JOURNAL OF NEUROCHEMISTRY
| 2015 | 134 | 969–977
1
doi: 10.1111/jnc.13191
1
College of Life Sciences, University of Chinese Academy of Science, Beijing, China
Abstract The protein arginine methyltransferase 5 (PRMT5) controls cell growth and apoptosis by catalyzing mono and symmetric dimethylation of arginine residues. In human brain tissue, PRMT5 is predominantly expressed in neuronal cells. There is evidence that PRMT5 provides protection against cell death, but the impact of PRMT5 on neuronal apoptosis during the evolution of Alzheimer’s disease has not been tested. In the present study, we show that PRMT5 is down-regulated by bamyloid (Ab) in primary neurons and SH-SY5Y cells, and this is associated with the up-regulation of the PRMT5 target protein E2F-1. Furthermore, knockdown of PRMT5 in SHSY5Y cells over-expressing the Swedish mutant form of human amyloid-b precursor protein caused activation of E2F1/p53/Bax, NF-jB, and GSK-3b pathways, which coincided
with increased apoptosis. Co-depletion of E2F-1 reduced the activation of p53/Bax, NF-jB, and GSK-3b, and limited cell apoptosis. In addition, inhibiting NF-jB and GSK-3b activity by specific inhibitors also attenuated cell apoptosis, suggesting that E2F-1/NF-jB/GSK-3b pathways mediate for apoptosis induced by PRMT5 depletion. More importantly, knockdown of PRMT5 resulted in more paralysis in a transgenic Caenorhabditis elegans strain CL2006, indicating that PRMT5 provides protection against Ab toxicity in vivo. Collectively, our findings identify PRMT5 as a novel regulator of Ab toxicity and suggest that strategies aimed at activating PRMT5 in the neuron may represent a potential therapeutic approach for the prevention of Alzheimer’s disease. Keywords: Alzheimer’s disease, Apoptosis, E2F-1, PRMT5. J. Neurochem. (2015) 134, 969–977.
Protein methylation is increasingly recognized as an important type of post-translational modification that regulates protein function (Bedford and Richard 2005; Bedford and Clarke 2009). The methylation of arginine residues is catalyzed by a family of intracellular enzymes termed protein arginine methyltransferase (PRMT), which have been classified by their specific catalytic activities: type I enzymes catalyze formation of asymmetric modifications, while type II enzymes catalyze symmetric modifications (Bedford and Clarke 2009; Wolf 2009). As a type II enzyme, PRMT5 functions in the nucleus as well as in the cytoplasm and its substrates include histones, spliceosomal proteins, transcription factors, and proteins involved in PIWI-interacting RNA (piRNA) biogenesis (Bedford and Clarke 2009; Karkhanis et al. 2011). While PRMT5 can be detected in many human organs (e.g., heart, muscle, brain, and testis) (Pollack et al. 1999), it is particularly up-regulated in various forms of cancer, including leukemia and lymphoma (Pal et al. 2007; Wang et al. 2008), breast cancer (Powers et al. 2011), and
colorectal cancer (Cho et al. 2012). Importantly, PRMT5 up-regulation in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival (Yan et al. 2014). In addition, genetic depletion of PRMT5 was found to induce cell-cycle arrest, apoptosis, and loss of cell migratory activity in a variety of cancer cells (Cho et al. 2012; Chung et al. 2013; Yan et al. 2014), suggesting targeting PRMT5 may have therapeutic potential for the treatment of cancer. Received April 1, 2015; revised manuscript received May 16, 2015; accepted May 28, 2015. Address correspondence and reprint requests to Zhongbing Lu, College of Life Science, University of Chinese Academy of Science, 19A Yuquanlu, Beijing 100049, China. E-mail: luzhongbing@ ucas.ac.cn 1 These authors contributed equally to this work. Abbreviations used: AD, Alzheimer’s disease; APP, amyloid-b precursor protein; PRMT5, protein arginine methyltransferase 5; TUDCA, tauroursodeoxycholic acid.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
969
970
X. Quan et al.
Alzheimer’s disease (AD) is one of the most common progressive and neurodegenerative disorders affecting the elderly. Although the exact etiology of AD remains elusive, excessive neuronal cell death induced by beta amyloid (Ab) is one of the most important pathological characteristic in AD brain (Haass and Selkoe 2007). Immunohistochemistry revealed high PRMT5 expression in human neuronal cells, but not in glial cells (Han et al. 2014). However, the functional role of PRMT5 in the neuron has not been previously examined. In this study, we examined the impact of PRMT5 on AD pathogenesis in human SH-SY5Y cell line stably over-expressing the Swedish mutant of human amyloid-b precursor protein (APPsw) and in a transgenic Caenorhabditis elegans strain CL2006. We show that knockdown of PRMT5 significantly increased cell apoptosis in APPsw cells, and increased the paralysis of CL2006 worms, suggesting that PRMT5 is an important regulator of Ab neurotoxicity.
Material and methods Chemicals and antibodies Synthetic Ab25-35 peptide was purchased from Qiangyao Peptide Company (Jiangsu, China). To induce soluble oligomeric forms of Ab peptide, the peptides were dissolved in distilled water and incubated for 1 week at 37°C. Then the Ab solution was centrifuged at 12 000 g for 20 min at 4°C. The supernatant was used for experiment. Antibodies used in this study were listed in Table S1. Cell cultures and plasmids transfection Primary cortical neuronal cells from neonatal mice (C57BL/6 background) were isolated and maintained in neurobasal medium supplemented with 1% B27 supplement and 2 mM glutamine. The animal-related experiments and protocols were approved by the Animal Care and Use Committee of the University of Chinese Academy of Science. Human neuroblastoma SH-SY5Y cells stably expressing human APPsw or empty vector pCLNCXv.2 (neo) were cultured as previously reported (Wan et al. 2011). The pLKO.1TRC Cloning Vector (Addgene Plasmid 10878, Addgene, Cambridge, MA, USA) was used to construct clone shRNA against human PRMT5 (pLKO.1-PRMT5, target sequence is GCCATCTA TAAATGTCTGCTA) and E2F-1 (E2F Transcription Factor 1) (pLKO-E2F-1, target sequence is ACCTCTTCGACTGTGAC TTTG). PLKO.1-scramble shRNA was used as control. The pIRES2-Zsgreen1-PRMT5 plasmid was used to over-express PRMT5 protein and pIRES2-Zsgreen1 was used as control. Plasmids were transfected into APPsw cells using lipofectamine 2000 (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instruction. Puromycin was used to generate stably PLKO and PLKO-PRMT5 transfected clones in APPsw cells.
assay kit, respectively, (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). SYTOX green stains (Life Technologies), which do not cross intact cell membranes and exhibit increased fluorescence upon dsDNA binding, were used to visualize the dead cell. Worm strains and maintenance The wild type Bristol N2, CL2006 (dvIs2 [unc-54::human bamyloid 1-42; pRF4]) and GMC101 dvIs100 [unc-54p::A-beta-142::unc-54 30 -UTR + mtl-2p::green fluorescent protein (GFP)] were obtained from Caenorhabditis Genetics Center (University of Minnesota, Minnesota, MN, USA). Strains were maintained at 20°C and 40% relative to humidity condition on Nematode Growth Medium plates with Escherichia coli stain OP50 as a food source as previously reported (Luo et al. 2011). RNA interference and paralysis assay Briefly, egg-laid gravid adult worms were treated by the NaClO method for progeny synchronization. After 12–24 h, the L1 larvae were grown on RNA interference (RNAi) plates seeded with bacteria HT115 (DE3) expressing the dsRNA of the corresponding target gene. After reaching the young adult stage, the worms were maintained on the RNAi plates until assays were performed. The control and PRMT5 RNAi constructs were kindly provided by Professor Chonglin Yang (Institute of Genetics and Developmental Biology, CAS). The worms were tested for paralysis by tapping their noses with a platinum wire. Worms that moved their noses but failed to move their bodies were scored as ‘paralyzed’. To avoid scoring old worms as paralyzed, the paralysis assay was terminated on day 12 of adulthood (Cohen et al. 2006) for N2 and CL2006 worms. The paralysis of GMC101 strain was induced by shifting worms to a 25°C incubator. The final results represent the average of three independent experiments. Western blotting The cells were washed with ice-cold phosphate-buffered saline and then lysed in buffer (50 mM Tris-Cl, 150 mM NaCl, 100 lg/mL phenylmethylsulfonyl fluoride, protease, and phosphatase inhibitor cocktail from Roche (South San Francisco, CA, USA) and 1% Triton X-100) on ice for 30 min. After centrifugation at 12 000 g for 20 min at 4°C, the supernatant was used for western blot analysis as previous described (Luo et al. 2015). Statistical analysis All values are expressed as mean SE. Statistical significance was defined as p < 0.05. One-way ANOVA was used to test each variable for differences among the treatment groups with StatView (SAS Institute Inc., Cary, NC, USA). If ANOVA demonstrated a significant effect, pair-wise post hoc comparisons were made with Fisher’s least significant difference test.
