Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction

Author’s Accepted Manuscript Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction Ming-Chang Chiang, Yi-Chuan Cheng, ShiangJiuun Chen, Chia-Hui Yen, Rong-Nan Huang www.elsevier.com/locate/yexcr

PII: DOI: Reference:

S0014-4827(16)30242-7 http://dx.doi.org/10.1016/j.yexcr.2016.08.013 YEXCR10316

To appear in: Experimental Cell Research Received date: 26 May 2016 Revised date: 15 August 2016 Accepted date: 17 August 2016 Cite this article as: Ming-Chang Chiang, Yi-Chuan Cheng, Shiang-Jiuun Chen, Chia-Hui Yen and Rong-Nan Huang, Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-betainduced mitochondrial dysfunction, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2016.08.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction

Ming-Chang Chianga*, Yi-Chuan Chengb, Shiang-Jiuun Chenc, Chia-Hui Yend, Rong-Nan Huange

a

Department of Life Science, College of Science and Engineering, Fu Jen Catholic

University, New Taipei City 242, Taiwan b

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung

University, Taoyuan, Taiwan c

Department of Life Science and Institute of Ecology and Evolutionary Biology,

College of Life Science, National Taiwan University, Taipei 106, 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

*

Corresponding author: Ming-Chang Chiang, Department of Life Science, College of

Science and Engineering, Fu Jen Catholic, University, New Taipei City 242, Taiwan, Tel: 886-2-29052467, fax: 886-2-29052193, E-mail: [email protected]

Abstract Alzheimer's disease (AD) is the general consequence of dementia and is diagnostic neuropathology by the cumulation of amyloid-beta (A) protein aggregates, which are thought to promote mitochondrial dysfunction processes leading to neurodegeneration. 1

AMP-activated protein kinase (AMPK), a critical regulator of energy homeostasis and a major player in lipid and glucose metabolism, is potentially implied in the mitochondrial deficiency of AD. Metformin, one of the widespread used antimetabolic disease drugs, use its actions in part by stimulation of AMPK. While the mechanisms of AD are well established, the neuronal roles for AMPK in AD are still not well understood. In the present study, human neural stem cells (hNSCs) exposed to A had significantly reduced cell viability, which correlated with decreased AMPK, neuroprotective genes (Bcl-2 and CREB) and mitochondria associated genes (PGC1, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3/9 activity and cytosolic cytochrome c. Co-treatment with metformin distinct abolished the A-caused actions in hNSCs. Metformin also signficantly rescued hNSCs from A-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Importantly, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology in the hNSCs with A. These findings extend our understanding of the central role of AMPK in Aβ-related neuronal impairment. Thus, a better understanding of AMPK might assist in both the recognition of its critical effects and the implementation of new therapeutic strategies in the treatment of AD.

Keywords: AMPK, PGC1, mitochondrial function, hNSCs, A 1. Introduction

Alzheimer's disease (AD) is a serious neurodegenerative disorder of brain in the aged population. AD is the progressive neurodegenerative disease of aging and the general form of dementia [1, 2]. AD causes difficulty for patients, containing loss of 2

independence, emotional suffering, cognitive impairment such as memory loss, and changes in behavioral symptoms [3, 4]. AD is correlated with numerous cellular alter in the brain, including the loss of synaptic structures, mitochondrial function, oxidative stress, inflammation, amyloid beta (Aβ) deposits, and neurofibrillary tangles [5-14]. The gross pathology of AD described by A peptide deposition into cerebral amyloid plaques [15]. A peptides have a major role in the pathogenesis of the AD brains and illustrate the lesions invariably found in the hippocampus and cortex, and the subsequent neurodegeneration that follows [3, 15-19]. Aggregates of A progress in extracellular plaques and are correlated with neurodegeneration in AD [20-22]. Several reports have indicated that Aβ is accountable for damaging synapses and mitochondria in neurons influenced by AD [23-25]. Aβ have been exhibited in mitochondrial membranes and to interact with mitochondrial proteins, change mitochondrial enzymes, disorganize electron transport chain, suppress ATP task, elicit free radical damage, and impair mitochondrial biogenesis [23-27].

