Cell Mol Neurobiol DOI 10.1007/s10571-015-0222-6
ORIGINAL RESEARCH
Ab25–35 Suppresses Mitochondrial Biogenesis in Primary Hippocampal Neurons Weiguo Dong1 • Feng Wang1 • Wanqing Guo2 • Xuehua Zheng1 • Yue Chen1 Wenguang Zhang1 • Hong Shi1
•
Received: 15 March 2015 / Accepted: 2 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Mitochondrial biogenesis is involved in the regulation of mitochondrial content, morphology, and function. Impaired mitochondrial biogenesis has been observed in Alzheimer’s disease. Amyloid-b (Ab) has been shown to cause mitochondrial dysfunction in cultured neurons, but its role in mitochondrial biogenesis in neurons remains poorly defined. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are key energy-sensing molecules regulating mitochondrial biogenesis. In addition, peroxisome proliferatoractivated receptor-c coactivator 1-alpha (PGC-1a), the master regulator of mitochondrial biogenesis, is a target for SIRT1 deacetylase activity. In this study, we investigated the effects of Ab25–35 on mitochondrial biogenesis in cultured hippocampal neurons and the underlying mechanisms. In primary hippocampal neurons, we found that 24-h incubation with Ab25–35 suppressed both phosphorylations of AMPK and SIRT1 expression and increased PGC-1a acetylation expression. In addition, Ab25–35 also resulted in a decrease in mitochondrial DNA copy number, as well as decreases in the expression of mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam). Taken together, these data show that Ab25–35 suppresses mitochondrial biogenesis in hippocampal neurons. Ab25–35-induced impairment of mitochondrial biogenesis may be associated with the inhibition of the AMPK-SIRT1-PGC-1a pathway.
& Weiguo Dong [email protected] 1
Department of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian, People’s Republic of China
2
The Third People’s Hospital of Fujian Province, Fuzhou 350122, Fujian, People’s Republic of China
Keywords Alzheimer’s disease Mitochondrial biogenesis Amyloid-b AMPK SIRT1 PGC-1a
Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the aged population. The pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles (Selkoe 2004). Increasing evidence has shown that mitochondrial dysfunction is a prominent feature of AD (Wang et al. 2014). Amyloid-b peptide (Ab), which is the major component of senile plaques, elicits neurotoxicity and has a pivotal function in AD pathogenesis (Deshpande et al. 2006). Ab has been reported to interfere with multiple aspects of mitochondrial function. Xie et al. have observed severe structural and functional abnormalities of mitochondria in the immediate vicinity of Ab plaques (Xie et al. 2013). Studies using neuronal cell culture demonstrated that Ab causes morphological alteration of mitochondria, ATP depletion, and production of ROS (Casley et al. 2002; Cha et al. 2012; Chen and Yan 2007; Dong et al. 2010). The exposure of isolated mitochondria to Ab reduces complex IV activity (Canevari et al. 1999) and induces the formation of the permeability transition pore (Du et al. 2008). In addition, Ab progressively accumulates within mitochondria of both human AD brain and Tg mouse models for AD (Chen and Yan 2007) and interacts with the mitochondrial proteins, including cyclophilin D, amyloid binding alcohol dehydrogenase, and Drp1 (Benek et al. 2015). This interaction has a causative role in impairing mitochondrial physiological functions. Mitochondrial biogenesis can be defined as the growth and division of pre-existing mitochondria, which plays an
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essential role in maintaining healthy mitochondria in eukaryotic cells during the life cycle of mitochondria. Recently, impaired mitochondrial biogenesis is observed in the brain of AD patients, a mouse model of AD, and APPswe M17 cells (Calkins et al. 2011; Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012). Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in AD (Sheng et al. 2012). Moreover, mitochondrial biogenesis is severely affected and significantly decreases after injection of Ab in hippocampal CA1 area of rats (Shaerzadeh et al. 2014). However, whether Ab suppresses mitochondrial biogenesis in primary hippocampal neurons remains unclear. AMP-activated protein kinase (AMPK) is a critical regulator of mitochondrial biogenesis which regulates intracellular energy metabolism in response to acute energy crises (Hardie 2007). AMPK activates sirtuin 1 (SIRT1) by increasing cellular NAD? levels, resulting in the deacetylation and modulation of the activity of peroxisome proliferator-activated receptor-c coactivator 1-alpha (PGC-1a) (Canto et al. 2009; Fulco and Sartorelli 2008). AMPK can also directly activate PGC-1a (Jager et al. 2007). SIRT1, the class III NAD?-dependent histone deacetylase, has been shown to combat aging, extend lifespan, and regulate metabolism (Kwon and Ott 2008). In addition, SIRT1 has been proposed as an important regulator of mitochondrial biogenesis (Canto et al. 2009; Gerhart-Hines et al. 2007). SIRT1 physically interacts with and deacetylates PGC-1a (Nemoto et al. 2005; Rodgers et al. 2005). The peroxisome proliferator-activated receptor a (PPARa) is a fatty acidactivated nuclear receptor that plays a key role in the transcriptional regulation of genes involved in energy metabolism. PGC-1 is a coactivator of PPARa and binds PPARa in a ligand-influenced manner (Vega et al. 2000). PGC-1a belongs to the PGC-1 family of regulated coactivators, including PGC-1b and PRC which play a central role in a regulatory network governing the transcriptional control of mitochondrial biogenesis (Lin et al. 2005; Scarpulla 2011). PGC-1a targets multiple transcription factors including nuclear respiratory factor 1 (NRF 1) and nuclear respiratory factor 2 (NRF 2), which activate mitochondrial transcription factor A (Tfam) (Rohas et al. 2007; Wu et al. 1999). Tfam drives transcription and replication of mitochondrial DNA (mtDNA) (Virbasius and Scarpulla 1994). Additionally, previous studies showed that AMPK activity and the expression of SIRT1 and PGC1a were reduced in AD brain (Julien et al. 2009; Qin et al. 2009; Sheng et al. 2012). We propose that Ab-induced mitochondrial dysfunction may be associated with mitochondrial biogenesis. We hypothesize that Ab can suppress AMPK and SIRT1, both of which further downregulate PGC-1a and the
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downstream mitochondrial biogenesis program. To test this hypothesis, we used primary hippocampal neurons as a model system to study the effects of Ab25–35 on mitochondrial biogenesis by measuring the expression of the AMPK-SIRT1-PGC-1a pathway, as well as the levels of transcription factors involved in mitochondrial biogenesis and mitochondrial components such as mtDNA copy number.
