Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Aug 2019Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells Hongshuang Qin, Jiasi Wang, Jinsong Ren and Xiaogang Qu Hongshuang Qin Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China) Google Scholar More articles by this author , Jiasi Wang Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China) University of Chinese Academy of Sciences, Beijing 100039 (China) Google Scholar More articles by this author , Jinsong Ren Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China) Google Scholar More articles by this author and Xiaogang Qu *Corresponding author: E-mail Address: [email protected] Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022 (China) Google Scholar More articles by this author https://doi.org/10.31635/ccschem.019.20180034 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The biological functions of amyloid β (Aβ) peptides have received much attention, playing a central role in the pathogenesis of Alzheimer’s disease (AD). Recent studies have demonstrated that AD is associated with cancer. However, it is still unknown if there is a correlation between Aβ and cancer. In this report, we investigated the effect of Aβ42, the more fibrillogenic form of Aβ peptides, on telomere and telomerase sustenance in tumor cells. Telomere maintenance and telomerase reactivation are the early events in carcinogenesis and play essential roles for cell immortality and tumor initiation. Our study found that janus-faced Aβ42 could target telomeres and induce classical telomere dysfunction, including telomere DNA damage, telomere uncapping, chromosome fusion, and telomere shortening. Furthermore, Aβ42 could induce telomerase reverse transcriptase (TERT) downregulation, TERT translocation, inhibition of telomerase activity, as well as cell senescence and apoptosis. To our knowledge, this is the first report that provides a link between janus-faced Aβ and tumorigenesis. Our work would help in gaining a better understanding of the correlation between AD and cancer. Download figure Download PowerPoint Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease and features synaptic dysfunction and neuronal loss in the brain.1 Cancer belongs to a group of diseases characterized by uncontrolled cell proliferation, and at times, spreading of the abnormal cells.2 Both AD and cancer increase exponentially with age, and their inverse correlation in many aspects have been proposed.3,4 Recent studies have indicated that AD is associated with a lower risk of various cancers and vice versa.5–7 Several investigations have been performed to expound the association between AD and cancer.8–11 These include genome-wide association study, which suggests that there is a genetic correlation between AD and cancer,9 and transcriptomic meta-analyses reveal significant numbers of genes with inverse patterns of expression in AD and lung cancer10. Further, functional analyses show that most of these genes are associated with mitochondrial metabolism.10 Different expressions of Pin1, BRCA1, and some common microRNAs (miRNAs) such as the miR-9, miR-29, and miR-34 family provide additional support for the association between AD and cancer.11–13 Despite these findings, the details of the relationship between AD and cancer remain unknown. We envisioned that insight into the connections between these two diseases would provide new opportunities for a better understanding of their pathogenesis. The β-amyloid peptide (Aβ) plays an essential role in the pathogenesis of AD.14 Aβ deposits extracellularly in AD brain, forming amyloid plaques, which is one of the hallmarks of AD.15 Interestingly, Aβ is also found within the neuronal cells. Intraneuronal Aβ, predominantly Aβ42, is believed to be the main trigger of the pathological cascade of events leading to neurodegeneration at present.16–18 For a long time, most studies focused on the effect of Aβ on neurons, microglia, and astrocytes in the central nervous system (CNS).16,19,20 Besides the brain, Aβ can cross the blood-brain barrier (BBB) and has been detected in blood, urine, cerebrospinal fluid (CSF), and various peripheral tissues such as the liver, kidney, and adrenal gland.21–26 Recently, Wang’s group reported that plasma levels of Aβ in various patients with cancer were higher than those in healthy controls, indicating that Aβ elevation may correlate with cancer development.12 Also, several studies have indicated that Aβ can internalize into various cancer cells, interact with tubulin, disrupt mitochondrial