AMP-activated protein kinase (AMPK) is a Ser/Thr kinase which shows a crucial role in the maintenance of energy balance at both the cellular and whole-body, senses degrees of adenosine triphosphate (ATP) [28-32]. When ATP levels decrease, AMPK is stimulated to provide the cells to modulate to the metabolic modify in the cell [33-35]. As a cellular energy sensor responding to low ATP levels, AMPK regulates several intracellular systems including the lipid metabolism, cellular glucose uptake by glucose transporter 4 (GLUT4), and mitochondria biogenesis [30, 36-41]. AMPK is activated in response by consume cellular ATP, including low glucose, nutrient deprivation, oxidative stress, and hypoxia in effect to an increase in the AMP/ATP ratio [42-45]. Raised Ca2+ levels and cytoplasmic AMP are the critical activators of neuronal AMPK signaling [46]. 3

Research studies demonstrate that the AMPK are constantly showed in neurons and thus can be capability activated [46]. AMPK has demonstrated as a major sensor of cellular energy balance, and is potentially involved in a wide range of states, including AD [47, 48]. AMPK action has been shown to reduce with age, which may provide to decreased mitochondrial function with aging-related diabetes and AD [49-54]. Diabetes major raise cognitive decline, indicating the association that cellular mechanisms of energy homeostasis are connect to AD pathogenesis [55]. Therefore, AMPK is a potential target to ameliorate disturbed brain energy biogenesis that is activated, might alter AD pathology and is a potential therapeutic target for AD [48, 56-59]. Through activation of AMPK explained that it inhibits A accumulation, meliorates mitochondrial dysfunction and suppress oxidative stress, and plays a beneficial effect in mouse brain [60]. Several studies exhibit that AMPK is a neuroprotective element against metabolic stress, involving a critical role in the prevention of AD pathogenesis [61-63]. Therefore, further studies required to explain the function of AMPK in either pathogenesis or treatment of AD.

2. Materials and methods 2.1. Cell culture GIBCO® human neural stem cells (hNSCs) were originally obtained from National Institutes of Health (NIH) approved H9 (WA09) human embryonic stem cells. Complete StemPro NSC serum free medium (SFM) was used for optimal growth and expansion of GIBCO hNSCs, and kept the hNSCs undifferentiated as

4

described previously [64]. Complete StemPro NSC SFM consists of KnockOut™ D-MEM/F-12 with 2% StemPro Neural Supplement, 20 ng/mL of EGF, 20 ng/mL of bFGF, and 2 mM of GlutaMAX™-I.

2.2. Evaluation of cell growth Cell viability was assayed by MTT (3 -[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) absorbance and cell count as reported elsewhere [64]. Synchronized hNSCs were treated as described with or without A1-42 synthetic peptide (Millipore Billerica, MA, USA; 5 M), AMPK agonist metformin (1 mM) or AMPK pharmacological inhibitor Compound C (10 M) (both from Cayman Chemicals, Ann Arbor, MI, USA) for 3 days. MTT solution (Sigma, Austin, TX, USA) was added to the culture medium for incubating the cells, and absorbance at 570 nm was measured in solubilized cells using an EZ Read 400 ELISA Reader (Biochrom, Holliston, USA). The cell growth rate was expressed as a percentage of values obtained in vehicle control.

2.3. Caspase activity assay Caspase activity assay was carried out using caspase-3-like (DEVD-AFC) and caspase-9-like (LEHD-FMK) Fluorometric Protease Assay Kit (Chemicon, Michigan USA) as described previously [65]. In brief, hemisected fresh cells were homogenized in the lysis buffer for 10 min. The cellular lysate (standardized to protein concentration) was incubated with an equal volume of 2  reaction buffer (with 0.01 M of dithiothreitol) for an additional 1 h at 37 °C with caspase-3 substrates (DEVD-AFC) or caspase-9 (LEHD-FMK) at a final concentration of 50 M. 5

Fluorescence was measured by a microplate reader with an excitation filter of 390 ± 22 nm and an emission filter of 510 ± 10 nm.