Materials and Methods Primary Hippocampal Neuron Culture and Treatment Primary cultures of hippocampal neurons were prepared as described in our previous study (Dong et al. 2010). Briefly, we dissected newborn (P0, 0–24 h) Sprague–Dawley rats to obtain primary hippocampal neurons. We explanted, plated, and maintained the neurons in serum-free DMEM/F12 medium supplemented with 2 % B27 and 1 % penicillin– streptomycin. Half of the growth medium was changed every 3 days thereafter. All experiments were performed according to ‘‘National Institute of Health Guide for the Care and Use of Laboratory Animals’’ (NIH Publications No. 80-23) and were approved by the Committee on Ethical Use of Animals of Fujian University of Traditional Chinese Medicine. Ab25–35 (Sigma-Aldrich, USA) stock solution of 1 mM was prepared in sterile distilled water and stored at -20 °C. To obtain the fibrillar form of Ab25–35, an aliquot of the stock solution was incubated at 37 °C for 4 days before use (Tohda et al. 2004). Ab25–35 was used at a final concentration of 25 lM according to the previous studies (Bulbarelli et al. 2009; Dong et al. 2010; Resende et al. 2007). Cells were treated with 25 lM Ab25–35 for 24 h on 10 days in vitro (DIV). After that, treatment cells were harvested for subsequent assays. Measurement of Cell Viability After 24-h incubation with Ab25–35, cell viability was determined using the MTT assay as previously described (Mosmann 1983). In brief, MTT was dissolved in phosphate-buffered saline (PBS) and was added to culture medium. Hippocampal neurons were incubated with MTT (0.5 mg/ml) for 4-h incubation at 37 °C. Next, the medium containing MTT was removed followed by the addition of 150 ll DMSO per well (96-well plate) to dissolve the formazan crystals. The absorbance was measured at 570 nm using an ELISA plate reader. Results were expressed as percentage (%) of MTT reduction, assuming the absorbance of control cells as 100 %.
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Total Protein Extraction
Real-Time Quantitative RT-PCR
Primary hippocampal neurons were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors cocktail (Sigma-Aldrich, USA) and sonicated. The cell lysates were centrifuged at 15,0009g for 10 min at 4 °C and supernatants were collected. Protein concentrations were determined using the BCA protein assay (Thermo Scientific).
Total RNA was isolated from hippocampal neurons using the RNeasy RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA concentrations and quality were measured by Nanodrop Spectrophotometer ND1000 (Thermo Scientific, Wilminton, DE). We synthesized cDNA from 0.5 lg of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Real-time quantitative RT-PCR (qRT-PCR) was performed using SYBR Green PCR master mix (Applied Biosystems) and a 7300 real-time PCR system (Applied Biosystems, CA, USA). Sequences of specific primers are the following: SIRT1 forward, 50 -TCGTGGAG ACATTTTTAATCAGG-3 and reverse 50 -GCTTCATGAT GGCAAGTGG-3; PGC-1a forward, 50 -GTGCAGCCAAG ACTCTGTATGG-30 and reverse 50 -GTCCAGGTCATTCA CATCAAGTTC-30 ; NRF 1 forward, 50 -TTACTCTGCTGT GGCTGATGG-30 and reverse 50 -CCTCTGATGCTTGCGT CGTCT-30 ; NRF 2a forward, 50 -AGGTGAGGAGATGGG CTGC-30 and reverse 50 -CGTTGTCCCCATTTTTGCG-3; Tfam forward, 50 -GAAAGCACAAATCAAGAGGAG-30 and reverse 50 -CTGCTTTTCATCATGAGACAG-30 ; 18S forward, 50 -TCCCTAGTGATCCCCG AGAAGT-30 and reverse 50 -CCCTTAATGGCAGTGATAGCGA-30 . The data from qRT-PCR were analyzed with delta–delta threshold cycle (DDCt) method using 18S as the internal control gene. Each DCt value was determined by subtracting 18S Ct value from the target gene Ct value. The DDCt was calculated by subtracting the DCt value of the control from the DCt value of the Ab25–35 sample. 2DDCt represented the average relative amount of mRNA for each target gene.
Western Blotting Analysis Equal amounts of proteins (30 lg) were separated using SDS-PAGE and transferred to nitrocellulose membranes (Millipore). Membranes were blocked in 5 % milk in 19 Tris-buffered saline with 0.05 % Tween 20 (TBS-T) for 1 h at room temperature with shaking. Membranes were incubated with Abs against AMPK (1:1000, Cell Signaling), phosphorylated AMPK (1:1000, Cell Signaling), SIRT1 (1:1000, Abcam), PGC-1a (1:1000, Millipore), NRF 1 (1:1000, Santa Cruz, CA, USA), NRF 2a (1:1000, Santa Cruz, CA, USA), NRF 2b (1:1000, Santa Cruz, CA, USA), and Tfam (1:1000, Santa Cruz, CA, USA). Membranes were washed three times in TBS-T for 5 min each wash. We applied appropriate horseradish peroxidaseconjugated secondary antibody at a concentration of 1:2000 for 1 h at room temperature with shaking. We washed membranes three times in TBS-T and applied the Supersignal West Pico chemiluminescent reagent (Thermo Scientific) for 3 min. The signals of the membrane were scanned using the FluorChem Scanner and quantified with the NIH Image J software. These results were normalized with b-actin expression levels and confirmed by triplicate measurements of the same sample. Immunoprecipitation For examination of PGC-1a acetylation, immunoprecipitation was performed according to the immunoprecipitation method provided by Abcam. The protein samples were precleared with 20 ll of protein G (Sigma-Aldrich, USA) for 3 h and then centrifuged to obtain supernatant at 15,0009g for 10 min. PGC-1a antibody (1:1000, Millipore, USA) and 30 ll of protein G were added to the precleared supernatant and incubated for 12 h at 4 °C. The protein G agarose was washed three times with cold PBS for 15 min. The immunoprecipitated protein was visualized and blotted using the Western blotting method. PGC-1a acetylation was measured using acetyl-lysine antibody (Cell Signaling).
Mitochondrial DNA Quantification Total cellular DNA in hippocampal neurons was extracted using Blood & Cell Culture DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. mtDNA copy number was measured by real-time PCR method using the Step one plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with the SYBR Green detection method as previously described (Sheng et al. 2012). Each real-time PCR (20 ll total volume) contained 3 ll of template DNA, 10 ll of 29 SYBR Green Real-time PCR Master (Applied Biosystems), 1 ll of each of the forward and reverse primers, and 5 ll of ultrapure water. The primers for cytochrome b were forward, 50 -AAAGCCACCT TGACCCGATT-30 and reverse 50 -GATTCGTAGGGCCG CGATAGG-30 (Casuso et al. 2014). Primers for 18S were
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forward, 50 -GGTGCATGGCCGTTCTTA-30 and reverse 50 -TCGTTCGTTATCGGAATTAACC-30 . The ratio of cytochrome b to 18S represents the relative mitochondrial copy number. Statistical Analysis Results are presented as the mean ± SEM. Statistical significance of Ab25–35-induced differences was evaluated by Student’s t test assuming equal variance, and a value of P \ 0.05 was considered significant.