function, induce lamin fragmentation in tumor cells, and inhibit angiogenesis in glioblastoma and lung adenocarcinoma tumors.27–33 Therefore, it is essential to decipher how Aβ can induce carcinogenesis. Investigating Aβ behaviors in the molecular processes related to tumorigenesis may provide new insights into the link between AD and cancer. Telomere sustaining and telomerase reactivation occur in the early stage of carcinogenesis and are necessary for tumor initiation and the immortality of cancerous cells.34–39 Telomere consists of a series of noncoding repeats (TTAGGG), located at the end of the chromosome, and protects chromosome integrity from illegitimate recombination, fusion, and degradation.40,41 Telomere DNA includes a duplex region (2–15 kb) and a single-stranded overhang (50–400 nucleotides), named the 3′-overhang. The 3′-overhang can loop back, invade the duplex region, and form the T-loop/D-loop structure to protect the end of chromosome.42–45 Several specific telomeric shelterin proteins (TRF1, TRF2, POT1, TIN2, TPP1, and Rap1) bind and protect the 3′-overhang and maintain the telomere structure.40 The progressive shortening of the telomere length or damage to the telomere structure can lead to telomere dysfunction and further result in p53-dependent cellular senescence and apoptosis.46–53 For avoiding telomere shortening, telomerase, a specialized reverse transcriptase consisting of a protein catalytic subunit telomerase reverse transcriptase (TERT) and an RNA template (telomerase RNA component, TERC), is reexpressed to maintain telomere length.54,55 Telomerase can add TTAGGG repeats to the ends of telomeres using its RNA component as a template.56,57 In addition to telomere extension, TERT is essential in malignant transformation.34 In this report, we intend to decipher the mechanism of Aβ effect on the status of telomere and telomerase system in tumor cells. Herein, Aβ42, the most aggregation prone and most toxic form of Aβ, was transfected into pheochromocytoma (PC12) cells to establish a tumor cell model expressing Aβ42 (PC12-Aβ42 cells). We found that Aβ42 rapidly entered the nucleus and localized at the telomeres. Further studies suggested that Aβ42 interacted with telomeric DNA and triggered a classical telomere dysfunction, including telomere DNA damage, telomere uncapping, elevated frequencies of end-to-end chromosome fusion, and telomere shortening. Moreover, our results indicated that Aβ42 could not only disrupt telomere but also induce TERT translocation from the nucleus to the cytoplasm and reduce the expression of TERT. These effects resulted in cell senescence and apoptosis. Overexpression of the telomere-associated protein, TERT, TRF2, and POT1 could efficiently protect the cells from Aβ42-induced cell death. This further indicates that intracellular Aβ could induce telomere dysfunction, thereby, providing new insights into understanding the correlation between AD and cancer. Results Aβ42 has been found in many intracellular sites, such as mitochondrion, endoplasmic reticulum, Golgi complexes, endosomes, multivesicular bodies, and cytosol.58 Recently, the nuclear localization of Aβ acting as a putative transcription factor has been reported in AD brain samples and Chinese hamster ovary cells.59–63 To delineate the distribution of Aβ42 in our experimental system, we performed immunofluorescence experiments. As shown in , Aβ42 was observed clearly in cytoplasm and nuclei after transfection. The nuclear localization of Aβ42 motivated us to study whether Aβ42 could induce DNA damage. Strikingly, significant phosphorylation of H2AX (γ-H2AX), which is believed to be the marker of DNA double-strand break,40,64 was observed at 12 h after the transfection (,). The DNA damage became stronger at 24 h, which was also confirmed by the Western-blot experiment (). These results demonstrated that Aβ42 could translocate from cytosol to nucleus and trigger a DNA damage response. We also overexpressed an unrelated protein (green fluorescence protein) as a control and did not observe appreciable DNA damage response up to 36 h after transfection (). We next investigated whether the DNA damage occurred at the telomeres. Immunofluorescence staining for γ-H2AX and TRF1, the telomere-binding protein that has been considered a telomere marker,50 was performed in PC12-Aβ42 cells, using the PC12 cells transfected with vector pcDNA3.1 as control unless otherwise specified. As shown in Figure 1a, colocalization of the γ-H2AX foci and TRF1, called the telomere dysfunction-induced foci (TIFs), was