Results Cell death assay Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) proliferation kit method. Cell apoptosis and death were determined by Annexin V-FITC apoptosis detection kit and lactate dehydrogenase (LDH) release
The expression of PRMT5 in aged Ab-treated neurons and APPsw cells To examine the role of PRMT5 in apoptotic neuronal death, we firstly treated mouse primary cortical neurons with 20 lg/
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
mL soluble oligomeric forms of Ab peptide for 48 h, which decreased the cell viability about 31.3% relative to control (Figure S1). We then measured the expression of PRMT5 in both control and oligomeric Ab25-35-treated neuronal cells by western blot. We found that oligomeric Ab25-35 induced about 54% decrease in PRMT5, as well as 70% increase in E2F-1(Fig. 1a and b). Similar results were also observed in SH-SY5Y cells treated with 20 lg/mL oligomeric Ab25-35 (Fig. 1c and d). In agreement with previous studies (Zhang et al. 2006; Wan et al. 2011), APPsw cells exhibited significantly higher levels of Ab1-42 as compared to neo cells (Figure S2a). The increased Ab1-42 secretion in APPsw cells significantly upregulated E2F-1 expression, but had no obvious effect on PRMT5 expression and translocation (Fig. 1e, f, and Figure S3). PRMT5 knockdown leads to apoptosis in APPsw cells Previous studies have demonstrated that PRMT5 depletion had little effect on SH-SY5Y cells (Park et al. 2015). Therefore, we transfected PLKO-PRMT5 plasmid into APPsw cells and then used puromycin to generate a stably transfected PRMT5 knockdown cell line. The significant reduction in PRMT5 expression in PLKO-PRMT5 cells was confirmed by western blot (Fig. 2a and b). Another APPsw cell line stably transfected with PLKO.1-scramble shRNA was used as control. After seeding in 96-well plates for 24 h, quantitation of viable cell numbers (MTT OD595 nm value) revealed that PLKO-PRMT5 cell number was only 61% that of the control group (0.371 0.019 vs. 0.599 0.024,
n = 8, p < 0.05). Similar results were also observed at 48 and 72 h, suggesting PRMT5 depletion triggered a greater reduction in the proliferative rate in APPsw cells (Fig. 2c). Using the propidium iodide (PI) and Annexin V-FITC, double-staining kit and flow cytometry assay, we measured cell apoptosis rates in both cell lines after 24 h of culture. Consistent with the MTT value, the apoptosis rate was almost doubled in the PLKO-PRMT5 cells and was significantly higher than that of the control cells (21.07 3.23% vs. 10.73 1.52%, n = 3, p < 0.05) (Fig. 2d). The increased cell death in PLKO-PRMT5 cells was confirmed by SYTOX staining (Fig. 2e), and also supported by the observation of increased levels of cleaved caspase-3 (Fig. 2a and b). When caspase-3 inhibitor Ac-DEVD-FMK (20 lM) were applied to PRMT5 deficient cells, the level of cell death was diminished (Fig. 2e), suggesting the apoptosis induced by PRMT5 depletion is caspase-3 dependent. PRMT5 regulates E2F-1/p53/bax and GSK-3b pathways in APPsw cells To investigate the mechanism by which PRMT5 depletion induces apoptosis in APPsw cells, we measured the expression of PRMT5’s target proteins in control and PRMT5 knockdown cells. As shown in Fig. 3(a) and (b), cells stably transfected with PLKO-PRMT5 shRNA exhibited a 71% increase in E2F-1 expression, as well as significant increases of p53 and Bax expression (50% and 60%, respectively). At the same time, glycogen synthase kinase (GSK-3b) activation appeared to be increased by PRMT5 knockdown, as indicated by reduced phosphorylation of its inhibitory
(a)
(c)
(e)
(b)
(d)
(f)
Fig. 1 Regulation of protein arginine methyltransferase 5 (PRMT5)/ E2F-1 pathway by oligomeric forms of Ab25-35 or secreted Ab in neuronal cells. Primary neuronal cells were treated with 20 lg/mL oligomeric Ab25-35 for 48 h, and then cell lysates were examined by western blot for expression of PRMT5, E2F-1. GAPDH was used as a
971
loading control (a and b). The PRMT5/E2F-1 pathway was also examined in control and 20 lg/mL oligomeric Ab25-35 treated SH-SY5Y cells (c and d), as well as in neo and Swedish mutant of human amyloid-b precursor protein (APPsw) cells (e and f). *p < 0.05 comparing control and aged Ab25-35 treated or APPsw cells.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
X. Quan et al.
972
(a)
(d)
(b)
(c)
(e)
Fig. 2 Protein arginine methyltransferase 5 (PRMT5) depletion in Swedish mutant of human amyloid-b precursor protein (APPsw) cells induced cell death. The expression of PRMT5 and cleaved caspase-3 was determined by western blot (a and b). Cells were seeded in 96well plates and cell viability was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 0, 24, 48 and
(a)
(c)
72 h (c). After seeding in 6-well plates for 24 h, cells were stained with PI and Annexin V-FITC and cell apoptosis rate was determined by flow cytometry (d). Ac-DEVD-FMK was used to inhibit Caspase-3 activity in PLKO-PRMT5 cells and cell death was indicated by SYTOX staining (e). n = 3–8, *p < 0.05 comparing control and PRMT5 knockdown cells.
(b)
Fig. 3 Protein arginine methyltransferase 5 (PRMT5) regulates E2F-1/p53/bax and GSK-3b pathways in Swedish mutant of human amyloid-b precursor protein (APPsw) cells. Lysates of control and PLKO-PRMT5 stably transfected APPsw cells were examined by western blot for expression of APP, PRMT5, E2F-1, p53, Bax, phosphorylated and total GSK-3b (a and b). The levels of those proteins were also determined in lysates of pIRES2Zsgreen1 and pIRES2-Zsgreen1-PRMT5 plasmid transfected APPsw cells (c and d). *p < 0.05 comparing control and PRMT5 knockdown or overexpression cells.
(d)
phosphorylation site (Ser9) in PLKO-PRMT5 cells (Fig. 3a and b). Conversely, over-expression of PRMT5 in APPsw cells resulted in the opposite effect on those pathways, as demonstrated by significantly reduced expression of E2F-1/
p53/Bax expression and increased GSK-3bSer9 phosphorylation as compared to control cells (Fig. 3c and d). To determine the effect of PRMT5 on Ab secretion, the levels of Ab1-42 in the culture medium were measured using
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
a sandwich ELISA kit. The results showed that neither knockdown nor over-expression of PRMT5 had significant effects on Ab1-42 secretion in APPsw cells (Figure S2). Western blot also revealed that the expression of APP was not affected by PRMT5 expression (Fig. 3). PRMT5 depletion induced IKK activation and IjB degradation PRMT5 has been shown to increase nuclear factor-jB (NFjB) activity through dimethylation of R30 of the p65 subunit (Wei et al. 2013). There is also evidence that NF-jB activation is associated with neuronal cell death (Huang et al. 2012). Therefore, we analyzed whether NF-jB signaling was involved in cell apoptosis induced by PRMT5 depletion. Western blot analysis of phosphorylated and total IKKa/b and IjBa revealed that phosphorylated IKKa/b (ser176/180) and IjBa (ser32) levels were significantly increased in PRMT5 knockdown cells as compared to control cells (Fig. 4). IKK levels were not changed, but IjBa levels were decreased by 30% in PRMT5 knockdown cells (Fig. 4). These data suggest that PRMT5 depletion induced IKK activation and IjB degradation, thereby increasing NFjB activation. Knockdown/inhibition of E2F-1 attenuated cell apoptosis Since E2F-1 is a direct target of PRMT5 and up-regulation of E2F-1 expression is associated with cell apoptosis (Cho et al. 2012), we examined the impact of E2F-1 expression in apoptosis by knocking down E2F-1 expression in PRMT5 deficient cells. Transfection of shRNA plasmid PLKO-E2F-1 into PRMT5 deficient cells for 72 h decreased E2F-1 expression by 75% (Fig. 5a and b). As compared with control cells, cleaved caspase-3, p53, Bax, and phosphorylated IjBa were markedly diminished in E2F-1deficient cells. We also found that E2F-1 knockdown significantly increased IjBa and p-GSK-3bSer9 levels, suggesting IjB degradation and GSK-3b activity were inhibited (Fig. 5a and b).
(a)
Fig. 4 Protein arginine methyltransferase 5 (PRMT5) depletion induced IjB kinase (IKK) activation and IjB degradation in Swedish mutant of human amyloid-b precursor protein (APPsw) cells. Lysates of control
973
To further verify the role of E2F-1 in cell apoptosis, we treated PRMT5 deficient cells with tauroursodeoxycholic acid (TUDCA), which has been shown to reduce Ab induced apoptosis of PC12 cells by inhibition of the E2F-1/p53/Bax pathway (Ramalho et al. 2004). As shown in Fig. 5(c), TUDCA increased cell viability in a dose-dependent manner. In particular, TUDCA at 50 lM concentration increased cell viability by 50% (p < 0.05) (Fig. 5c). The protective effects of E2F-1 knockdown or TUDCA (50 lM) on cell death were also confirmed by SYTOX staining (Fig. 5d). Inhibitors of NF-jB or GSK-3b rescue the cell death Since PRMT5 knockdown activates NF-jB and GSK-3b pathways in APPsw cells, we tested whether inhibiting NFjB or GSK-3b activity in PLKO-PRMT5 stably transfected APPsw cells could rescue the cell death phenotype. We first treated cells with different concentrations of NF-jB specific inhibitor (Pyrrolidinedithiocarbamic acid, ammonium salt, PDTC), or GSK-3b inhibitor III for 24 h and measured cell viability with the MTT method. Relative to control cells, PDTC (50 lM) and GSK-3b inhibitor III (25 lM) increased cell viability by 37% and 62%, respectively (p < 0.05) (Fig. 6a). The protective effect of the two inhibitors on cell death was further determined by quantifying LDH levels in the culture medium and western blot analysis. After treatment with PDTC (50 lM) or GSK-3b inhibitor III (25 lM) for 24 h, the LDH level in the culture medium and cleaved caspase-3 levels was significantly decreased as compared to control cells (Fig. 6b–d). Interestingly, inhibition of NF-jB or GSK-3b activity also down-regulated E2F1, suggesting additional crosstalk between E2F-1 and NF-jB or GSK-3b (Fig. 6c and d). PRMT5 knockdown exacerbates Ab induced paralysis in CL2006 worms In the CL2006 worms, the expression and subsequent aggregation of human Ab1-42 protein in the muscle leads to progressive paralysis of these worms (Keowkase et al.