Results Ab25–35 Induces Cytotoxicity in Primary Hippocampal Neurons It has been reported that Ab25–35 aggregates and retains the neurotoxic activities just like the full-length Ab (Pike et al. 1993). Thus, we first investigated the neurotoxic effect of Ab25–35 in primary culture of rat brain hippocampal neurons using the MTT assay. Cell viability decreased to 70.5 % after 24-h incubation with 25 lM Ab25–35 at DIV 10 (Fig. 1). Ab25–35 Downregulates Phosphorylation of AMPK and SIRT1 Expression AMPK is a key regulatory factor for mitochondrial biogenesis (Fulco et al. 2008). The degree of AMPK phosphorylation (p-AMPK) was used as a measure of AMPK
activation. It has been reported that AMPK activity was reduced in the hippocampus of a mouse model of AD (Pedros et al. 2014). Thus, we investigated whether Ab25–35 suppressed AMPK activity in cultured hippocampal neurons. It is evident from Fig. 2 that a significant decrease in p-AMPK/t-AMPK was observed after Ab25–35 treatment, which indicates that Ab25–35 suppresses phosphorylation of AMPK. However, there was no significant difference in t-AMPK protein levels after 24-h incubation with Ab25–35 (Fig. 2b, P [ 0.05). AMPK has been known to regulate SIRT1 expression (Canto et al. 2009). Thus, we further measured SIRT1 expression after Ab25–35 treatment. Our data showed that the SIRT1 protein levels were decreased after Ab25–35 treatment (Fig. 2). Additionally, we also found that the SIRT1 mRNA level was reduced after 24 h of Ab25–35 treatment (Fig. 4). Ab25–35 Decreases PGC-1a Expression and Increases PGC-1a Acetylation AMPK could directly activate PGC-1a (Jager et al. 2007). Moreover, SIRT1 interacts with and deacetylates PGC-1a (Nemoto et al. 2005; Rodgers et al. 2005). Thus, we next evaluated the effect of Ab25–35 on PGC-1a expression and PGC-1a deacetylation in primary hippocampal neurons. In our studies of PGC-1a expression level after Ab25–35 treatment, a significant decrease in the mRNA level and protein level of PGC-1a was observed (Figs. 2, 4). In accordance with decreased protein levels of SIRT1, there was an increased in the acetylation state of PGC-1a, a target for SIRT1 deacetylase activity (Fig. 3). Ab25–35 Suppresses Mitochondrial Biogenesis Program
Fig. 1 Effect of Ab25–35 on the viability of primary cultured hippocampal neurons. Cultured hippocampal neurons were treated with 25 lM Ab25–35 for 24 h. After that, cell viability was evaluated using the MTT assay. *P \ 0.05 compared with the control
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To examine whether mitochondrial biogenesis program is downregulated in response to Ab25–35, qRT-PCR and Western blotting were performed to compare the expression of downstream genes of PGC-1a. We first analyzed the gene expression of NRF 1, NRF 2, and Tfam. The mRNA levels of NRF 1, NRF 2a, and Tfam were significantly reduced after Ab25–35 treatment (Fig. 4). Similar to mRNA levels, a significant decrease was observed in protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam (Fig. 5). To directly determine whether mitochondrial biogenesis is affected by Ab25–35, we measured mtDNA relative to nDNA (mtDNA/nDNA) by real-time PCR and found that mtDNA/nDNA was significantly reduced in cultured hippocampal neurons treated with Ab25–35 (Fig. 6). In summary, these data demonstrate that the mitochondrial biogenesis program is downregulated by Ab25–35.
Cell Mol Neurobiol Fig. 2 Ab25–35 suppressed phosphorylation of AMPK and the protein levels of SIRT1 and PGC-1a in hippocampal neurons. a Western blotting analysis of total and phosphorylated AMPK (t-AMPK and p-AMPK), SIRT1, and PGC-1a protein in cultured hippocampal neurons after 24 h of Ab25–35 treatment. b Quantification of immunoblots in (a). c The ratio between p-AMPK and t-AMPK. b-actin was used as an internal loading control. *P \ 0.05 compared with the control
Fig. 3 Ab25–35 increased acetylation levels of PGC-1a in hippocampal neurons. Hippocampal neurons were treated with Ab25–35, and total lysates were used to test PGC1a acetylation. Relative acetylation levels of PGC-1a are shown on the right. *P \ 0.05 compared with the control
Discussion This study provides in vitro an evidence in support of the hypothesis that Ab suppresses mitochondrial biogenesis in hippocampal neurons. Two types of evidences were obtained. First, key molecules of the mitochondrial biogenesis pathway, including energy-sensing AMPK and SIRT1 as well as their downstream targets such as PGC-1a,
are downregulated by Ab25–35. Second, mtDNA levels are decreased after Ab25–35 treatment. The hippocampus is a key brain structure for learning and memory and among the first brain regions to be affected in AD, which arises on the pathological background of amyloid b plaques, neurofibrillary tangles, and synaptic and neuronal loss (Ittner and Gotz 2010). In addition, a number of studies have used cultured
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Fig. 4 Ab25–35 inhibited the mRNA levels of SIRT1, PGC-1a, NRF 1, NRF 2a, and Tfam in hippocampal neurons. The mRNA levels of SIRT1, PGC-1a, NRF 1, NRF 2a, and Tfam were determined using qRT-PCR and normalized to 18S. Results are expressed as mean ± SEM of triplicate samples in one representative experiment from 3 independent trials. *P \ 0.05 compared with the control
Fig. 6 Ab25–35 decreased mtDNA copy number. Hippocampal neurons were exposed to 24 h Ab25–35 and harvested for total DNA extraction. Using real-time PCR, the relative mitochondrial DNA copy number was determined by the ratio of cytochrome b to 18S. *P \ 0.05 versus the control
hippocampal neurons to explore the pathophysiological mechanism of AD (Filipcik et al. 2009; Joshi et al. 2014). Thus, we used cultured hippocampal neurons in this study. Ab is believed to play a central role in the development of AD (Hardy and Selkoe 2002). Ab25–35, a synthetic peptide corresponding to amino acids 25-35 in Ab1-40 and Ab1-42, possesses the same b-sheet structure (Kaminsky et al. 2010). Ab25–35 has been used in a number of studies and found to have many effects in common with full-length Ab1-40/42, including its toxicity (Pike et al. 1993; Yankner et al. 1990). Additionally, it has been reported that Ab25–35 is more toxic for cellular survival than Ab1-42 in cultured cortical neurons (Arancibia et al. 2008). Our data showed that Ab25–35 induced toxicity in cultured hippocampal neurons, which was consistent with previous studies (Arancibia et al. 2008; Dong et al. 2010; Resende
