldehyde dissolved in PBS (10 mM, pH 7.4). Then thebrains were dissected and fixed in 4 % paraformaldehydeat 4 °C overnight followed by 30 % sucrose in PBS. Thebrains were coronally cut into 25 μm slices using afreezing microtome. The SVZ and SGZ sections werecollected on slides and blocked with 10 % normal goatserum in PBS, followed by overnight incubation withmice anti-neuron antibody (1:500; Novus) and rabbitanti-GFAP antibody (1:500; Boster). After washes, theslides were incubated with FITC or TRITC-conjugatedsecondary antibodies (1:100; Bioworld) at room tempera-ture for 1.5 h, and then mounted in antifade mountingmedium (Beyotime). Fluorescence images were capturedusing an OLYMPUS IX71 microscope.

Western Blotting

Brain were homogenized in lysis buffer (20 mM Tris,pH 7.5, 150 mM NaCl, 1 % NP-40, 10 % glycerol, 1 mMEDTA, 1.5 mM MgCl2, 20 mM NaF, and 20 mM glycero-phosphate) supplemented with protease inhibitors (1 mg/mlleupeptin, 1 mg/m l aprotinin, 1 mM PMSF, and 1 mMNa3VO4). The protein concentrations were determined bythe Pierce BCA Protein Assay Kit (Beyotime). Equalamounts of protein samples were boiled for 5 min andsubjected to SDS-polyacrylamide gel electrophoresis. Theproteins were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) and blocked in 3 % BSA in PBS-Tbuffer (10 mM PBS plus 0.05 % Tween-20) at room tem-perature for 4 h. The membrane was incubated at 4 °Covernight with antibodies against IGF-1 (1:200; Santacruz), FEF-2 (1:200; Santa Cruz), phospho-ERK 1/2(1:1,000; Bioworld), phospho-Akt (1:1,000; Bioworld),and β-actin (1:1,000; Santa Cruz), followed by secondantibodies 926–32210 IRDye 800 CW goat anti-mice (LI-COR) and 926–68021 IRDye 680 RD goat anti-rabbit (LI-COR). Detection was performed using an Odessey scanner.Densitometric analysis was performed using ImageJsoftware.

Drug Microinfusion

The C57BL/6J mice and transgenic mice under chloralhydrate anesthesia were placed in a stereotaxic frame.

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Then 10 μM U0126 (Beyotime) and 20 μM LY294002(Beyotime) in DMSO were injected into caudate-putamen (Cpu; stereotaxic coordinates: AP, 0.02 mM;ML, 18.00 mM; and DV, 27.50 mm) and SGZ (stereo-taxic coordinates: AP, −2.18 mM; ML, 12.50 mm; andDV, 22.00 mm) through microinfusion catheters in avolume of 2 μl/side, which were removed 5 min later.

Cell Counting

BrdU-positive (BrdU+) cells in the ipsilateral SVZ werecounted along ventricle 400 μm and along corpus callosum300 μm. BrdU + cells in the ipsilateral SGZ were countedall maked cells. NeuN-positive (NeuN+) cells in the ipsi-lateral SVZ were counted in an area of 100×100 μm, andthose in the ipsilateral SGZ were counted as IOD of fluo-rescence intensity in an area of 100×50 μm. GFAP-positive(GFAP+) cells in the ipsilateral SVZ and SGZ were countedin an area of 100×100 μm. The data were presented as totalcounted cells.

Statistical Analysis

All the results are represented as mean ± SEM. Eachdata represents at least three separate experiments per-formed in duplicate or triplicate, unless otherwise indi-cated. Statistical differences of the experimental datawere assessed by one- or two-way analysis of variancefollowed by Tukey’s test. A value of P<0.05 was con-sidered statistically significant.

Results

Exogenous IGF-1 Expression in NSCs of Adult TransgenicMice

In adult mice, NSCs are mainly located in the SGZ and SVZ.As a secretory protein, exogenous IGF-1 expression can en-hance the IGF-1 protein concentrations in the SGZ and theSVZ. The results indicated that compared with the wild-typemice, there was a significant increased in expression of IGF-1in NSCs of the SGZ and SVZ in adult transgenic mice (Fig. 1).

The Expression of FGF-2 in Adult Mice

IGF-2 is secreted by brain astrocytes and of importance forregulating NSCs. Here, we measured the levels of FGF-2 inthe SGZ and SVZ of both adult transgenic and wild-type miceusing Western blotting. The results showed the expression ofFGF-2 in the SGZ and SVZ of the transgenic mice, whichhowever reached a similar extent to that in the wild-type mice(Fig. 2).

