AMP-activated kinase mediates adipose stem cell-stimulated neuritogenesis of PC12 cells

Neuroscience 181 (2011) 40 – 47

AMP-ACTIVATED KINASE MEDIATES ADIPOSE STEM CELL-STIMULATED NEURITOGENESIS OF PC12 CELLS B. TAN,a1 Z. LUAN,b1 X. WEI,a Y. HE,a G. WEI,a B. H. JOHNSTONE,c M. FARLOWa AND Y. DUa,d*

Pluripotent cells residing in the stroma or non-adipocyte fraction of adipose tissues are a plentiful source of readily available cells for autologous cell therapies to regenerate damaged or diseased tissues (Zuk et al., 2001, 2002). Adipose-derived stem cells (ASC) from mice, rats, nonhuman primates, and humans were demonstrated to exhibit differentiation into neural and glial cells in vivo and in vitro (Safford et al., 2002, 2004; Zuk et al., 2002; Kang et al., 2003, 2004; Tholpady et al., 2003; Yang et al., 2004; Fujimura et al., 2005; Ning et al., 2006). In a rat middle cerebral artery occlusion model (MCAO) of ischemic brain injury, transplanted pre-differentiated human ASC migrated to areas of ischemic injury and expressed neuronal specific markers in conjunction with functional benefit (Kang et al., 2003). However, recently, we have shown that it is possible that functional improvement of these animals suffered from hypoxia-ischemic (HI) injury may be due to trophic support provided to host cells from factors released by ASC in conditioned medium from cultured rat ASC (ASC-CM) (Wei et al., 2009a). It is increasingly recognized that this paracrine effect of ASC, as well as that of mesenchymal stem cells originating from other tissues, may be the predominant, if not exclusive, mechanisms of action. The proposed functional benefits of these factors are most likely promotion of cell survival and stimulation of endogenous progenitor and stem cell differentiation (Yanez et al., 2006; Bochev et al., 2008; Cai et al., 2009; Wei et al., 2009b; Yoo et al., 2009). Interestingly, the beneficial effects were observed even with treatment at 24 h after HI injury. Given that neuronal apoptosis in the HI model begins at 4 h, peaking at 16 h (Wei et al., 2006), this finding suggests that ASC-CM may not only be neuroprotective, but may also stimulate the neurogenesis. We have observed ASC-CM induced neuronal regeneration in neonatal rats. ASC have been reported previously to express mRNAs for brain-derived neurotrophic factor (BDNF), glialderived neurotrophic factor (GDNF), and nerve growth factor (NGF) at levels higher than those normally detected in rat brain (Tholpady et al., 2003; Ning et al., 2006). However, although it was suggested that ASC-CM promotes tissue regeneration as well as neuroprotection, the functional activity and specific effects of ASC-CM on neuronal differentiation have not been elucidated. The rat pheochromocytoma cell line PC12 has been widely used to investigate molecular mechanisms underlying neuronal differentiation (Greene and Tischler, 1976). Cell cycle arrest and differentiation occur with NGF treatment (Byrd and Alho, 1987; Ebert et al., 1997). The signaling pathways contributing to NGF-induced PC12 cell neuritogenesis are not fully understood; although, it has

a Department of Neurology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA b

Department of Pediatrics, Navy General Hospital, NO. 6, Fucheng Road, Beijing, China c Department of Medicine, Krannert Institute of Cardiology, School of Medicine, Indiana University, Indianapolis, IN 46202, USA d School of Health Sciences, Purdue University, West Lafayette, IN 47907, USA

