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Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10 Xiao-liang Wang a, Qing Liu a, *, Guo-Jun Chen b, Mei-ling Li a, Yan-hui Ding a a b
Department of Clinical Research Center, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Chongqing Key Laboratory of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2016 Accepted 24 November 2016 Available online xxx
Alzheimer's disease (AD) is characterized by the deposition of b-amyloid (Ab) peptide in the brain, which is produced by the proteolysis of b-amyloid precursor protein (APP). Recently, the mitochondrial transcription factor 4 (MTERF4), a member of the MTERF family, was implicated in regulating mitochondrial DNA transcription and directly in controlling mitochondrial ribosomal translation. The present study identified a novel role for MTERF4 in shifting APP processing toward the amyloidogenic pathway. The levels of MTERF4 protein were significantly increased in the hippocampus of APP/PS1 mice. In addition, the overexpression of MTERF4 induced a significant increase in the levels of APP protein and secreted Ab42 in HEK293-APPswe cells compared with control cells. Further, MTERF4 overexpression shifted APP processing from a-to b-cleavage, as indicated by decreased C83 levels and elevated C99 levels. Finally, the MTERF4 overexpression suppressed a disintegrin and metalloproteinase 10 (ADAM10) expression via a transcriptional mechanism. Taken together, these results suggest that MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10 in HEK293-APPswe cells; therefore, MTERF4 may play an important role in the pathogenesis of AD. © 2016 Elsevier Inc. All rights reserved.
Keywords: Alzheimer's disease Amyloid precursor protein MTERF4 ADAM10 BACE1
1. Introduction Alzheimer's disease (AD), which is associated with memory deterioration and other cognitive domains and eventually leads to death, is the most common form of dementia in the elderly [1]. Existing in both familial and sporadic forms, AD is characterized by two major pathological hallmarks: the extracellular deposition of amyloid-b (Ab) peptide in senile plaques, and the intracellular accumulation of neurofibrillary tangles (NFTs) that are composed of hyperphosphorylated tau protein. Several morphological and functional changes are associated with these lesions in the diseased brain, such as neuronal and synaptic alterations, astrogliosis, and microglial cell activation [2]. Although the precise physiologic changes that trigger the development of AD largely remain unknown, the abnormal metabolism of amyloid precursor protein (APP) into Ab plays a major causative role in AD pathogenesis [3]. The cleavage and processing of APP is achieved by groups of
* Corresponding author. Department of Clinical Research Center, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400016, China. E-mail address: [email protected] (Q. Liu).
enzymes or enzyme complexes termed a-, b- and g-secretases; the processing can be divided into an amyloidogenic pathway and a non-amyloidogenic pathway. In the amyloidogenic pathway, cleavage by b-site APP-cleaving enzyme 1 (BACE1) releases the soluble APP N-terminal fragment (sAPPb), thereby leaving a 99amino-acid C-terminal stub (C99) in the membrane, which is the direct precursor to Ab. Intracellularly, the subsequent actions of gsecretase on C99 produce Ab40/42 peptides and APP intracellular domain (AICD). In the non-amyloidogenic pathway, most of the APP is cleaved at the plasma membrane by a-secretase, which precludes Ab formation but produces soluble sAPPa and small membranebound 83-amino-acid C-terminal fragment (C83). Furthermore, C83 is cleaved by the g-secretase complex, resulting in the release of P3 peptide and AICD [4]. Several proteases have been suggested as AD-relevant a-secretases, many of which belong to the family of a disintegrin and metalloprotease (ADAM), including ADAM9, ADAM10, and ADAM17 [5]. Strong evidence supports a role for ADAM10, but not ADAM9 or -17, as the constitutive and inducible APP a-secretase in neurons [6]. The neuronal overexpression of ADAM10 increased the secretion of sAPPa, prevented plaque formation and alleviated cognitive defects in a transgenic AD mouse model [7]. Blocking the transport of ADAM10 to the postsynaptic
http://dx.doi.org/10.1016/j.bbrc.2016.11.135 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135
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membrane shifted APP metabolism toward amyloidogenesis and generated a model of sporadic AD (SAD) [8]. Therefore, ADAM10 was identified as the major a-secretase in vivo and dysregulation of ADAM10 may play a significant role in the establishment of Ab pathology. Mitochondrial dysfunction induced by various factors has been strongly implicated in the pathogenesis of AD [9]. In recent years, mitochondrial transcription termination factors (MTERFs) have been implicated in regulating of mitochondrial transcription, replication, and translation. Four MTERFs have been identified in vertebrates, designated MTERF1e4 [10]. To date, MTERF4 is the least studied MTERF family member; however, recent studies provided the first insight into the function of this protein in mitochondria. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4, and is necessary for recruiting of this factor to the large ribosomal subunit [11]. MTERF4 may play an important role in regulating mitochondrial dysfunction in cellular models of Parkinson's disease [12]. These findings highlight the importance of MTERF4 in regulating mitochondrial function. Increasing evidence suggests that mitochondrial defects play a key role in AD pathogenesis [13]. It is therefore plausible that abnormal MTERF4 activity could contribute to the pathogenesis of AD. In vivo and in vitro studies support a major role for mitochondria and bioenergetics in the fate of APP processing and cellular trafficking. No attempt has been made to elucidate the relationship between MTERF4 and APP processing. In this study, we show that MTERF4 has a novel role in reducing the a-secretase-mediated proteolysis of APP by suppressing the transcription of ADAM10. This mechanism may play an important role in AD pathogenesis. 2. Materials and methods
M596L), were grown in the same culture medium with the addition of 1 mg/ml geneticin (G418). The cells were maintained in a humidified 5% CO2 atmosphere at 37 C and transfected with the designed constructs and empty vectors using Lipofectamine 2000 (Invitrogen). 2.4. Quantitative real-time PCR Total RNA was extracted from transfected cells using the Trizol total RNA extraction kit (Sangon) and then 1 mg total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) according to the manufacturer's protocol. Quantitative PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) with a Prefect Real Time SYBR Premix Ex Taq Kit (TaKaRa). The primers used are listed in Table 1. 2.5. Western blotting Total protein was extracted from frozen mice hippocampal tissue or HEK293-APPswe cells using lysis buffer. The samples (cell or tissue extract) were boiled in the presence of b-mercaptoethanol for 10 min and loaded onto 10% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were incubated individually with primary antibodies against MTERF4 (1:1000, Abcam), C-terminal APP (1:1000, Sigma), ADAM10 (1:500, Abcam), BACE1 (1:500, Abcam), or GAPDH (1:1000, Abcam) overnight at 4 C. The membranes were then incubated with peroxidaseconjugated goat anti-mouse IgG antibodies or goat anti-rabbit IgG antibodies (1:5000, Abcam) for 1 h. Finally, the blots were exposed to ECL detection reagent (Millipore). Images were captured using a e, Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Valle France) and the bands were quantified using ImageJ software.