et al. 2007). We and other groups have further demonstrated Ab25–35 caused mitochondrial dysfunction in cultured neurons (Casley et al. 2002; Cha et al. 2012; Dong et al. 2010). In the present study, we found a decrease in mtDNA copy number in cultured hippocampal neurons treated with Ab25–35. Our results also showed that mRNA levels and protein levels of mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam) were reduced after 24-h incubation with Ab25–35. Similarly, intrahippocampal injection of Ab1-42 in rats resulted in decreases in PGC-1a, NRF 1, and Tfam (Shaerzadeh et al. 2014). Taken together, these findings indicate that Ab suppresses mitochondrial biogenesis both in vitro and in vivo. AMPK is a key regulatory factor not only for energy metabolism but also for mitochondrial biogenesis (Fulco et al. 2008). AMPK has been shown to regulate SIRT1 and PGC-1a, causing incremental elevations in mitochondrial
Fig. 5 Effects of Ab25–35 on the protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam in cultured hippocampal neurons. Western blotting analysis showed that the protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam were reduced in hippocampal neurons after 24 h of Ab25–35 treatment. b-actin was used as an internal control. *P \ 0.05 compared with the control
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biogenesis (Canto et al. 2009; Ruderman et al. 2010). In addition, SIRT1 is required for AMPK activation in resveratrol treatment on mitochondrial biogenesis (Price et al. 2012). AMPK activity was decreased in AD animal models (Pedros et al. 2014). Activation of AMPK can enhance mitochondrial biogenesis and improve mitochondrial function in cell lines (Dong et al. 2013; Shin et al. 2009) and animal models (Moran et al. 2013; Ng et al. 2012). Conversely, the inhibition of AMPK leads to a decrease in mitochondrial biogenesis (Wang and Brautigan 2013). Our data clearly show that Ab25–35 reduces AMPK activity in vitro, which indicates that Ab25–35 results in a decrease in mitochondrial biogenesis. In fact, our present results demonstrate that Ab25–35 suppresses mitochondrial biogenesis. Additionally, our previous study showed that treatment with 25 lM Ab25–35 for 24 h caused mitochondrial dysfunction in cultured hippocampal neurons (Dong et al. 2010). Taken together, Ab25–35 results in both impairment of mitochondrial biogenesis and mitochondrial dysfunction, which further supports the notion that impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in AD (Sheng et al. 2012). SIRT1 and AMPK have been shown to play many similar roles, including their ability to induce mitochondrial biogenesis and regulate the activity of PGC-1a (Fulco and Sartorelli 2008). SIRT1 promotes mitochondrial biogenesis through deacetylation and activation of PGC-1a (Gerhart-Hines et al. 2007; Rodgers et al. 2005). SIRT1 confers significant neuroprotection in a mouse model of AD likely through inhibition of PGC-1a acetylation and promotion of PGC-1a activity (Kim et al. 2007). The acetylation status of PGC-1a has been implicated as a fundamental post-translational modification that regulates its transcriptional activity (Canto et al. 2009; Nemoto et al. 2005). We clearly show that the expression levels of SIRT1 and PGC-1a are reduced after Ab25–35 treatment, which is consistent with the data showed a decrease in SIRT1 expression in SK-N-SH cells treated with Ab1-42 (Sun et al. 2014). Moreover, reduced SIRT1 expression subsequently suppressed PGC-1a deacetylation. These data indicate that Ab25–35 decreases not only PGC-1a expression but also PGC-1a activity. Previous studies showed that mtDNA copy number and the expression levels of PGC-1a, NRF 1, NRF 2, and Tfam were significantly reduced in AD hippocampus and APPswe M17 cells (Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012), which suggests that the mitochondrial biogenesis signaling is impaired in AD. Moreover, PGC-1a overexpression was shown to completely rescue whereas knockdown of PGC-1a exacerbated impaired mitochondrial biogenesis in APPswe M17 cells (Sheng et al. 2012). Based on previous findings and those of the current study, there is
a clear evidence that SIRT1 and PGC-1a play an important role in impaired mitochondrial biogenesis caused by Ab25–35. Consistent with previous studies (Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012), our data show that Ab25–35 suppresses mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam). We further show that Ab25–35 downregulates phosphorylation of AMPK and SIRT1 expression. NRF 2a protein levels remained unchanged in APPswe M17 cells compared with M17 cells (Sheng et al. 2012). Interestingly, we showed that NRF 2a protein levels were decreased after 24-h incubation with Ab25–35. The discrepancy may be due to different cells and different treatment between our study and their study. It has been reported that AMPK-SIRT1-PGC-1a signaling is implicated in energy metabolism, mitochondrial function, and apoptosis (Chau et al. 2010; Wei et al. 2011). Activation of AMPK-SIRT1-PGC-1a pathway has been shown to play an important role in exercise and temperature-induced mitochondrial biogenesis (Li et al. 2011; Liu and Brooks 2012). Conversely, inhibition of AMPK-SIRT1-PGC-1a signaling is involved in impaired mitochondrial biogenesis caused by Ab25–35. Overall, AMPK-SIRT1-PGC-1a signaling is associated with mitochondrial biogenesis. Mitochondrial biogenesis is an important and exciting area of cell biology. Lack of normal mitochondrial function has a negative impact in insulin resistance and aging (Petersen et al. 2003). In the current study, we show that mitochondrial biogenesis is downregulated in response to Ab25–35 in hippocampal neurons. The inhibition of AMPK-SIRT1-PGC-1a pathway and its downstream targets (NRF 1, NRF 2, and Tfam) is associated with the Ab25–35-induced changes in mitochondrial biogenesis. One limitation of our study is that hippocampal neurons culture system may not entirely reflect what occurs in vivo. Our findings need to be confirmed in animal models to see whether the Ab25–35-induced effects are reproducible in vivo. Future studies are also needed to test the hypothesis of a causal link between AMPK-SIRT1-PGC-1a inhibition and Ab25–35-induced mitochondrial biogenesis in hippocampus. If so, then, Ab25–35 may have the effect of decreasing the background levels of metabolic gene expression. The results presented here suggest that stimulation of pathways upstream of mitochondrial biogenesis and increased mitochondrial biogenesis may be a potential therapeutic approach for AD. Acknowledgments This work was supported by the National Natural Science Foundation of China (Project 81102625), the Natural Science Foundation of Fujian Province Grants (Project 2012J05154), and study abroad scholarships of Fujian Province. Conflict of interest The authors declare no financial or other conflicts of interest related to this study.
