Beta-adrenoceptor pathway enhances mitochondrial function in human neural stem cells via rotary cell culture system

Journal of Neuroscience Methods 207 (2012) 130–136

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Basic Neuroscience

Beta-adrenoceptor pathway enhances mitochondrial function in human neural stem cells via rotary cell culture system Ming-Chang Chiang a,∗ , Heng Lin b , Yi-Chuan Cheng c , Chia-Hui Yen d , Rong-Nan Huang e , Kuan-Hung Lin f a

Department of Life Science, Fu Jen Catholic University, New Taipei City 242, Taiwan Institute of Physiology, Taipei Medical University, Taipei 110, Taiwan c Department of Biochemistry and Molecular Biology, Chang Gung University, Tao Yuan 333, Taiwan d Department of International Business, Ming Chuan University, Taipei 111, Taiwan e Department of Entomology and Research Center for Plant-Medicine, National Taiwan University, Taipei 106, Taiwan f Graduate Institute of Biotechnology, Chinese Culture University, Taipei 111, Taiwan b

a r t i c l e

i n f o

Article history: Received 18 January 2012 Received in revised form 4 April 2012 Accepted 5 April 2012 Keywords: Rotary cell culture system Human neural stem cells PGC1␣ Mitochondrial function

a b s t r a c t The structure and function of the human nervous system are altered in space when compared with their state on earth. To investigate directly the influence of simulated microgravity conditions which may be beneficial for cultivation and proliferation of human neural stem cells (hNSCs), the rotary cell culture system (RCCS) developed at the National Aeronautics and Space Administration (NASA) was used. RCCS allows the creation of a unique microgravity environment of low shear force, high-mass transfer and enables three-dimensional (3D) cell culture of dissimilar cell types. The results show that simulated microgravity using an RCCS would induce ␤-adrenoceptor, upregulate cAMP formation and activate both PKA and CREB (cAMP response element binding protein) pathways. The expression of intracellular mitochondrial genes, including PGC1␣ (PPAR coactivator 1␣), nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam), regulated by CREB, were all significantly increased at 72 h after the onset of microgravity. Accordingly and importantly, the ATP level and amount of mitochondrial mass were also increased. These results suggest that exposure to simulated microgravity using an RCCS would induce cellular proliferation in hNSCs via an increased mitochondrial function. In addition, the RCCS bioreactor would support hNSCs growth, which may have the potential for cell replacement therapy in neurological disorders. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Many neurological conditions, such as stroke, Parkinson’s disease and Huntington’s disease, cause severe functional deficits, and the currently available cell therapy has become a hopeful restorative treatment choice that promotes anatomical reconstruction and functional recovery in animal models of neurological disorders (Lindvall and Kokaia, 2006; Roberts et al., 2006). Inevitably, neural stem cells (NSCs) have become a subject of intensive investigation in translational research for common degenerative diseases. In fact, an important goal is to achieve neuroregeneration by transplantation of exogenous cells via cell-mediated therapy. Most self-renewal is dependent on NSCs that can be isolated from embryonic brains. NSCs are multipotent and self-renewal progenitor cells that can be differentiated into neurons, oliogdendrocytes, and

∗ Corresponding author. Tel.: +886 2 2905 2467; fax: +886 2 2905 2193. E-mail address: [email protected] (M.-C. Chiang). 0165-0270/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneumeth.2012.04.005

astrocytes (Cai et al., 2006; Reh, 2002; Song et al., 2002; Temple, 2001). Thus, NSCs have been implicated in processes which lead to neural regeneration following neurodegenerative diseases and neurological disorders, raising the possibility that brain injury might be relieved (Cameron and McKay, 2001; Hallbergson et al., 2003; Kruger et al., 2002; Lie et al., 2004). However, the source of NSCs is very limited due to ethical concerns and the restricted number of embryos available. The small number of adult NSCs identified thus needs to have a comprehensive device or protocol to amplify the NSCs population in vitro. In this study, we examined the inductive effect of microgravity environments (in vitro 3D models) in hNSCs by a RCCS bioreactor (Schwarz et al., 1992). The RCCS, developed by NASA at the Johnson Space Center, with adjustable rotational speed, permits the growth of cells and tissues suspended in culture fluid without colliding with the vessel walls. The continuous rotation alters the cell’s comprehension of gravitational direction, generating an average microgravity of 1.0−2 × g (Chen et al., 2007; Cummings and Waters, 2007; Waters et al., 2006). Therefore, RCCS is an effective