frequently observed in PC12-Aβ42 cells. On average, 72.6% of PC12-Aβ42 cells were TIF positive (displaying more than five γ-H2AX/TRF1 colocalizations per nucleus) much higher than that in control cells (Figure 1b). To further determine the telomere DNA damage induced by Aβ42, a positive control experiment using cells transfected with TRF2ΔBΔM mutation, which results in dysfunctional telomeres,46,50,65 was also carried out. As shown in Figure 1b,c, the percentage of cells overexpressing Aβ42 with TIFs positive was comparable with that measured in cells overexpressing the TRF2ΔBΔM allele. These results demonstrated that Aβ42-induced DNA damage primarily occurred at the telomere. Figure 1 | Aβ42 interacted with telomeric DNA and induced telomere DNA damage. (a) Representative immunofluorescence images of merged γ-H2AX (red) and TRF1 (green) in PC12 cells expressing Aβ42 or TRF2ΔBΔM. Enlarged merged signals are exhibited on the right. Scale bar = 5 μm. (b) The percentage of TIF-positive cells was calculated in PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM. Cells with five or more γ-H2AX/TRF1 foci were scored as TIF positive. More than 100 nuclei were scored for each sample. Error bars indicate SD. **p < 0.001 vs. control cells. (c) Average number of TIFs per nucleus in PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM. More than 100 nuclei were scored. **p < 0.001 vs. control cells. (d) Telomeric DNA ChIP assay with Aβ42 antibody in PC12-Aβ42 cells. The percentage of telomeric DNA was normalized to the input sample. Data represent triplicate ChIP experiments. **p < 0.005 vs. positive cells. Download figure Download PowerPoint Therefore, we asked whether the telomere DNA damage induced by Aβ42 was caused by the interaction between Aβ42 and telomere DNA. To document the evidence for Aβ42 interacting with telomeric DNA, we performed quantitative real-time PCR (qRT-PCR) for telomere repeats following Aβ42 chromatin immunoprecipitation (ChIP) in PC12-Aβ42 cells. As shown in Figure 1d, a robust telomere signal (average ninefold higher) was detected in the cells, demonstrating the association between Aβ42 and telomeric DNA. The nonspecific IgG was also used as the antibody control to confirm the specific association between Aβ42 and telomeric DNA. These results demonstrated that Aβ42 was a telomere target peptide and triggered a DNA damage response at the telomeres. Telomere-targeting agents often induce the dissociation of telomere-binding proteins. Therefore, we tested the effect of Aβ42 on the localization of TRF1, POT1, and TRF2, which were suggested to play key roles in telomere structure maintenance.40,52 A qRT-PCR-based ChIP assay indicated that Aβ42 affected the binding of POT1 and TRF2 to telomeres, rather than that of TRF1 (Figure 2a), implying that telomeres could be represented by TRF1 foci. Confocal microscopy results showed that TRF2 and POT1 were colocalized with TRF1 foci in control cells. However, they were delocalized specifically from TRF1 foci in PC12-Aβ42 cells (Figure 2b,c). Quantitative analysis by counting the number of TRF2/TRF1 and POT1/TRF1 colocalizations revealed that the percentage of nuclei with more than six colocalizations decreased in PC12-Aβ42 cells (Figure 2d). We also examined the expression of TRF2, POT1, and TRF1. The results are shown in , which demonstrated that the removal of these proteins from telomeres was independent of their expression. Figure 2 | Delocalization of TRF2 and POT1 from telomeres in PC12-Aβ42 cells. (a) ChIP assay to determine the binding of POT1, TRF2, and TRF1 with telomeres in PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM. **p < 0.005 vs. control cells. (b) Representative confocal images, revealing TRF2 (red) and TRF1 (green) in PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM. Enlarged merged signals were exhibited. Scale bar = 5 μm. (c) Representative confocal images of merged POT1 (red) and TRF1 (green) in PC12 cells expressing Aβ42 or TRF2ΔBΔM. Enlarged merged signals are shown on the right. Scale bar = 5 μm. (d) The percentage of cells with more than 6 colocalizations of TRF2/TRF1 or POT1/TRF1 per nucleus. More than 100 nuclei were scored. **p < 0.001 vs. control cells. Download figure Download PowerPoint Telomere forms “a cap” at the chromosome end to protect it from being recognized as a double-strand break. Removal of shelterin components leads to telomere-capping alteration, evidenced by the emergence of micronuclei, anaphase bridges, and fused telomeres.66 We, therefore, analyzed the telomere status