(b)
and PLKO-PRMT5 stably transfected APPsw cells were examined by western blot for expression of phosphorylated and total IKKa/b and IjBa (a and b). *p < 0.05 comparing control and PRMT5 knockdown cells.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
974
(a)
X. Quan et al.
(b)
(c) (d)
Fig. 5 Knockdown or inhibition of E2F-1 rescued cell death induced by protein arginine methyltransferase 5 (PRMT5) depletion. Lysates of control and PLKO-E2F-1 transfected cells were examined by western blot for expression of E2F-1, cleaved caspase-3, p53, Bax, p-IjBa and p-GSK-3b. GAPDH was used as a loading control (a and b). After 12 h of seeding, PLKO-PRMT5 stably transfected Swedish mutant of human amyloid-b precursor protein (APPsw) cells were incubated
with different concentration of tauroursodeoxycholic acid (TUDCA) for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) method (c). The SYTOX fluorescence in control, E2F-1 knockdown and TUDCA treated cells was imaged using fluorescence microscopy (d). Results were collected from three to eight independent experiments. *p < 0.05 comparing control and E2F-1 knockdown or TUDCA treated cells.
2010). To investigate whether PRMT5 specifically affects Ab-induced toxicity in vivo, N2 and CL2006 worms were grown on plates seeded with bacteria HT115 (DE3), expressing control or dsRNA of PRMT5 for 12 days. We found that knockdown of PRMT5 significantly increased paralysis in CL2006 worms (34.45 1.38% vs. 18.86 1.43%, n = 3, p < 0.05), but had no effect on N2 worms (9.16 0.76% vs. 8.35 0.33%) (Fig. 7). Similar results were also observed in GMC101 worms (Figure S5).
noticed that PRMT5 mRNA dramatically decreased in five samples from patients with severe AD (about 11–30% of mean value of control samples), suggesting that decreased PRMT5 mRNA may be an important phenomenon in patients with severe AD.
Global gene profiling indicates PRMT5 mRNA was moderately decreased in hippocampus tissue of AD patients To understand the alteration of PRMT5 mRNA in hippocampus of AD patients, we downloaded an original microarray data set (GDS810; Reference Series: GSE1297) used for investigation of global gene profiles of hippocampus samples from the normal donor (n = 9), and patients with incipient (n = 7) or moderate (n = 8), and severe AD (n = 7)(Blalock et al. 2004). These microarray data revealed that hippocampal PRMT5 mRNA slightly decreased in AD patients (Table S2). In addition, we
Discussion The present study provides the first direct evidence that knockdown of PRMT5 exacerbates the neurotoxicity of Ab. PRMT5 depletion did not induce apoptosis in SH-SY5Y cells or exacerbate paralysis in N2 worms, but did increase cell apoptosis as well as paralysis in CL2006 worms that over-express Ab. The underlying mechanism for apoptosis induced by PRMT5 depletion involved with activation of E2F-1/p53/Bax, NF-jB, and GSK-3b pathways. The finding that PRMT5 over-expression inhibits E2F-1/p53/Bax and GSK-3b pathways in APPsw cells suggests that strategies aimed at activating PRMT5 in the neuron may represent a potential therapeutic approach for the prevention of neurodegenerative disorder.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
(a)
(b)
(c)
(d)
Fig. 6 Pyrrolidinedithiocarbamic acid, ammonium salt (PDTC) and GSK-3b inhibitor III rescued cell death induced by protein arginine methyltransferase 5 (PRMT5) depletion. 12 h after seeding, PLKOPRMT5 stably transfected Swedish mutant of human amyloid-b precursor protein (APPsw) cells were incubated with different concentration of PDTC or GSK-3b Inhibitor III for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (a). LDH release was determined in the culture
Fig. 7 Knockdown of protein arginine methyltransferase 5 (PRMT5) aggravated Ab-induced paralysis in C. elegans. On the 12th day of adulthood, paralysis was determined in N2 and CL2006 worms. *p < 0.05 comparing N2 to CL2006 worms. #p < 0.05 comparing control to PRMT5 knockdown worms.
Neuronal cell apoptosis is believed to contribute to the pathogenesis and progression of AD (Wirths et al. 2004; Lee et al. 2006). E2F-1 is a well-recognized pro-apoptotic factor
975
medium from control and 50 lM PDTC or 25 lM GSK-3b inhibitor III treated groups (b). Lysates of control and 50 lM PDTC or 25 lM GSK3b inhibitor III treated cells were examined by western blot for expression of cleaved caspase-3 and E2F-1. GAPDH was used as a loading control (c and d). Results were collected from three to eight independent experiments. *p < 0.05 comparing control and inhibitor treated cells.
that links DNA damage to apoptosis. Neurons lacking E2F-1 were significantly protected from death because of the Ab peptide (Giovanni et al. 2000). Conversely, over-expression of E2F-1 in PC12 cells was sufficient to induce cell apoptosis (Ramalho et al. 2004). In the present study, we observed a significant reduction in PRMT5 as well as a marked increase in E2F-1 protein levels in neuronal cells exposed to high dose Ab. The inverse relationship between PRMT5 and E2F1 could also be found in hippocampus tissue of control and AD patients according to the microarray data (Blalock et al. 2004) (Figure S4). In addition, the findings that the knockdown of PRMT5 increased E2F-1 expression, whereas overexpression of PRMT5 decreased E2F-1 expression in APPsw cells that identified PRMT5 as a negative regulator of neuronal E2F-1 expression. This is consistent with previous reports that PRMT5 regulates E2F-1 stability and target gene expression through arginine methylation (Cho et al. 2012). As PRMT5 expression was not changed, we speculated that the increased intracellular reactive oxygen species was responsible for the E2F-1 induction in APPsw cells. Significantly, caspase-3-mediated cell apoptosis and death in PLKO-PRMT5 stably transfected APPsw cells was rescued by knockdown or inhibition of E2F-1, indicating PRMT5 depletion promotes neuronal cell apoptosis through an E2F1-dependent pathway.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
976
X. Quan et al.
The tumor suppressor p53 is also up-regulated/activated in Ab-trigged neuronal cell apoptosis (Zhang et al. 2002; Morel et al. 2009). Here p53 can be methylated by PRMT5 at selected arginine residues (Jansson et al. 2008). Several studies have demonstrated that PRMT5 deficiency triggers p53-dependent apoptosis in response to DNA damage (Jansson et al. 2008; Yang et al. 2009). However, deficiency in PRMT5 has also been found to have no effect on the transcription or mRNA stability or protein stability of cep-1 (C. elegans p53 homolog)(Yang et al. 2009) reduce p53 levels and p53 transcriptional activity (Jansson et al. 2008), or inhibit p53 protein synthesis (Scoumanne et al. 2009). We found that PRMT5 depletion increases p53 levels and co-depletion of E2F-1 decreased p53 levels. Because E2F-1 can stabilize p53 protein through tumor suppressor protein p14ARF (Bates et al. 1998), it is possible that the increase in p53 protein levels in PRMT5 depleting cells was not related to arginine methylation, but rather, was dependent on E2F-1 induction. The transcription factor NF-jB is widely expressed in the nervous system and activation of NF-jB by Ab potentiates apoptosis in neuronal cells (Huang et al. 2012). In the present study, we found that PRMT5 depletion not only induced apoptosis and E2F-1 up-regulation, but also activated NF-jB, as evidenced by IKK activation and IjB degradation (Fig. 4). These findings are in contrast to a recent study which demonstrated that PRMT5 over-expression increases NF-jB activity though arginine dimethylation of the p65 subunit (Wei et al. 2013). Our findings that codepletion of E2F-1 inhibited IjB degradation (Fig. 5), suggests PRMT5 depletion-mediated E2F-1 induction may be responsible for NF-jB activation. GSK-3b is also involved in a common neuronal apoptotic pathway. Sustained pharmacological GSK-3b inhibition robustly decreases the amount of total Ab plaques and oligomeric Ab deposits in the brain of APPsw–tauvlw mice (DaRocha-Souto et al. 2012). GSK-3b inhibition regulates the cell cycle in primary cerebellar granule cells by inhibiting Rb phosphorylation and E2F-1 activity (Yeste-Velasco et al. 2007). Our results indicate that GSK-3b activity is regulated by PRMT5 through the E2F-1-dependent pathway and that GSK3b inhibiton significantly attenuates cell apoptosis and E2F-1 induction in PRMT5-depleted cells, suggesting a crosstalk between GSK-3b and E2F-1 in regulating neuronal apoptosis. Interestingly, there is convincing evidence that patients with a history of cancer have a reduced risk of developing AD (Nudelman et al. 2014). It has also been well established that aberrant expression of PRMT5 is associated with many cancer types such as lymphoma (Chung et al. 2013), glioblastoma multiform (Han et al. 2014) and testicular tumors (Liang et al. 2007). The finding that PRMT5 overexpression decreases E2F-1 expression and GSK-3b activity in APPsw cells suggests a potential mechanism for the protective effects of PRMT5 in Ab neurotoxicity. Thus, it will be interesting to examine whether susceptibility to
cancer is associated with globally increased PRMT5 expression, as this may suggest whether a PRMT5-related mechanism underlies the inverse association between cancer and protection against AD. Meanwhile, whether inducing PRMT5 for therapeutic purposes to treat AD would increase the risk of developing cancer still needs extensive study. In summary, our study demonstrates for the first time that PRMT5 regulates Ab neurotoxicity. PRMT5 depletion increases expression of E2F-1, thereby increasing neural cell susceptibility to apoptosis. Because PRMT5 is predominantly expressed in neurons and over-expression of PRMT5 attenuates E2F-1 and GSK-3b activation, strategies to increase PRMT5 activity may have therapeutic potential for the treatment of AD.