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tool to produce simulated microgravity conditions which may be beneficial for cultivation and proliferation (Dabos et al., 2001; Di Loreto et al., 2006). Moreover, 3D models more closely mimic in vivo conditions, allowing the expression of many views of normal cell behaviors (Fournier and Martin, 2006), including more normal cellular morphology and structure (Kleinman et al., 1987; Stoker et al., 1990). The investigation of in vitro 3D models has contributed to our knowledge of basic cellular responses involved in hNSCs’ function. The RCCS creating simulated microgravity culture conditions was conducive for maintaining cultures of functional hNSCs in the study. Analysis of gene and protein expression is an important tool in biological research into microgravity, since any changes in the physiology of an organism or a cell are accompanied by changes in the pattern of gene and protein expressions. Our results demonstrate that microgravity induces the cellular proliferation of hNSCs via the upregulation of mitochondrial function, and the effects of microgravity on cellular proliferation would further link to hNSCs in the nervous system.

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was performed using a TaqMan kit (PE Applied Biosystems, Foster City, CA, USA) on a StepOne quantitative PCR machine (PE Applied Biosystems) using heat-activated TaqDNA polymerase (Amplitaq Gold; PE Applied Biosystems). The sequences of primers were as follows: ␤-adrenoceptor (5 -GTCTCCTTCTACGTTCC3 and 5 -AAGAAGGGCATCCAGCAGAG-3 ), PKA (5 -AGGCGACGAGGTGCTATC-3 and 5 -AAGCGGCCATTGTCTTGT-3 ), CREB (5 -CCAAGCTTATGGATCCTCCTGGAGAGAAGATGG-3 and 5 GCCTCGAGA AGCACATTGACGCTCCTGAC-3 ), PGC1␣ (5 -TGAGAGGGCCAAGCAAAG-3 and 5 -ATAAATCACACGGCGCTCTT-3 ), NRF1 (5 -CCATCTGGTGGCCTGAAG-3 and 5 -GTGCCTGGGTCCATGAAA3 ), NRF2 (5 -CAAGAACGCCTTGGGATACC-3 and 5 -AAACCACCCAATGCAGGACTT-3 ), Tfam (5 -GAACAACTACCCATATTTAAAGCTCA-3 and 5 -GAATCAGGAAGTTCCCTCCA-3 ), and GAPDH (5 -TGCACCACCAACTGCTTAGC-3 and 5 -GGCATGGACTGTGGTCATGAG-3 ). Independent reverse-transcription PCRs were performed as described previously (Chiang et al., 2011). The relative transcript amount of the target gene, calculated for standard curves of serial RNA dilutions, was normalized to that of GAPDH of the same RNA sample.