in PC12-Aβ42 cells via staining with 4′,6-diamidino-2-phenylindole (DAPI). The results in Figure 3a,b showed that there was an apparent increase of micronuclei and anaphase bridges in PC12-Aβ42 cells, which indicated the uncapping of telomeres. We further generated metaphase spreads from the cells and stained with DAPI. A marked increase in end-to-end joining was also noticeable in the cells (Figure 3c,d). Similar results were observed in the cells transfected with TRF2ΔBΔM, which further indicated telomere uncapping in PC12-Aβ42 cells. Figure 3 | Telomere uncapping induced by Aβ42. (a) PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM were stained with DAPI, and the representative images of micronuclei and anaphase bridges are shown. Scale bar = 10 μm. (b) Telomere abnormity frequency was calculated (a minimal 50 anaphase cells were counted). **p < 0.001 vs. control cells. (c) Metaphase spreads were stained with DAPI in PC12-Aβ42 cells or PC12 cells expressing TRF2ΔBΔM, and the representative images of telomere fusion are shown (original magnification: ×100 objective). Scale bar equals = 10 μm. (d) The number of chromosome end-to-end fusions per metaphase was shown. **p < 0.001 vs. control cells. Download figure Download PowerPoint To further confirm and label the unprotected telomeres directly, we performed a terminal deoxy-transferase (TdT) assay to add Cy3-conjugated deoxyuridine to the naked ends of telomeres.67 Obvious colocalization of the TdT signals with TRF1 foci was observed in PC12-Aβ42 cells (), indicating telomere uncapping. Quantitative analysis revealed that 58% of TdT signals colocalized with telomeres in PC12-Aβ42 cells and 70% for TRF2ΔBΔM expressing cells (). However, specific nuclear staining was not detectable in the control cells. These results strongly supported the hypothesis that Aβ42 could target telomeres and, subsequently, cause telomere-capping alteration. We also investigated the telomere length in PC12-Aβ42 cells. After 48 h of transfection, the telomere length in PC12-Aβ42 cells did not decrease significantly, compared with the control cells (), suggesting that the effect induced by Aβ depended on the structure of the telomere but not telomere length. However, we found a visible telomere shortening () in the stable-transfected PC12-Aβ42 cell line (ST-PC12-Aβ42 cells). These results indicated that long-term production of Aβ42 could destroy telomere structure and induce telomere shortening. We next determined the effect of Aβ42 on telomerase activity, as well as the expression and location of TERT, employing a telomeric repeat amplification protocol (TRAP) assay. Aβ42 aggregation was evident, but not Aβ42 monomers, which significantly inhibited the activity of telomerase in vitro (Figure 4a). Furthermore, the activity of telomerase in PC12-Aβ42 cells was dramatically decreased, compared with the control cells (Figure 4b). To explore the reason for the decreased telomerase activity in PC12-Aβ42 cells, we next examined the expression of TERT, the amount of which was highly associated with the telomerase activity. Results in Figure 4c revealed the decline of TERT expression in PC12-Aβ42 cells, which were confirmed by the results of an immunofluorescence assay (Figure 4d). Figure 4 | TERT expression was downregulated and translocated from the nucleus to cytoplasm in PC12-Aβ42 cells. (a) Aβ42 aggregates, but not monomeric forms, inhibited the telomerase activity in vitro in a dose-dependent manner. (b) TRAP assay for telomerase activity in control cells and PC12-Aβ42 cells. (c) Western blot assay of TERT expression in control cells and PC12-Aβ42 cells. The level of β-actin was used to verify equivalent protein loading. (d) Representative images for TERT (red) and cell nucleus (blue) are shown. Scale bar equals = 10 μm. Download figure Download PowerPoint Moreover, we found more detectable cytoplasmic TERT in PC12-Aβ42 cells, indicating its translocation from the nucleus to the cytosol (Figure 4d). It was reported that reactive oxygen species (ROS) induce the nuclear export of TERT.68,69 Thus, we investigated intracellular ROS in PC12-Aβ42 cells. The ROS assay revealed that Aβ42 expression significantly increased intracellular ROS in PC12-Aβ42 cells (). Clearance of ROS using antioxidants (N-acetylcysteine) partially reversed TERT translocation, indicating that Aβ42-induced oxidative stress was involved in the nuclear export of TERT (). Collectively, these results suggested that Aβ42 induced the reduction of TERT expression and its nuclear translocation, resulting