2.4. Live/Dead Cell Viability Assays Cell toxicity was detected by measuring the fluorescence-based LIVE/DEAD® assays (Thermo Fisher Scientific Inc) according to the manufacturer’s instructions. Briefly, hNSCs were collected following treatments and stained using a two-color assay for microphotographs of live (calcein-AM, green) or dead (ethidium homodimer-1, red) cells.

2.5. Western blot assays Equal amounts of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels. The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) for Western blot analyses as reported elsewhere [66]. Primary antibodies of cytochrome c (1:1000; GeneTex, Inc, Irvine, CA), Voltage-Dependent Anion Channel (1:2000; GeneTex), AMPK (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA), PGC1 (1:2000; GeneTex), and actin (1:3000; GeneTex) were utilized as manufacturer’s instructions. 2.6. Measurements of cytochrome c release For measurement of cytochrome c release, mitochondria and cytosolic fractions were prepared from hNSCs as described previously [65]. Briefly, cells were washed 6

with cold PBS and resuspended in ice-cold buffer (10 mM Hepes, 1 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 1 mM Na3VO4, 10 mM NaF, 1 M okadaic acid, 0.5% Nonidet P-40 and protease inhibitors-complete cocktail). After 30 min of incubation on ice, cells were homogenized (Dounce, 20 strokes) in buffer. Homogenates were then centrifuged at 650 g for 5 min at 4 °C, and the supernatants were re-centrifuged at 9,000 g for 30 min at 4 °C to collect the mitochondrial fraction. The supernatants were again centrifuged at 95,000 g for 1h at 4 °C, and the final supernatant was used as a cytosolic fraction. Cytochrome c levels in the mitochondrial and cytosolic fraction were determined by western blot analysis.

2.7. RNA isolation and quantitative real-time polymerase chain reaction (Q-PCR) Total RNA was isolated and cDNA synthesis reactions were performed as reported elsewhere [67]. A real-time quantitative PCR 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: AMPK (5′-GGGTGAAGATCGGACACTACGT-3′ and 5′-TTGATGTTCAATCTTCACTTTG-3′), for Bcl-2 (5’-GGCTGGGATGCCTTTGTG-3’ and 5’-CAGCCAGGAGAAATCAAACAGA-3’), for CREB (5’-CCAAGCTTATGGATCCTCCTGGAGAGAAGATGG-3’ and 5’-GCCTCGAGAAGCACATTGACGCTCCTGAC-3’), for PGC1 (5’-TGAGAGGGCCAAGCAAAG-3’ and 5’-ATAAATCACACGGCGCTCTT-3’), for NRF-1 (5’-CCATCTGGTGGCCTGAAG-3’ and 5’-GTGCCTGGGTCCATGAAA-3’), for 7

Tfam (5’-GAACAACTACCCATATTTAAAGCTCA-3’ and 5’-GAATCAGGAAGTTCCCTCCA-3’), for D-loop (5’- GGTTCTTACTTCAGGGCCATCA-3’ and 5’GATT-AGACCCGTTACCATCGAGAT-3’), and GAPDH (5'-TGCACCACCAACTGCTTAGC-3' and 5'-GGCATGGACTGTGGTCATGAG-3'). Independent reversetranscription PCRs were performed as described previously [67]. The relative transcript amount of the target gene, calculated using standard curves of serial RNA dilutions, is normalized to GAPDH expression in the same RNA sample.

2.8. Measurement of intracellular ATP concentration To determine ATP levels, hNSCs were collected in a lysis buffer (0.1 M Tris, 0.04M EDTA, pH 7.2), and boiled for 3 min. Samples were then centrifuged (112g for 5min), and the supernatants were used for the luciferin/luciferase assay as described previously [64]. 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% of bovine serum albumin, and 0.2 M of Tris (pH 7.5).