2.1. Animal experiments All animal studies were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University. Male and female APP/PS1 transgenic B6C3-Tg (APPswe/PSEN1dE9) mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). Wild-type (WT) mice in the C57BL/6 background were purchased from Chongqing Medical University and used as controls. All mice were provided with a standard diet and housed in an approved facility with climate control and a 12-h light/12-h dark cycle. Six-month-old APP/PS1 mice and WT counterparts were used for all experiments. 2.2. Plasmid construction The full-length open reading frame (ORF) of MTERF4 was generated as described previously [14]. The fragment containing the human MTERF4 gene ORF was sub-cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) to generate the recombinant plasmid pcDNA-MTERF4. All sequences were confirmed by DNA sequencing and matched with reference sequences found in the NCBI database.
2.6. Human Ab42 quantitation (ELISA) The effects of MTERF4 on Ab42 levels in the culture medium were assayed using a human Ab42-specific sandwich ELISA kit (Invitrogen). HEK293-APPswe cells were transfected with the designed constructs and empty vectors for 24 and 48 h; the supernatant was then collected and a protease inhibitor cocktail was added. The concentrations of Ab42 in diluted samples were determined according to the manufacturer's protocols. 2.7. Dual-luciferase reporter assay A DNA fragment comprising nucleotides from 2095 to 39 upstream of the human ADAM10 translation initiation site was amplified using the primers 5'-CAGCACTACCACAAGAGAT-3' and 5'TTAACAGCAGCACATCGA-3'. The fragment was sub-cloned into the firefly luciferase reporter vector pGL3-basic (Promega, Madison, WI, USA) to generate pGL3-ADAM10. The Renilla luciferase vector pRL-SV40 (Promega) was cotransfected to normalize the transfection efficiency. HEK293 cells were seeded in 24-well plates and transfected with the indicated vector. The transfected cells were
2.3. Cell culture and transfection HEK 293 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sangon, Shanghai, China), 100 IU/ml penicillin, and 100 mg/ml streptomycin. HEK293APPswe cells, the human embryonic kidney 293 cells transfected with human APP bearing the Swedish mutation (APPsw, K595N/
Table 1 Primers for quantitative real-time PCR. Gene
Forward primer
Reverse primer
MTERF4 GAPDH APP BACE1 ADAM10
50 -CGTAACTGCTGCCATCTT-30 5-CCATCACGCCACAGTTTC-30 50 -CAACCAACCAGTGACCAT-30 50 -GCCATCTCACAGTCATCC-30 50 -AGACCGAACTCTGCCATT-30
50 -GTCGTCCTTCTCTGTTCTC-30 50 -ATCCCATCACCATCTTCCAG-30 50 -GTGTGCCAGTGAAGATGA-30 50 -TGTAGCCACAGTCTTCCA-30 50 -TTCCTCTACACCAGTCATCT-30
Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135
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cultivated for 48 h, washed twice with phosphate-buffered saline (PBS), and then lysed with lysis buffer. Firefly and Renilla luciferase activities were measured sequentially using a dual luciferase reporter assay system and the GloMax-Multi Jr Single Tube Multimode Reader (Promega). 2.8. Cell cycle analysis HEK293-APPswe cells were transfected with pcDNA-MTERF4 and vector control for 24 and 48 h. The cells were trypsinized and washed twice with cold PBS. After fixing in 70% ethanol at 4 C overnight, the cells were washed and stained with propidium iodide (PI) by incubation with PI/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocols. The DNA content in individual samples was quantified on a FACSCalibur flow cytometer (BD Biosciences). 2.9. Cell proliferation assay Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assays (Sangon) according to the manufacturer's instructions. HEK293-APPswe cells were seeded in 96-well plates at a density of 2 103 cells per well. After 24 and 48 h incubation, the cell monolayer was rinsed twice in PBS, and then diluted CCK-8 solution (diluted 1:10 in DMEM) was added to the cells and incubated for 2 h at 37 C. The absorbance in each well at 450 nm was measured using a microplate reader. 2.10. Statistical analysis The data are presented as the means ± SEM of at least three independent experiments. Statistical significance was determined using Student's t-tests or ANOVA using SPSS 19.0 software. A p value <0.05 was considered significant. 3. Results 3.1. MTERF4 expression is upregulated in the hippocampus of APP/ PS1 mice To determine whether the expression of MTERF4 differed in APP/PS1 mice compared with control, hippocampal samples obtained from 6-month-old APP/PS1 mice and age-matched nontransgenic C57BL/6 controls were compared. Western blotting showed that the levels of APP protein were dramatically increased by 159% in the hippocampus of APP/PS1 mice compared with WT control (Fig. 1A and B). Furthermore, the levels of MTERF4 protein were significantly upregulated by 68% in APP/PS1 mice hippocampus compared with WT control (Fig. 1A, C). 3.2. MTERF4 promotes the amyloidogenic processing of APP in HEK293-APPswe cells The recombinant plasmid pcDNA-MTERF4 and vector control were transiently transfected into HEK293-APPswe cells. The levels of the MTERF4 protein were significantly increased by 67% and 258%, respectively, when cells were transfected for 24 and 48 h compared with control (Fig. 1D and E). Next, quantitative real-time PCR showed that MTERF4 mRNA levels were increased 235 and 216-fold at 24 and 48 h in pcDNA-MTERF4-transfected cells compared with control (Fig. 1F). Thus, MTERF4 was highly expressed in pcDNA-MTERF4 transfected cells. The overexpression of MTERF4 induced a significant increase (186%) in the levels of APP protein in MTERF4-overexpressing cells