in a decrease of telomerase activity and interference with telomere maintenance. We further investigated whether Aβ42 cause of telomere dysfunction could lead to apoptosis. As shown in Figure 5a,b, Aβ42 triggered a rapid induction of apoptosis in PC12-Aβ42 cells. Further investigation demonstrated the cellular senescence phenotype in PC12-Aβ42 cells, clarified by the occurrence of β-galactosidase activity (Figure 5c,d). Figure 5 | Senescence and apoptosis induced by telomere dysfunction in PC12-Aβ42 cells. (a) Flow cytometry analysis of the apoptotic cell in PC12-Aβ42 cells with indicated transfection time, based on staining with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate. (b) The percentages of apoptotic cells measuring by flow cytometry are shown. *p < 0.05, **p < 0.001. (c) Expression of β-galactosidase (SA-β-gal) associated with senescence in PC12-Aβ42 cells. Scale bar equals 15 μm. (d) The percentage of senescent cells. **p < 0.001. (e) Western blot assay of the expression of p16, p21, and p53 in PC12-Aβ42 cells. Download figure Download PowerPoint To evaluate the molecular mechanism responsible for the Aβ42-induced apoptosis and senescence, we examined the expressions of p16, p21, and p53, which are the key regulators in cellular apoptosis or senescence.48,51,70,71 As indicated in Figure 5e, 48 h after the transfection, significant upregulations of p16, p21 and p53 were observed in PC12-Aβ42 cells, which indeed indicated the involvement of these proteins in Aβ42-induced cellular apoptosis and senescence. To further verify the cellular senescence and apoptosis induced by Aβ42-caused telomere dysfunction, we overexpressed the telomere-associated proteins, TERT, TRF2, and POT1, to explore whether they could prevent Aβ42-triggered cell death. Results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that overexpression of TERT reversed the cell death induced by Aβ42 (Figure 6a), which was consistent with previous studies.72,73 Moreover, we also found that overexpression of TRF2 and POT1 significantly alleviated the cell death induced by Aβ42, although lesser than the effect of TERT (Figure 6a). These results were further evidenced by Annexin-V staining and β-galactosidase activity, which indicated reduced apoptosis and senescence in TRF2 and POT1 overexpressing cells (Figure 6b,c). These strongly supported the hypothesis that Aβ42-induced cell apoptosis and senescence resulted from telomere dysfunction and that the enhancement of telomere-capping function by TERT, POT1, and TRF2 could counteract the effect of Aβ42 on telomeres. Figure 6 | Overexpression of TERT, TRF2, and POT1 protects cells from Aβ42-induced cell death, cellular apoptosis, and senescence. PC12 cells were transfected with TRF2, POT1, and TERT expression plasmids. Twenty-four hours later, the cells were transfected with Aβ42. After 48 h of Aβ42 transfection, the cells were subjected to MTT assay, apoptosis analysis, and SA-β-Gal assay. Shown are the percentages of (a) cell death, (b) cell apoptosis, and (c) cellular senescence. Data represent the means of three separate experiments with SD. **p < 0.001 vs. control cells. *p < 0.05 vs. Aβ expressing cells. Download figure Download PowerPoint Discussion Until now, the detailed association between Aβ and cancer remained unknown. In this study, we focused on the effect of Aβ on the status of telomere and the telomerase system. We found four lines of evidence to support the conclusion that: (1) Aβ42 rapidly entered the nucleus and induced telomeric DNA damage, (2) ChIP experiment demonstrated Aβ42 interacted with telomeric DNA, (3) a rapid dissociation of telomere-binding proteins (TRF2 and POT1) from telomeres occurred, (4) telomere uncapping occurred, evidenced by the emergence of micronuclei, anaphase bridges, and fused telomeres. Furthermore, our results showed that Aβ42 inhibited telomerase activity, induced TERT expression, and promoted TERT translocation. Taken together, Aβ42 regulated telomere and the telomerase system in a multisynergistic way. AD is conventionally considered a CNS disorder. However, increasing evidence from experimental, epidemiological, and clinical studies indicate that manifestations of AD extend beyond the brain. AD is accompanied by systemic abnormalities such as systemic immunity disorders, blood abnormalities, metabolic disorders, cardiovascular disease, hepatic dysfunction, renal dysfunction, respiratory and sleep disorders, gut microbiota disturbance and infection, and systemic inflammation.21 These alterations are not just the