Abstract—Adipose tissue stroma contains a population of mesenchymal stem cells, which support repair of damaged tissues through the protective effects of secreted trophic factors. Neurotrophic factors, including nerve growth factor (NGF) have been identified in media collected from cultured adipose-derived stem cells (ASC). We previously demonstrated that administration of cell-free ASC conditioned medium (ASC-CM) at 24 h after injury reduced lesion volume and promoted functional recovery in a rat model of neonatal brain hypoxic-ischemic (HI) injury. The timing of administration well after the peak in neural cell apoptosis in the affected region suggests that regeneration of lost neurons is promoted by factors in ASC-CM. In this study, we determined which of the factors in ASC-CM could induce neurogenesis by testing the ability of the mixture, either whole or after inactivating specific components, to stimulate neurite outgrowth in vitro using the neurogenic cell line PC12. Neuritogenesis in PC12 cells treated with ASC-CM was observed at a level comparable to that observed with purified recombinant NGF. It was observed that NGF in ASC-CM was mainly responsible for inducing PC12 cell neuritogenesis. Interestingly, both ASC-CM and NGF induced PC12 cell neuritogenesis through activation of the AMP-activated kinase (AMPK) pathway which is the central protein involved in controlling many critical functions in response to changes in the cellular energy status. Pharmacological and genetic inhibition of AMPK activity greatly reduced neuritogenesis in PC12 cells. These results suggest that, in addition to possessing neuroprotective properties, ASC-CM mediates repair of damaged tissues through inducing neuronal differentiation via NGFinduced AMPK activation. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: adipose-derived stromal cell, ASC conditioned medium, nerve growth factor, PC-12, differentiation, neurons. 1 Both authors contributed equally to this work. *Corresponding author. Tel: ⫹1-317-278-0220; fax: ⫹1-317-274-3587. E-mail address: [email protected] (Y. Du). Abbreviations: ACC, acetylCoA carboxylase; AMPK, AMP-activated kinase; ASC, adipose-derived stem cells, ASC-CM, ASC conditioned medium; HI, hypoxia-ischemic; MAPK, mitogen-activated protein kinases; NGF, nerve growth factor; PI3Kinase, phosphotidylinositol-3kinases; siRNA, small interfering RNA.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.02.038

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B. Tan et al. / Neuroscience 181 (2011) 40 – 47

been suggested that activation of phosphotidylinositol-3kinases (PI3Kinase) and mitogen-activated protein kinases (MAPK) may be involved (Jackson et al., 1996; Kobayashi et al., 1997; Santos et al., 2007). There is evidence to suggest that AMP-activated kinase (AMPK), the central energy sensor in all cells, also may be a key regulator of neuronal stress responses and neural progenitor commitment (Bergeron et al., 2001; Wang et al., 2005; Kukidome et al., 2006). AMPK is a heterotrimeric enzyme complex which consists of ␣, ␤, and ␥ subunits. The ␣ subunit contains the catalytic domain, including the important regulatory Thr172 residue, which can be phosphorylated by upstream kinases. Phosphorylation of threonine 172 on AMPK␣ is able to activate AMPK (Tschape et al., 2002). AMPK links neuronal function with energy supply and plays a key function in hypothalamic control of food intake and peripheral energy expenditure (Xue and Kahn, 2006). In Drosophila, AMPK helps maintain genomic integrity in neural precursors as well as promotes structural and functional stability in mature neurons (Lee et al., 2007). Loss of AMPK function induces neurodegeneration in Drosophila (Tschape et al., 2002); correspondingly, activation in mice protects hippocampal neurons against metabolic, excitotoxic, and oxidative insults (Culmsee et al., 2001). It was recently shown that AMPK might be involved in resveratrol-induced neuronal regeneration; however, astroglial differentiation did not occur (Dasgupta and Milbrandt, 2007); thus, the exact regulatory function of AMPK in neural stem cell differentiation is not clear. In the present study, we used PC12 cells to investigate whether ASC-CM induced neural cell differentiation and if AMPK might be involved in this process. We discovered that ASC-CM markedly stimulated neuritogenesis in PC12 cells and that NGF, through activation of AMPK, is the main mediator of this effect.

EXPERIMENTAL PROCEDURES Materials AMPK inhibitor, Compound C was purchased from Toronto Research Chemicals (North York, Ontario, Canada). PI3Kinase inhibitor 294002 and P38 MAPK inhibitor SB203580 were purchased from Calbiochem (San Diego, CA, USA). K252a was purchased from MP Biomedicals, LLC (Solon, OH, USA). Rat ␤-NGF and Neutralizing NGF antibody (anti-rat ␤-NGF antibody cat# AF-556-NA) were obtained from R&D systems (Minneapolis, MN, USA). Antibodies, antiAMPK, anti-phospho-AMPKa (thr172) antibody, and anti-phospho AcetylCoA carboxylase (ACC) (Ser79) antibody were purchased from Millipore (Billerica, MA, USA). AMPK siRNA or scrambled control were purchased from Invitrogen (Rockville, MD, USA) and resuspended in DEPC-treated water. Fetal Bovine Serum (FBS) and horse serum (HS) were from Lonza (Allendale, NJ, USA) and DMEM from Invitrogen (Carlsbad, CA, USA).