Acknowledgments and conflict of interest disclosure This study was supported by grants from the National Natural Science Foundation of China (81270319 and 81470520) and the Hundred Talents Program of Chinese Academy of Sciences. N2 and CL2006 strains were provided by the CGC, which was funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.
Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Table S1. The detail information of primary antibody. Table S2. Hippocampal PRMT5 mRNA levels in control and AD patients, data were extracted from the original microarray data set (GDS810; Reference Series: GSE1297). Figure S1. Aged Ab25-35decreased primary cortical neuronal cell viability in a dose-dependent manner. Figure S2. PRMT5 expression has no significantly effect on Ab secretion. Figure S3. The expressions of PRMT5 in cytoplasmand nucleuswere almost identical in neo and APPsw cells. Figure S4. PRMT5 is significant negative correlated with E2F-1 in hippocampus tissue of control and AD patients. Figure S5. Knockdown of PRMT5 aggravated Ab-induced paralysis in GMC101 strains. Figure S6. Knockdown of PRMT1 decreased E2F-1 expression in APPsw cells.
References Bates S., Phillips A. C., Clark P. A., Stott F., Peters G., Ludwig R. L. and Vousden K. H. (1998) p14ARF links the tumour suppressors RB and p53. Nature 395, 124–125. Bedford M. T. and Clarke S. G. (2009) Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1–13. Bedford M. T. and Richard S. (2005) Arginine methylation an emerging regulator of protein function. Mol. Cell 18, 263–272.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
Blalock E. M., Geddes J. W., Chen K. C., Porter N. M., Markesbery W. R. and Landfield P. W. (2004) Incipient Alzheimer’s disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc. Natl Acad. Sci. USA 101, 2173–2178. Cho E. C., Zheng S., Munro S. et al. (2012) Arginine methylation controls growth regulation by E2F-1. EMBO J. 31, 1785–1797. Chung J., Karkhanis V., Tae S. et al. (2013) Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing. J. Biol. Chem. 288, 35534–35547. Cohen E., Bieschke J., Perciavalle R. M., Kelly J. W. and Dillin A. (2006) Opposing activities protect against age-onset proteotoxicity. Science 313, 1604–1610. DaRocha-Souto B., Coma M., Perez-Nievas B. G. et al. (2012) Activation of glycogen synthase kinase-3 beta mediates betaamyloid induced neuritic damage in Alzheimer’s disease. Neurobiol. Dis. 45, 425–437. Giovanni A., Keramaris E., Morris E. J., Hou S. T., O’Hare M., Dyson N., Robertson G. S., Slack R. S. and Park D. S. (2000) E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3. J. Biol. Chem. 275, 11553–11560. Haass C. and Selkoe D. J. (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid betapeptide. Nat. Rev. Mol. Cell Biol. 8, 101–112. Han X., Li R., Zhang W. et al. (2014) Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro. J. Neurooncol. 118, 61–72. Huang X., Chen Y., Zhang H., Ma Q., Zhang Y. W. and Xu H. (2012) Salubrinal attenuates beta-amyloid-induced neuronal death and microglial activation by inhibition of the NF-kappaB pathway. Neurobiol. Aging 33(1007), e1009–e1017. Jansson M., Durant S. T., Cho E. C., Sheahan S., Edelmann M., Kessler B. and La Thangue N. B. (2008) Arginine methylation regulates the p53 response. Nat. Cell Biol. 10, 1431–1439. Karkhanis V., Hu Y. J., Baiocchi R. A., Imbalzano A. N. and Sif S. (2011) Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem. Sci. 36, 633–641. Keowkase R., Aboukhatwa M., Adam B. L., Beach J. W., Terry A. V., Jr, Buccafussco J. J. and Luo Y. (2010) Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1-84-1 and JAY 2-22-33 in neuronal culture and Caenorhabditis elegans. Mol. Neurodegener. 5, 59. Lee K. W., Im J. Y., Song J. S. et al. (2006) Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein. Neurobiol. Dis. 22, 10–24. Liang J. J., Wang Z., Chiriboga L. et al. (2007) The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors. J. Urol. 177, 1918–1922. Luo Y., Zhang J., Liu N. and Zhao B. (2011) Copper ions influence the toxicity of beta-amyloid (1-42) in a concentration-dependent manner in a Caenorhabditis elegans model of Alzheimer’s disease. Sci. China Life Sci. 54, 527–534. Luo Y., Yue W., Quan X., Wang Y., Zhao B. and Lu Z. (2015) Asymmetric dimethylarginine exacerbates Abeta-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radic. Biol. Med. 79, 117–126. Morel M., Couturier J., Pontcharraud R., Gil R., Fauconneau B., Paccalin M. and Page G. (2009) Evidence of molecular links between PKR and mTOR signalling pathways in Abeta neurotoxicity: role of p53, Redd1 and TSC2. Neurobiol. Dis. 36, 151–161.
977
Nudelman K. N., Risacher S. L., West J. D., McDonald B. C., Gao S. and Saykin A. J. (2014) Association of cancer history with Alzheimer’s disease onset and structural brain changes. Front. Physiol. 5, 423. Pal S., Baiocchi R. A., Byrd J. C., Grever M. R., Jacob S. T. and Sif S. (2007) Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J. 26, 3558–3569. Park J. H., Szemes M., Vieira G. C. et al. (2015) Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells. Mol. Oncol. 9, 617–627. Pollack B. P., Kotenko S. V., He W., Izotova L. S., Barnoski B. L. and Pestka S. (1999) The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity. J. Biol. Chem. 274, 31531–31542. Powers M. A., Fay M. M., Factor R. E., Welm A. L. and Ullman K. S. (2011) Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 71, 5579–5587. Ramalho R. M., Ribeiro P. S., Sola S., Castro R. E., Steer C. J. and Rodrigues C. M. (2004) Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid beta-peptide-induced apoptosis of PC12 cells. J. Neurochem. 90, 567–575. Scoumanne A., Zhang J. and Chen X. (2009) PRMT5 is required for cell-cycle progression and p53 tumor suppressor function. Nucleic Acids Res. 37, 4965–4976. Wan L., Nie G., Zhang J., Luo Y., Zhang P., Zhang Z. and Zhao B. (2011) beta-Amyloid peptide increases levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radic. Biol. Med. 50, 122–129. Wang L., Pal S. and Sif S. (2008) Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells. Mol. Cell. Biol. 28, 6262–6277. Wei H., Wang B., Miyagi M., She Y., Gopalan B., Huang D. B., Ghosh G., Stark G. R. and Lu T. (2013) PRMT5 dimethylates R30 of the p65 subunit to activate NF-kappaB. Proc. Natl Acad. Sci. USA 110, 13516–13521. Wirths O., Multhaup G. and Bayer T. A. (2004) A modified betaamyloid hypothesis: intraneuronal accumulation of the betaamyloid peptide–the first step of a fatal cascade. J. Neurochem. 91, 513–520. Wolf S. S. (2009) The protein arginine methyltransferase family: an update about function, new perspectives and the physiological role in humans. Cell. Mol. Life Sci. 66, 2109–2121. Yan F., Alinari L., Lustberg M. E. et al. (2014) Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res. 74, 1752–1765. Yang M., Sun J., Sun X., Shen Q., Gao Z. and Yang C. (2009) Caenorhabditis elegans protein arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. PLoS Genet. 5, e1000514. Yeste-Velasco M., Folch J., Trullas R., Abad M. A., Enguita M., Pallas M. and Camins A. (2007) Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells. Neuropharmacology 53, 295–307. Zhang Y., McLaughlin R., Goodyer C. and LeBlanc A. (2002) Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons. J. Cell Biol. 156, 519–529. Zhang J., Mori A., Chen Q. and Zhao B. (2006) Fermented papaya preparation attenuates beta-amyloid precursor protein: betaamyloid-mediated copper neurotoxicity in beta-amyloid precursor protein and beta-amyloid precursor protein Swedish mutation overexpressing SH-SY5Y cells. Neuroscience 143, 63–72.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
| 2015 | 134 | 969–977
1
doi: 10.1111/jnc.13191
1
College of Life Sciences, University of Chinese Academy of Science, Beijing, China
Abstract The protein arginine methyltransferase 5 (PRMT5) controls cell growth and apoptosis by catalyzing mono and symmetric dimethylation of arginine residues. In human brain tissue, PRMT5 is predominantly expressed in neuronal cells. There is evidence that PRMT5 provides protection against cell death, but the impact of PRMT5 on neuronal apoptosis during the evolution of Alzheimer’s disease has not been tested. In the present study, we show that PRMT5 is down-regulated by bamyloid (Ab) in primary neurons and SH-SY5Y cells, and this is associated with the up-regulation of the PRMT5 target protein E2F-1. Furthermore, knockdown of PRMT5 in SHSY5Y cells over-expressing the Swedish mutant form of human amyloid-b precursor protein caused activation of E2F1/p53/Bax, NF-jB, and GSK-3b pathways, which coincided
with increased apoptosis. Co-depletion of E2F-1 reduced the activation of p53/Bax, NF-jB, and GSK-3b, and limited cell apoptosis. In addition, inhibiting NF-jB and GSK-3b activity by specific inhibitors also attenuated cell apoptosis, suggesting that E2F-1/NF-jB/GSK-3b pathways mediate for apoptosis induced by PRMT5 depletion. More importantly, knockdown of PRMT5 resulted in more paralysis in a transgenic Caenorhabditis elegans strain CL2006, indicating that PRMT5 provides protection against Ab toxicity in vivo. Collectively, our findings identify PRMT5 as a novel regulator of Ab toxicity and suggest that strategies aimed at activating PRMT5 in the neuron may represent a potential therapeutic approach for the prevention of Alzheimer’s disease. Keywords: Alzheimer’s disease, Apoptosis, E2F-1, PRMT5. J. Neurochem. (2015) 134, 969–977.