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compared with control at 48 h (Fig. 2A and B). In addition, MTERF4 shifted APP processing from a-to b-cleavage 48 h after transfection, as indicated by decreased C83 levels (Fig. 2A, D) and clearly elevated C99 levels (Fig. 2A, C). Next, APP mRNA levels were assessed, which revealed that the overexpression of MTERF4 did not influence APP expression (Fig. 2E). Because a-secretase not only increases the secretion of sAPPa but also decreases the production of neurotoxic Ab, we next measured the effect of MTERF4 on Ab42 peptide levels in the medium. Consistent with the suppressive effects of MTERF4 on a-secretase, a stimulatory effect on Ab42 peptide was observed. Forty-eight hours after transfection, the secretion of Ab42 peptide into the medium was 58.1% higher than that of control (Fig. 2F). 3.3. ADAM10 expression is suppressed by MTERF4 We hypothesized that the decreased C83 levels and the elevated C99 levels in MTERF4-overexpressing cells might be caused by the dysregulated expression of APP-relevant secretases. To test this hypothesis, the protein expression of the a-secretase ADAM10 and the b-secretase BACE1 was measured. Quantification revealed a 27% decrease in ADAM10 levels in cells transfected with pcDNAMTERF4 at 48 h (Fig. 3A and B). As shown in Fig. 3D, ADAM10 mRNA expression was significantly reduced by 68% in MTERF4overexpressing cells at 48 h compared with control. However, MTERF4 had no effect on the expression of BACE1. At 24 and 48 h after transfection, the levels of BACE1 mRNA and protein were unchanged in MTERF4-overexpressing cells compared with control (Fig. 3C, E). Therefore, MTERF4 plays a role in APP processing by directly inhibiting the ADAM10-mediated a-cleavage of APP. 3.4. ADAM10 promoter activity is suppressed by MTERF4 To provide further evidence that MTERF4 regulates the expression of the a-secretase ADAM10 via a transcriptional mechanism, 2056 bp of the 5'-region upstream of the ADAM10 gene was subcloned into the pGL3-basic plasmid. Compared with the empty pGL3-Basic control, pGL3-ADAM10-transfected cells exhibited a significantly increased luciferase activity (Fig. 3F), suggesting that the region 2097 to 39 upstream of the ADAM10 translation site contains a human ADAM10 functional promoter. In addition, overexpressing MTERF4 for 48 h decreased the promoter activity compared with control cells transfected with pGL3-ADAM10 (Fig. 3F). Taken together, these results suggest that MTERF4 suppresses ADAM10 promoter activity. 3.5. MTERF4 overexpression has no effect on the cell cycle and proliferation To investigate the effects of MTERF4 overexpression on cell cycle progression, flow cytometry analysis was performed. The data revealed that there was no significant difference between control and MTERF4-overexpressing cells (Fig. 4A). The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle 24 and 48 h after transfection with pcDNA-MTERF4 were similar to controls in all groups (Fig. 4B). Consistent with the cell cycle results, no obvious differences in cell proliferation were observed at 24 and 48 h after transfection compared with control (Fig. 4C). Taken together, these data suggest that MTERF4 overexpression does not alter cell cycle distribution and proliferation in HEK293-APPswe cells. 4. Discussion One of the pathologic hallmarks of AD is the presence of
Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135
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Fig. 1. MTERF4 expression in the hippocampus of APP/PS1 mice and in HEK293-APPswe cells. (A) Representative Western blot showing APP, MTERF4 protein expression in the hippocampus of 6-month-old APP/PS1 mice, as compared with age-matched wild-type (WT) mice (n ¼ 3 for each group). (BeC) Quantification of protein levels of APP, MTERF4 in the hippocampus of APP/PS1 mice and WT mice. (D) Representative Western blot showing MTERF4 protein expression in control and MTERF4 overexpression cells. (E) Quantification of protein levels of MTERF4 in MTERF4 overexpression cells compared with control. (F) The relative mRNA expression of MTERF4 detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. Data are presented as means ± SEM. **p < 0.01 vs. control.
Fig. 2. MTERF4 overexpression promotes the amyloidogenic processing of APP in HEK293-APPswe cells. (A) Representative Western blot showing full length APP, C99 and C83 protein expression in control and MTERF4 overexpression cells. (BeD) Quantification of protein levels of APP, C99 and C83 in MTERF4 overexpression cells compared with control. (E) The relative mRNA expression of APP detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. (F) The amounts of Ab42 peptides in the culture media were analyzed by ELISA. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control.
extracellular amyloid plaques containing Ab, which is a peptidic fragment derived from the amyloidogenic proteolytic processing of APP by sequential cleavages involving b- and g-secretases [15]. APP can also be cleaved by a-secretase via a nonamyloidogenic pathway, which precludes the formation of pathogenesisassociated peptides and gives rise to a neurotrophic and neuroprotective fragment [16]. Activation of the amyloidogenic pathway is one mechanism behind the accumulation of Ab, as observed in cases of familial AD (FAD) that are associated with APP and presenilin (PS) mutations [17]. However, unlike autosomal-dominant FAD, SAD patients generally lack APP mutations [18]. Therefore, identifying the molecular mechanisms underlying pathogenesis of
SAD is essential for the development of novel therapeutics to treat or prevent this disorder. Significantly, the current study revealed that MTERF4 overexpression yielded similar impacts on human APP processing to those observed in SAD. First, MTERF4 expression was upregulated in the hippocampus of APP/PS1 mice. After transfecting HEK293-APPswe cells with MTERF4 for 48 h, APP expression was increased and Ab42 levels were increased in the media. Compared with Ab40, Ab42 is a highly fibrillogenic peptide that favors the formation of neurotoxic species [19]. Accordingly, the overexpression of MTERF4 reduced the production of C83 and increased the production of C99. This suggests that MTERF4 overexpression suppresses the a-site cleavage of APP.
Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135
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Fig. 3. ADAM10 Expression is suppressed in cells overexpressing MTERF4. (A) Representative Western blot showing ADAM10 and BACE1 protein expression in control and MTERF4 overexpression cells. (BeC) Quantification of protein levels of ADAM10 and BACE1 in MTERF4 overexpression cells compared with control. (D-E) Quantification of mRNA levels of ADAM10 and BACE1 detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. (F) Luciferase activity in cells transfected with the promoterless pGL3-Basic, ADAM10 promoter-luciferase construct pGL3-ADAM10 and recombinant plasmid pcDNA-MTERF4. Data are presented as means ± SEM. *p < 0.05, **p < 0.01 vs. control; #p < 0.05 vs. pGL-ADAM10 transfected cells; ##p < 0.01 vs. pGL3-Basic transfected cells.
Fig. 4. MTERF4 overexpression has no effect on the cell cycle and proliferation. (A) Cell cycle was measured by flow cytometry in control and MTERF4 overexpression cells. (B) Representative histograms for cell cycle distribution. Results are calculated distribution for cells in G0/G1, S, and G2/M phases. (C) Cell proliferation was measured using the CCK-8 assay. Data are presented as means ± SEM.
Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135
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Then, we investigated how MTERF4 affected APP processing. The current study further revealed that this a-cleavage inhibiting activity was due to the direct suppression of ADAM10 expression. There was a reduction in ADAM10 expression in cells transfected with MTERF4 for 48 h. In contrast, there were no differences in BACE1 expression. We hypothesized that the reduction in ADAM10 expression occurs via a transcriptional mechanism. Previous studies demonstrated that the region 2179 to 1 upstream of the ADAM10 translation initiation site possesses functional promoter activity and that the proximal region 508 bp to 300 bp is essential for constitutive ADAM10 promoter activity [20]. Therefore, we cloned 2056 bp of the 50 -region upstream of the human ADAM10 gene and demonstrated that this genomic DNA fragment contained a functional ADAM10 promoter. The promoter activity of this DNA fragment was decreased after transfection with MTERF4 for 48 h. Furthermore, MTERF4 overexpression had no significant effect on cell cycle distribution and proliferation in HEK293APPswe cells. Thus, these results may have important implications for both the initiation and progression of AD. Insights into MTERF4 function have been reported only recently, and the precise role of MTERF4 in mitochondrial regulation is unclear. MTERF4 is necessary for cell proliferation because MTERF4 knockdown in HeLa cells resulted in sub-G1 cell accumulation and cell death [14]. In vivo and in vitro studies confirmed that MTERF4 localizes to the mitochondria and is essential for embryonic development in the mouse [11,21]. MTERF4 forms a stable complex with a mitochondrial rRNA methyltransferase, NSUN4, and is required for the recruitment of NSUN4 to the large ribosomal subunit in an essential step in ribosomal biogenesis [22,23]. Based on the finding that MTERF4 was upregulated in the hippocampus of APP/PS1 mice, we investigated whether MTERF4 could modulate APP processing and thereby contribute to the pathogenesis of AD. Several studies revealed that mitochondrial dysfunction is one of the earliest pathological features of AD [24,25]. The current study demonstrated that MTERF4 is detrimentally associated with several pathological aspects of AD, including Ab generation and the dysregulation of ADAM10. Several lines of evidence suggest that Ab itself leads to mitochondrial dysfunction and increased ROS levels [26]. We propose that the upregulation of MTERF4 initiates a vicious cycle beginning with enhanced amyloidogenic APP processing. ADAM10 is the major a-secretase in vivo; therefore, it offers a new target for therapeutic manipulation in AD. The current study reveals a pathway for the downregulation of ADAM10 involving MTERF4. Several studies have shown a role for retinoid X-receptor and retinoic acid receptor in the induction of ADAM10 transcription in neuronal cell lines [20,27]. In addition to the retinoic acid-related signaling pathways, various other effectors have been established as ADAM10 transcription enhancers [28]. A G-rich region in the ADAM10 5'-UTR forms a highly stable G-quadruplex secondary structure, which contributes to the translational repression of ADAM10 [29]. The translation of ADAM10 mRNA into protein is also controlled by several microRNAs [30,31]. We demonstrated that ADAM10 is downregulated at the transcriptional level by MTERF4 overexpression, which reveals a possible pathway that accelerates the pathology of AD. In conclusion, the present study identified a novel role for MTERF4 in shifting APP processing toward the amyloidogenic pathway. Overexpression of MTERF4 increased APP protein levels, downregulated ADAM10 expression, and promoted amyloidogenesis. These data demonstrated that MTERF4 reduced ADAM10 levels via a transcriptional mechanism. The current findings suggest that the upregulation of MTERF4 may represent a novel risk factor for the onset and progression of AD. Further studies should help clarify whether MTERF4 has the potential to prevent Ab
generation and slow down the progression of AD. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by NSFC grants (numbers 81171197 & 81220108010) to G-J Chen. References [1] H.W. Querfurth, F.M. LaFerla, Alzheimer's disease, N. Engl. J. Med. 362 (2010) 329e344. [2] A. Serrano-Pozo, M.P. Frosch, E. Masliah, et al., Neuropathological alterations in Alzheimer disease, Cold Spring Harb. Perspect. Med. 1 (2011) a006189. [3] Y.W. Zhang, R. Thompson, H. Zhang, et al., APP processing in Alzheimer's disease, Mol. Brain 4 (2011) 3. [4] F.M. LaFerla, K.N. Green, S. Oddo, Intracellular amyloid-beta in Alzheimer's disease, Nature reviews, Neuroscience 8 (2007) 499e509. [5] J. Prox, A. Rittger, P. Saftig, Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and presenilin, Exp. Brain Res. 217 (2012) 331e341. [6] P.H. Kuhn, H. Wang, B. Dislich, et al., ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons, EMBO J. 29 (2010) 3020e3032. [7] R. Postina, A. Schroeder, I. Dewachter, et al., A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model, J. Clin. Investig. 113 (2004) 1456e1464. [8] R. Epis, E. Marcello, F. Gardoni, et al., Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer's disease, Brain J. Neurol. 133 (2010) 3323e3335. [9] Q. Cai, P. Tammineni, Alterations in mitochondrial quality control in Alzheimer's disease, Front. Cell. Neurosci. 10 (2016) 24. [10] M. Roberti, P.L. Polosa, F. Bruni, et al., MTERF factors: a multifunction protein family, Biomol. Concepts 1 (2010) 215e224. [11] Y. Camara, J. Asin-Cayuela, C.B. Park, et al., MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian mitochondrial ribosome, Cell metab. 13 (2011) 527e539. [12] X. Ye, Y. Han, L. Zhang, et al., MTERF4 regulates the mitochondrial dysfunction induced by MPP(þ) in SH-SY5Y cells, Biochem. Biophys. Res. Commun. 464 (2015) 214e220. [13] P.H. Reddy, R. Tripathi, Q. Troung, et al., Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer's disease: implications to mitochondria-targeted antioxidant therapeutics, Biochimica Biophys. Acta 1822 (2012) 639e649. [14] M. Yu, J. Dai, W. Huang, et al., hMTERF4 knockdown in HeLa cells results in sub-G1 cell accumulation and cell death, Acta Biochimica Biophys. Sinica 43 (2011) 372e379. [15] G. Evin, C. Hince, BACE1 as a therapeutic target in Alzheimer's disease: rationale and current status, Drugs & Aging 30 (2013) 755e764. [16] K. Endres, F. Fahrenholz, Regulation of alpha-secretase ADAM10 expression and activity, Exp. Brain Res. 217 (2012) 343e352. [17] R.E. Tanzi, L. Bertram, Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective, Cell 120 (2005) 545e555. [18] S. Lista, S.E. O'Bryant, K. Blennow, et al., Biomarkers in sporadic and familial Alzheimer's disease, J. Alzheimer's Dis. JAD 47 (2015) 291e317. [19] I. Kuperstein, K. Broersen, I. Benilova, et al., Neurotoxicity of Alzheimer's disease Abeta peptides is induced by small changes in the Abeta42 to Abeta40 ratio, EMBO J. 29 (2010) 3408e3420. [20] C. Prinzen, U. Muller, K. Endres, et al., Genomic structure and functional characterization of the human ADAM10 promoter, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 19 (2005) 1522e1524. [21] Q. Xu, F. Zhang, H. He, et al., Expression profile of mouse Mterfd2, a novel component of the mitochondrial transcription termination factor (MTERF) family, Genes & Genet. Syst. 86 (2011) 269e275. [22] H. Spahr, B. Habermann, C.M. Gustafsson, et al., Structure of the human MTERF4-NSUN4 protein complex that regulates mitochondrial ribosome biogenesis, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 15253e15258. [23] M.D. Metodiev, H. Spahr, P. Loguercio Polosa, et al., NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly, PLoS Genet. 10 (2014) e1004110. [24] H. Du, L. Guo, S. Yan, et al., Early deficits in synaptic mitochondria in an Alzheimer's disease mouse model, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 18670e18675. [25] S. Hauptmann, I. Scherping, S. Drose, et al., Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice, Neurobiol. aging 30 (2009) 1574e1586. [26] K. Leuner, T. Schutt, C. Kurz, et al., Mitochondrion-derived reactive oxygen species lead to enhanced amyloid beta formation, Antioxid. Redox Signal. 16
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Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10 Xiao-liang Wang a, Qing Liu a, *, Guo-Jun Chen b, Mei-ling Li a, Yan-hui Ding a a b
Department of Clinical Research Center, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Chongqing Key Laboratory of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 November 2016 Accepted 24 November 2016 Available online xxx
Alzheimer's disease (AD) is characterized by the deposition of b-amyloid (Ab) peptide in the brain, which is produced by the proteolysis of b-amyloid precursor protein (APP). Recently, the mitochondrial transcription factor 4 (MTERF4), a member of the MTERF family, was implicated in regulating mitochondrial DNA transcription and directly in controlling mitochondrial ribosomal translation. The present study identified a novel role for MTERF4 in shifting APP processing toward the amyloidogenic pathway. The levels of MTERF4 protein were significantly increased in the hippocampus of APP/PS1 mice. In addition, the overexpression of MTERF4 induced a significant increase in the levels of APP protein and secreted Ab42 in HEK293-APPswe cells compared with control cells. Further, MTERF4 overexpression shifted APP processing from a-to b-cleavage, as indicated by decreased C83 levels and elevated C99 levels. Finally, the MTERF4 overexpression suppressed a disintegrin and metalloproteinase 10 (ADAM10) expression via a transcriptional mechanism. Taken together, these results suggest that MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10 in HEK293-APPswe cells; therefore, MTERF4 may play an important role in the pathogenesis of AD. © 2016 Elsevier Inc. All rights reserved.