Isolation of rat ASCs and collection of ASC-CM Rat adipocytes were prepared from the epididymal fat-pads. Briefly, adult Sprague-Dawley rats weighing 100 –140 g were anesthetized with CO2 and shaved. The inguinal and perirenal fat pads were harvested and extensively washed with phosphate-buffered saline, and then were excised, finely minced, and incubated with 1 mg/ml

41

collagenase type I (Worthington Biochemical Corporation, Cat # 4196, Lakewood, NJ, USA) and dispase (BD Biosciences, Cat# 354235, San Diego, CA) under gentle agitation for 1 h at 37 °C or until the fat was thoroughly digested. Sample volume of DMEM was added to the mixture and then centrifuged at 1000 rpm for 5 min. The supernatant was then removed and the cell pellet was re-suspended with 5 ml red blood cell lysis buffer and incubated at 37 °C for 5 min. Then, 20 ml of fresh DMEM was added and mixed completely before being filtered by a 100 ␮m cell strainer (BD Biosciences) to remove debris. The filtrate was centrifuged at 1000 rpm for 5 min to separate the stromal cell fraction (pellet) from adipocytes. The cell pellet was then re-suspended in the appropriate EGM-2 culture medium supplemented with EGM2-MV (Lonza), plated in a T75 tissue culture flask at a density of 4⫻106 cells/cm2, and incubated in a humidified chamber at 37 °C in an atmosphere of 5% CO2. Media were replenished every 3 days and cultures were serially passaged at 95% confluence at a 1:3 ratio. To collect ASC conditioned medium, when the ASC’s confluence reached 95% after passage, the medium was removed and fresh basal media (BME, Invirogen) was added to the cells after being washed three times with 1 XPBS without Ca2⫹ Mg2⫹. After 24 h incubation, the BME was then collected as ASC conditioned medium.

PC12 cell culture and determination of neurites growth cell PC12 cells were cultured and neurite outgrowth was scored according to the method described by Greene et al. (Greene and Tischler, 1976). Briefly, PC12 cells were cultured in DMEM media (Invitrogen) containing 10% FBS and 1% penicillin and streptomycin (Invitrogen). Cells were passaged 1:3 every 2 days. For the experiment study, PC12 cells were plated onto a six- or 24-well plate (Falcon, Fisher Scientific, Pittsburg, PA, USA) which was previously coated with poly-L-lysine and maintained in DMEM media containing 5% FBS and 5% horse serum. Cell viability was confirmed by the use of Trypan Blue (⬎95% of cells counted excluded the dye). PC12 cells were treated with BME or different doses of ASCCM, NGF, or ASC-CM pre-treated with the NGF antibody. For signaling pathway studies, 10 ␮m Compound C (an AMPK inhibitor), 10 ␮m LY294002 (a PI3Kinase inhibitor), or 10 ␮m SB203580 (a P38 inhibitor) was used to pretreat PC12 cells for 2 h before cells were subject to ASC-CM or NGF treatments. For PC12 cell neuritogenesis images of six fields per well were randomly selected, with at least 500 cells per well. Differentiated cells were counted by visual examination of the field; only cells that had at least one neurite with a length twice the cell body diameter were counted, and were then expressed as a percentage of the total cells in the well. The counting was performed by two persons in a blinded manner.

Western blot analysis Proteins were extracted from treated cell using cold lysis buffer (10 mM tetra sodium pyrophosphate, 20 mM HEPES, 1% TritonX-100, 100 mM NaCl, 2 ␮g/ml ptotinin, 2 ␮g/ml leupeptin, and 100 ␮g/ml phenylmethylsulfonyl fluorides). Protein concentrations were determined using the Bradford protein assay. Equal amounts of protein were placed in 2⫻ sample buffer (0.125 M Tris–HCI, pH 6.8, 2% glycerol, 0.2 mg/ml Bromophanol Blue dye, 2% SDS, and 10% ␤-mercaptoethanol) and electrophoresed on 10% SDS-polyacrylamide gel. Proteins were then transferred to nitrocellulose membrane by electroblotting. Membranes were blocked for 1 h at room temperature in TBST and 5% non-fat-milk. Primary antibodies (1:1000) at the appropriate dilution were incubated overnight at 4 °C temperature. Blots were then washed and incubated with a peroxidase-conjugated secondary antibody (1:2000) for 1 h in TBST. The chemiluminescent substrate for secondary antibody was developed with the ECL detection system.