Protein methylation is increasingly recognized as an important type of post-translational modification that regulates protein function (Bedford and Richard 2005; Bedford and Clarke 2009). The methylation of arginine residues is catalyzed by a family of intracellular enzymes termed protein arginine methyltransferase (PRMT), which have been classified by their specific catalytic activities: type I enzymes catalyze formation of asymmetric modifications, while type II enzymes catalyze symmetric modifications (Bedford and Clarke 2009; Wolf 2009). As a type II enzyme, PRMT5 functions in the nucleus as well as in the cytoplasm and its substrates include histones, spliceosomal proteins, transcription factors, and proteins involved in PIWI-interacting RNA (piRNA) biogenesis (Bedford and Clarke 2009; Karkhanis et al. 2011). While PRMT5 can be detected in many human organs (e.g., heart, muscle, brain, and testis) (Pollack et al. 1999), it is particularly up-regulated in various forms of cancer, including leukemia and lymphoma (Pal et al. 2007; Wang et al. 2008), breast cancer (Powers et al. 2011), and
colorectal cancer (Cho et al. 2012). Importantly, PRMT5 up-regulation in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival (Yan et al. 2014). In addition, genetic depletion of PRMT5 was found to induce cell-cycle arrest, apoptosis, and loss of cell migratory activity in a variety of cancer cells (Cho et al. 2012; Chung et al. 2013; Yan et al. 2014), suggesting targeting PRMT5 may have therapeutic potential for the treatment of cancer. Received April 1, 2015; revised manuscript received May 16, 2015; accepted May 28, 2015. Address correspondence and reprint requests to Zhongbing Lu, College of Life Science, University of Chinese Academy of Science, 19A Yuquanlu, Beijing 100049, China. E-mail: luzhongbing@ ucas.ac.cn 1 These authors contributed equally to this work. Abbreviations used: AD, Alzheimer’s disease; APP, amyloid-b precursor protein; PRMT5, protein arginine methyltransferase 5; TUDCA, tauroursodeoxycholic acid.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
969
970
X. Quan et al.
Alzheimer’s disease (AD) is one of the most common progressive and neurodegenerative disorders affecting the elderly. Although the exact etiology of AD remains elusive, excessive neuronal cell death induced by beta amyloid (Ab) is one of the most important pathological characteristic in AD brain (Haass and Selkoe 2007). Immunohistochemistry revealed high PRMT5 expression in human neuronal cells, but not in glial cells (Han et al. 2014). However, the functional role of PRMT5 in the neuron has not been previously examined. In this study, we examined the impact of PRMT5 on AD pathogenesis in human SH-SY5Y cell line stably over-expressing the Swedish mutant of human amyloid-b precursor protein (APPsw) and in a transgenic Caenorhabditis elegans strain CL2006. We show that knockdown of PRMT5 significantly increased cell apoptosis in APPsw cells, and increased the paralysis of CL2006 worms, suggesting that PRMT5 is an important regulator of Ab neurotoxicity.
Material and methods Chemicals and antibodies Synthetic Ab25-35 peptide was purchased from Qiangyao Peptide Company (Jiangsu, China). To induce soluble oligomeric forms of Ab peptide, the peptides were dissolved in distilled water and incubated for 1 week at 37°C. Then the Ab solution was centrifuged at 12 000 g for 20 min at 4°C. The supernatant was used for experiment. Antibodies used in this study were listed in Table S1. Cell cultures and plasmids transfection Primary cortical neuronal cells from neonatal mice (C57BL/6 background) were isolated and maintained in neurobasal medium supplemented with 1% B27 supplement and 2 mM glutamine. The animal-related experiments and protocols were approved by the Animal Care and Use Committee of the University of Chinese Academy of Science. Human neuroblastoma SH-SY5Y cells stably expressing human APPsw or empty vector pCLNCXv.2 (neo) were cultured as previously reported (Wan et al. 2011). The pLKO.1TRC Cloning Vector (Addgene Plasmid 10878, Addgene, Cambridge, MA, USA) was used to construct clone shRNA against human PRMT5 (pLKO.1-PRMT5, target sequence is GCCATCTA TAAATGTCTGCTA) and E2F-1 (E2F Transcription Factor 1) (pLKO-E2F-1, target sequence is ACCTCTTCGACTGTGAC TTTG). PLKO.1-scramble shRNA was used as control. The pIRES2-Zsgreen1-PRMT5 plasmid was used to over-express PRMT5 protein and pIRES2-Zsgreen1 was used as control. Plasmids were transfected into APPsw cells using lipofectamine 2000 (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instruction. Puromycin was used to generate stably PLKO and PLKO-PRMT5 transfected clones in APPsw cells.
assay kit, respectively, (Beyotime Institute of Biotechnology, Nantong, Jiangsu, China). SYTOX green stains (Life Technologies), which do not cross intact cell membranes and exhibit increased fluorescence upon dsDNA binding, were used to visualize the dead cell. Worm strains and maintenance The wild type Bristol N2, CL2006 (dvIs2 [unc-54::human bamyloid 1-42; pRF4]) and GMC101 dvIs100 [unc-54p::A-beta-142::unc-54 30 -UTR + mtl-2p::green fluorescent protein (GFP)] were obtained from Caenorhabditis Genetics Center (University of Minnesota, Minnesota, MN, USA). Strains were maintained at 20°C and 40% relative to humidity condition on Nematode Growth Medium plates with Escherichia coli stain OP50 as a food source as previously reported (Luo et al. 2011). RNA interference and paralysis assay Briefly, egg-laid gravid adult worms were treated by the NaClO method for progeny synchronization. After 12–24 h, the L1 larvae were grown on RNA interference (RNAi) plates seeded with bacteria HT115 (DE3) expressing the dsRNA of the corresponding target gene. After reaching the young adult stage, the worms were maintained on the RNAi plates until assays were performed. The control and PRMT5 RNAi constructs were kindly provided by Professor Chonglin Yang (Institute of Genetics and Developmental Biology, CAS). The worms were tested for paralysis by tapping their noses with a platinum wire. Worms that moved their noses but failed to move their bodies were scored as ‘paralyzed’. To avoid scoring old worms as paralyzed, the paralysis assay was terminated on day 12 of adulthood (Cohen et al. 2006) for N2 and CL2006 worms. The paralysis of GMC101 strain was induced by shifting worms to a 25°C incubator. The final results represent the average of three independent experiments. Western blotting The cells were washed with ice-cold phosphate-buffered saline and then lysed in buffer (50 mM Tris-Cl, 150 mM NaCl, 100 lg/mL phenylmethylsulfonyl fluoride, protease, and phosphatase inhibitor cocktail from Roche (South San Francisco, CA, USA) and 1% Triton X-100) on ice for 30 min. After centrifugation at 12 000 g for 20 min at 4°C, the supernatant was used for western blot analysis as previous described (Luo et al. 2015). Statistical analysis All values are expressed as mean SE. Statistical significance was defined as p < 0.05. One-way ANOVA was used to test each variable for differences among the treatment groups with StatView (SAS Institute Inc., Cary, NC, USA). If ANOVA demonstrated a significant effect, pair-wise post hoc comparisons were made with Fisher’s least significant difference test.