Keywords: Alzheimer's disease Amyloid precursor protein MTERF4 ADAM10 BACE1
1. Introduction Alzheimer's disease (AD), which is associated with memory deterioration and other cognitive domains and eventually leads to death, is the most common form of dementia in the elderly [1]. Existing in both familial and sporadic forms, AD is characterized by two major pathological hallmarks: the extracellular deposition of amyloid-b (Ab) peptide in senile plaques, and the intracellular accumulation of neurofibrillary tangles (NFTs) that are composed of hyperphosphorylated tau protein. Several morphological and functional changes are associated with these lesions in the diseased brain, such as neuronal and synaptic alterations, astrogliosis, and microglial cell activation [2]. Although the precise physiologic changes that trigger the development of AD largely remain unknown, the abnormal metabolism of amyloid precursor protein (APP) into Ab plays a major causative role in AD pathogenesis [3]. The cleavage and processing of APP is achieved by groups of
* Corresponding author. Department of Clinical Research Center, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400016, China. E-mail address: [email protected] (Q. Liu).
enzymes or enzyme complexes termed a-, b- and g-secretases; the processing can be divided into an amyloidogenic pathway and a non-amyloidogenic pathway. In the amyloidogenic pathway, cleavage by b-site APP-cleaving enzyme 1 (BACE1) releases the soluble APP N-terminal fragment (sAPPb), thereby leaving a 99amino-acid C-terminal stub (C99) in the membrane, which is the direct precursor to Ab. Intracellularly, the subsequent actions of gsecretase on C99 produce Ab40/42 peptides and APP intracellular domain (AICD). In the non-amyloidogenic pathway, most of the APP is cleaved at the plasma membrane by a-secretase, which precludes Ab formation but produces soluble sAPPa and small membranebound 83-amino-acid C-terminal fragment (C83). Furthermore, C83 is cleaved by the g-secretase complex, resulting in the release of P3 peptide and AICD [4]. Several proteases have been suggested as AD-relevant a-secretases, many of which belong to the family of a disintegrin and metalloprotease (ADAM), including ADAM9, ADAM10, and ADAM17 [5]. Strong evidence supports a role for ADAM10, but not ADAM9 or -17, as the constitutive and inducible APP a-secretase in neurons [6]. The neuronal overexpression of ADAM10 increased the secretion of sAPPa, prevented plaque formation and alleviated cognitive defects in a transgenic AD mouse model [7]. Blocking the transport of ADAM10 to the postsynaptic
http://dx.doi.org/10.1016/j.bbrc.2016.11.135 0006-291X/© 2016 Elsevier Inc. All rights reserved.
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membrane shifted APP metabolism toward amyloidogenesis and generated a model of sporadic AD (SAD) [8]. Therefore, ADAM10 was identified as the major a-secretase in vivo and dysregulation of ADAM10 may play a significant role in the establishment of Ab pathology. Mitochondrial dysfunction induced by various factors has been strongly implicated in the pathogenesis of AD [9]. In recent years, mitochondrial transcription termination factors (MTERFs) have been implicated in regulating of mitochondrial transcription, replication, and translation. Four MTERFs have been identified in vertebrates, designated MTERF1e4 [10]. To date, MTERF4 is the least studied MTERF family member; however, recent studies provided the first insight into the function of this protein in mitochondria. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4, and is necessary for recruiting of this factor to the large ribosomal subunit [11]. MTERF4 may play an important role in regulating mitochondrial dysfunction in cellular models of Parkinson's disease [12]. These findings highlight the importance of MTERF4 in regulating mitochondrial function. Increasing evidence suggests that mitochondrial defects play a key role in AD pathogenesis [13]. It is therefore plausible that abnormal MTERF4 activity could contribute to the pathogenesis of AD. In vivo and in vitro studies support a major role for mitochondria and bioenergetics in the fate of APP processing and cellular trafficking. No attempt has been made to elucidate the relationship between MTERF4 and APP processing. In this study, we show that MTERF4 has a novel role in reducing the a-secretase-mediated proteolysis of APP by suppressing the transcription of ADAM10. This mechanism may play an important role in AD pathogenesis. 2. Materials and methods
M596L), were grown in the same culture medium with the addition of 1 mg/ml geneticin (G418). The cells were maintained in a humidified 5% CO2 atmosphere at 37 C and transfected with the designed constructs and empty vectors using Lipofectamine 2000 (Invitrogen). 2.4. Quantitative real-time PCR Total RNA was extracted from transfected cells using the Trizol total RNA extraction kit (Sangon) and then 1 mg total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit (TaKaRa, Tokyo, Japan) according to the manufacturer's protocol. Quantitative PCR was performed using a Mastercycler ep realplex (Eppendorf, Hamburg, Germany) with a Prefect Real Time SYBR Premix Ex Taq Kit (TaKaRa). The primers used are listed in Table 1. 2.5. Western blotting Total protein was extracted from frozen mice hippocampal tissue or HEK293-APPswe cells using lysis buffer. The samples (cell or tissue extract) were boiled in the presence of b-mercaptoethanol for 10 min and loaded onto 10% SDS-polyacrylamide gels. The separated proteins were transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were incubated individually with primary antibodies against MTERF4 (1:1000, Abcam), C-terminal APP (1:1000, Sigma), ADAM10 (1:500, Abcam), BACE1 (1:500, Abcam), or GAPDH (1:1000, Abcam) overnight at 4 C. The membranes were then incubated with peroxidaseconjugated goat anti-mouse IgG antibodies or goat anti-rabbit IgG antibodies (1:5000, Abcam) for 1 h. Finally, the blots were exposed to ECL detection reagent (Millipore). Images were captured using a e, Fusion FX7 imaging system (Vilber Lourmat, Marne-la-Valle France) and the bands were quantified using ImageJ software.