B. Tan et al. / Neuroscience 181 (2011) 40 – 47

siRNA transfection assay 6

PC12 cells were cultured in six wells at 1⫻10 density. The next day 20 ␮mol siRNA of AMPK or Random siRNA of AMPK (Invitrogen) were mixed with lipofectamine (Invitrogen) and incubated at room temperature for 20 min. The mixture was then used to transfect PC12 cells for 24 h. After 24 h 5 ng/ml NGF was added to media. The transfection efficiency was checked under fluorescence microscope (Nikon, Melville, NY, USA). The transfected PC12 cells expressed GFP protein. The PC12 cell neuritogenesis was measured at day 7 and neurite outgrowth of PC12 cell with GFP was counted, also protein lysis was harvested for Western blot.

Statistical analysis Unless indicated otherwise, data are given as mean⫾SEM representing separate experiments carried out independently using triplicate or quadruple samples. Data were evaluated using ANOVA followed by a post-hoc t-test, and a P-value of less than 0.05 was considered significant.

RESULTS Induction of PC12 cell neuritogenesis by ASC-CM was reduced by neutralizing NGF The addition of both ASC-CM and NGF to the medium induced neurite outgrowth of PC12 cells (Fig. 1). The effect

a

of ASC-CM was dose-dependent with 20⫾11% and 36⫾15% of the cells having neurites after culturing for 8 days in medium containing 30% and 100%, respectively, of ASC-CM (Fig. 1e). The neurite outgrowth of PC12 cell in ASC-CM was significantly greater than controls grown in basal medium in which only 2⫾0.5% of the PC12 cells were associated with neurites. Promotion of cell cycle arrest and differentiation of PC12 cells by NGF have been well characterized; predictably, we measured a concentration of 0.68 ng/ml NGF in ASC-CM, which is similar to previously published values for these cells (Peeraully et al., 2004). Induction of neuritogenesis in the presence of ASC-CM was similar to that observed when cells were treated with 5 ng/ml purified recombinant NGF (Fig. 1g). To investigate the importance of NGF contained in ASC-CM in ASC-CM-induced neuritogenesis, ASC-CM was pre-incubated with a neutralizing antibody before adding to plated PC12 cells. Neurite outgrowth of PC12 cells was reduced by 2.7-fold after inactivation of NGF in ASCCM. Additionally, PC12 cell neuritogenesis was also markedly reduced to a similar degree by K252a, a potent inhibitor of the NGF receptor (Fig. 1f). Furthermore, neutralization function of this antibody was examined. As expected,

b

cells with neurites

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60% 50% 40% 30% 20% 10% 0%

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Fig. 1. Adipose stem cell-conditioned medium (ASC-CM) induced neurite outgrowth in PC12 cells. (a– d) ASC-CM and NGF induced PC12 cell neuritogenesis. Phase contrast images (20⫻ magnification) of PC12 cells cultured for 8 d in basal media (BME) alone (a), basal media with 30% ASC-CM (b), basal media supplemented with 5 ng/ml NGF (c), basal media with 30% ASC-CM that had been pre-treated with 2 ␮g/ml of a neutralizing NGF antibody (d) were for demonstration of cells with different morphologies only. Scale bar, 50 ␮m. (e, f) ASC-CM-induced PC12 cell neurite growth was dose dependent and markedly inhibited by the NGF antibody and K252a. The percentage of cells exhibiting neurite outgrowth was quantitated by counting at least 500 cells per six fields in each well. All cells were separated well in the field. An NGF receptor antagonist, K252a (50 nM) was used to pre-treat PC12 cells for 2 h before ASC-CM treatments. (g) NGF induced PC12 cell neurite growth in a dose fashion and the NGF antibody specifically neutralized the NGF function. Data represent the mean⫾SEM of triplicate wells from single experiments repeated at least three times independently with similar results. (* P⬍0.05, ** P⬍0.01; compared with the cultures treated with BME (e) or ASC-CM (f), as analyzed by ANOVA followed by a post-hoc t-test). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