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ORIGINAL RESEARCH
Ab25–35 Suppresses Mitochondrial Biogenesis in Primary Hippocampal Neurons Weiguo Dong1 • Feng Wang1 • Wanqing Guo2 • Xuehua Zheng1 • Yue Chen1 Wenguang Zhang1 • Hong Shi1
•
Received: 15 March 2015 / Accepted: 2 June 2015 Ó Springer Science+Business Media New York 2015
Abstract Mitochondrial biogenesis is involved in the regulation of mitochondrial content, morphology, and function. Impaired mitochondrial biogenesis has been observed in Alzheimer’s disease. Amyloid-b (Ab) has been shown to cause mitochondrial dysfunction in cultured neurons, but its role in mitochondrial biogenesis in neurons remains poorly defined. AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) are key energy-sensing molecules regulating mitochondrial biogenesis. In addition, peroxisome proliferatoractivated receptor-c coactivator 1-alpha (PGC-1a), the master regulator of mitochondrial biogenesis, is a target for SIRT1 deacetylase activity. In this study, we investigated the effects of Ab25–35 on mitochondrial biogenesis in cultured hippocampal neurons and the underlying mechanisms. In primary hippocampal neurons, we found that 24-h incubation with Ab25–35 suppressed both phosphorylations of AMPK and SIRT1 expression and increased PGC-1a acetylation expression. In addition, Ab25–35 also resulted in a decrease in mitochondrial DNA copy number, as well as decreases in the expression of mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam). Taken together, these data show that Ab25–35 suppresses mitochondrial biogenesis in hippocampal neurons. Ab25–35-induced impairment of mitochondrial biogenesis may be associated with the inhibition of the AMPK-SIRT1-PGC-1a pathway.
& Weiguo Dong [email protected] 1
Department of Integrated Traditional Chinese and Western Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian, People’s Republic of China
2
The Third People’s Hospital of Fujian Province, Fuzhou 350122, Fujian, People’s Republic of China
Keywords Alzheimer’s disease Mitochondrial biogenesis Amyloid-b AMPK SIRT1 PGC-1a
Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the aged population. The pathological hallmarks of AD are amyloid plaques and neurofibrillary tangles (Selkoe 2004). Increasing evidence has shown that mitochondrial dysfunction is a prominent feature of AD (Wang et al. 2014). Amyloid-b peptide (Ab), which is the major component of senile plaques, elicits neurotoxicity and has a pivotal function in AD pathogenesis (Deshpande et al. 2006). Ab has been reported to interfere with multiple aspects of mitochondrial function. Xie et al. have observed severe structural and functional abnormalities of mitochondria in the immediate vicinity of Ab plaques (Xie et al. 2013). Studies using neuronal cell culture demonstrated that Ab causes morphological alteration of mitochondria, ATP depletion, and production of ROS (Casley et al. 2002; Cha et al. 2012; Chen and Yan 2007; Dong et al. 2010). The exposure of isolated mitochondria to Ab reduces complex IV activity (Canevari et al. 1999) and induces the formation of the permeability transition pore (Du et al. 2008). In addition, Ab progressively accumulates within mitochondria of both human AD brain and Tg mouse models for AD (Chen and Yan 2007) and interacts with the mitochondrial proteins, including cyclophilin D, amyloid binding alcohol dehydrogenase, and Drp1 (Benek et al. 2015). This interaction has a causative role in impairing mitochondrial physiological functions. Mitochondrial biogenesis can be defined as the growth and division of pre-existing mitochondria, which plays an
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essential role in maintaining healthy mitochondria in eukaryotic cells during the life cycle of mitochondria. Recently, impaired mitochondrial biogenesis is observed in the brain of AD patients, a mouse model of AD, and APPswe M17 cells (Calkins et al. 2011; Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012). Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in AD (Sheng et al. 2012). Moreover, mitochondrial biogenesis is severely affected and significantly decreases after injection of Ab in hippocampal CA1 area of rats (Shaerzadeh et al. 2014). However, whether Ab suppresses mitochondrial biogenesis in primary hippocampal neurons remains unclear. AMP-activated protein kinase (AMPK) is a critical regulator of mitochondrial biogenesis which regulates intracellular energy metabolism in response to acute energy crises (Hardie 2007). AMPK activates sirtuin 1 (SIRT1) by increasing cellular NAD? levels, resulting in the deacetylation and modulation of the activity of peroxisome proliferator-activated receptor-c coactivator 1-alpha (PGC-1a) (Canto et al. 2009; Fulco and Sartorelli 2008). AMPK can also directly activate PGC-1a (Jager et al. 2007). SIRT1, the class III NAD?-dependent histone deacetylase, has been shown to combat aging, extend lifespan, and regulate metabolism (Kwon and Ott 2008). In addition, SIRT1 has been proposed as an important regulator of mitochondrial biogenesis (Canto et al. 2009; Gerhart-Hines et al. 2007). SIRT1 physically interacts with and deacetylates PGC-1a (Nemoto et al. 2005; Rodgers et al. 2005). The peroxisome proliferator-activated receptor a (PPARa) is a fatty acidactivated nuclear receptor that plays a key role in the transcriptional regulation of genes involved in energy metabolism. PGC-1 is a coactivator of PPARa and binds PPARa in a ligand-influenced manner (Vega et al. 2000). PGC-1a belongs to the PGC-1 family of regulated coactivators, including PGC-1b and PRC which play a central role in a regulatory network governing the transcriptional control of mitochondrial biogenesis (Lin et al. 2005; Scarpulla 2011). PGC-1a targets multiple transcription factors including nuclear respiratory factor 1 (NRF 1) and nuclear respiratory factor 2 (NRF 2), which activate mitochondrial transcription factor A (Tfam) (Rohas et al. 2007; Wu et al. 1999). Tfam drives transcription and replication of mitochondrial DNA (mtDNA) (Virbasius and Scarpulla 1994). Additionally, previous studies showed that AMPK activity and the expression of SIRT1 and PGC1a were reduced in AD brain (Julien et al. 2009; Qin et al. 2009; Sheng et al. 2012). We propose that Ab-induced mitochondrial dysfunction may be associated with mitochondrial biogenesis. We hypothesize that Ab can suppress AMPK and SIRT1, both of which further downregulate PGC-1a and the
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downstream mitochondrial biogenesis program. To test this hypothesis, we used primary hippocampal neurons as a model system to study the effects of Ab25–35 on mitochondrial biogenesis by measuring the expression of the AMPK-SIRT1-PGC-1a pathway, as well as the levels of transcription factors involved in mitochondrial biogenesis and mitochondrial components such as mtDNA copy number.