2. Materials and methods 2.1. Cell culture GIBCO® human neural stem cells (H9 hESC-derived) were originally obtained from NIH approved H9 (WA09) human embryonic stem cells (hESCs). The medium Complete StemPro® NSC SFM (serum free medium) was used for optimal growth and expansion of GIBCO® hNSCs, as well as keeping the NSCs undifferentiated. StemPro® NSC SFM complete medium consists of KnockOutTM DMEM/F-12 with 2% StemPro® Neural Supplement, 20 ng/mL of EGF, 20 ng/mL of bFGF, and 2 mM of GlutaMAXTM -I. 2.2. RCCS The NASA-designed RCCS was purchased from Synthecon (Houston, TX, USA). This model of RCCS was designed to grow cell cultures into a 3D condition. The RCCS contains a gas exchange membrane and provides a low shear stress, bubble free microenvironment for growing cells into a 3D tissue-like condition, which mimics the structure and function of the parent tissues. The rotator bases were placed inside a humidified, 37 ◦ C, 5% CO2 incubator and connected to power supplies set up externally to the incubator. hNSCs were seeded onto 50 mg microcarrier beads (Sigma, St Louis, MO, USA) and incubated with StemPro® NSC SFM complete medium, pretreated according to the manufacturer’s instructions. hNSCs (at a density of 5 × 105 cells) were placed into disposable 4 mL RCCS cassettes after the hNSCs adherence to the microcarrier, with a rotation speed of 15 rpm for 72 h. Fresh medium was supplemented daily. A set of hNSCs with microcarrier beads were placed in the incubator in a standard 6-well plate to serve as static control cultures (CON) for 72 h without rotation. When harvesting cells, the microcarriers were allowed to settle, and the culture medium was removed, followed by washing for 5 min in 5 mL of PBS (pH 7.6). The PBS was then removed and replaced by 5 mL of 0.5% trypsin/EDTA and incubated at 37 ◦ C. After 10 min, the action of the trypsin was stopped by addition of StemPro® NSC SFM complete medium. The separated hNSCs were separated from the microcarriers by filtering through a 100 ␮m filter. The hNSCs’ experiments were collected from the CON and RCCS culture conditions. 2.3. RNA isolation and quantitative real-time polymerase chain reaction (PCR) Total RNA was isolated and reverse-transcribed as detailed elsewhere (Chiang et al., 2011). A real-time quantitative PCR

2.4. Western blot assays Equal amounts of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels according to the method of Laemmli (1970). The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) for Western blot analyses as reported elsewhere (Chiang et al., 2012). Primary antibodies of ␤-adrenoceptor (1:1000; Abcam, Cambridge, MA, USA), PKA (1:2000; Santa Cruz Biotechnology, CA, USA), CREB (1:2000; UpState, Charlottesville, VA, USA), phosphorylated CREB (1:1000; UpState), PGC1␣ (1:2000; Cell Signaling Technology, Beverly, MA, USA), phosphorylated PGC1␣ (1:1000; Cell Signaling Technology), NRF1 (1:2000; Santa Cruz Biotechnology), NRF2 (1:2000; Santa Cruz Biotechnology), Tfam (1:2000; Abcam), nestin (1:2000; Santa Cruz Biotechnology), SOX2 (1:2000; Santa Cruz Biotechnology), and actin (1:3000; Chemicon International, Temecula, CA, USA) were utilized as recommended by the mentioned corresponding manufacturers. 2.5. cAMP assay The intracellular cAMP content was prepared as described previously (Chiang et al., 2009). hNSCs were washed twice with Ca2+ -free Locke’s solution (150 mM of NaCl, 5.6 mM of KCl, 5 mM of glucose, 1 mM of MgCl2 and 10 mM of Hepes; pH 7.4) containing 0.5 mM of isobutylmethylxanthine (Sigma) and then treated with the indicated reagent(s) for 20 min at 25 ◦ C. The cAMP content was assayed using the cAMP assay system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). 2.6. Measurement of intracellular ATP concentration To determine the ATP levels, hNSCs were collected in a lysis buffer (0.1 M Tris, 0.04 M EDTA, pH 7.2) and boiled for 3 min. Samples were then centrifuged (112 × g for 5 min), and the supernatants were used for the luciferin/luciferase assay. The ATP levels were normalized to the protein content in the samples. Protein concentrations were determined by the Bradford analysis, and used to calculate protein content in the number of samples used for the ATP assay (Promega, Madison, WI, USA). The reaction buffer for this assay contained 60 ␮M of luciferin, 0.06 ␮g/mL of luciferase, 0.01 M of magnesium acetate, 0.05% bovine serum albumin, and 0.2 M of Tris (pH 7.5).