B. Tan et al. / Neuroscience 181 (2011) 40 – 47

the NGF antibody used here almost completely abolished the neuritogenic effect of NGF at doses from 5 to 50 ng/ml (Fig. 1g). Thus, of the many potential candidate factors found in ASC-CM, NGF appears to be the major stimulator of neurite outgrowth. However, other factors not examined may also contribute to neuritogenesis of PC12 cells since a significant amount of neurite outgrowth was observed with NGF-depleted ASC-CM. NGF induced AMPK phosphorylation in PC12 cell Phosphorylation of AMPK was examined in NGF-treated and non-treated PC12 cells. NGF treatment resulted in a marked increase in AMPK Thr172 phosphorylation within 2 h that peaked at approximately 8 h. For further confirmation we examined phosphorylation levels of the downstream AMPK substrate ACC. Consistent with the AMPK activation data, induction of ACC phosphorylation started from 4 h, peaked at 8 h, and persisted for up to 24 h after NGF treatment. The data demonstrated that the NGF treatment led to phosphorylation of AMPK and ACC in PC12 cells (Fig. 2a– c). AMPK phosphorylation was essential for NGF and ASC-CM induction of PC12 cell neuritogenesis Because NGF both activated AMPK and induced PC12 cell neuritogenesis, the contribution of AMPK activity to NGF-induced neurite outgrowth was examined. PC12 cells were treated with the specific AMPK inhibitor Compound C before adding NGF to the culture medium and neurite outgrowth was compared to cultures treated with NGF only or basal medium for control (Fig. 3a– c). The inhibitor significantly reduced NGF-induced neurite outgrowth in a

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dose fashion starting from 2 ␮M and by four-fold (P⬍0.01) at 10 – 40 ␮M concentrations (Fig. 3d). Additionally, phosphorylation levels of the downstream AMPK substrate ACC were also inhibited by Compound C in a similar dose fashion (Fig. 3e). Furthermore, specific knock down of AMPK expression by using the siRNA directed to AMPK markedly reduced AMPK/ACC phosphorylation (Fig. 4a, b) and inhibited PC12 cell neuritogenesis by almost four-fold in the presence of NGF and ASC-CM (Fig. 4c, d) compared to untreated or non-specific siRNA control treated cells. Therefore, as expected, both Compound C and siRNA of AMPK treatments significantly inhibited ASC-CM-induced PC12 cell neuritogenesis. Taken together, our data demonstrate that AMPK is involved in both NGF- and ASC-CM-induced PC12 cell neuritogenesis. AMPK played a more significant role in NGF- and ASC-CM-induced neuritogenesis compared to other known pathways Both P38 MAPK and PI3Kinase were previously shown to be crucial signaling molecules in PC12 cell neuritogenesis (Yung et al., 2008). Our data confirmed these reports by showing that both P38 MAPK and PI3Kinase inhibitors reduced NGF-induced PC12 cell neuritogenesis (Fig. 5a). However, neuritogenesis was attenuated to a greater degree by inhibition of AMPK with Compound C. Compared to purified NGF, the involvement of AMPK in PC12 cell neuritogenesis induced by ASC-CM is less but still significant, and greater than either P38 MAPK or PI3Kinase (Fig. 5b).

b

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Fig. 2. NGF induced AMP-activated kinase (AMPK) activation in differentiating PC12 cells. PC12 cells were treated with 5 ng/ml NGF in basal media. Protein lysates were prepared at the indicated times and 50 ␮g protein of each lysate was analyzed by Western blot with phosphorylated protein-specific antibodies to AMPK or acetylCoA carboxylase (ACC) and protein-specific antibodies to AMPK or ACC. ␤-Actin bands were used as the loading control. Representative images of visualized protein species are demonstrated in (a). Western blot bands were quantitated by densitometric analysis and presented as ratios of phospho-AMPK/total AMPK or phospho-ACC/total ACC (b, c). Data represent the mean⫾SEM of triplicate wells from single experiments repeated at least three times independently with similar results. (* P⬍0.05, ** P⬍0.01; compared with the time point 0, as analyzed by ANOVA followed by a post-hoc t-test). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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B. Tan et al. / Neuroscience 181 (2011) 40 – 47

b

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Fig. 3. NGF induction of PC12 cell neuritogenesis required AMPK activity. The specific AMPK inhibitor Compound C (CC) attenuated NGF-induced PC12 cell neuritogenesis. Phase contrast representative micrographs (20⫻ magnification) of PC12 cells cultured in basal media alone (a), basal media with NGF (5 ng/ml, b), or basal media with NGF and 10 ␮m CC (c) showed that neurites induced in part of PC12 cells by NGF were almost completely blocked by CC. Differentiated PC12 cells in each group treated with different doses of CC were counted and analyzed (d). Agreed with cell data that showed NGF-induced neuritogenesis was inhibited by different doses of CC, NGF-induced ACC phosphorylation was also inhibited by CC in a dose fashion (e). Data represent the mean⫾SEM of quadruplicate wells from single experiments repeated four times independently with similar results. (* P⬍0.05, ** P⬍0.01; compared with the cultures treated with NGF only, as analyzed by ANOVA followed by a post-hoc t-test). Scale bar, 50 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