2.1. Animal experiments All animal studies were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University. Male and female APP/PS1 transgenic B6C3-Tg (APPswe/PSEN1dE9) mice were obtained from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). Wild-type (WT) mice in the C57BL/6 background were purchased from Chongqing Medical University and used as controls. All mice were provided with a standard diet and housed in an approved facility with climate control and a 12-h light/12-h dark cycle. Six-month-old APP/PS1 mice and WT counterparts were used for all experiments. 2.2. Plasmid construction The full-length open reading frame (ORF) of MTERF4 was generated as described previously [14]. The fragment containing the human MTERF4 gene ORF was sub-cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) to generate the recombinant plasmid pcDNA-MTERF4. All sequences were confirmed by DNA sequencing and matched with reference sequences found in the NCBI database.
2.6. Human Ab42 quantitation (ELISA) The effects of MTERF4 on Ab42 levels in the culture medium were assayed using a human Ab42-specific sandwich ELISA kit (Invitrogen). HEK293-APPswe cells were transfected with the designed constructs and empty vectors for 24 and 48 h; the supernatant was then collected and a protease inhibitor cocktail was added. The concentrations of Ab42 in diluted samples were determined according to the manufacturer's protocols. 2.7. Dual-luciferase reporter assay A DNA fragment comprising nucleotides from 2095 to 39 upstream of the human ADAM10 translation initiation site was amplified using the primers 5'-CAGCACTACCACAAGAGAT-3' and 5'TTAACAGCAGCACATCGA-3'. The fragment was sub-cloned into the firefly luciferase reporter vector pGL3-basic (Promega, Madison, WI, USA) to generate pGL3-ADAM10. The Renilla luciferase vector pRL-SV40 (Promega) was cotransfected to normalize the transfection efficiency. HEK293 cells were seeded in 24-well plates and transfected with the indicated vector. The transfected cells were
2.3. Cell culture and transfection HEK 293 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Sangon, Shanghai, China), 100 IU/ml penicillin, and 100 mg/ml streptomycin. HEK293APPswe cells, the human embryonic kidney 293 cells transfected with human APP bearing the Swedish mutation (APPsw, K595N/
Table 1 Primers for quantitative real-time PCR. Gene
Forward primer
Reverse primer
MTERF4 GAPDH APP BACE1 ADAM10
50 -CGTAACTGCTGCCATCTT-30 5-CCATCACGCCACAGTTTC-30 50 -CAACCAACCAGTGACCAT-30 50 -GCCATCTCACAGTCATCC-30 50 -AGACCGAACTCTGCCATT-30
50 -GTCGTCCTTCTCTGTTCTC-30 50 -ATCCCATCACCATCTTCCAG-30 50 -GTGTGCCAGTGAAGATGA-30 50 -TGTAGCCACAGTCTTCCA-30 50 -TTCCTCTACACCAGTCATCT-30
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cultivated for 48 h, washed twice with phosphate-buffered saline (PBS), and then lysed with lysis buffer. Firefly and Renilla luciferase activities were measured sequentially using a dual luciferase reporter assay system and the GloMax-Multi Jr Single Tube Multimode Reader (Promega). 2.8. Cell cycle analysis HEK293-APPswe cells were transfected with pcDNA-MTERF4 and vector control for 24 and 48 h. The cells were trypsinized and washed twice with cold PBS. After fixing in 70% ethanol at 4 C overnight, the cells were washed and stained with propidium iodide (PI) by incubation with PI/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocols. The DNA content in individual samples was quantified on a FACSCalibur flow cytometer (BD Biosciences). 2.9. Cell proliferation assay Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assays (Sangon) according to the manufacturer's instructions. HEK293-APPswe cells were seeded in 96-well plates at a density of 2 103 cells per well. After 24 and 48 h incubation, the cell monolayer was rinsed twice in PBS, and then diluted CCK-8 solution (diluted 1:10 in DMEM) was added to the cells and incubated for 2 h at 37 C. The absorbance in each well at 450 nm was measured using a microplate reader. 2.10. Statistical analysis The data are presented as the means ± SEM of at least three independent experiments. Statistical significance was determined using Student's t-tests or ANOVA using SPSS 19.0 software. A p value <0.05 was considered significant. 3. Results 3.1. MTERF4 expression is upregulated in the hippocampus of APP/ PS1 mice To determine whether the expression of MTERF4 differed in APP/PS1 mice compared with control, hippocampal samples obtained from 6-month-old APP/PS1 mice and age-matched nontransgenic C57BL/6 controls were compared. Western blotting showed that the levels of APP protein were dramatically increased by 159% in the hippocampus of APP/PS1 mice compared with WT control (Fig. 1A and B). Furthermore, the levels of MTERF4 protein were significantly upregulated by 68% in APP/PS1 mice hippocampus compared with WT control (Fig. 1A, C). 3.2. MTERF4 promotes the amyloidogenic processing of APP in HEK293-APPswe cells The recombinant plasmid pcDNA-MTERF4 and vector control were transiently transfected into HEK293-APPswe cells. The levels of the MTERF4 protein were significantly increased by 67% and 258%, respectively, when cells were transfected for 24 and 48 h compared with control (Fig. 1D and E). Next, quantitative real-time PCR showed that MTERF4 mRNA levels were increased 235 and 216-fold at 24 and 48 h in pcDNA-MTERF4-transfected cells compared with control (Fig. 1F). Thus, MTERF4 was highly expressed in pcDNA-MTERF4 transfected cells. The overexpression of MTERF4 induced a significant increase (186%) in the levels of APP protein in MTERF4-overexpressing cells
