BRES : 44692
pp: 128ðcol:fig: : NILÞ
Model7 brain research ] (]]]]) ]]]–]]]
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research report
Q2
Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells
Q1
Jamileh Saberzadeh, Rita Arabsolghar, Mohammad Ali Takhshidn Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
art i cle i nfo
ab st rac t
Article history:
Increased expression and aggregation of α-synuclein (α-syn) protein plays a critical role in
Accepted 22 January 2016
mediating the toxic effects of a number of neurodegenerative substances including metals.
Keywords:
Parkinson disease (PD). Aluminum (Al) is a neurotoxic metal that contributes to pathogen-
Thus, knockdown expression of α-syn is proposed as a possible modality for treatment of Alpha synuclein Aluminum maltolate
esis of PD. The aim of this study was to investigate the role of α-syn protein in mediating Al-induced toxicity in PC12 cell. Specific α-syn small interference RNA (siRNA) was applied
Oxidative stress
to knockdown the expression of α-syn protein in PC12 cells. The effects of different
Apoptosis
concentrations of Al-maltolate (Almal) were then evaluated on cell viability and oxidative stress in the α-syn downregulated cells. The results showed that Almal dose dependently induced apoptosis and increased malondialdehyde (MDA) and catalase activity in PC12 cells. Downregulation of α-syn protein significantly increased cell viability and decreased oxidative markers in Almal-treated cells. These findings suggest that α-syn protein may mediate Al-induced apoptosis and oxidative stress in PC12 cells. & 2016 Published by Elsevier B.V.
1.
Introduction
Numerous studies have indicated the possible role of Al in development of neurodegenerative disease including PD and
PD is a neurodegenerative disorder which is caused by
Alzheimer's disease. The association of Al with increased risk
degeneration of dopaminergic neurons in the substantia
of PD was firstly suggested on the basis of epidemiological
nigra. A complex contribution of several genetic and environ-
studies which revealed higher prevalence of PD in the sub-
mental risk factors is necessary for the initiation and pro-
jects with chronic occupational exposure to Al (Zayed et al.,
gression of PD (Pan-Montojo and Reichmann, 2014; Spatola
1990). The accumulation of Al in neurons of the substantia
and Wider, 2014). Al is a non-essential and toxic metal that
nigra of PD patients (Hirsch et al., 1991), presence of Al in the
can enter the human body through ingestion, inhalation and/
lewy bodies of PD patients (Uversky et al., 2001, 2002),
or during hemodialysis (Peto, 2010; Verstraeten et al., 2008).
decreased locomotor activity in the Al-treated rats (Erazi
n
Corresponding author. Fax: þ98 711 2270301. E-mail address: [email protected] (M.A. Takhshid).
http://dx.doi.org/10.1016/j.brainres.2016.01.037 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
BRES : 44692
2
198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
brain research ] (]]]]) ]]]–]]]
et al., 2011), and Al-induced apoptosis and -oxidative stress in the neurons and neural cell lines further indicated the possible role of Al in the pathogenesis of PD (Kumar et al., 2009; Sanchez-Iglesias et al., 2009; Tsubouchi et al., 2001; Satoh et al., 2005). α-syn is a presynaptic protein which is normally expressed in the brain and is proposed to be involved in the regulation of neurotransmitters release, synaptic plasticity, and neuroprotection (Bendor et al., 2013; Cheng et al., 2011). Several lines of evidence have documented the role of α-syn in the pathogenesis of PD (Martin et al., 2011; Seidl et al., 2014; Spatola and Wider, 2014; Tolleson and Fang, 2013; Trinh and Farrer, 2013; Tsuboi, 2012). Firstly, mutations in the gene encoding α-syn are among well-known genetic risk factors for both sporadic and familial PD (Kay et al., 2008). In addition, αsyn protein is a major component of cytoplasmic lewy bodies which is known as a pathological hallmark in the brain of patients with PD (Mizuno et al., 2008). Moreover, it has been demonstrated that overexpression of α-syn can induce progressive neurodegenerative pathological changes in nigrostriatal dopaminergic neurons (Wills et al., 2010). Finally, it has also been reported that toxic effects of several substances like 6-hydroxydopamine (Banerjee et al., 2014), 1-methyl-4phenylpyridinium (Chen et al., 2014), pesticides (Chorfa et al., 2013) and metals such as manganese (Prabhakaran et al., 2011) mediated through increasing expression of α-syn gene. An in vitro study has shown that Al accelerates aggregation of α-syn (Uversky et al., 2001). Nonetheless, the role of α-syn expression in Al-induced neurotoxicity is not well defined. The goal of this study was to investigate the role of cellular α-syn expression in Al-induced cytotoxicity in PC12 cells. In this study, we used Almal to transport Al3þ into the cells. Previous studies have revealed that it can readily transport across the cell membrane and release Al3þ in the cells (Martin, 1991).The findings of the present study revealed that knockdown expression of α-syn using a specific siRNA ameliorated Almal-induced oxidative stress and cell death in PC12 cells.