IGF-1 Induces Proliferation and Differentiation of NeuralStem Cells in Adult Mice

To investigate the role of IGF-1 in the proliferation and differ-entiation of NSCs in adult mice, an immunofluorescent assaywas performed to evaluate the number of BrdU+, GFAP+, andNeuN + cells in the SGZ and SVZ of transgenic and wild-typemice. The number of BrdU+, GFAP+ andNeuN+ cells reflectsthe proliferation and differentiation of NSCs, respectively. The

Fig. 1 Expression of exogenousIGF-1 in the SGZ and SVZ ofadult mice. a The expression ofIGF-1 was detected in the SVZand SGZ of the transgenic andwild-type mice using Westernblotting. b The expression of IGF-1 was significantly increased inthe SGZ and SVZ of the adulttransgenic mice than that in thewild type. Data are expressed asmean ± SD. **P<0.01 comparedwith the wild type

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Fig. 2 Expression of FGF-2 inthe SGZ and SVZ of adult mice. aThe expression of FGF-2 wasdetected in the SGZ and SVZ ofthe transgenic and wild-type miceusing Western blotting. b Thephotodensitometry assayindicated no significant differencein FGF-2 protein expressionbetween the adult transgenic andwild-type mice. Data areexpressed as mean ± SD. P>0.05compared with the wild type

Fig. 3 IGF-1 inducesproliferation and differentiation ofNSCs in the SGZ and SVZ ofadult mice. a BrdU was injectedcavities (twice/day), into adulttransgenic and wild-type mice.After 3 days, the brains werecoronally sectioned at a thicknessof 25 μm and immunofluorescentstaining was performed. BrdU +cells were evident in the SGZ andSVZ. b, c NeuN + and GFAP +cells were observed in the SGZand SVZ followingimmunofluorescent staining. dEvaluation of BrdU + cells in theSGZ and SVZ showed thepresence of NeuN + cells in theSVZ (e) and GFAP + cells in theSGZ and SVZ (g). f Fluorescenceintensity of NeuN + cells in theSGZ. The results suggest that intransgenic mice, the numbers ofneural stem cells, astrocytes, andneurons were all significantlyhigher than in wild-type mice.Data are expressed as mean ± SD;**P<0.01 compared with wild-type mice; bar, 100 μm

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results indicate that in adult transgenic mice, the number ofNSCs, astrocytes, and neurons was significantly higher than inwild-type mice (Fig. 3). These results suggest that IGF-1 canpromote cell proliferation and differentiation in adult mice.

IGF-1 Activates the PI3K/Akt and MEK/ERK Pathwaysin NSCs of Adult Mice

The activated PI3K/Akt and MEK/ERK pathways are charac-terized in the phosphorylation of Akt and ERK1/2. Here,Western blotting analysis was performed to compare the

amounts of phospho-Akt and phospho-ERK1/2 between theSGZ and SVZ of the adult transgenic and wild-type mice(Fig. 4). The result suggested that excessive IGF-1 upregulatethe phosphorylation of Akt and ERK1/2 in NSCs, triggeringthe PI3K/Akt and MEK/ERK cascades.

IGF-1 Activates the MEK/ERK Pathways to Promotethe Proliferation of NSCs in Adult Mice

The MEK/ERK inhibitor U0126 was microinjected intothe brains of adult transgenic mice and was found to

Fig. 4 Overexpression of IGF-1in the adult transgenic mice brainactivates the MAPK/ERK andPI3K/Akt pathways. a, b Theexpression of phosphor-ERK andphosph-Akt were detected in theSVZ and SGZ of the transgenicand wild-type mice usingWesternblotting. c, d Thephotodensitometry assayindicated that the p-ERK and p-Akt in the transgenic mice wassignificantly higher than that inthe wile type, suggesting theactivation of the MAPK/ERK andPI3K/Akt pathways by IGF-1.Data are expressed as mean ± SD.**P<0.01; *P<0.05 comparedwith the wild type

Fig. 5 The specific inhibitorsU0126 and LY294002 inhibit thephosphorylation of ERK and Akt,respectively. a, b U0126 (10 μM)or LY294002 (20 μM) wasmicroinjected into the Cpu andSGZ of adult transgenic mice,while the same dose of DMSOwas injected into the samelocation in transgenic mice as thecontrol group. Five days later, thetissues were collected, andWestern blotting was performedto detect the expression of p-ERKand p-Akt in the SVZ and SGZ. c,d A photodensitometry assaysuggested that in the SVZ andSGZ, U1026 and LY294002significantly inhibit theexpression of ERK and Akt,respectively. Data are expressedas mean ± SD; **P<0.01;*P<0.05 compared with thecontrol group