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Fig. 1. RCCS elevated the expression of ␤-adrenoceptor and cAMP in the hNSCs. (A) Normal gravity control hNSCs (CON) were cultured on microcarriers without rotation for 72 h. In contrast, microgravity hNSCs (RCCS) were maintained on microcarriers with rotation for 72 h. (B) Total RNA was collected and used for the Q-PCR (quantitative real-time PCR) analysis. The expression level of the ␤-adrenoceptor was normalized to that of the GAPDH. (C) Lysates (20 ␮g) were collected from the indicated condition and subjected to Western blot analysis. Levels of the ␤-adrenoceptor protein were normalized to the corresponding internal control (actin), and the normalized level for the control sample was set to 1.0 for allowing quantitative comparison of the sample from RCCS. The resulting values were shown under the protein analyzed. (D) Assay of cAMP in hNSCs. Data are expressed as mean ± SEM from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

2.7. Mitochondrial mass The fluorescent probe Mitotracker GreenTM dye (MitoGreen, Invitrogen, Carlsbad, CA, USA) binds the mitochondrial membrane lipids regardless of mitochondrial membrane potential or oxidant status (Chiang et al., 2012). To determine the mass of mitochondria, cells were loaded with 0.2 (M/mL of Mitotracker GreenTM dye in the medium for 30 min at 37 ◦ C. Fluorescence measurements were made with excitation at 490 nm and emission at 516 nm using an Axiovert 200 live cell observation system (Carl Ziess Inc., Germany). Amounts were determined by comparing the means of the fluorescent signals.

paraformaldehyde, 4% sucrose in PBS for 30 min at room temperature, followed by permeabilizing with 0.05% Nonidet P-40 in PBS at room temperature three times for 20 min each time. After a 1h blocking in 2% PBS, bovine serum albumin and 2% normal goat serum, cells were stained with the desired primary antibody reconstituted in PBS and 2% goat serum at 4 ◦ C for 14–16 h. Dilutions of the anti-SOX2 antibody (Santa Cruz Biotechnology) and anti-MAP2 antibody (Chemicon International, Temecula, CA) were 1:500. After extensive washing, the slides were incubated with Avidin-Alexa Fluor® 488 (green) and 568 (red)-conjugated secondary antibodies (1:500; Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature, followed by washing and analysis with the aid of a laser confocal microscope.

2.8. Evaluation of cell growth 2.10. Statistical analysis Cell viability was assayed by MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) absorbance and cell count. After synchronized hNSCs were treated with vehicle control or RCCS for 3 days, MTT solution (Sigma) was added to the culture medium and the cells were incubated. The absorbance at 570 nm was measured in solubilized cells using an Epoch plate reader (BioTek). The cell viability rate was expressed as a percentage of values obtained in vehicle control. Moreover, the number of cells was directly counted by a 0.4% solution of trypan blue (Life Technologies Corporation). Cell growth was calculated as the number of viable cells divided by the total number of cells within the grids on the microscope. The following procedure enabled the determination of the success rate of cell viability. 2.9. Immunofluorescence The hNSCs were treated with vehicle control or RCCS for 3 days. The cells were then grown on glass coverslips and fixed with 4%

All data were expressed as means ± SEM. To establish significance, data were subjected to unpaired one-way ANOVA using the Sigma Stat 3.5 software statistical package (Systat SigmaStat V3.5.0.54 Software; San Jose, CA, USA). The criterion for significance was set at p < 0.001. Differences between groups were assessed with Student’s t tests or one-way analysis of variance (one-way ANOVA) as indicated. The significance level was set at 0.001. 3. Results 3.1. RCCS elevated ˇ-adrenoceptor and its pathway expression in the hNSCs In the present study, hNSCs were maintained in RCCS bioreactors for up to 3 days. This model was generated by culturing hNSCs in the RCCS, a unique culture environment (Fig. 1A). Since activation of neurogenic precursors and stem cells via ␤-adrenoceptor could