2.
Results
2.1.
Almal treatment reduced viability of PC12 cells
MTT assay was used to evaluate Almal-induced cytotoxicity in PC12 cells. The cells were treated with increasing concentration of Almal and/or AlCl3 (250–1000 mM) for 48 h and cell viability was determined using MTT assay. The results revealed that the toxic effect of Almal on cell viability was more stronger than AlCl3. Treatment with increasing concentration of AlCl3 for 48 h had no significant effect on cell viability up to 500 mM and reduced cell viability slightly at concentrations higher than 750 mM ,whereas Almal treatment at 250, 500,750 and 1000 μM for 48 h reduced cell viability dose dependently to 75.3374.04%, 4872.5%, 4473.6% ,and 3072% of untreated control values, respectively which showed significant differences (Po0.001) compared to control group (One way ANOVA followed by Tukey's post-hoc test) (Fig. 1).
Fig. 1 – The effect of 48 h treatment with different concentrations (250, 500, 750 and 1000 μM) of AlCl3 (□) and Almal (■) on viability of PC12 cells. Cell viability was determined using MTT assay. The results are the mean7S.D of at least three independent experiments. *Significant difference (# po0.05, * Po0.001) compared to the control group (One way ANOVA followed by Tukey's post-hoc test).
2.2.
Almal treatment induced apoptosis of PC12 cells
Annexin V-FITC/PI flow cytometric analysis was used to explore the effects of Almal on PC12 cells apoptosis. The findings showed that treatment of PC12 cells with Almal resulted in a dose-dependent increase in the percentage of apoptotic cells. 48 h after treatment of PC12 cells with 250, 500, 750 and 1000 μM of Almal, the percentage of apoptotic cells were 23.0171.4%, 27.870.81%, 55.0770.66% and 6171.03%, respectively which showed significant differences compared to the control group (Fig. 2) (Po0.001 , One way ANOVA followed by Tukey's post-hoc test).
2.3.
Almal caused DNA fragmentation
To investigate the effect of Almal on cellular DNA fragmentation, PC12 cells were treated with 500 μM Almal for 48 h. As can be seen in Fig. 3, Almal induced DNA fragmentation in PC12 cells.
2.4. The role of α-syn in Almal-induced PC12 cell cytotoxicity To assess the possible role of α-syn in Almal-induced cell cytotoxicity, PC12 cells were treated with Almal (500 μM) for 48 h and the cellular level of α-syn were then evaluated using western blotting analysis. As can be seen in Fig. 4, western blot analysis showed that exposure with Almal had no significant effect on α-syn accumulation and the level of αsyn in Almal-treated PC12 cells was similar to the basal levels observed in untreated control cells. In a separate experiment, the effect of knocking down expression of α-syn on Almal cytotoxicity was investigated. Thus, PC12 cells were firstly treated with siRNA against α-syn to knocking down expression of α-syn and then treated with Almal. Western blotting analysis showed a significant downregulation of α-syn opon using siRNA (Fig. 5A and B). After treatment with 500 μM of Almal for 48 h, cell viability in α-syn-knocking down cells was
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317
BRES : 44692 brain research ] (]]]]) ]]]–]]]
318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
3
Fig. 2 – The effect of Almal on apoptosis of PC12 cells. PC12 cells were treated with 0(A), 250(B), 500(C), 750(D), and 1000(E) μM of Almal for 48 h and the percentage of apoptotic cells was measured using AnnexinV-FITC/PI flocytometric method. Data were expressed as percentage of apoptotic cells in 105 events per sample and were mean7S.D of three experiments (F). Asterisks (*) show significant difference (po0.001) compared to the control group (One way ANOVA followed with Tukey's post-hoc test).
significantly higher than siRNA-untreated cells (62.0372.5% and 46.6773.6% compared to the control, respectively) (Fig. 5B). Knocking down expression of α-syn also reduced Almal-induced apoptosis in α-syn-knocking down cells compare to siRNA-untreated cells (Fig. 5A).
by specific siRNA significantly inhibited the effect of Almal on increasing catalase activity (Fig. 7A) and MDA levels (Fig. 7B). These results suggest that α-syn may mediate Almal-induced oxidative stress.