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compared with control at 48 h (Fig. 2A and B). In addition, MTERF4 shifted APP processing from a-to b-cleavage 48 h after transfection, as indicated by decreased C83 levels (Fig. 2A, D) and clearly elevated C99 levels (Fig. 2A, C). Next, APP mRNA levels were assessed, which revealed that the overexpression of MTERF4 did not influence APP expression (Fig. 2E). Because a-secretase not only increases the secretion of sAPPa but also decreases the production of neurotoxic Ab, we next measured the effect of MTERF4 on Ab42 peptide levels in the medium. Consistent with the suppressive effects of MTERF4 on a-secretase, a stimulatory effect on Ab42 peptide was observed. Forty-eight hours after transfection, the secretion of Ab42 peptide into the medium was 58.1% higher than that of control (Fig. 2F). 3.3. ADAM10 expression is suppressed by MTERF4 We hypothesized that the decreased C83 levels and the elevated C99 levels in MTERF4-overexpressing cells might be caused by the dysregulated expression of APP-relevant secretases. To test this hypothesis, the protein expression of the a-secretase ADAM10 and the b-secretase BACE1 was measured. Quantification revealed a 27% decrease in ADAM10 levels in cells transfected with pcDNAMTERF4 at 48 h (Fig. 3A and B). As shown in Fig. 3D, ADAM10 mRNA expression was significantly reduced by 68% in MTERF4overexpressing cells at 48 h compared with control. However, MTERF4 had no effect on the expression of BACE1. At 24 and 48 h after transfection, the levels of BACE1 mRNA and protein were unchanged in MTERF4-overexpressing cells compared with control (Fig. 3C, E). Therefore, MTERF4 plays a role in APP processing by directly inhibiting the ADAM10-mediated a-cleavage of APP. 3.4. ADAM10 promoter activity is suppressed by MTERF4 To provide further evidence that MTERF4 regulates the expression of the a-secretase ADAM10 via a transcriptional mechanism, 2056 bp of the 5'-region upstream of the ADAM10 gene was subcloned into the pGL3-basic plasmid. Compared with the empty pGL3-Basic control, pGL3-ADAM10-transfected cells exhibited a significantly increased luciferase activity (Fig. 3F), suggesting that the region 2097 to 39 upstream of the ADAM10 translation site contains a human ADAM10 functional promoter. In addition, overexpressing MTERF4 for 48 h decreased the promoter activity compared with control cells transfected with pGL3-ADAM10 (Fig. 3F). Taken together, these results suggest that MTERF4 suppresses ADAM10 promoter activity. 3.5. MTERF4 overexpression has no effect on the cell cycle and proliferation To investigate the effects of MTERF4 overexpression on cell cycle progression, flow cytometry analysis was performed. The data revealed that there was no significant difference between control and MTERF4-overexpressing cells (Fig. 4A). The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle 24 and 48 h after transfection with pcDNA-MTERF4 were similar to controls in all groups (Fig. 4B). Consistent with the cell cycle results, no obvious differences in cell proliferation were observed at 24 and 48 h after transfection compared with control (Fig. 4C). Taken together, these data suggest that MTERF4 overexpression does not alter cell cycle distribution and proliferation in HEK293-APPswe cells. 4. Discussion One of the pathologic hallmarks of AD is the presence of
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Fig. 1. MTERF4 expression in the hippocampus of APP/PS1 mice and in HEK293-APPswe cells. (A) Representative Western blot showing APP, MTERF4 protein expression in the hippocampus of 6-month-old APP/PS1 mice, as compared with age-matched wild-type (WT) mice (n ¼ 3 for each group). (BeC) Quantification of protein levels of APP, MTERF4 in the hippocampus of APP/PS1 mice and WT mice. (D) Representative Western blot showing MTERF4 protein expression in control and MTERF4 overexpression cells. (E) Quantification of protein levels of MTERF4 in MTERF4 overexpression cells compared with control. (F) The relative mRNA expression of MTERF4 detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. Data are presented as means ± SEM. **p < 0.01 vs. control.
Fig. 2. MTERF4 overexpression promotes the amyloidogenic processing of APP in HEK293-APPswe cells. (A) Representative Western blot showing full length APP, C99 and C83 protein expression in control and MTERF4 overexpression cells. (BeD) Quantification of protein levels of APP, C99 and C83 in MTERF4 overexpression cells compared with control. (E) The relative mRNA expression of APP detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. (F) The amounts of Ab42 peptides in the culture media were analyzed by ELISA. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control.
extracellular amyloid plaques containing Ab, which is a peptidic fragment derived from the amyloidogenic proteolytic processing of APP by sequential cleavages involving b- and g-secretases [15]. APP can also be cleaved by a-secretase via a nonamyloidogenic pathway, which precludes the formation of pathogenesisassociated peptides and gives rise to a neurotrophic and neuroprotective fragment [16]. Activation of the amyloidogenic pathway is one mechanism behind the accumulation of Ab, as observed in cases of familial AD (FAD) that are associated with APP and presenilin (PS) mutations [17]. However, unlike autosomal-dominant FAD, SAD patients generally lack APP mutations [18]. Therefore, identifying the molecular mechanisms underlying pathogenesis of
SAD is essential for the development of novel therapeutics to treat or prevent this disorder. Significantly, the current study revealed that MTERF4 overexpression yielded similar impacts on human APP processing to those observed in SAD. First, MTERF4 expression was upregulated in the hippocampus of APP/PS1 mice. After transfecting HEK293-APPswe cells with MTERF4 for 48 h, APP expression was increased and Ab42 levels were increased in the media. Compared with Ab40, Ab42 is a highly fibrillogenic peptide that favors the formation of neurotoxic species [19]. Accordingly, the overexpression of MTERF4 reduced the production of C83 and increased the production of C99. This suggests that MTERF4 overexpression suppresses the a-site cleavage of APP.
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Fig. 3. ADAM10 Expression is suppressed in cells overexpressing MTERF4. (A) Representative Western blot showing ADAM10 and BACE1 protein expression in control and MTERF4 overexpression cells. (BeC) Quantification of protein levels of ADAM10 and BACE1 in MTERF4 overexpression cells compared with control. (D-E) Quantification of mRNA levels of ADAM10 and BACE1 detected by quantitative real-time PCR in MTERF4 overexpression cells compared with control. (F) Luciferase activity in cells transfected with the promoterless pGL3-Basic, ADAM10 promoter-luciferase construct pGL3-ADAM10 and recombinant plasmid pcDNA-MTERF4. Data are presented as means ± SEM. *p < 0.05, **p < 0.01 vs. control; #p < 0.05 vs. pGL-ADAM10 transfected cells; ##p < 0.01 vs. pGL3-Basic transfected cells.
Fig. 4. MTERF4 overexpression has no effect on the cell cycle and proliferation. (A) Cell cycle was measured by flow cytometry in control and MTERF4 overexpression cells. (B) Representative histograms for cell cycle distribution. Results are calculated distribution for cells in G0/G1, S, and G2/M phases. (C) Cell proliferation was measured using the CCK-8 assay. Data are presented as means ± SEM.