2.9. Mitochondrial mass The fluorescent probe Mitotracker GreenTM dye (MitoGreen, Invitrogen, Carlsbad, CA, USA) binds mitochondrial membrane lipids regardless of mitochondrial membrane potential or oxidant status. To determine the mass of mitochondria [67], 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 8

emission at 516 nm using fluorescence microscopy (OPTIMA). Amounts were determined by comparing the means of the fluorescent signals.

2.10. Mitochondrial functional parameters The maximal respiratory function, COX activity, and mitochondrial membrane potential were used for mitochondrial functional parameters as reported elsewhere [65]. For mitochondrial respiratory studies cells were grown on a 10 cm plate, treated with AGEs (0.5 mg/ml) for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h, then trypsinized, and suspended in 0.5 ml of mitochondrial isolated buffer. Respiratory measurements of mitochondria were isolated using the mitochondria isolation kit utilized as manufacturer’s instructions (Thermo Fisher Scientific, Waltham, USA). 50 g of mitochondria were suspended in a sealed and continually stirred at 37 ℃ containing 0.3 ml of respiration buffer (100 mM KCl, 2 mM MgCl2, 4 mM KH2PO4, 10 mM pyruvate, 5 mM malate, 250 M EGTA, 10 mM HEPES). The maximal respiratory rate was gained following the addition of 10 M FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone). The respiratory function of isolated mitochondria (0.2 mg/ml final concentration) was measured using a miniature Clark-type oxygen electrode (MT200 Mitocell Miniature Respirometer, Hamden, CT, USA).

9

The cytochrome oxidase (COX) activity was measured using the assay kit from Sigma (St. Louis, USA) using isolated mitochondrial fractions from the isolation kit (Thermo Fisher Scientific) for hNSCs. The mitochondrial fraction (2 g) was added to 1 ml of the reaction solution, then assayed with the COX activity assay; reactions were set up following the procedures provided by the manufacturer. The absorbance readings were detected by a spectrophotometer at 550 nm and COX activity was measured as Unit/mg of mitochondrial protein.

The measurement of mitochondrial membrane potential was detected using the JC-1 dye from Life Technologies (Waltham, USA) for hNSCs. Briefly, cells were treated with 1 M JC-1 for 30 min in Earle’s balanced salt solution (EBSS) at 37 °C. The cells were then washed three times in EBSS before fluorescence values were detected. The fluorescence was calculated by a microplate reader with an excitation filter of 530 ± 25 nm and an emission filter of 590 ± 30 nm.

2.11. Transmission electron microscopy hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. Cells were washed with 1xPBS, collected in 1ml of 0.05 % Trypsin-ETDA. After centrifugation at 2000 rpm for 5 min at 4 °C, the cell pellet was then washed in 0.1M PBS. Cells were fixed with 1% osmium tetroxide at room temperature for 2h. The pellet was 10

washed in 0.1M PBS, then addition of ethanol is dehydrated 15 minutes, after addition of 100% acetone is dehydrated 15 minutes. Followed by 100% acetone / spurr's resin (3:1) overnight were embedded. Finally Pure spurr's resin embedding for 4h, placed in an 70 ℃ oven overnight, then processed by the ultrathin section, and after the copper mesh to collect sections in cells. High- magnification views of hNSCs by transmission electron microscopy (JEOL JEM-1400).

2.12. Statistical analysis All reactions were run in triplicate from each independent experiment. All data were expressed as means ± SEM from three independent experiments. To establish significance, data were subjected to unpaired one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls Method using the Sigma Stat 3.5 software statistical package (Systat SigmaStat V3.5.0.54 Software; San Jose, California, USA). The criterion for significance was set at p < 0.001.

3.