Materials and Methods Primary Hippocampal Neuron Culture and Treatment Primary cultures of hippocampal neurons were prepared as described in our previous study (Dong et al. 2010). Briefly, we dissected newborn (P0, 0–24 h) Sprague–Dawley rats to obtain primary hippocampal neurons. We explanted, plated, and maintained the neurons in serum-free DMEM/F12 medium supplemented with 2 % B27 and 1 % penicillin– streptomycin. Half of the growth medium was changed every 3 days thereafter. All experiments were performed according to ‘‘National Institute of Health Guide for the Care and Use of Laboratory Animals’’ (NIH Publications No. 80-23) and were approved by the Committee on Ethical Use of Animals of Fujian University of Traditional Chinese Medicine. Ab25–35 (Sigma-Aldrich, USA) stock solution of 1 mM was prepared in sterile distilled water and stored at -20 °C. To obtain the fibrillar form of Ab25–35, an aliquot of the stock solution was incubated at 37 °C for 4 days before use (Tohda et al. 2004). Ab25–35 was used at a final concentration of 25 lM according to the previous studies (Bulbarelli et al. 2009; Dong et al. 2010; Resende et al. 2007). Cells were treated with 25 lM Ab25–35 for 24 h on 10 days in vitro (DIV). After that, treatment cells were harvested for subsequent assays. Measurement of Cell Viability After 24-h incubation with Ab25–35, cell viability was determined using the MTT assay as previously described (Mosmann 1983). In brief, MTT was dissolved in phosphate-buffered saline (PBS) and was added to culture medium. Hippocampal neurons were incubated with MTT (0.5 mg/ml) for 4-h incubation at 37 °C. Next, the medium containing MTT was removed followed by the addition of 150 ll DMSO per well (96-well plate) to dissolve the formazan crystals. The absorbance was measured at 570 nm using an ELISA plate reader. Results were expressed as percentage (%) of MTT reduction, assuming the absorbance of control cells as 100 %.
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Total Protein Extraction
Real-Time Quantitative RT-PCR
Primary hippocampal neurons were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors cocktail (Sigma-Aldrich, USA) and sonicated. The cell lysates were centrifuged at 15,0009g for 10 min at 4 °C and supernatants were collected. Protein concentrations were determined using the BCA protein assay (Thermo Scientific).
Total RNA was isolated from hippocampal neurons using the RNeasy RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. RNA concentrations and quality were measured by Nanodrop Spectrophotometer ND1000 (Thermo Scientific, Wilminton, DE). We synthesized cDNA from 0.5 lg of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Real-time quantitative RT-PCR (qRT-PCR) was performed using SYBR Green PCR master mix (Applied Biosystems) and a 7300 real-time PCR system (Applied Biosystems, CA, USA). Sequences of specific primers are the following: SIRT1 forward, 50 -TCGTGGAG ACATTTTTAATCAGG-3 and reverse 50 -GCTTCATGAT GGCAAGTGG-3; PGC-1a forward, 50 -GTGCAGCCAAG ACTCTGTATGG-30 and reverse 50 -GTCCAGGTCATTCA CATCAAGTTC-30 ; NRF 1 forward, 50 -TTACTCTGCTGT GGCTGATGG-30 and reverse 50 -CCTCTGATGCTTGCGT CGTCT-30 ; NRF 2a forward, 50 -AGGTGAGGAGATGGG CTGC-30 and reverse 50 -CGTTGTCCCCATTTTTGCG-3; Tfam forward, 50 -GAAAGCACAAATCAAGAGGAG-30 and reverse 50 -CTGCTTTTCATCATGAGACAG-30 ; 18S forward, 50 -TCCCTAGTGATCCCCG AGAAGT-30 and reverse 50 -CCCTTAATGGCAGTGATAGCGA-30 . The data from qRT-PCR were analyzed with delta–delta threshold cycle (DDCt) method using 18S as the internal control gene. Each DCt value was determined by subtracting 18S Ct value from the target gene Ct value. The DDCt was calculated by subtracting the DCt value of the control from the DCt value of the Ab25–35 sample. 2DDCt represented the average relative amount of mRNA for each target gene.
Western Blotting Analysis Equal amounts of proteins (30 lg) were separated using SDS-PAGE and transferred to nitrocellulose membranes (Millipore). Membranes were blocked in 5 % milk in 19 Tris-buffered saline with 0.05 % Tween 20 (TBS-T) for 1 h at room temperature with shaking. Membranes were incubated with Abs against AMPK (1:1000, Cell Signaling), phosphorylated AMPK (1:1000, Cell Signaling), SIRT1 (1:1000, Abcam), PGC-1a (1:1000, Millipore), NRF 1 (1:1000, Santa Cruz, CA, USA), NRF 2a (1:1000, Santa Cruz, CA, USA), NRF 2b (1:1000, Santa Cruz, CA, USA), and Tfam (1:1000, Santa Cruz, CA, USA). Membranes were washed three times in TBS-T for 5 min each wash. We applied appropriate horseradish peroxidaseconjugated secondary antibody at a concentration of 1:2000 for 1 h at room temperature with shaking. We washed membranes three times in TBS-T and applied the Supersignal West Pico chemiluminescent reagent (Thermo Scientific) for 3 min. The signals of the membrane were scanned using the FluorChem Scanner and quantified with the NIH Image J software. These results were normalized with b-actin expression levels and confirmed by triplicate measurements of the same sample. Immunoprecipitation For examination of PGC-1a acetylation, immunoprecipitation was performed according to the immunoprecipitation method provided by Abcam. The protein samples were precleared with 20 ll of protein G (Sigma-Aldrich, USA) for 3 h and then centrifuged to obtain supernatant at 15,0009g for 10 min. PGC-1a antibody (1:1000, Millipore, USA) and 30 ll of protein G were added to the precleared supernatant and incubated for 12 h at 4 °C. The protein G agarose was washed three times with cold PBS for 15 min. The immunoprecipitated protein was visualized and blotted using the Western blotting method. PGC-1a acetylation was measured using acetyl-lysine antibody (Cell Signaling).
Mitochondrial DNA Quantification Total cellular DNA in hippocampal neurons was extracted using Blood & Cell Culture DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. mtDNA copy number was measured by real-time PCR method using the Step one plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with the SYBR Green detection method as previously described (Sheng et al. 2012). Each real-time PCR (20 ll total volume) contained 3 ll of template DNA, 10 ll of 29 SYBR Green Real-time PCR Master (Applied Biosystems), 1 ll of each of the forward and reverse primers, and 5 ll of ultrapure water. The primers for cytochrome b were forward, 50 -AAAGCCACCT TGACCCGATT-30 and reverse 50 -GATTCGTAGGGCCG CGATAGG-30 (Casuso et al. 2014). Primers for 18S were
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forward, 50 -GGTGCATGGCCGTTCTTA-30 and reverse 50 -TCGTTCGTTATCGGAATTAACC-30 . The ratio of cytochrome b to 18S represents the relative mitochondrial copy number. Statistical Analysis Results are presented as the mean ± SEM. Statistical significance of Ab25–35-induced differences was evaluated by Student’s t test assuming equal variance, and a value of P \ 0.05 was considered significant.