DISCUSSION In this study, we investigated whether and how ASC-CM contributes to neuronal differentiation, which may explain

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NGF CsiRNA siRNA

the molecular mechanism underlying induction of endogenous neuronal progenitor or stem cell differentiation when ASC-CM was administered to a neonatal rat HI encephalopathy model. The robust effect of ASC-CM provides

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Phospho-AMPK

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Fig. 4. Knockdown of AMPK expression by siRNA blocked NGF induced PC12 cell neuritogenesis. Western blots showed levels of phosphor-ACC, phosphor-AMPK, total AMPK, and total ACC in cell lysates from PC12 cells treated with AMPK specific siRNA, a scrambled control siRNA (csiRNA), or nothing (NT) for 24 h followed by adding 5 ng/ml NGF to the culture media. AMPK siRNA significantly inhibited phosphorylation of AMPK and ACC by markedly knocking down AMPK expression in cell lysates (a, b). The percentage of PC12 cell neuritogenesis induced by NGF or ASC-CM was also decreased by knockdown of AMPK expression with siRNA (c, d). Data represent the mean⫾SEM of at least triplicate wells from single experiments repeated more than three times independently with similar results. (* P⬍0.05, ns: no statistic significance; compared with the cultures treated with ASC-CM or NGF, as analyzed by ANOVA followed by a post-hoc t-test). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

B. Tan et al. / Neuroscience 181 (2011) 40 – 47 50%

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Fig. 5. The contribution of AMPK to PC12 cell neuritogenesis induced by NGF and ASC-CM was more significant than either P38 MAPK or PI3Kinase. PC12 cells were pre-incubated for 2 h with or without 10 ␮m of either AMPK inhibitor Compound C, P38 MAPK inhibitor SB203580, or PI3Kinase inhibitor LY29842002, and then incubated with either NGF (10 ng/ml) (a) or ASC-CM (b) for 8 d before quantitating the percentage of cells bearing outgrown neuritis. Data represent the mean⫾SEM of triplicate wells from single experiments repeated three times independently with similar results. (* P⬍0.05, ** P⬍0.01; compared with the cultures treated with NGF or ASC-CM, as analyzed by ANOVA followed by a post-hoc t-test). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

indirect evidence for paracrine support being the predominant effect of ASC on injured tissues; although, it does not exclude a possible direct tissue replacement by pluripotent ASC, which have been observed to differentiate into neural and glial cells in vivo and in vitro (Safford et al., 2002; Ashjian et al., 2003). It should be noted that it has not been demonstrated that ASC are capable of homing to specific areas of the brain and differentiating into functional neurons to replace specialized neural cells found in the affected regions. In a Parkinson’s disease model ASC delivered directly to the injured area induced repair and regeneration of depleted dopaminergic neurons. The inability to detect engrafted ASC suggested trophic factor stimulated recovery in a paracrine manner. Given that only a very small fraction of transplanted cells apparently survive in host tissues, it may be difficult to attain sufficient and sustained levels of paracrine factors to stimulate meaning-