Results Cell death assay Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) proliferation kit method. Cell apoptosis and death were determined by Annexin V-FITC apoptosis detection kit and lactate dehydrogenase (LDH) release
The expression of PRMT5 in aged Ab-treated neurons and APPsw cells To examine the role of PRMT5 in apoptotic neuronal death, we firstly treated mouse primary cortical neurons with 20 lg/
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
mL soluble oligomeric forms of Ab peptide for 48 h, which decreased the cell viability about 31.3% relative to control (Figure S1). We then measured the expression of PRMT5 in both control and oligomeric Ab25-35-treated neuronal cells by western blot. We found that oligomeric Ab25-35 induced about 54% decrease in PRMT5, as well as 70% increase in E2F-1(Fig. 1a and b). Similar results were also observed in SH-SY5Y cells treated with 20 lg/mL oligomeric Ab25-35 (Fig. 1c and d). In agreement with previous studies (Zhang et al. 2006; Wan et al. 2011), APPsw cells exhibited significantly higher levels of Ab1-42 as compared to neo cells (Figure S2a). The increased Ab1-42 secretion in APPsw cells significantly upregulated E2F-1 expression, but had no obvious effect on PRMT5 expression and translocation (Fig. 1e, f, and Figure S3). PRMT5 knockdown leads to apoptosis in APPsw cells Previous studies have demonstrated that PRMT5 depletion had little effect on SH-SY5Y cells (Park et al. 2015). Therefore, we transfected PLKO-PRMT5 plasmid into APPsw cells and then used puromycin to generate a stably transfected PRMT5 knockdown cell line. The significant reduction in PRMT5 expression in PLKO-PRMT5 cells was confirmed by western blot (Fig. 2a and b). Another APPsw cell line stably transfected with PLKO.1-scramble shRNA was used as control. After seeding in 96-well plates for 24 h, quantitation of viable cell numbers (MTT OD595 nm value) revealed that PLKO-PRMT5 cell number was only 61% that of the control group (0.371 0.019 vs. 0.599 0.024,
n = 8, p < 0.05). Similar results were also observed at 48 and 72 h, suggesting PRMT5 depletion triggered a greater reduction in the proliferative rate in APPsw cells (Fig. 2c). Using the propidium iodide (PI) and Annexin V-FITC, double-staining kit and flow cytometry assay, we measured cell apoptosis rates in both cell lines after 24 h of culture. Consistent with the MTT value, the apoptosis rate was almost doubled in the PLKO-PRMT5 cells and was significantly higher than that of the control cells (21.07 3.23% vs. 10.73 1.52%, n = 3, p < 0.05) (Fig. 2d). The increased cell death in PLKO-PRMT5 cells was confirmed by SYTOX staining (Fig. 2e), and also supported by the observation of increased levels of cleaved caspase-3 (Fig. 2a and b). When caspase-3 inhibitor Ac-DEVD-FMK (20 lM) were applied to PRMT5 deficient cells, the level of cell death was diminished (Fig. 2e), suggesting the apoptosis induced by PRMT5 depletion is caspase-3 dependent. PRMT5 regulates E2F-1/p53/bax and GSK-3b pathways in APPsw cells To investigate the mechanism by which PRMT5 depletion induces apoptosis in APPsw cells, we measured the expression of PRMT5’s target proteins in control and PRMT5 knockdown cells. As shown in Fig. 3(a) and (b), cells stably transfected with PLKO-PRMT5 shRNA exhibited a 71% increase in E2F-1 expression, as well as significant increases of p53 and Bax expression (50% and 60%, respectively). At the same time, glycogen synthase kinase (GSK-3b) activation appeared to be increased by PRMT5 knockdown, as indicated by reduced phosphorylation of its inhibitory
(a)
(c)
(e)
(b)
(d)
(f)
Fig. 1 Regulation of protein arginine methyltransferase 5 (PRMT5)/ E2F-1 pathway by oligomeric forms of Ab25-35 or secreted Ab in neuronal cells. Primary neuronal cells were treated with 20 lg/mL oligomeric Ab25-35 for 48 h, and then cell lysates were examined by western blot for expression of PRMT5, E2F-1. GAPDH was used as a
971
loading control (a and b). The PRMT5/E2F-1 pathway was also examined in control and 20 lg/mL oligomeric Ab25-35 treated SH-SY5Y cells (c and d), as well as in neo and Swedish mutant of human amyloid-b precursor protein (APPsw) cells (e and f). *p < 0.05 comparing control and aged Ab25-35 treated or APPsw cells.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
X. Quan et al.
972
(a)
(d)
(b)
(c)
(e)
Fig. 2 Protein arginine methyltransferase 5 (PRMT5) depletion in Swedish mutant of human amyloid-b precursor protein (APPsw) cells induced cell death. The expression of PRMT5 and cleaved caspase-3 was determined by western blot (a and b). Cells were seeded in 96well plates and cell viability was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 0, 24, 48 and
(a)
(c)
72 h (c). After seeding in 6-well plates for 24 h, cells were stained with PI and Annexin V-FITC and cell apoptosis rate was determined by flow cytometry (d). Ac-DEVD-FMK was used to inhibit Caspase-3 activity in PLKO-PRMT5 cells and cell death was indicated by SYTOX staining (e). n = 3–8, *p < 0.05 comparing control and PRMT5 knockdown cells.
(b)
Fig. 3 Protein arginine methyltransferase 5 (PRMT5) regulates E2F-1/p53/bax and GSK-3b pathways in Swedish mutant of human amyloid-b precursor protein (APPsw) cells. Lysates of control and PLKO-PRMT5 stably transfected APPsw cells were examined by western blot for expression of APP, PRMT5, E2F-1, p53, Bax, phosphorylated and total GSK-3b (a and b). The levels of those proteins were also determined in lysates of pIRES2Zsgreen1 and pIRES2-Zsgreen1-PRMT5 plasmid transfected APPsw cells (c and d). *p < 0.05 comparing control and PRMT5 knockdown or overexpression cells.
(d)
phosphorylation site (Ser9) in PLKO-PRMT5 cells (Fig. 3a and b). Conversely, over-expression of PRMT5 in APPsw cells resulted in the opposite effect on those pathways, as demonstrated by significantly reduced expression of E2F-1/
p53/Bax expression and increased GSK-3bSer9 phosphorylation as compared to control cells (Fig. 3c and d). To determine the effect of PRMT5 on Ab secretion, the levels of Ab1-42 in the culture medium were measured using
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
a sandwich ELISA kit. The results showed that neither knockdown nor over-expression of PRMT5 had significant effects on Ab1-42 secretion in APPsw cells (Figure S2). Western blot also revealed that the expression of APP was not affected by PRMT5 expression (Fig. 3). PRMT5 depletion induced IKK activation and IjB degradation PRMT5 has been shown to increase nuclear factor-jB (NFjB) activity through dimethylation of R30 of the p65 subunit (Wei et al. 2013). There is also evidence that NF-jB activation is associated with neuronal cell death (Huang et al. 2012). Therefore, we analyzed whether NF-jB signaling was involved in cell apoptosis induced by PRMT5 depletion. Western blot analysis of phosphorylated and total IKKa/b and IjBa revealed that phosphorylated IKKa/b (ser176/180) and IjBa (ser32) levels were significantly increased in PRMT5 knockdown cells as compared to control cells (Fig. 4). IKK levels were not changed, but IjBa levels were decreased by 30% in PRMT5 knockdown cells (Fig. 4). These data suggest that PRMT5 depletion induced IKK activation and IjB degradation, thereby increasing NFjB activation. Knockdown/inhibition of E2F-1 attenuated cell apoptosis Since E2F-1 is a direct target of PRMT5 and up-regulation of E2F-1 expression is associated with cell apoptosis (Cho et al. 2012), we examined the impact of E2F-1 expression in apoptosis by knocking down E2F-1 expression in PRMT5 deficient cells. Transfection of shRNA plasmid PLKO-E2F-1 into PRMT5 deficient cells for 72 h decreased E2F-1 expression by 75% (Fig. 5a and b). As compared with control cells, cleaved caspase-3, p53, Bax, and phosphorylated IjBa were markedly diminished in E2F-1deficient cells. We also found that E2F-1 knockdown significantly increased IjBa and p-GSK-3bSer9 levels, suggesting IjB degradation and GSK-3b activity were inhibited (Fig. 5a and b).
(a)
Fig. 4 Protein arginine methyltransferase 5 (PRMT5) depletion induced IjB kinase (IKK) activation and IjB degradation in Swedish mutant of human amyloid-b precursor protein (APPsw) cells. Lysates of control
973
To further verify the role of E2F-1 in cell apoptosis, we treated PRMT5 deficient cells with tauroursodeoxycholic acid (TUDCA), which has been shown to reduce Ab induced apoptosis of PC12 cells by inhibition of the E2F-1/p53/Bax pathway (Ramalho et al. 2004). As shown in Fig. 5(c), TUDCA increased cell viability in a dose-dependent manner. In particular, TUDCA at 50 lM concentration increased cell viability by 50% (p < 0.05) (Fig. 5c). The protective effects of E2F-1 knockdown or TUDCA (50 lM) on cell death were also confirmed by SYTOX staining (Fig. 5d). Inhibitors of NF-jB or GSK-3b rescue the cell death Since PRMT5 knockdown activates NF-jB and GSK-3b pathways in APPsw cells, we tested whether inhibiting NFjB or GSK-3b activity in PLKO-PRMT5 stably transfected APPsw cells could rescue the cell death phenotype. We first treated cells with different concentrations of NF-jB specific inhibitor (Pyrrolidinedithiocarbamic acid, ammonium salt, PDTC), or GSK-3b inhibitor III for 24 h and measured cell viability with the MTT method. Relative to control cells, PDTC (50 lM) and GSK-3b inhibitor III (25 lM) increased cell viability by 37% and 62%, respectively (p < 0.05) (Fig. 6a). The protective effect of the two inhibitors on cell death was further determined by quantifying LDH levels in the culture medium and western blot analysis. After treatment with PDTC (50 lM) or GSK-3b inhibitor III (25 lM) for 24 h, the LDH level in the culture medium and cleaved caspase-3 levels was significantly decreased as compared to control cells (Fig. 6b–d). Interestingly, inhibition of NF-jB or GSK-3b activity also down-regulated E2F1, suggesting additional crosstalk between E2F-1 and NF-jB or GSK-3b (Fig. 6c and d). PRMT5 knockdown exacerbates Ab induced paralysis in CL2006 worms In the CL2006 worms, the expression and subsequent aggregation of human Ab1-42 protein in the muscle leads to progressive paralysis of these worms (Keowkase et al.