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Fig. 2. RCCS increased the expression of PKA and CREB in the hNSCs. (A) The PKA and CREB transcripts in the indicated hNSCs were analyzed using the Q-PCR technique. RNA of the indicated hNSCs was collected and reverse-transcribed into cDNA. Q-PCR technique of the indicated gene was performed and normalized to that of GAPDH. (B) Lysates (20 ␮g) were collected from the indicated condition and subjected to Western blot analysis. Quantification of the levels of PKA, CREB and p-CREB was similar to that described in Fig. 1C. Results in A and B are presented as mean ± SEM from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

be a potent mechanism to increase neuronal production (Jhaveri et al., 2010), whether RCCS directly elevated the ␤-adrenoceptor expression in the hNSCs were tested accordingly. As shown in Fig. 1B and C, both transcript and protein levels of ␤-adrenoceptor were elevated by RCCS. To assess the signal transduction mediating the action of the ␤-adrenoceptor, the levels of cAMP were then assayed, and the result shows that RCCS elevated cellular cAMP level (Fig. 1D). Because stimulation of the ␤-adrenoceptor has been shown to increase cAMP in hNSCs, examinations whether the cAMP/PKA/CREB pathway (Schenk and Snaar-Jagalska, 1999) playing an important role in the RCCS effect was conducted. Indeed, the transcript and protein levels of PKA and CREB in the hNSCs were effectively enhanced by RCCS (Fig. 2A and B). Mayr and Montminy (2001) reported that one of the major mechanisms which CREB is activated by phosphorylation at a key conserved serine residue, Ser133, allowing interaction with the transcriptional coactivators (Mayr and Montminy, 2001). We found that the amounts of activated CREB, determined by assessing the phosphorylation level at Ser133, were increased in hNSCs by RCCS (Fig. 2B).

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Fig. 3. RCCS enhanced the expression of PGC1␣ in the hNSCs. (A)The PGC-1␣ transcripts in the indicated hNSCs were analyzed using the Q-PCR technique. RNA of the indicated hNSCs was collected and reverse-transcribed into cDNA. Q-PCR technique of the indicated gene was performed and normalized to that of GAPDH. (B) Lysates (20 ␮g) were collected from the indicated condition and subjected to Western blot analysis. Quantification of the levels of PGC1␣ and p-PGC1␣ protein was similar to that of Fig. 1C. Results in A and B are presented as mean ± SEM from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

notion, the transcript and protein levels of PGC1␣ in the hNSCs were effectively enhanced by RCCS (Fig. 3A and B). Stimulation of PKA may directly phosphorylate the PGC1␣ protein, resulting in its activation and stabilization (Puigserver and Spiegelman, 2003). Importantly, the amount of activated PGC1␣, determined by measuring the phosphorylation level, was increased in hNSCs by RCCS (Fig. 3B). PGC1␣ has also been implicated in mitochondrial biogenesis by modulating a number of genes such as NRF1, NRF2 and Tfam (McGill and Beal, 2006; Puigserver and Spiegelman, 2003). These transcript and protein levels in the hNSCs were also effectively enhanced by RCCS (Fig. 4A and B). 3.3. RCCS enhanced ATP level and mitochondrial mass in the hNSCs The ATP level was assayed for analyzing the consequence of mitochondrial biogenesis, and found that the ATP level in the hNSCs was increased by RCCS (Fig. 5A). Therefore, whether RCCS mediated mitochondrial biogenesis in the hNSCs was evaluated. The mitochondrial activity assay using MitoGreen has been used to determine mitochondrial mass (Quintanilla et al., 2008; WilsonFritch et al., 2003). As shown in Fig. 5B and C, the mitochondrial mass in RCCS was markedly higher than that observed under the control (CON) conditions. 3.4. RCCS increased hNSCs viability