Results

3.1. Metformin rescued cell viability in hNSCs treated with A via the AMPK pathway

The effects of AGEs on cell viability and caspase 3/9 (a marker of caspase cascade activation) activity in hNSCs were initially assessed. Compared to vehicle controls, hNSCs treated with A for 72 h had significantly reduced cell viability (p<0.001) (Fig. 1A). In addition, hNSCs caspase 3 and 9 activities, detected after A 11

treatment for 72 h, were significantly increased 2-fold compared to their respective controls (p<0.001) (Fig. 1B, C). Furthermore, treatment with a AMPK agonist (metformin) significantly (p<0.001) normalized both cell viability (Fig. 1A) and caspase 3/9 activities (Fig 1B, C), although this protective effect was blocked by co-treatment with an antagonist of AMPK (Compound C). Moreover, hNSCs were collected to show typical microphotographs of using two-color assay to with either live (for calcein-AM, green) or dead (for ethidium homodimer-1, red) cells. Metformin rescued the A decreased hNSCs survivability (Fig. 2), although this protective effect was blocked by co-treatment with Compound C.

Release of cytochrome c from mitochondria is one of the major initial processes in the induction of the apoptosis containing activation of caspase 9 [68]. To test whether the effect of caspase in A induced cell death needs cytochrome c, hNSCs were treated with A. The results exhibited in Fig. 3 distinctly demonstrate that previous to A treatment, the mass of cytochrome c is localized in the mitochondria and scarcely discoverable levels were detected in the cytosol. In contrast, incubation of cells to A effected release of cytochrome c from the mitochondria into the cytosol (Fig. 3). Importantly, treatment with metformin significantly prevented cytochrome c from the mitochondria into the cytosol which was blocked by Compound C.

3.2.Metformin increases AMPK, Bcl-2 and CREB gene expression levels in A-treated hNSCs

To evaluate whether activation of AMPK in hNSCs treated with A exerts a 12

neuroprotective effect, the expression levels of two genes (Bcl-2 and CREB) that are implicated as important in hNSCs survival were assessed. This is of particular interest because two genes (Bcl-2 and CREB) are downstream targets of AMPK [65, 69]. The mRNA transcript levels of AMPK, Bcl-2 and CREB were significantly lower in A-treated hNSCs compared to respective vehicle controls (p<0.001) (Fig. 4). Co-treatment with metformin significantly increased transcript levels in hNSC compared to A-treatment alone, but this protective effect was absent in the presence of Compound C (Fig. 4).

3.3. Metformin rescued PGC1, NRF-1, and Tfam gene expression levels suppressed in hNSCs treated with A

Since, stimulation of AMPK directly actives the PPAR coactivator-1α (PGC1), which in turn induces genes associated to mitochondrial function and biogenesis [65]. Interestingly, the mRNA transcripts of PGC1, nuclear respiratory factor-1 (NRF-1), and mitochondrial transcription factor A (Tfam) were significantly decreased by more than 50% in A-treated hNSCs compared to respective controls (p<0.001) (Fig. 5). Co-treatment with metformin significantly enhanced PGC1, NRF-1, and Tfam mRNA levels, which were blocked by the presence of Compound C (Fig. 5). To further evaluate the protein levels of AMPK and PGC1 in hNSCs, we analyzed the expression by Western blot assay. Both the protein levels of AMPK and 13

PGC1 were significantly reduced in hNSCs treated with A compared to vehicle controls (p<0.001) (Fig. 6). In contrast, the addition of metformin significantly enhanced AMPK and PGC1 expression, but this rescue was blocked in the presence of Compound C (Fig. 6).