(b)
and PLKO-PRMT5 stably transfected APPsw cells were examined by western blot for expression of phosphorylated and total IKKa/b and IjBa (a and b). *p < 0.05 comparing control and PRMT5 knockdown cells.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
974
(a)
X. Quan et al.
(b)
(c) (d)
Fig. 5 Knockdown or inhibition of E2F-1 rescued cell death induced by protein arginine methyltransferase 5 (PRMT5) depletion. Lysates of control and PLKO-E2F-1 transfected cells were examined by western blot for expression of E2F-1, cleaved caspase-3, p53, Bax, p-IjBa and p-GSK-3b. GAPDH was used as a loading control (a and b). After 12 h of seeding, PLKO-PRMT5 stably transfected Swedish mutant of human amyloid-b precursor protein (APPsw) cells were incubated
with different concentration of tauroursodeoxycholic acid (TUDCA) for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) method (c). The SYTOX fluorescence in control, E2F-1 knockdown and TUDCA treated cells was imaged using fluorescence microscopy (d). Results were collected from three to eight independent experiments. *p < 0.05 comparing control and E2F-1 knockdown or TUDCA treated cells.
2010). To investigate whether PRMT5 specifically affects Ab-induced toxicity in vivo, N2 and CL2006 worms were grown on plates seeded with bacteria HT115 (DE3), expressing control or dsRNA of PRMT5 for 12 days. We found that knockdown of PRMT5 significantly increased paralysis in CL2006 worms (34.45 1.38% vs. 18.86 1.43%, n = 3, p < 0.05), but had no effect on N2 worms (9.16 0.76% vs. 8.35 0.33%) (Fig. 7). Similar results were also observed in GMC101 worms (Figure S5).
noticed that PRMT5 mRNA dramatically decreased in five samples from patients with severe AD (about 11–30% of mean value of control samples), suggesting that decreased PRMT5 mRNA may be an important phenomenon in patients with severe AD.
Global gene profiling indicates PRMT5 mRNA was moderately decreased in hippocampus tissue of AD patients To understand the alteration of PRMT5 mRNA in hippocampus of AD patients, we downloaded an original microarray data set (GDS810; Reference Series: GSE1297) used for investigation of global gene profiles of hippocampus samples from the normal donor (n = 9), and patients with incipient (n = 7) or moderate (n = 8), and severe AD (n = 7)(Blalock et al. 2004). These microarray data revealed that hippocampal PRMT5 mRNA slightly decreased in AD patients (Table S2). In addition, we
Discussion The present study provides the first direct evidence that knockdown of PRMT5 exacerbates the neurotoxicity of Ab. PRMT5 depletion did not induce apoptosis in SH-SY5Y cells or exacerbate paralysis in N2 worms, but did increase cell apoptosis as well as paralysis in CL2006 worms that over-express Ab. The underlying mechanism for apoptosis induced by PRMT5 depletion involved with activation of E2F-1/p53/Bax, NF-jB, and GSK-3b pathways. The finding that PRMT5 over-expression inhibits E2F-1/p53/Bax and GSK-3b pathways in APPsw cells suggests that strategies aimed at activating PRMT5 in the neuron may represent a potential therapeutic approach for the prevention of neurodegenerative disorder.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
(a)
(b)
(c)
(d)
Fig. 6 Pyrrolidinedithiocarbamic acid, ammonium salt (PDTC) and GSK-3b inhibitor III rescued cell death induced by protein arginine methyltransferase 5 (PRMT5) depletion. 12 h after seeding, PLKOPRMT5 stably transfected Swedish mutant of human amyloid-b precursor protein (APPsw) cells were incubated with different concentration of PDTC or GSK-3b Inhibitor III for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (a). LDH release was determined in the culture
Fig. 7 Knockdown of protein arginine methyltransferase 5 (PRMT5) aggravated Ab-induced paralysis in C. elegans. On the 12th day of adulthood, paralysis was determined in N2 and CL2006 worms. *p < 0.05 comparing N2 to CL2006 worms. #p < 0.05 comparing control to PRMT5 knockdown worms.
Neuronal cell apoptosis is believed to contribute to the pathogenesis and progression of AD (Wirths et al. 2004; Lee et al. 2006). E2F-1 is a well-recognized pro-apoptotic factor
975
medium from control and 50 lM PDTC or 25 lM GSK-3b inhibitor III treated groups (b). Lysates of control and 50 lM PDTC or 25 lM GSK3b inhibitor III treated cells were examined by western blot for expression of cleaved caspase-3 and E2F-1. GAPDH was used as a loading control (c and d). Results were collected from three to eight independent experiments. *p < 0.05 comparing control and inhibitor treated cells.
that links DNA damage to apoptosis. Neurons lacking E2F-1 were significantly protected from death because of the Ab peptide (Giovanni et al. 2000). Conversely, over-expression of E2F-1 in PC12 cells was sufficient to induce cell apoptosis (Ramalho et al. 2004). In the present study, we observed a significant reduction in PRMT5 as well as a marked increase in E2F-1 protein levels in neuronal cells exposed to high dose Ab. The inverse relationship between PRMT5 and E2F1 could also be found in hippocampus tissue of control and AD patients according to the microarray data (Blalock et al. 2004) (Figure S4). In addition, the findings that the knockdown of PRMT5 increased E2F-1 expression, whereas overexpression of PRMT5 decreased E2F-1 expression in APPsw cells that identified PRMT5 as a negative regulator of neuronal E2F-1 expression. This is consistent with previous reports that PRMT5 regulates E2F-1 stability and target gene expression through arginine methylation (Cho et al. 2012). As PRMT5 expression was not changed, we speculated that the increased intracellular reactive oxygen species was responsible for the E2F-1 induction in APPsw cells. Significantly, caspase-3-mediated cell apoptosis and death in PLKO-PRMT5 stably transfected APPsw cells was rescued by knockdown or inhibition of E2F-1, indicating PRMT5 depletion promotes neuronal cell apoptosis through an E2F1-dependent pathway.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
976
X. Quan et al.
The tumor suppressor p53 is also up-regulated/activated in Ab-trigged neuronal cell apoptosis (Zhang et al. 2002; Morel et al. 2009). Here p53 can be methylated by PRMT5 at selected arginine residues (Jansson et al. 2008). Several studies have demonstrated that PRMT5 deficiency triggers p53-dependent apoptosis in response to DNA damage (Jansson et al. 2008; Yang et al. 2009). However, deficiency in PRMT5 has also been found to have no effect on the transcription or mRNA stability or protein stability of cep-1 (C. elegans p53 homolog)(Yang et al. 2009) reduce p53 levels and p53 transcriptional activity (Jansson et al. 2008), or inhibit p53 protein synthesis (Scoumanne et al. 2009). We found that PRMT5 depletion increases p53 levels and co-depletion of E2F-1 decreased p53 levels. Because E2F-1 can stabilize p53 protein through tumor suppressor protein p14ARF (Bates et al. 1998), it is possible that the increase in p53 protein levels in PRMT5 depleting cells was not related to arginine methylation, but rather, was dependent on E2F-1 induction. The transcription factor NF-jB is widely expressed in the nervous system and activation of NF-jB by Ab potentiates apoptosis in neuronal cells (Huang et al. 2012). In the present study, we found that PRMT5 depletion not only induced apoptosis and E2F-1 up-regulation, but also activated NF-jB, as evidenced by IKK activation and IjB degradation (Fig. 4). These findings are in contrast to a recent study which demonstrated that PRMT5 over-expression increases NF-jB activity though arginine dimethylation of the p65 subunit (Wei et al. 2013). Our findings that codepletion of E2F-1 inhibited IjB degradation (Fig. 5), suggests PRMT5 depletion-mediated E2F-1 induction may be responsible for NF-jB activation. GSK-3b is also involved in a common neuronal apoptotic pathway. Sustained pharmacological GSK-3b inhibition robustly decreases the amount of total Ab plaques and oligomeric Ab deposits in the brain of APPsw–tauvlw mice (DaRocha-Souto et al. 2012). GSK-3b inhibition regulates the cell cycle in primary cerebellar granule cells by inhibiting Rb phosphorylation and E2F-1 activity (Yeste-Velasco et al. 2007). Our results indicate that GSK-3b activity is regulated by PRMT5 through the E2F-1-dependent pathway and that GSK3b inhibiton significantly attenuates cell apoptosis and E2F-1 induction in PRMT5-depleted cells, suggesting a crosstalk between GSK-3b and E2F-1 in regulating neuronal apoptosis. Interestingly, there is convincing evidence that patients with a history of cancer have a reduced risk of developing AD (Nudelman et al. 2014). It has also been well established that aberrant expression of PRMT5 is associated with many cancer types such as lymphoma (Chung et al. 2013), glioblastoma multiform (Han et al. 2014) and testicular tumors (Liang et al. 2007). The finding that PRMT5 overexpression decreases E2F-1 expression and GSK-3b activity in APPsw cells suggests a potential mechanism for the protective effects of PRMT5 in Ab neurotoxicity. Thus, it will be interesting to examine whether susceptibility to
cancer is associated with globally increased PRMT5 expression, as this may suggest whether a PRMT5-related mechanism underlies the inverse association between cancer and protection against AD. Meanwhile, whether inducing PRMT5 for therapeutic purposes to treat AD would increase the risk of developing cancer still needs extensive study. In summary, our study demonstrates for the first time that PRMT5 regulates Ab neurotoxicity. PRMT5 depletion increases expression of E2F-1, thereby increasing neural cell susceptibility to apoptosis. Because PRMT5 is predominantly expressed in neurons and over-expression of PRMT5 attenuates E2F-1 and GSK-3b activation, strategies to increase PRMT5 activity may have therapeutic potential for the treatment of AD.