3.2. Enhancement of the expression of PGC1˛ and mitochondrial genes in the hNSCs by RCCS PKA phosphorylates CREB transcription factor, which is involved in the induction of PGC1␣ gene expression (Handschin et al., 2003; Herzig et al., 2003; Karamitri et al., 2009). Consistent with this

To evaluate the proliferation potential and viability of the hNSCs treated in RCCS, MTT assay and cell counting were conducted. As shown in Fig. 6A, the RCCS environment significantly promoted the proliferation potential of hNSCs when compared with cells in CON condition throughout the experiment in MTT assay. In addition,

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Fig. 6. RCCS environment statistically promoted the hNSCs’ viability. (A) hNSCs’ viability was expressed as a percentage of the MTT activity measured in the CON group. (B) Lysates (20 ␮g) were collected from the indicated condition, and subjected to a Western blot analysis. Quantification of the levels of nestin and Sox2 proteins was similar to that of Fig. 1C. Data are expressed as mean ± SEM from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

Fig. 4. Improvement in the expression of mitochondrial genes in the hNSCs with RCCS. (A) The NRF1, NRF2 and Tfam transcripts in the indicated hNSCs were analyzed using the Q-PCR technique. RNA of the indicated hNSCs was collected and reversetranscribed into cDNA. Q-PCR technique of the indicated gene was performed and normalized to that of GAPDH. (B) Lysates (20 ␮g) were collected from the indicated condition and subjected to Western blot analysis. Quantification of the levels of the NRF1, NRF2 and Tfam proteins was similar to that of Fig. 1C. Results in A and B are presented as mean ± SEM values from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

hNSCs in the RCCS environment had a higher number of cells than cells in CON condition throughout the experiment in cell counting analysis (Fig. S1). Furthermore, RCCS treated cells showed an increase in the success rate of hNSCs proliferation potential and viability. More importantly, cells grown on the RCCS bioreactor showed a normal amount of neural stem cell-type specific markers nestin and SOX2 by Western blot analysis (Fig. 6B). The high

Fig. 5. RCCS enhanced ATP level and mitochondrial mass in the hNSCs. (A) Lysates harvested from the indicated condition were subjected to ATP assay. (B) hNSCs were collected to determine the level of mitochondrial mass using Mitotracker GreenTM dye (green). Scale bar: 100 ␮m. (C) The levels of mitochondrial mass were normalized to cell numbers. Data are expressed as mean ± SEM from three independent experiments. a Specific comparison to CON environment (p < 0.001; one-way ANOVA).

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magnification images of hNSCs were treated with vehicle control or RCCS for 3 days, after cells were stained with SOX2 (green) and neuronal marker (MAP2, red) by immunostaining (Fig. S2). The proliferative capacity, combined with the neuronal mitochondrial function of hNSCs in 3D culture, suggests their potential use in attempts to promote neuronal regeneration in vivo.