3.4. Metformin enhanced D-loop levels, mitochondrial mass, mitochondrial functional parameters and morphology in A-treated hNSCs To further evaluate mitochondrial DNA (mtDNA) copy number in the consequence of potential mitochondrial biogenesis, we analyzed the expression of D-loop [70] by quantitative PCR. D-loop levels in A-treated hNSCs were significantly decreased compared to vehicle controls (p<0.001). However, co-treatment with metformin significantly restored D-loop levels to almost normal levels, but only in the absence of Compound C (Fig. 7A). Stimulation of AMPK promotes mitochondrial biogenesis and remodeling via the induction of PGC1 and its downstream target genes [65, 71]. Therefore, we evaluated whether metformin regulated mitochondrial capacity via an AMPK-dependent pathway in A-treated hNSCs. The mitochondrial biogenesis assay using MitoGreen [67], and reflective of mitochondrial mass, was used to assess the effects of A treatment on hNSCs. As shown in representative images (Fig 7B), and when quantitated by mean fluorescence 14

(Fig 7C), these data show treatment with A significantly decreases hNSC mitochondrial mass by almost 60% compared to vehicle controls (p<0.001), and that co-treatment with metformin significantly abrogated this effect in the absence of Compound C.

To further verify the role of A in mitochondrial functional parameters, we used assays of maximal respiratory function, COX activity, and mitochondrial membrane potential. We found that mitochondrial ability in hNSCs with metformin attenuated the A-induced reduction of maximal respiratory function (Fig 8A), COX activity (Fig 8B), and mitochondrial membrane potential (Fig 8C), which were blocked by Compound C. These mitochondrial functional parameters confirm that metformin elevated mitochondrial function in the hNSCs with A via AMPK.

We used transmission electron microscopy to determine the morphology of mitochondria in the cell bodies of hNSCs. Morphological assessment of hNSCs demonstrated normal mitochondrial morphology by transmission electron microscopy (Fig 9). These fragmented mitochondria (white arrow) in A-treated hNSCs were significantly increased compared to vehicle controls (Fig 9). However, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology (black arrow), but only in the absence of Compound C (Fig 9). 15

Discussion Since, the identification of AD as mitochondrial diseases has been causing elevating attention [14, 72]. Moreover, experiments guided in animal as well as cellular models of AD demonstrate the benefit of mitochondrial protection for reduction neuronal degeneration [73]. AMPK activity has been shown to reduce with age, which may provide to decreased survivability and mitochondrial biogenesis with aging-correlated AD (Jornayvaz and Shulman, 2010). Here we exhibit our result of the central role of AMPK in A-related neuronal impairment, and discuss how AMPK may protect against AD through attenuation of the A-mediated toxic effects on mitochondrial dysfunction in the hNSCs. The protective role of metformin in mediating the hNSCs has not been explored in A before. In the present study, we found a metformin mediates the cell viability of hNSCs via downregulation of caspase 3/9 activities and cytosolic cytochrome c (Figs. 1, 2 and 3). Here, we found a lower level of AMPK, Bcl-2, and CREB in the hNSCs with A (Figs. 4 and 6). In support of this study, AMPK can also prevent apoptotic cell death by activating AMPK, Bcl-2, and CREB [32, 65]. In addition to control the cellular defense, AMPK has an major function in neuroprotection via its downstream network of signaling pathways including PGC-1 [46, 57, 74, 75]. The poor PGC-1 activity observed in the hNSCs with A led to the inevitable downregulation of two important downstream targets of PGC-1 (e.g., NRF1 and Tfam), and might result in a decrease in the D-loop level and mitochondrial mass (Figs. 5, 6 and 7). Importantly, AD is related with neuronal abnormalities in energy metabolism, for example, mitochondrial dysfunctions [76]. Considering the capabilities of AMPK, these mitochondrial dysfunctions of AD might be associated to functional defects in AMPK signaling. Our study also demonstrated 16

that impair these mitochondrial homeostasis function and morphology observed in the hNSCs with A (Figs. 8 and 9). This rescue effect of metformin was effectively blocked by Compound C, demonstrating that the action of metformin in mitochondrial dysfunction was mediated by AMPK pathway. Our findings led to the conclusion that deficient expression of AMPK plays an important role in the hNSCs with A.