Acknowledgments and conflict of interest disclosure This study was supported by grants from the National Natural Science Foundation of China (81270319 and 81470520) and the Hundred Talents Program of Chinese Academy of Sciences. N2 and CL2006 strains were provided by the CGC, which was funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). All experiments were conducted in compliance with the ARRIVE guidelines. The authors have no conflict of interest to declare.
Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Table S1. The detail information of primary antibody. Table S2. Hippocampal PRMT5 mRNA levels in control and AD patients, data were extracted from the original microarray data set (GDS810; Reference Series: GSE1297). Figure S1. Aged Ab25-35decreased primary cortical neuronal cell viability in a dose-dependent manner. Figure S2. PRMT5 expression has no significantly effect on Ab secretion. Figure S3. The expressions of PRMT5 in cytoplasmand nucleuswere almost identical in neo and APPsw cells. Figure S4. PRMT5 is significant negative correlated with E2F-1 in hippocampus tissue of control and AD patients. Figure S5. Knockdown of PRMT5 aggravated Ab-induced paralysis in GMC101 strains. Figure S6. Knockdown of PRMT1 decreased E2F-1 expression in APPsw cells.
References Bates S., Phillips A. C., Clark P. A., Stott F., Peters G., Ludwig R. L. and Vousden K. H. (1998) p14ARF links the tumour suppressors RB and p53. Nature 395, 124–125. Bedford M. T. and Clarke S. G. (2009) Protein arginine methylation in mammals: who, what, and why. Mol. Cell 33, 1–13. Bedford M. T. and Richard S. (2005) Arginine methylation an emerging regulator of protein function. Mol. Cell 18, 263–272.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
PRMT5 regulates Ab neurotoxicity
Blalock E. M., Geddes J. W., Chen K. C., Porter N. M., Markesbery W. R. and Landfield P. W. (2004) Incipient Alzheimer’s disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc. Natl Acad. Sci. USA 101, 2173–2178. Cho E. C., Zheng S., Munro S. et al. (2012) Arginine methylation controls growth regulation by E2F-1. EMBO J. 31, 1785–1797. Chung J., Karkhanis V., Tae S. et al. (2013) Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing. J. Biol. Chem. 288, 35534–35547. Cohen E., Bieschke J., Perciavalle R. M., Kelly J. W. and Dillin A. (2006) Opposing activities protect against age-onset proteotoxicity. Science 313, 1604–1610. DaRocha-Souto B., Coma M., Perez-Nievas B. G. et al. (2012) Activation of glycogen synthase kinase-3 beta mediates betaamyloid induced neuritic damage in Alzheimer’s disease. Neurobiol. Dis. 45, 425–437. Giovanni A., Keramaris E., Morris E. J., Hou S. T., O’Hare M., Dyson N., Robertson G. S., Slack R. S. and Park D. S. (2000) E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3. J. Biol. Chem. 275, 11553–11560. Haass C. and Selkoe D. J. (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid betapeptide. Nat. Rev. Mol. Cell Biol. 8, 101–112. Han X., Li R., Zhang W. et al. (2014) Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro. J. Neurooncol. 118, 61–72. Huang X., Chen Y., Zhang H., Ma Q., Zhang Y. W. and Xu H. (2012) Salubrinal attenuates beta-amyloid-induced neuronal death and microglial activation by inhibition of the NF-kappaB pathway. Neurobiol. Aging 33(1007), e1009–e1017. Jansson M., Durant S. T., Cho E. C., Sheahan S., Edelmann M., Kessler B. and La Thangue N. B. (2008) Arginine methylation regulates the p53 response. Nat. Cell Biol. 10, 1431–1439. Karkhanis V., Hu Y. J., Baiocchi R. A., Imbalzano A. N. and Sif S. (2011) Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem. Sci. 36, 633–641. Keowkase R., Aboukhatwa M., Adam B. L., Beach J. W., Terry A. V., Jr, Buccafussco J. J. and Luo Y. (2010) Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1-84-1 and JAY 2-22-33 in neuronal culture and Caenorhabditis elegans. Mol. Neurodegener. 5, 59. Lee K. W., Im J. Y., Song J. S. et al. (2006) Progressive neuronal loss and behavioral impairments of transgenic C57BL/6 inbred mice expressing the carboxy terminus of amyloid precursor protein. Neurobiol. Dis. 22, 10–24. Liang J. J., Wang Z., Chiriboga L. et al. (2007) The expression and function of androgen receptor coactivator p44 and protein arginine methyltransferase 5 in the developing testis and testicular tumors. J. Urol. 177, 1918–1922. Luo Y., Zhang J., Liu N. and Zhao B. (2011) Copper ions influence the toxicity of beta-amyloid (1-42) in a concentration-dependent manner in a Caenorhabditis elegans model of Alzheimer’s disease. Sci. China Life Sci. 54, 527–534. Luo Y., Yue W., Quan X., Wang Y., Zhao B. and Lu Z. (2015) Asymmetric dimethylarginine exacerbates Abeta-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radic. Biol. Med. 79, 117–126. Morel M., Couturier J., Pontcharraud R., Gil R., Fauconneau B., Paccalin M. and Page G. (2009) Evidence of molecular links between PKR and mTOR signalling pathways in Abeta neurotoxicity: role of p53, Redd1 and TSC2. Neurobiol. Dis. 36, 151–161.
977
Nudelman K. N., Risacher S. L., West J. D., McDonald B. C., Gao S. and Saykin A. J. (2014) Association of cancer history with Alzheimer’s disease onset and structural brain changes. Front. Physiol. 5, 423. Pal S., Baiocchi R. A., Byrd J. C., Grever M. R., Jacob S. T. and Sif S. (2007) Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma. EMBO J. 26, 3558–3569. Park J. H., Szemes M., Vieira G. C. et al. (2015) Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells. Mol. Oncol. 9, 617–627. Pollack B. P., Kotenko S. V., He W., Izotova L. S., Barnoski B. L. and Pestka S. (1999) The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity. J. Biol. Chem. 274, 31531–31542. Powers M. A., Fay M. M., Factor R. E., Welm A. L. and Ullman K. S. (2011) Protein arginine methyltransferase 5 accelerates tumor growth by arginine methylation of the tumor suppressor programmed cell death 4. Cancer Res. 71, 5579–5587. Ramalho R. M., Ribeiro P. S., Sola S., Castro R. E., Steer C. J. and Rodrigues C. M. (2004) Inhibition of the E2F-1/p53/Bax pathway by tauroursodeoxycholic acid in amyloid beta-peptide-induced apoptosis of PC12 cells. J. Neurochem. 90, 567–575. Scoumanne A., Zhang J. and Chen X. (2009) PRMT5 is required for cell-cycle progression and p53 tumor suppressor function. Nucleic Acids Res. 37, 4965–4976. Wan L., Nie G., Zhang J., Luo Y., Zhang P., Zhang Z. and Zhao B. (2011) beta-Amyloid peptide increases levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease. Free Radic. Biol. Med. 50, 122–129. Wang L., Pal S. and Sif S. (2008) Protein arginine methyltransferase 5 suppresses the transcription of the RB family of tumor suppressors in leukemia and lymphoma cells. Mol. Cell. Biol. 28, 6262–6277. Wei H., Wang B., Miyagi M., She Y., Gopalan B., Huang D. B., Ghosh G., Stark G. R. and Lu T. (2013) PRMT5 dimethylates R30 of the p65 subunit to activate NF-kappaB. Proc. Natl Acad. Sci. USA 110, 13516–13521. Wirths O., Multhaup G. and Bayer T. A. (2004) A modified betaamyloid hypothesis: intraneuronal accumulation of the betaamyloid peptide–the first step of a fatal cascade. J. Neurochem. 91, 513–520. Wolf S. S. (2009) The protein arginine methyltransferase family: an update about function, new perspectives and the physiological role in humans. Cell. Mol. Life Sci. 66, 2109–2121. Yan F., Alinari L., Lustberg M. E. et al. (2014) Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res. 74, 1752–1765. Yang M., Sun J., Sun X., Shen Q., Gao Z. and Yang C. (2009) Caenorhabditis elegans protein arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. PLoS Genet. 5, e1000514. Yeste-Velasco M., Folch J., Trullas R., Abad M. A., Enguita M., Pallas M. and Camins A. (2007) Glycogen synthase kinase-3 is involved in the regulation of the cell cycle in cerebellar granule cells. Neuropharmacology 53, 295–307. Zhang Y., McLaughlin R., Goodyer C. and LeBlanc A. (2002) Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons. J. Cell Biol. 156, 519–529. Zhang J., Mori A., Chen Q. and Zhao B. (2006) Fermented papaya preparation attenuates beta-amyloid precursor protein: betaamyloid-mediated copper neurotoxicity in beta-amyloid precursor protein and beta-amyloid precursor protein Swedish mutation overexpressing SH-SY5Y cells. Neuroscience 143, 63–72.
© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 969--977