2.5.
3.
Almal induced oxidative stress in PC12 cells
To investigate the role of oxidative stress in Almal-induced cell death, PC12 cells were treated with different concentrations of Almal for 48 h and markers of oxidative stress including MDA levels and catalase activity were then determined. The results showed that treatment with Almal increased MDA levels (Fig. 6A) and catalase activity (Fig. 6B) dose dependently. In addition, silencing of α-syn expression
Discussion
The critical role of α-syn aggregation in the pathogenesis of PD has been clearly documented (Martin et al., 2011; Seidl et al., 2014; Spatola and Wider, 2014; Tolleson and Fang, 2013; Trinh and Farrer, 2013). Increased intracellular accumulation and structural modifications of α-syn protein are two main causes of α-syn aggregation. Accumulation of α-syn is determined by the level of α-syn gene expression and protein
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
BRES : 44692
4
438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497
brain research ] (]]]]) ]]]–]]]
Fig. 3 – Agarose gel electrophoresis of Almal-induced genomic DNA fragmentation of PC12 cell. PC12 cells were treated with 500 lM of Almal (A) or vehicle (B) for 48 h. Cellular DNA were then extracted, analyzed, by a 1% agarose gel electrophoresis stained with ethidium bromide, and viewed under UV light. Lane C displays 50 bp DNA ladder.
Fig. 4 – The effect of Almal on α-syn accumulation in PC12 cells. Almal treatment had no significant effect on α-syn accumulation in PC12 cells. PC12 cells were treated with Almal (500 μM) for 48 h and the level of α-syn was then evaluated by western blotting analysis. β actin was probed as a protein loading control.
degradation. Point mutations, oxidative stress and metal ions are among the factors that induced structural modifications in the α-syn protein (Minami et al., 2015). The associations between α-syn overexpression and cell death and conversely, down regulation of α-syn gene with cell protection and
survival against neurotoxin have been indicated in several studies (Banerjee et al., 2014; Chen et al., 2014; Chorfa et al., 2013; Prabhakaran et al., 2011). The findings of the present study showed that knocking down of α-syn gene expression using a specific siRNA increased the cell viability and ameliorated the oxidative stress in Almal-treated PC12 cells. To the best of our knowledge, this was the first study which showed the role of α-syn protein in Al-induced cell death and oxidative stress. Consistent with the results of previous studies (Satoh et al., 2005; Satoh et al., 2007), the findings of the current study showed that Almal dose dependently increased cell death in the PC12 cells. However, AlCl3 up to concentration of 500 mM had no significant effect on viability of PC12 cell and at higher concentrations slightly reduced PC12 cell viability which is in accordance with the results of previous studies (Johnson et al., 2005; Ohyashiki et al., 2002; Tsubouchi et al., 2001). Higher toxicity of Almal may be due to higher stability and solubility of Almal than AlCl3 in aqueous solutions (Vasudevaraju et al., 2008; Campbell et al., 1999; Savory et al., 2006). In addition, Almal is an electro-neutral and lipophilic compound, mediating its more efficient transport across the cell membrane and releasing of Al3þ into the cells (Lévesque et al., 2000; Ohyashiki et al., 2002; Savory et al., 1999). Flow cytometric evaluation and DNA fragmentation assay indicated that apoptosis was the main cause of Al-induced cell death. A number of studies have suggested the critical role of α-syn in metals-induced cytotoxicity (Brown, 2009; Ostrerova-Golts et al., 2000; Uversky et al., 2001). As increased intracellular accumulation of α-syn, arising from increased αsyn gene expression or reduced proteosomal degradation of the protein can be lead to formation of aggregated forms of αsyn which is the only forms of the protein with cytotoxic effects (Junn et al., 2009; Zabrocki et al., 2005; Wan and Chung, 2012). Thus, to elucidate the role of α-syn in Alinduced cell death, we first investigated the effect of Almal treatment on intracellular accumulation of α-syn. However, findings from western blot analysis showed that Almal treatment had no significant effect on the accumulation of α-syn in PC12 cells. These data indicate that increase accumulation of α-syn may not contribute to Almal-induced cytotoxicity in PC12 cells. We then applied specific α-syn siRNA to evaluate the effect of α-syn downregulation on Alinduced cell death. The results revealed that knockdown of αsyn expression increased viability and decreased Al-induced apoptosis in PC12 cells. These findings imply that α-syn at its basal cellular level, similar to that present in the untreated cells, may mediate Al-induced cell toxicity. The exact mechanism linking α-syn to Al-induced cell death is not clear. However, oxidative stress can be a possible mechanism. Our data showed that Almal treatment increased levels of markers of oxidative stress, including MDA level and activity of catalase in PC12 cells. It has been suggested that a cause-effect relationship and vicious cycle may exist between reactive oxygen species production and αsyn aggregation so that increased oxidative stress can lead to aggregation of α-syn in the cells and vice versa α-syn can induce oxidative stress (Borza, 2014). In this study, the knockdown of α-syn expression reduced the level of MDA and
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557
BRES : 44692 brain research ] (]]]]) ]]]–]]]
558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617
5
Fig. 5 – The effects of α-syn knocking down by α-syn siRNA on Almal-induced cell cytotoxicity. Western blotting analysis showed a significant downregulation of α-syn opon using α-syn siRNA. α-syn downregulation ameliorated Almal-induced apoptosis (A) and protected against Almal-mediated cell death (B). Data are mean7SD of 3 independent experiments. Asterisks indicate significant statistical difference between group (***Po 0.001, ** Po0.01, and *Po0.05, one way ANOVA followed by Tukey's post-hoc test).