4. Discussion Microgravity is a physical force that alters the function of cells and whole organisms (Hahn et al., 2008; Norsk, 2005; VunjakNovakovic et al., 2002). Recent studies suggest that microgravity affects cellular functions such as proliferation (Dai et al., 2007), signal transduction (Vincent et al., 2005), and gene expression (Clement et al., 2008; Nistor et al., 2010). A gene chip microarray analysis revealed microgravity-induced changes in the expression of genes belonging to various functional categories, including those directly related to immune response, cell proliferation and differentiation, protein folding, transport and degradation, as well as apoptosis (Ward et al., 2006). Nevertheless, the molecular mechanism of microgravity on hNSCs is still not well understood. In the present study, an RCCS bioreactor elevated ␤-adrenoceptor and its pathway (e.g. cAMP/PKA/CREB) expression in the hNSCs (Figs. 1 and 2) were identified. Our study further demonstrated that RCCS enhancing PGC1␣ and mitochondrial gene expression may contribute to the elevated ATP levels and mitochondrial mass in the hNSCs (Figs. 3–5). In the study, RCCS was used to create an in vitro environment that included biochemical signals (e.g. cAMP and ATP), biologically derived genes and mitochondrial expression that triggered hNSCs proliferation (Fig. 6). How ␤-adrenoceptors mediate activation and proliferation of hNSCs is currently unknown. However, given that ␤-adrenoceptors are seven transmembrane receptors coupled to heterotrimeric Gproteins that signal via multiple intracellular pathways, including activation of adenylyl cyclase and cAMP-dependent phosphorylation (Ursino et al., 2009), and that increase in the intracellular level of cAMP regulates the proliferation of hippocampal precursors in vivo (Nakagawa et al., 2002). It is possible that ␤-adrenoceptor driven activation of neural precursors may also use this cAMP mediated signaling mechanism. In addition to target genes that are directly neuroprotective, CREB-induced transcription factors or cofactors may also contribute to neuron survival by regulating downstream gene batteries controlled by elevated cAMP levels in a transcription factor cascade initiated by activated CREB. Nakagawa et al. (2002) demonstrated that cAMP plays a role in hippocampal neural progenitor cells proliferation in vivo (Nakagawa et al., 2002). Moreover, one study showed that activated CREB, in the form of pCREB is constitutively expressed in neurogenic cells of the embryonic mouse brain (Mantamadiotis et al., 2002). Indeed, CREB synthesis and phosphorylation act as a potent survival signal to neurons (Walton and Dragunow, 2000). In the present study, stimulation of ␤-adrenoceptors could activate a cAMP/PKA/CREB pathway (Figs. 1 and 2), alter the expression of genes and regulate the proliferative events in hNSCs. The master transcription factor CREB is known to be an important regulator of PGC1␣ transcription (St-Pierre et al., 2006). PGC1␣ activates the expression of the subunits of the respiratory chain and Tfam through the expression of NRFs and the coactivation of NRF1mediated transcription. Tfam subsequently translocates into the mitochondria and directly increases the transcription and replication of mitochondrial DNA (Puigserver and Spiegelman, 2003). Thus, PGC1␣ and its functions provide a plausible molecular basis for the connection between environmental/hormonal stimuli and mitochondrial biogenesis and respiration when the organism has an altered energy or neurogenesis requirement. Our results show

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that upregulation of PGC1␣ (Fig. 3) led to a subsequent increase in mitochondrial genes (Fig. 4) and function (e.g. ATP and mitochondrial mass, Fig. 5), and probably rescued a wide variety of mechanisms that suffered from energy demand in hNSCs. Recently, the microgravity mediated proliferation and differentiation of neuronal cells have become the topic of considerable investigation (Di Loreto et al., 2006; Lin et al., 2004; Wang and Good, 2001). Our suggestion is that the RCCS environment could promote the proliferation of hNSCs (Fig. 6). This information should be helpful for the development of bioreactors to regulate the proliferation of NSCs and their prospective manipulation to replace the lost or dysfunctional neurons following trauma or disease. 5. Conclusions The hNSCs culture in RCCS show two major advantages, improving mitochondrial genes and function, and promoting the growth of hNSCs. The importance of CREB and PGC1␣ in the regulation of target genes was involved in the mitochondrial biogenesis and growth of hNSCs. Although the exploration of hNSCs using RCCS for neuroregenerative purposes is still a long-term goal, the present results provide fundamental information in hNSCs’ biology and plasticity. Acknowledgements We thank Chia-Nan, Yen for proofreading and editing the manuscript. This work was supported by grants from the National Science Council (NSC97-2320-B-034-001-MY2 and NSC99-2320-B030-008-MY3). Appendix A. Supplementary data Supplementary data associated with cle can be found, in the online http://dx.doi.org/10.1016/j.jneumeth.2012.04.005

this artiversion, at

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