It is common agreed that metformin, the first-line drug of select for the treatment of type 2 diabetes, is an activator of AMPK and that many of its signals are through AMPK pathway [77, 78]. AMPK is believed to be a critical therapeutic target for the obesity, diabetes mellitus, aging, and neurodegeneration diseases [58, 79-82]. Gupta and his collaborators have described metformin ameliorates AD-associated neuropathological changes in differentiated N2A cells [83]. Kickstein and colleagues demonstrated that metformin reduced tau protein phosphorylation in cultured neurons and in mouse brain [84]. A number of other studies have described potential mechanisms through which metformin might act to neuroprotection.

In 2010, a

study revealed that chronic metformin can lead to increase neuronal viability in mice model of ischemia [85]. Moreover, El-Mir and colleagues (2008) showed that metformin has been shown as a protection against apoptotic cell death in primary cortical neurons [86]. Hwang and colleagues also demonstrated that metformin normalized type 2 diabetes-induced decrease in cell proliferation and neuroblast differentiation in rat hippocampal dentate gyrus [87]. Our recently study showed that activation of AMPK by metformin protects hNSCs against advanced glycation end products (AGEs) induced cytotoxicity, further suggesting that AMPK may be involved in cell survival [65].

17

On the other hand, Kwon et al. demonstrated that AMPK activator (resveratrol) acts as an effective treatment for AD by attenuating Aβ-induced oxidative stress and the AMPK-dependent pathway in murine HT22 hippocampal cell line [58]. They also observed that AMPK signaling with resveratrol controls A metabolism and mediates the anti-amyloidogenic effect of resveratrol in N2A cells and mouse primary neurons [88]. Vingtdeux and his collaborators have described activators (novel synthetic small-molecule) of AMPK as enhancers of autophagy and A degradation in primary neurons [48]. Importantly, Vingtdeux also indicate that resveratrol, orally executed in mice, arrived the brain where it stimulated AMPK and distinct decreased A levels in the cerebral cortex [89]. AMPK activator like AICAR decreases A production, and have even suggested AMPK activators to be potential therapeutics against in vivo AD models [63].

In this study, hNSCs treated with A, with or without metformin, were examined for changes in cell death and mitochondrial dysfunction. Our findings provide important new evidence of the mechanisms by which A mediate hNSCs degeneration, and metformin confers AMPK-dependent neuroprotection. Together, these data support AMPK as a potential therapeutic drug target to treat neurodegeneration in AD patients.

Conflict of interest The authors have declared no conflict of interest.

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Acknowledgements The authors thank Chia-Nan Yen for proof-reading and editing the article. This work was supported by grants from the Ministry of Science and Technology (MOST 105-2314-B-030-005), Fu Jen Catholic University (A0104020 and A0204104), and Terry Whole Brain & Potential Development Center (Terry 105-11-01). The authors wish to thank for technical assistance of Electron Microscope Laboratory of Tzong Jwo Jang, collage of Medicine, Fu Jen Catholic University.

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Figure legends Fig. 1. Metformin rescues the A-mediated decrease in hNSCs viability via the AMPK pathway. (A) hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h, and cell viability was detected by MTT assay. (B, C) Caspase activities were detected by a fluorometric protease assay using substrates for caspase-3-like (DEVD-AFC) and caspase-9-like (LEHD-FMK). All reactions were run in triplicate from each independent experiment. Values A, B and C are expressed as a percentage of the indicated transcript in CON and is presented as the mean ± SEM values from three independent experiments. a Specific comparison to the indicated hNSCs with A (p < 0.001; one-way ANOVA). Fig. 2. Metformin rescued the A decreased hNSCs survivability. hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. hNSCs were collected to show typical microphotographs of using two-color assay to with either live (for calcein-AM, green) or dead (for ethidium homodimer-1, red) cells. A representative image of three independent experiments is shown. Scale bar: 200 μm. Fig.3. Metformin prevented A induced cytochrome c release from mitochondria into cytosol in the hNSCs. hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 27