Results Ab25–35 Induces Cytotoxicity in Primary Hippocampal Neurons It has been reported that Ab25–35 aggregates and retains the neurotoxic activities just like the full-length Ab (Pike et al. 1993). Thus, we first investigated the neurotoxic effect of Ab25–35 in primary culture of rat brain hippocampal neurons using the MTT assay. Cell viability decreased to 70.5 % after 24-h incubation with 25 lM Ab25–35 at DIV 10 (Fig. 1). Ab25–35 Downregulates Phosphorylation of AMPK and SIRT1 Expression AMPK is a key regulatory factor for mitochondrial biogenesis (Fulco et al. 2008). The degree of AMPK phosphorylation (p-AMPK) was used as a measure of AMPK
activation. It has been reported that AMPK activity was reduced in the hippocampus of a mouse model of AD (Pedros et al. 2014). Thus, we investigated whether Ab25–35 suppressed AMPK activity in cultured hippocampal neurons. It is evident from Fig. 2 that a significant decrease in p-AMPK/t-AMPK was observed after Ab25–35 treatment, which indicates that Ab25–35 suppresses phosphorylation of AMPK. However, there was no significant difference in t-AMPK protein levels after 24-h incubation with Ab25–35 (Fig. 2b, P [ 0.05). AMPK has been known to regulate SIRT1 expression (Canto et al. 2009). Thus, we further measured SIRT1 expression after Ab25–35 treatment. Our data showed that the SIRT1 protein levels were decreased after Ab25–35 treatment (Fig. 2). Additionally, we also found that the SIRT1 mRNA level was reduced after 24 h of Ab25–35 treatment (Fig. 4). Ab25–35 Decreases PGC-1a Expression and Increases PGC-1a Acetylation AMPK could directly activate PGC-1a (Jager et al. 2007). Moreover, SIRT1 interacts with and deacetylates PGC-1a (Nemoto et al. 2005; Rodgers et al. 2005). Thus, we next evaluated the effect of Ab25–35 on PGC-1a expression and PGC-1a deacetylation in primary hippocampal neurons. In our studies of PGC-1a expression level after Ab25–35 treatment, a significant decrease in the mRNA level and protein level of PGC-1a was observed (Figs. 2, 4). In accordance with decreased protein levels of SIRT1, there was an increased in the acetylation state of PGC-1a, a target for SIRT1 deacetylase activity (Fig. 3). Ab25–35 Suppresses Mitochondrial Biogenesis Program
Fig. 1 Effect of Ab25–35 on the viability of primary cultured hippocampal neurons. Cultured hippocampal neurons were treated with 25 lM Ab25–35 for 24 h. After that, cell viability was evaluated using the MTT assay. *P \ 0.05 compared with the control
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To examine whether mitochondrial biogenesis program is downregulated in response to Ab25–35, qRT-PCR and Western blotting were performed to compare the expression of downstream genes of PGC-1a. We first analyzed the gene expression of NRF 1, NRF 2, and Tfam. The mRNA levels of NRF 1, NRF 2a, and Tfam were significantly reduced after Ab25–35 treatment (Fig. 4). Similar to mRNA levels, a significant decrease was observed in protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam (Fig. 5). To directly determine whether mitochondrial biogenesis is affected by Ab25–35, we measured mtDNA relative to nDNA (mtDNA/nDNA) by real-time PCR and found that mtDNA/nDNA was significantly reduced in cultured hippocampal neurons treated with Ab25–35 (Fig. 6). In summary, these data demonstrate that the mitochondrial biogenesis program is downregulated by Ab25–35.
Cell Mol Neurobiol Fig. 2 Ab25–35 suppressed phosphorylation of AMPK and the protein levels of SIRT1 and PGC-1a in hippocampal neurons. a Western blotting analysis of total and phosphorylated AMPK (t-AMPK and p-AMPK), SIRT1, and PGC-1a protein in cultured hippocampal neurons after 24 h of Ab25–35 treatment. b Quantification of immunoblots in (a). c The ratio between p-AMPK and t-AMPK. b-actin was used as an internal loading control. *P \ 0.05 compared with the control
Fig. 3 Ab25–35 increased acetylation levels of PGC-1a in hippocampal neurons. Hippocampal neurons were treated with Ab25–35, and total lysates were used to test PGC1a acetylation. Relative acetylation levels of PGC-1a are shown on the right. *P \ 0.05 compared with the control
Discussion This study provides in vitro an evidence in support of the hypothesis that Ab suppresses mitochondrial biogenesis in hippocampal neurons. Two types of evidences were obtained. First, key molecules of the mitochondrial biogenesis pathway, including energy-sensing AMPK and SIRT1 as well as their downstream targets such as PGC-1a,
are downregulated by Ab25–35. Second, mtDNA levels are decreased after Ab25–35 treatment. The hippocampus is a key brain structure for learning and memory and among the first brain regions to be affected in AD, which arises on the pathological background of amyloid b plaques, neurofibrillary tangles, and synaptic and neuronal loss (Ittner and Gotz 2010). In addition, a number of studies have used cultured
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Fig. 4 Ab25–35 inhibited the mRNA levels of SIRT1, PGC-1a, NRF 1, NRF 2a, and Tfam in hippocampal neurons. The mRNA levels of SIRT1, PGC-1a, NRF 1, NRF 2a, and Tfam were determined using qRT-PCR and normalized to 18S. Results are expressed as mean ± SEM of triplicate samples in one representative experiment from 3 independent trials. *P \ 0.05 compared with the control
Fig. 6 Ab25–35 decreased mtDNA copy number. Hippocampal neurons were exposed to 24 h Ab25–35 and harvested for total DNA extraction. Using real-time PCR, the relative mitochondrial DNA copy number was determined by the ratio of cytochrome b to 18S. *P \ 0.05 versus the control
hippocampal neurons to explore the pathophysiological mechanism of AD (Filipcik et al. 2009; Joshi et al. 2014). Thus, we used cultured hippocampal neurons in this study. Ab is believed to play a central role in the development of AD (Hardy and Selkoe 2002). Ab25–35, a synthetic peptide corresponding to amino acids 25-35 in Ab1-40 and Ab1-42, possesses the same b-sheet structure (Kaminsky et al. 2010). Ab25–35 has been used in a number of studies and found to have many effects in common with full-length Ab1-40/42, including its toxicity (Pike et al. 1993; Yankner et al. 1990). Additionally, it has been reported that Ab25–35 is more toxic for cellular survival than Ab1-42 in cultured cortical neurons (Arancibia et al. 2008). Our data showed that Ab25–35 induced toxicity in cultured hippocampal neurons, which was consistent with previous studies (Arancibia et al. 2008; Dong et al. 2010; Resende