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Then, we investigated how MTERF4 affected APP processing. The current study further revealed that this a-cleavage inhibiting activity was due to the direct suppression of ADAM10 expression. There was a reduction in ADAM10 expression in cells transfected with MTERF4 for 48 h. In contrast, there were no differences in BACE1 expression. We hypothesized that the reduction in ADAM10 expression occurs via a transcriptional mechanism. Previous studies demonstrated that the region 2179 to 1 upstream of the ADAM10 translation initiation site possesses functional promoter activity and that the proximal region 508 bp to 300 bp is essential for constitutive ADAM10 promoter activity [20]. Therefore, we cloned 2056 bp of the 50 -region upstream of the human ADAM10 gene and demonstrated that this genomic DNA fragment contained a functional ADAM10 promoter. The promoter activity of this DNA fragment was decreased after transfection with MTERF4 for 48 h. Furthermore, MTERF4 overexpression had no significant effect on cell cycle distribution and proliferation in HEK293APPswe cells. Thus, these results may have important implications for both the initiation and progression of AD. Insights into MTERF4 function have been reported only recently, and the precise role of MTERF4 in mitochondrial regulation is unclear. MTERF4 is necessary for cell proliferation because MTERF4 knockdown in HeLa cells resulted in sub-G1 cell accumulation and cell death [14]. In vivo and in vitro studies confirmed that MTERF4 localizes to the mitochondria and is essential for embryonic development in the mouse [11,21]. MTERF4 forms a stable complex with a mitochondrial rRNA methyltransferase, NSUN4, and is required for the recruitment of NSUN4 to the large ribosomal subunit in an essential step in ribosomal biogenesis [22,23]. Based on the finding that MTERF4 was upregulated in the hippocampus of APP/PS1 mice, we investigated whether MTERF4 could modulate APP processing and thereby contribute to the pathogenesis of AD. Several studies revealed that mitochondrial dysfunction is one of the earliest pathological features of AD [24,25]. The current study demonstrated that MTERF4 is detrimentally associated with several pathological aspects of AD, including Ab generation and the dysregulation of ADAM10. Several lines of evidence suggest that Ab itself leads to mitochondrial dysfunction and increased ROS levels [26]. We propose that the upregulation of MTERF4 initiates a vicious cycle beginning with enhanced amyloidogenic APP processing. ADAM10 is the major a-secretase in vivo; therefore, it offers a new target for therapeutic manipulation in AD. The current study reveals a pathway for the downregulation of ADAM10 involving MTERF4. Several studies have shown a role for retinoid X-receptor and retinoic acid receptor in the induction of ADAM10 transcription in neuronal cell lines [20,27]. In addition to the retinoic acid-related signaling pathways, various other effectors have been established as ADAM10 transcription enhancers [28]. A G-rich region in the ADAM10 5'-UTR forms a highly stable G-quadruplex secondary structure, which contributes to the translational repression of ADAM10 [29]. The translation of ADAM10 mRNA into protein is also controlled by several microRNAs [30,31]. We demonstrated that ADAM10 is downregulated at the transcriptional level by MTERF4 overexpression, which reveals a possible pathway that accelerates the pathology of AD. In conclusion, the present study identified a novel role for MTERF4 in shifting APP processing toward the amyloidogenic pathway. Overexpression of MTERF4 increased APP protein levels, downregulated ADAM10 expression, and promoted amyloidogenesis. These data demonstrated that MTERF4 reduced ADAM10 levels via a transcriptional mechanism. The current findings suggest that the upregulation of MTERF4 may represent a novel risk factor for the onset and progression of AD. Further studies should help clarify whether MTERF4 has the potential to prevent Ab
generation and slow down the progression of AD. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by NSFC grants (numbers 81171197 & 81220108010) to G-J Chen. References [1] H.W. Querfurth, F.M. LaFerla, Alzheimer's disease, N. Engl. J. Med. 362 (2010) 329e344. [2] A. Serrano-Pozo, M.P. Frosch, E. Masliah, et al., Neuropathological alterations in Alzheimer disease, Cold Spring Harb. Perspect. Med. 1 (2011) a006189. [3] Y.W. Zhang, R. Thompson, H. Zhang, et al., APP processing in Alzheimer's disease, Mol. Brain 4 (2011) 3. [4] F.M. LaFerla, K.N. Green, S. Oddo, Intracellular amyloid-beta in Alzheimer's disease, Nature reviews, Neuroscience 8 (2007) 499e509. [5] J. Prox, A. Rittger, P. Saftig, Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and presenilin, Exp. Brain Res. 217 (2012) 331e341. [6] P.H. Kuhn, H. Wang, B. Dislich, et al., ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons, EMBO J. 29 (2010) 3020e3032. [7] R. Postina, A. Schroeder, I. Dewachter, et al., A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model, J. Clin. Investig. 113 (2004) 1456e1464. [8] R. Epis, E. Marcello, F. Gardoni, et al., Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer's disease, Brain J. Neurol. 133 (2010) 3323e3335. [9] Q. Cai, P. Tammineni, Alterations in mitochondrial quality control in Alzheimer's disease, Front. Cell. Neurosci. 10 (2016) 24. [10] M. Roberti, P.L. Polosa, F. Bruni, et al., MTERF factors: a multifunction protein family, Biomol. Concepts 1 (2010) 215e224. [11] Y. Camara, J. Asin-Cayuela, C.B. Park, et al., MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian mitochondrial ribosome, Cell metab. 13 (2011) 527e539. [12] X. Ye, Y. Han, L. Zhang, et al., MTERF4 regulates the mitochondrial dysfunction induced by MPP(þ) in SH-SY5Y cells, Biochem. Biophys. Res. Commun. 464 (2015) 214e220. [13] P.H. Reddy, R. Tripathi, Q. Troung, et al., Abnormal mitochondrial dynamics and synaptic degeneration as early events in Alzheimer's disease: implications to mitochondria-targeted antioxidant therapeutics, Biochimica Biophys. Acta 1822 (2012) 639e649. [14] M. Yu, J. Dai, W. Huang, et al., hMTERF4 knockdown in HeLa cells results in sub-G1 cell accumulation and cell death, Acta Biochimica Biophys. Sinica 43 (2011) 372e379. [15] G. Evin, C. Hince, BACE1 as a therapeutic target in Alzheimer's disease: rationale and current status, Drugs & Aging 30 (2013) 755e764. [16] K. Endres, F. Fahrenholz, Regulation of alpha-secretase ADAM10 expression and activity, Exp. Brain Res. 217 (2012) 343e352. [17] R.E. Tanzi, L. Bertram, Twenty years of the Alzheimer's disease amyloid hypothesis: a genetic perspective, Cell 120 (2005) 545e555. [18] S. Lista, S.E. O'Bryant, K. Blennow, et al., Biomarkers in sporadic and familial Alzheimer's disease, J. Alzheimer's Dis. JAD 47 (2015) 291e317. [19] I. Kuperstein, K. Broersen, I. Benilova, et al., Neurotoxicity of Alzheimer's disease Abeta peptides is induced by small changes in the Abeta42 to Abeta40 ratio, EMBO J. 29 (2010) 3408e3420. [20] C. Prinzen, U. Muller, K. Endres, et al., Genomic structure and functional characterization of the human ADAM10 promoter, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 19 (2005) 1522e1524. [21] Q. Xu, F. Zhang, H. He, et al., Expression profile of mouse Mterfd2, a novel component of the mitochondrial transcription termination factor (MTERF) family, Genes & Genet. Syst. 86 (2011) 269e275. [22] H. Spahr, B. Habermann, C.M. Gustafsson, et al., Structure of the human MTERF4-NSUN4 protein complex that regulates mitochondrial ribosome biogenesis, Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 15253e15258. [23] M.D. Metodiev, H. Spahr, P. Loguercio Polosa, et al., NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly, PLoS Genet. 10 (2014) e1004110. [24] H. Du, L. Guo, S. Yan, et al., Early deficits in synaptic mitochondria in an Alzheimer's disease mouse model, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 18670e18675. [25] S. Hauptmann, I. Scherping, S. Drose, et al., Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice, Neurobiol. aging 30 (2009) 1574e1586. [26] K. Leuner, T. Schutt, C. Kurz, et al., Mitochondrion-derived reactive oxygen species lead to enhanced amyloid beta formation, Antioxid. Redox Signal. 16
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Please cite this article in press as: X.-l. Wang, et al., Overexpression of MTERF4 promotes the amyloidogenic processing of APP by inhibiting ADAM10, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.135