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ful levels of recovery in the much larger brains of humans. Administration of whole or fractionated cell-free ASC-CM to injured tissues to stimulate differentiation of endogenous stem and progenitor cells overcomes the multiple issues limiting stem cell therapies such as homing, productive engraftment, directed differentiation and functional replacement and, therefore, may be a more optimal therapy. The discovery that NGF secreted by ASC accumulates to levels capable of inducing neuronal differentiation in vitro could likely explain the reduced lesion volumes observed in the neonatal rat HI encephalopathy model where ASC-CM was administered at 24 h after injury, well after apoptosis peaked. However, other factors not identified are apparently responsible for a considerable fraction of this activity since inactivation of NGF reduced, but did not abolish, neurite outgrowth from PC12 cells. It is possible that the combination of NGF and other inducers of differentiation or cell survival present in ASC-CM may produce a greater effect than each used individually. We are presently testing this hypothesis by first identifying factors in the ASC-CM milieu and then recombining the purified form of these factors to produce a similar effect. Previous work has documented the involvement of multiple signaling pathways in NGF induced PC12 cell neuritogenesis, including those involving P38 MAPK and PI3Kinase. Inhibition of the P38 MAPK pathway suppressed NGF-induced PC12 cell neuritogenesis via G protein coupled receptors (Yung et al., 2008). Additionally, Akt, as a downstream target of PI3Kinase, was activated by NGF and inhibition of Akt activity by expressing dominant negative forms of Akt also suppressed NGF-induced PC12 cell neuritogenesis (Kim et al., 2004). In this study, consistence with these results, we showed that NGF and ASC-CM may have diverse functions in neurite out-growth of PC12 cells through P38 and AKT signaling pathways. The master regulator protein AMPK performs a central function in sensing cellular energy states; accordingly it is activated by a number of pathological stresses, including hypoxia, oxidative stress, glucose deprivation, as well as exercise- and dietary-induced hormones (Hardie, 2003). The proper function of AMPK is critical for neuronal energy metabolism and promoting neuroprotection during energy deprivation (Hawley et al., 2005). Loss of AMPK function leads to progressive neuron degeneration (Tschape et al., 2002). Recently it was reported that AMPK controls mammalian brain development through the retinoblastoma protein (Dasgupta and Milbrandt, 2007); as suggested by our data, AMPK also might play a role in neuronal differentiation. Specifically we demonstrate here that the AMPK signaling pathway is necessary for neurite out-growth of PC12 cells. Inhibition of AMPK activation by Compound C blocks the effects of NGF-induced neuritogenesisto a greater extent than that of ASC-CM. We speculate that the function of AMPK is less, although still significant, in ACMCM-induced PC12 cell neuritogenesis due to the effects of other undefined neuritogenic factors contained in this complex mixture. One major outcome of the finding in present study is that NGF and ASC-CM activate cellular AMPK and cause

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neuronal differentiation which may provide a novel therapeutic modality for treatment of a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and stroke.

REFERENCES Ashjian PH, Elbarbary AS, Edmonds B, DeUgarte D, Zhu M, Zuk PA, Lorenz HP, Benhaim P, Hedrick MH (2003) In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast Reconstr Surg 111:1922–1931. Bergeron R, Ren JM, Cadman KS, Moore IK, Perret P, Pypaert M, Young LH, Semenkovich CF, Shulman GI (2001) Chronic activation of AMP kinase results in NRF-1 activation and mitochondrial biogenesis. Am J Physiol Endocrinol Metab 281:E1340 –E1346. Bochev I, Elmadjian G, Kyurkchiev D, Tzvetanov L, Altankova I, Tivchev P, Kyurkchiev S (2008) Mesenchymal stem cells from human bone marrow or adipose tissue differently modulate mitogen-stimulated B-cell immunoglobulin production in vitro. Cell Biol Int 32:384 –393. Byrd JC, Alho H (1987) Differentiation of PC12 pheochromocytoma cells by sodium butyrate. Brain Res 428:151–155. Cai L, Johnstone BH, Cook TG, Tan J, Fishbein MC, Chen PS, March KL (2009) IFATS collection: human adipose tissue-derived stem cells induce angiogenesis and nerve sprouting following myocardial infarction, in conjunction with potent preservation of cardiac function. Stem Cells 27:230 –237. Culmsee C, Bondada S, Mattson MP (2001) Hippocampal neurons of mice deficient in DNA-dependent protein kinase exhibit increased vulnerability to DNA damage, oxidative stress and excitotoxicity. Brain Res Mol Brain Res 87:257–262. Dasgupta B, Milbrandt J (2007) Resveratrol stimulates AMP kinase activity in neurons. Proc Natl Acad Sci U S A 104:7217–7222. Ebert SN, Lindley SE, Bengoechea TG, Bain D, Wong DL (1997) Adrenergic differentiation potential in PC12 cells: influence of sodium butyrate and dexamethasone. Brain Res Mol Brain Res 47:24 –30. Fujimura J, Ogawa R, Mizuno H, Fukunaga Y, Suzuki H (2005) Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice. Biochem Biophys Res Commun 333:116 –121. Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73:2424 –2428. Hardie DG (2003) Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 144: 5179 –5183. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMPactivated protein kinase. Cell Metab 2:9 –19. Jackson TR, Blader IJ, Hammonds-Odie LP, Burga CR, Cooke F, Hawkins PT, Wolf AG, Heldman KA, Theibert AB (1996) Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase. J Cell Sci 109(2):289 – 300. Kang SK, Lee DH, Bae YC, Kim HK, Baik SY, Jung JS (2003) Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp Neurol 183:355–366. Kang SK, Putnam LA, Ylostalo J, Popescu IR, Dufour J, Belousov A, Bunnell BA (2004) Neurogenesis of rhesus adipose stromal cells. J Cell Sci 117:4289 – 4299. Kim Y, Seger R, Suresh Babu CV, Hwang SY, Yoo YS (2004) A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation. Mol Cells 18:353–359. Kobayashi M, Nagata S, Kita Y, Nakatsu N, Ihara S, Kaibuchi K, Kuroda S, Ui M, Iba H, Konishi H, Kikkawa U, Saitoh I, Fukui Y (1997) Expression of a constitutively active phosphatidylinositol