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remarkably reduce the level of phospho-ERK1/2 in boththe SGZ and SVZ (Fig. 5). In addition, we found that thenumber of NSCs, neurons, and astrocytes declined in theSVZ and SGZ (Fig. 6). These results indicate that IGF-1activates the MEK/ERK pathway to promote prolifera-tion of NSCs in adult mice.

IGF-1 Activates the PI3K/Akt Pathways and Promotesthe Differentiation of NSCs in Adult Mice

The PI3K/AKT inhibitor LY294002 was microinjected intothe brain of the adult transgenic mice, leading to substantialdecreases in phospho-Akt expression (Fig. 5) and the numberof astrocytes and neurons. However, no significant change

was seen in terms of the number of NSCs in SVZ and SGZ(Fig. 7). These results indicated that IGF-1 could facilitate thedifferentiation of NSCs through the PI3K/Akt cascade. Therewas no evidence confirming the crosstalk between thePI3K/Akt and MEK/ERK pathways in the adult mice inNSCs.

It has been reported that LY294002 can inhibit Akt phos-phorylation and simultaneously inhibit phosphorylation ofERK1/2 in NSCs. As a result, it is considered that a crosstalkexists between the PI3K and MEK/ERK pathways within theNSCs. However, in our study, LY294002 had no influence onthe phosphorylation level of ERK1/2 in the SGZ or the SVZof adult transgenic mice. Similarly, UO126 did not reduce thephosphorylation level of Akt (Fig. 8). These results indicate

Fig. 6 IGF-1 inducesproliferation of NSCs in adultmouse SGZ and SVZ via theMEK/ERK pathway. IGF-1transgenic mice weremicroinjected with a specificinhibitor of MEK1/2 (U0126)into the Cpu and SGZ, and 5 dayslater, the brain was coronallysectioned at a thickness of 25 μmand immunofluorescent stainingwas performed. a NSCs in theSGZ and SVZ are stained byBrdU. b, c The NeuN + andGFAP + cells in the SGZ andSVZ were examined byimmunofluorescent staining. dEvaluation of the BrdU + cells inthe SGZ and SVZ, eNeuN + cellsin the SVZ, f fluorescenceintensity of the NeuN + cells inthe SGZ, and g GFAP + cells inthe SGZ and SVZ. The resultssuggest that inhibition of theMEK/ERK pathway by U0126 inthe SGZ and SVZ significantlydecreases the number of NSCs,astrocytes, and neurons. Data areexpressed as mean ± SD;**P<0.01; *P<0.05 comparedwith the control group. Bar,100 μm

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that there is probably no crosstalk between the PI3K/Akt andMEK/ERK pathways.

Discussion

Exogenous IGF-1 Gene Was Specifically Expressed in NSCs

Nestin is a protein specifically expressed in NSCs. The5′ flanking sequence of nestin gene has a strong nonspe-cific regulatory capacity. However, after being integratedwith the second intron, it can regulate exogenous genesspecifically expressed in NSCs [21, 22]. In the currentstudy, the integrated sequences were adopted as a pro-moter to generate a transgenic mice overexpressing IGF-

1 in NSCs. The results showed that the levels of IGF-1were significantly enhanced in the SGZ and SVZ com-pared with the wild type.

IGF-1 Promote the Proliferation and Differentiation of NSCsin the SGZ and SVZ of Adult Mice

IGF-1 is a 7.6-kDa polypeptide that performs multiplefunctions during the development of the brain. One of itsmost fundamental functions in NSCs is regulation ofenergy metabolism [23]. IGF-1R is widely expressed inthe brain, where it transduces the signal transmitted byextracellular IGF-1, and subsequently regulates prolifera-tion, differentiation, survival of NSCs, and neurite out-growth [24, 25]. The role of IGF-1 in directly facilitating