h. Lysates (20 g) were collected from the indicated treatment group, and subjected to a western blot analysis. Levels of mitochondrial cytochrome c protein were normalized with the corresponding internal control (voltage-dependent anion channel; VDAC), compared with those in CON, shown at the bottom of the corresponding column. Levels of cytosolic cytochrome c protein were normalized with the corresponding internal control (actin), compared with those in CON, shown at the bottom of the corresponding column. All reactions were run in triplicate from each independent experiment. Values are expressed as a percentage of the indicated transcript in CON and are presented as the mean ± SEM values from three independent experiments. a Specific comparison to the indicated hNSCs with A (p < 0.001; one-way ANOVA). Fig. 4. Metformin increases AMPK, Bcl-2, and CREB gene expression levels in A-treated hNSCs. hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. The AMPK (A), Bcl-2 (B), and CREB (C) 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. All reactions were run in triplicate from each independent experiment. Values A, B and C are expressed as percentages of the indicated transcript in CON and are presented as the mean ± SEM values from three independent experiments. a Specific comparison to the indicated hNSCs with A (p < 0.001; one-way ANOVA). Fig. 5. Metformin rescued PGC1, NRF1, and Tfam gene expression levels suppressed in hNSCs treated with A.hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) 28

for another 48 h. The PGC1 (A), NRF1 (B) and Tfam (C) 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. All reactions were run in triplicate from each independent experiment. Values A, B and C are expressed as percentages of the indicated transcript in CON and are presented as the mean ± SEM values from three independent experiments.

a

Specific comparison to the

indicated hNSCs with A (p < 0.001; one-way ANOVA). Fig. 6. Metformin enhanced the levels of AMPK and PGC1 proteins in the hNSCs treated with A. hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. Lysates (20 g) were collected from the indicated treatment group, and subjected to a western blot analysis. Levels of AMPK and PGC1 proteins were normalized with the corresponding internal control (actin), compared with those in CON, shown at the bottom of the corresponding column. All reactions were run in triplicate from each independent experiment. Values are expressed as a percentage of the indicated transcript in CON and are presented as the mean ± SEM values from three independent experiments. a Specific comparison to the indicated hNSCs with A (p < 0.001; one-way ANOVA). Fig. 7. Metformin increased D-loop levels and mitochondrial mass in A-treated hNSCs.hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. (A) The D-loop 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 29

technique of the indicated gene was performed and normalized to that of GAPDH. (B) hNSCs were collected to determine the level of mitochondrial mass using Mitotracker GreenTM dye (green). Scale bar: 200 μm. (C) The expression levels of mitochondrial mass were normalized to those of cell numbers. All reactions were run in triplicate from each independent experiment. Values are expressed as a percentage of the indicated transcript in CON and are presented as the mean ± SEM values from three independent experiments. a Specific comparison to the indicated hNSCs with A (p < 0.001; o

n

e

-

w

a

y

A

N

O

V

A

)

.

Fig. 8. Metformin improved the performance of mitochondrial functions in the hNSCs treated with A. hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. (A) Maximal respiratory rate obtained from the indicated treatment were evaluated by the FCCP assay. (B) COX activity was assayed with the Cytochrome c Oxidase Assay Kit. (C) hNSCs were collected to determine the level of mitochondrial membrane potential using JC-1 dye. All reactions were run in triplicate from each independent experiment. Values are expressed as the mean ± SEM values from three independent experiments. a

Specific comparison to the indicated hNSCs with A (p < 0.001; one-way ANOVA).

Fig. 9. Metformin improved the mitochondrial morphology in the hNSCs treated with A by transmission electron microscopy.hNSCs were treated with 5 M A1-42 for 24 h, then treated with the indicated reagents (1 mM Metformin or 10 M Compound C) for another 48 h. High- magnification views of hNSCs by transmission electron microscopy (JEOL JEM-1400). These fragmented mitochondria (white arrow) in A-treated hNSCs were significantly increased compared to vehicle controls. However,

co-treatment

with

metformin 30

significantly

restored

fragmented

mitochondria to almost normal morphology (black arrow), but only in the absence of Compound C. A representative image of three independent experiments is shown. Scale bar: 0.5 μm.

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