Fig. 6 – The effect of Almal on MDA levels (A) and catalase activity (B) in PC12 cells. PC12 cells were treated with 0 (control), 250, 500. 750 and 1000 μM Almal for 48 h . MDA levels and catalase activity were then measured. *Significant difference (po0.001) compare to control group.
catalase activity to 50 and 45% of Almal-treated cells. This implies that α-syn lies upstream of oxidative stress induced by Almal exposure and is required for mediating the Almalinduced oxidative stress. Uversky et al. (2001) have reported that polyvalent metal ions, such as Al3þ, can directly accelerate aggregation of α-syn in vitro. Hence, Al-induced oxidative stress may arise in part from the effect of Al on α-syn aggregation.
3.1.
Conclusions
In conclusion, our finding showed that knocking down expression of α-syn ameliorated Al-induced cell apoptosis and oxidative stress in PC12 cells. Although further studies are needed to understand the exact potential mechanisms by
which α-syn modulates Al-induced cytotoxicity, our data suggest that oxidative stress may link α-syn to Al-induced neurotoxicity.
4.
Experimental procedure
4.1.
Cell culture
PC12 cell lines were obtained from Pasture institute (pasture institute, Iran) and cultured in DMEM (Gibco) with 10% Horse serum, 5% fetal bovine serum (Gibco) and 1% penicillin– streptomycin (Cinagen, Iran) at 37 1C and 5% CO2. Cells were seeded at 104 cells/ml for each experiment and allowed to grow for 24 h prior to treatment with Almal.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677
BRES : 44692
6
678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737
brain research ] (]]]]) ]]]–]]]
Fig. 7 – Effects of α-syn downregulation on Almal-induced oxidative stress in PC12 cells. MDA level (A) and catalase activity (B) were measured in PC12 cells after 48 h treatment with 500 μM of Almal in the presence or absence of α-syn gene silencing using α-syn siRNA. Data are mean7SD of three experiments. Asterisks (*) and hash symbols (#) show significant difference compared to control and Almal-treated cells, respectively (Po0.05, one way ANOVA followed by Tukey's post-hoc test).
4.2.
Almal preparation
Almal was prepared from Aluminum chloride hexahydrate (AlCl3 6H2O) and Maltol (3-hydroxy-2-methyl-4-pyrone) (Sigma) as described previously by Bertholf et al. (1989). Almal stock solution was prepared in double distilled water and sterilized using a 0.2 mm filter.
4.3.
Cell viability assay
The effect of Almal on cell viability was measured by MTT assay. In brief, 103 PC12 cells/100 ml of culture medium were seeded into each well of a 96-well plate and cultured for 24 h. The cells were then treated with various concentrations of Almal (final concentration 250, 500, 750 and 1000 μM) and or AlCl3 for 48 h. Next, 20 ml of MTT (5 g/L) was added to each well and incubated for 4 h at 37 1C. The culture medium was then replaced with 150 ml of dimethyl sulfoxide (DMSO). The absorbance rate at 570 nm was determined using ELISA reader. The rate of cell growth inhibition was calculated as the percentage of the control group.
4.4.
Flow cytometric apoptosis assay
Flow cytometric evaluation using AnnexinV–FITC/PI kit (Dako,cat no: k235011) was performed to identify the role of apoptosis in Almal cytotoxicity. Briefly, PC12 cells were cultured on T75 culture flasks for 24 h. The cells were then treated with various concentrations of Almal (final concentration 250, 500, 750 and 1000 μM) for 48 h. The cells were washed in cold PBS and suspended in 1 ml of ice cold binding buffer. 1 ml of Annexin V-FITC and 2.5 ml of PI were added to 96 ml of cell suspension and gently mixed, followed by keeping the tubes on ice and incubating for 10 min in dark. The cells were acquired within 5 min using a BD flow cytometer. The fluorochromes was excited using the 488 nm line of argon-ion laser, and annexinV and PI emissions were monitored at 519 and 620 nm, respectively. A total of 10,000 events were acquired for each sample.