Fig. 7 IGF-1 induces thedifferentiation of NSCs in adultmice through the PI3K/Aktpathways. Specific inhibitors ofPI3K/Akt pathways (LY294002)were microinjected into the Cpuand SGZ of the adult transgenicmice. Five days later, the brainwas cut into 25-μm sections forimmunohistochemical assay. aThe NSCs in SGZ and SVZ werestained by BrdU. b, c The neuronand GFAP + cells in the SGZ andSVZ were examined byimmunohistochemical staining. dEvaluation of the number of BrdU+ cells in the SGZ and SVZ. eNeuron-positive cells in SVZ. fThe fluorescence intensity ofneuron-positive cells in the SGZ.g GFAP-positive cells in the SGZand SVZ. The results indicatedthat microinjection withLY294002 could inhibit thedifferentiation of NSCs in adultmice and significantly reduce thenumber of neurons and astrocytesin the SGZ and SVZ. The numberof NSCs in the SGZ and SVZwasnot changed. Data are expressedas mean ± SD. **P<0.01,P>0.05. Compared with thecontrol, the number of NSCs intransgenic mice is not remarkablychanged after injection with LY294002. Bar, 100 μm

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mitogenic effects of NSCs is mediated via three mecha-nisms: increased expression of mitotic markers and short-ening of G0/G1 phase; acceleration of cell cycle re-entry;and upregulation of cyclin D1. In vivo studies haveconfirmed that IGF-1 promotes mitosis of embryoniccortical progenitors in the brain and is an importantregulator of proliferation during corticogenesis [26]. Invitro experiments have shown that under proliferativeculture conditions, IGF-1 can accelerate NSCs cycle pro-gression by upregulating cyclin D1 expression [11].Moreover, both in vivo and in vitro studies have dem-onstrated the ability of IGF-1 to stimulate the differenti-ation of NSCs into neurons, astrocytes, and oligodendro-cytes. Insufficiency of IGF-1 leads to decreased in adultmice neurogenesis and synaptic neuronal plasticity. Thesecretion of IGF-1 is stably decreased in adult mice braincompa red wi t h young mice and concomi t an tneurogenesis slowed down [27]. In astrocyte-specificIGF-I overexpression, the number of astrocytes is in-creased by 56 %, as well as increased production ofneurons and oligodendrocytes in the dentate gyrus [28].Recent studies have shown that in response to braindamage, cerebral ischemia, and neuronal apoptosis inadult mice, the endogenous secretion of IGF-1 is in-creased in the SGZ and SVZ, to facilitate therapeuticneurogenesis [29, 30]. Similarly, our study confirms thatNSCs-specific IGF-1 overexpression can promote

neurogenesis and obviously increase the number ofNSCs, neurones, and astrocytes in the SGZ and SVZ ofthe adult mice.

IGF-1 Can Activate the MEK/ERK Pathway and PromoteProliferation of NSCs in Adult Mice

ERK1/2 is an important mediator of extracellular signaltransduction and is widely expressed in the brain. It canphosphorylate and activate the MEK/ERK signalingpathway that is believed to facilitate cell proliferationduring the G1 phase of cell cycle [31, 32]. Moreover,recent studies have confirmed that ERK1/2 plays animportant role in regulating the survival and self-renewal of NSCs [33]. Both in vivo and in vitro exper-iments have shown that IGF-1 can improve the level ofphosphoration of ERK1/2 in the NSCs. Our results rein-force the thought that IGF-1 overexpression can activateMKE/ERK pathway and finally promote proliferation ofNSCs in the adult mice SGZ and SVZ. The MKE/ERKinhibitor U0126 can suppress phosphorylation ofERK1/2, further inhibiting proliferation of NSCs in adultmice SGZ and SVZ.

The MEK/ERK pathway is also known to contributeto the differentiation of postnatal NSCs [34, 35]. Bycontrast, some reports have claimed that ERK1/2 acti-vation can block the differentiation of NSCs found in

Fig. 8 The crosstalk between the PI3K/Akt and MEK/ERK pathways inthe NSCs of adult mice. a, b U0126 (10 μM) and LY294002 weremicroinjected into the Cpu and SGZ of transgenic mice, whereas DMSOwas adopted as the control group. Five days later, the tissues werecollected, and Western blotting was performed to detect the expressionof p-ERK and p-Akt in the SVZ and SGZ. c, d The photodensitometry

assay indicted that U0126 did not inhibit the phosphorylation of Akt.Meanwhile, the expression of p-ERK could not be inhibited byLY294002. In summary, there was no crosstalk between the PI3K/Aktand MEK/ERK pathways in NSCs of adult mice. Data are expressed asmean ± SD. P>0.05 compared with the control