secondary effects of the cerebral degeneration; they can reflect underlying processes linked to AD progression and might have connections with the outlined systemic abnormalities of Aβ metabolism.21 It has been suggested that Aβ derived from the brain can be transported into the peripheral pool through the blood–CSF barrier, BBB, arachnoid villi, and glymphatic–lymphatic pathway. More than 40% of brain Aβ is cleared via transportation to the periphery.74,75 In fact, there might be an equilibrium between the central and peripheral pools of Aβ. The Aβ can be found in blood, bile, urine, and various peripheral tissues and can be internalized by a variety of cells, such as monocytes, macrophages, neutrophils, lymphocytes, and hepatocytes.21 In view of the extensive distribution of Aβ, it could as well be involved in tumor initiation. The interaction between Aβ and DNA in vitro has been demonstrated for over 10 years.60,76,77 We previously have reported that Aβ could induce DNA condensation and conformation transition in vitro.78,79 Very recently, it has been reported that the interaction of Aβ with DNA had a sequence specificity and a “G”-rich consensus sequence, “KGGRKTGGGG,” has been determined to be the Aβ-interacting domain.61–63 The consecutive “G” sequence played a crucial part in the interaction of Aβ with DNA.62 This specific interaction is viewed as a novel mechanism for Aβ as a transcriptional factor. It is acknowledged that telomeric DNA with tandem TTAGGG repeats is “G” rich and also has a consecutive “G” sequence. Moreover, telomere consists of very long repeated DNA (2–15 kb). It is, therefore, reasonable to suggest that telomere is a highly probable target for Aβ. We observed not only the interaction of Aβ42 with telomeric DNA, but also the Aβ42-induced telomere uncapping, telomere DNA damage, with subsequent cellular apoptosis and senescence. These results indicate that telomeric DNA is a possible target for Aβ. Weinberg and colleagues34 reported that the alternative mechanism of telomere maintenance (ALT) cells, in which the expression of TERT was absent, could not accomplish transformation, suggesting that, apart from telomere elongation, an additional role of TERT was required for tumorigenesis. Subsequent studies showed that TERT possessed many novel molecular functions, including metabolic reprogramming, transcriptional regulation, inducing tumor-promoting inflammation, and genome instability.35,80–82 TERT is regarded as a central regulator of all the hallmarks of cancer.35 Our results showed that TERT expression was abrogated in the cells transfected with Aβ42. Therefore, the inhibition of carcinogenesis by Aβ42 was very potent. It was speculated that the reduction of TERT protein resulted from two causes. On the one hand, Aβ42 might interact with TERT promoter and inhibit its transcriptional activity, leading to a decrease of protein translation. This is because the above-mentioned “G”-rich consensus of “KGGRKTGGGG” could be found both in the basal core promoter (−234/−225, 5′-GGGACTGGGG-3′) and distal promoter (−1986/−1976 and −3585/−3575, 5′-GGGGGTGGGG-3′ and 5′-GGGGATGGGG-3′) regions of the TERT gene. On the other hand, the degradation of TERT in the cytoplasm might also contribute to the lower content of TERT. Aβ is known to increase free radical production and lead to oxidative stress.83 Although the specific molecular mechanism was not clear, TERT was reported to translocate to the cytoplasm under oxidative stress.68,84 Our results showed that ROS was increased in PC12 cells after transfection with Aβ42. Meanwhile, there was an observable TERT translocation from the nucleus to the cytoplasm. Further, it has been demonstrated that the shuttle of TERT between the nucleus and cytoplasm could promote TERT degradation.85,86 Therefore, we reasoned that the oxidative stress induced by Aβ42 resulted in the translocation of TERT from the nucleus to the cytoplasm and accelerated TERT degradation. However, further investigation is required to determine the specific factors involved in the lowering of TERT. We discovered a rapid removal of TRF2 and POT1 from the telomere. In the current telomere model, TRF2 and POT1 are suggested to play critical roles in the T-loop/D-loop structure formation for the protection of chromosome ends.40,52,87 Therefore, removal of the two proteins implied that Aβ targeted telomeres and destroyed the structure of the T-loop/D-loop. This hypothesis was supported by the formation of micronuclei, anaphase bridges, and fused telomeres, which indicated that