3-kinase induces process formation in rat PC12 cells. Use of Cre/loxP recombination system. J Biol Chem 272:16089 –16092. Kukidome D, Nishikawa T, Sonoda K, Imoto K, Fujisawa K, Yano M, Motoshima H, Taguchi T, Matsumura T, Araki E (2006) Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells. Diabetes 55:120 –127. Lee ES, Uhm KO, Lee YM, Han M, Lee M, Park JM, Suh PG, Park SH, Kim HS (2007) CAPE (caffeic acid phenethyl ester) stimulates glucose uptake through AMPK (AMP-activated protein kinase) activation in skeletal muscle cells. Biochem Biophys Res Commun 361:854 – 858. Ning H, Lin G, Lue TF, Lin CS (2006) Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. Differentiation 74:510 –518. Peeraully MR, Jenkins JR, Trayhurn P (2004) NGF gene expression and secretion in white adipose tissue: regulation in 3T3-L1 adipocytes by hormones and inflammatory cytokines. Am J Physiol Endocrinol Metab 287:E331–E339. Safford KM, Hicok KC, Safford SD, Halvorsen YD, Wilkison WO, Gimble JM, Rice HE (2002) Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 294:371–379. Safford KM, Safford SD, Gimble JM, Shetty AK, Rice HE (2004) Characterization of neuronal/glial differentiation of murine adiposederived adult stromal cells. Exp Neurol 187:319 –328. Santos SD, Verveer PJ, Bastiaens PI (2007) Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate. Nat Cell Biol 9:324 –330. Tholpady SS, Katz AJ, Ogle RC (2003) Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec A Discov Mol Cell Evol Biol 272:398 – 402. Tschape JA, Hammerschmied C, Muhlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D (2002) The neurodegeneration mutant lochrig interferes with cholesterol homeostasis and Appl processing. EMBO J 21:6367– 6376. Wang CZ, Wang Y, Di A, Magnuson MA, Ye H, Roe MW, Nelson DJ, Bell GI, Philipson LH (2005) 5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways. Biochem Biophys Res Commun 330:1073– 1079. Wei L, Han BH, Li Y, Keogh CL, Holtzman DM, Yu SP (2006) Cell death mechanism and protective effect of erythropoietin after focal ischemia in the whisker-barrel cortex of neonatal rats. J Pharmacol Exp Ther 317:109 –116. Wei X, Du Z, Zhao L, Feng D, Wei G, He Y, Tan J, Lee WH, Hampel H, Dodel R, Johnstone BH, March KL, Farlow MR, Du Y (2009a) IFATS collection: the conditioned media of adipose stromal cells protect against hypoxia-ischemia-induced brain damage in neonatal rats. Stem Cells 27:478 – 488. Wei X, Zhao L, Zhong J, Gu H, Feng D, Johnstone BH, March KL, Farlow MR, Du Y (2009b) Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis. Neurosci Lett 462: 76 –79. Xue B, Kahn BB (2006) AMPK integrates nutrient and hormonal signals to regulate food intake and energy balance through effects in the hypothalamus and peripheral tissues. J Physiol 574:73– 83. Yanez R, Lamana ML, Garcia-Castro J, Colmenero I, Ramirez M, Bueren JA (2006) Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease. Stem Cells 24:2582–2591. Yang LY, Liu XM, Sun B, Hui GZ, Fei J, Guo LH (2004) Adipose tissue-derived stromal cells express neuronal phenotypes. Chin Med J [Engl] 117:425– 429. Yoo KH, Jang IK, Lee MW, Kim HE, Yang MS, Eom Y, Lee JE, Kim YJ, Yang SK, Jung HL, Sung KW, Kim CW, Koo HH (2009)

B. Tan et al. / Neuroscience 181 (2011) 40 – 47 Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol 259:150 –156. Yung LY, Tso PH, Wu EH, Yu JC, Ip NY, Wong YH (2008) Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins. Cell Signal 20:1538 –1544.

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Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002) Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13: 4279 – 4295. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228.

(Accepted 16 February 2011)