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the adult hippocampus [36]. In addition to IGF-1, an-other growth factor, FGF-2 can also enhance ERK1/2phosphorylation, under the regulatory control of Hes1,and subsequently activate MEK/ERK signaling. FGF-2can thus facilitate proliferation and inhibit differentiationof NSCs [37, 20]. FGF-2 is secreted in astrocytes. Inour study, no significant difference is found in theexpression of FGF-2 in the SGZ and SVZ between theadult IGF-1 transgenic and wild-type mice. In otherwords, the MEK/ERK cascade may not involve in thedifferentiation of NSCs in the adult mice. Therefore,U0126 treatment can inhibit the MEK /ERK pathwayand decrease the number of neurons and astrocytes,which may be related with a reduced number of NSCsrather than suppressing the MEK/ERK pathway.

IGF-1 Can Activate the PI3K/Akt Pathway and PromoteDifferentiation of NSCs in Adult Mice

We found that the IGF-1 triggers Akt phosphorylation ofNSCs of adult mice, supporting the hypothesis that IGF-1 acts via the PI3K/Akt pathway. Akt signaling has beenknown to participate in the regulation of cell survival.However, there is now increasing evidence that it mightalso be involved in the regulation of G1/S transition,self-renewal of NSCs, and in controlling cell numberand cell-type composition in the brain [38]. In vivoexperiments have shown that the intrinsic Akt phosphor-ylation level in E16.5 cortical progenitors is very low,suppressing PI3K/Akt pathway, which leads to a reduc-tion in DNA synthesis by 12 %. This implies thatPI3K/Akt signaling plays a minor role in the prolifera-tion of early cortical progenitors [39]. However, otherstudies have indicated that activation of PI3K/Akt sig-naling can promote the survival, proliferation, and self-renewal of a subset of embryonic stem cells [40]. In vitrostudies suggest that PI3K/Akt signaling can process thecombination of information contained in multiple extra-cellular signals, to promote proliferation and inhibit dif-ferentiation of NSCs in the rat hippocampus [41, 42].These findings are contradictory to earlier reports whereIGF-1 was shown to activate the PI3K/Akt pathway topromote the differentiation of mice olfactory bulb NSCs[43]. In our study, IGF-1 is shown to activate PI3K/Aktpathway and promote differentiation of NSCs into neu-rons and astrocytes in adult mice SGZ and SVZ. We alsodemonstrate that PI3K/Akt signaling is not associatedwith NSCs proliferation in adult mice. Comparing ourresults with the findings of previous studies revealed atheme in that the contribution of PI3K/Akt pathway iscellular context dependent [44]. The PI3K/Akt signalingmay play a distinct role dependent on the cellular statesof different cells.

Lack of Evidence Confirming the CrosstalkBetween the PI3K/Akt and MEK/ERK Pathways in the AdultMice in NSCs

Interestingly, several studies have investigated and confirmedthe molecular crosstalk between PI3K/Akt and MEK/ERKsignaling. For instance, phosphorylation of Akt can inhibitIGF-1-induced activation of ERK1/2 in differentiatedmyotubes [45, 46]. In addition, some studies found that Aktsignaling might be involved in IGF-1-induced ERK1/2 acti-vation in rat in NSCs, based on the observation that the PI3Kinhibitor LY294002 can block the phosphorylation of ERK1/2[47, 48]. However, another study do not support suchcrosstalk between these two pathways in adult spinal cordstem cells [49]. Our results showed that LY294002 inhibitedAkt phosphorylation but has no effect on the phosphorylationof ERK1/2 or vice versa. The PI3K/Akt and MEK/ERKpathways crosstalk do not exist in NSCs of adult mice.

Conclusions

IGF-1 can stimulate neurogenesis in the SGZ and SVZ ofadult mice. IGF-1 can upregulate the expression of phospho-Akt to activate the PI3K/Akt pathway and promote the differ-entiation of NSCs. Meanwhile, it can enhance the level ofphospho-ERK1/2 activate the MEK/ERK cascades, and final-ly facilitate the proliferation of NSCs. However, no crosstalkhas been found between the PI3K/Akt and MEK/ERK path-ways in NSCs of adult mice.

Acknowledgments The research was financially supported by the Nat-ural Science Foundation of China (31172171), the Natural Science Foun-dation for youth in Jiangsu Province (BK2012138), the Natural ScienceFoundation of Jiangsu Province (BK2011209), and the President Foun-dation of Xuzhou Medical College (2012KJZ20). pNX7 vector wasprovided by Naihe Jing.

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