et al. 2007). We and other groups have further demonstrated Ab25–35 caused mitochondrial dysfunction in cultured neurons (Casley et al. 2002; Cha et al. 2012; Dong et al. 2010). In the present study, we found a decrease in mtDNA copy number in cultured hippocampal neurons treated with Ab25–35. Our results also showed that mRNA levels and protein levels of mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam) were reduced after 24-h incubation with Ab25–35. Similarly, intrahippocampal injection of Ab1-42 in rats resulted in decreases in PGC-1a, NRF 1, and Tfam (Shaerzadeh et al. 2014). Taken together, these findings indicate that Ab suppresses mitochondrial biogenesis both in vitro and in vivo. AMPK is a key regulatory factor not only for energy metabolism but also for mitochondrial biogenesis (Fulco et al. 2008). AMPK has been shown to regulate SIRT1 and PGC-1a, causing incremental elevations in mitochondrial
Fig. 5 Effects of Ab25–35 on the protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam in cultured hippocampal neurons. Western blotting analysis showed that the protein levels of NRF 1, NRF 2a, NRF 2b, and Tfam were reduced in hippocampal neurons after 24 h of Ab25–35 treatment. b-actin was used as an internal control. *P \ 0.05 compared with the control
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biogenesis (Canto et al. 2009; Ruderman et al. 2010). In addition, SIRT1 is required for AMPK activation in resveratrol treatment on mitochondrial biogenesis (Price et al. 2012). AMPK activity was decreased in AD animal models (Pedros et al. 2014). Activation of AMPK can enhance mitochondrial biogenesis and improve mitochondrial function in cell lines (Dong et al. 2013; Shin et al. 2009) and animal models (Moran et al. 2013; Ng et al. 2012). Conversely, the inhibition of AMPK leads to a decrease in mitochondrial biogenesis (Wang and Brautigan 2013). Our data clearly show that Ab25–35 reduces AMPK activity in vitro, which indicates that Ab25–35 results in a decrease in mitochondrial biogenesis. In fact, our present results demonstrate that Ab25–35 suppresses mitochondrial biogenesis. Additionally, our previous study showed that treatment with 25 lM Ab25–35 for 24 h caused mitochondrial dysfunction in cultured hippocampal neurons (Dong et al. 2010). Taken together, Ab25–35 results in both impairment of mitochondrial biogenesis and mitochondrial dysfunction, which further supports the notion that impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in AD (Sheng et al. 2012). SIRT1 and AMPK have been shown to play many similar roles, including their ability to induce mitochondrial biogenesis and regulate the activity of PGC-1a (Fulco and Sartorelli 2008). SIRT1 promotes mitochondrial biogenesis through deacetylation and activation of PGC-1a (Gerhart-Hines et al. 2007; Rodgers et al. 2005). SIRT1 confers significant neuroprotection in a mouse model of AD likely through inhibition of PGC-1a acetylation and promotion of PGC-1a activity (Kim et al. 2007). The acetylation status of PGC-1a has been implicated as a fundamental post-translational modification that regulates its transcriptional activity (Canto et al. 2009; Nemoto et al. 2005). We clearly show that the expression levels of SIRT1 and PGC-1a are reduced after Ab25–35 treatment, which is consistent with the data showed a decrease in SIRT1 expression in SK-N-SH cells treated with Ab1-42 (Sun et al. 2014). Moreover, reduced SIRT1 expression subsequently suppressed PGC-1a deacetylation. These data indicate that Ab25–35 decreases not only PGC-1a expression but also PGC-1a activity. Previous studies showed that mtDNA copy number and the expression levels of PGC-1a, NRF 1, NRF 2, and Tfam were significantly reduced in AD hippocampus and APPswe M17 cells (Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012), which suggests that the mitochondrial biogenesis signaling is impaired in AD. Moreover, PGC-1a overexpression was shown to completely rescue whereas knockdown of PGC-1a exacerbated impaired mitochondrial biogenesis in APPswe M17 cells (Sheng et al. 2012). Based on previous findings and those of the current study, there is
a clear evidence that SIRT1 and PGC-1a play an important role in impaired mitochondrial biogenesis caused by Ab25–35. Consistent with previous studies (Pedros et al. 2014; Rice et al. 2014; Sheng et al. 2012), our data show that Ab25–35 suppresses mitochondrial biogenesis factors (PGC-1a, NRF 1, NRF 2, and Tfam). We further show that Ab25–35 downregulates phosphorylation of AMPK and SIRT1 expression. NRF 2a protein levels remained unchanged in APPswe M17 cells compared with M17 cells (Sheng et al. 2012). Interestingly, we showed that NRF 2a protein levels were decreased after 24-h incubation with Ab25–35. The discrepancy may be due to different cells and different treatment between our study and their study. It has been reported that AMPK-SIRT1-PGC-1a signaling is implicated in energy metabolism, mitochondrial function, and apoptosis (Chau et al. 2010; Wei et al. 2011). Activation of AMPK-SIRT1-PGC-1a pathway has been shown to play an important role in exercise and temperature-induced mitochondrial biogenesis (Li et al. 2011; Liu and Brooks 2012). Conversely, inhibition of AMPK-SIRT1-PGC-1a signaling is involved in impaired mitochondrial biogenesis caused by Ab25–35. Overall, AMPK-SIRT1-PGC-1a signaling is associated with mitochondrial biogenesis. Mitochondrial biogenesis is an important and exciting area of cell biology. Lack of normal mitochondrial function has a negative impact in insulin resistance and aging (Petersen et al. 2003). In the current study, we show that mitochondrial biogenesis is downregulated in response to Ab25–35 in hippocampal neurons. The inhibition of AMPK-SIRT1-PGC-1a pathway and its downstream targets (NRF 1, NRF 2, and Tfam) is associated with the Ab25–35-induced changes in mitochondrial biogenesis. One limitation of our study is that hippocampal neurons culture system may not entirely reflect what occurs in vivo. Our findings need to be confirmed in animal models to see whether the Ab25–35-induced effects are reproducible in vivo. Future studies are also needed to test the hypothesis of a causal link between AMPK-SIRT1-PGC-1a inhibition and Ab25–35-induced mitochondrial biogenesis in hippocampus. If so, then, Ab25–35 may have the effect of decreasing the background levels of metabolic gene expression. The results presented here suggest that stimulation of pathways upstream of mitochondrial biogenesis and increased mitochondrial biogenesis may be a potential therapeutic approach for AD. Acknowledgments This work was supported by the National Natural Science Foundation of China (Project 81102625), the Natural Science Foundation of Fujian Province Grants (Project 2012J05154), and study abroad scholarships of Fujian Province. Conflict of interest The authors declare no financial or other conflicts of interest related to this study.
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