4.5.
DNA fragmentation assay
PC12 cells were treated with 500 mM of Almal for 48 h. The cells were then harvested and lysed in lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM EDTA and 1% Triton X-100). Cellular DNA was then extracted using DNA extraction kit (Cinagen, Iran) according to manufacturer's instruction. The extracted DNA was separated on 1% of agarose gel. The gel was then stained with ethidium bromide and visualized on an UVtrans illuminator and photographed. Fragmented DNA, shown as DNA ladder in the gel, indicates apoptotic cell death.
4.6.
Western blot
In order to identify the effect of Almal on expression-α synuclein, western blot analysis was conducted. PC12 cells were plated on T75 culture flasks for 24 h. They were then treated with 500 mM of Almal for 48 h. After treatments, the cells were harvested and frozen in micro-tubes containing PBS and stored at 801c until used. Almal-treated and nontreated PC12 cells were washed with PBS and lysed in 10 mM Tris–HCl pH 7.4, containing 0.825 M NaCl and 1% NP-40. For completing the lysis, sonication was used. Protein concentration was determined using Bradford method. Equal amounts of cell lysate were separated on 16% SDS–PAGE gel and transferred to a nitrocellulose membrane following the standard protocol. Immunoblotting was performed with anti-αsyn and anti-actin (ab1801; Abcam, Canada). A horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sigma, USA) and chemiluminescence substrates (ECL; Amersham Bioscience AB) were used to detect the immunolabeled bands.
4.7.
siRNA transfection
Knocking down of α-syn gene expression was performed with a specific siRNA molecule against α-syn mRNA. siRNA were selected based on the Genbank rat α-syn sequence NM019169 and the candidate sequence was blasted and confirmed for
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797
BRES : 44692 brain research ] (]]]]) ]]]–]]]
798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857
specificity (Leng and Chuang, 2006). The siRNA sense and antisense sequence were 50 GGGAGUCGUUCAUGGAGUG 30 and 50 CACUCCAUGAACGACUCCC 30 , respectively (Synthesized by Eurofins MWG operon, Germany). The sense and antisense oligonucleotides were annealed to make doublestranded siRNA. Electroporation was used for α-syn siRNA transfection. Briefly, 5 105 PC12 cells were added to a mixture of 50 μl of serum-free medium and 50 μl of sterile distilled water (DW) with (10 nM) or without annealed siRNA. The RNA-cells mixtures were transferred into 4 mm Biorad Gene Pulser cuvettes, and electroporated (960 μF and 250 V in 4 mm cuvettes) by Gene Pulser X cell Electroporation system (BioRad, US). After electreporation, the cells were re-plated onto T25 flask. 48 h after treatment, siRNA transfected cells were harvested to evaluate the α-syn protein using western blot analysis as previously described.
4.8.
Assay of catalase acticity and MDA concentration
Almal-treated cells and non-treated control cells were lysed by sonication in ice-cold 50 mM sodium phosphate buffer (pH 7.0). The cell lysates were then centrifuged at 13,000g for 10 min at 4 1C, and the supernatants were used for assay of catalase activity and MDA concentration. Catalase activity was measured based on the rate of decomposition of H2O2 at 25 1C, which was monitored by the decrease in absorbance at 240 nm. MDA concentration was estimated according to the method of thiobarbituric acid. The amount of protein in the supernatants was determined using method of Bradford. Catalase activities and MDA concentration are expressed as units/mg protein and nmol/ mg protein, respectively.
4.9.
Statistical Analysis
All experiments were performed for a minimum of three times and data have been expressed as mean7SD. Statistical analysis was performed using SPSS 15.0 statistical package (SPSS Inc., Chicago, IL, USA). Statistical significance of the results was calculated using one-way ANOVA followed by Tukey's multiple comparison tests. Differences were considered statistically significant at Po0.05.
Conflict of Interest None declared.
Acknowledgment This manuscript was extracted from the M.Sc. thesis of Jamileh Saberzadeh and was supported by Grant number 905970 from the Vice-Chancellor for Research Affairs of Shiraz University of Medical Sciences, Shiraz, Iran. We are also grateful to all staff of Diagnostic Laboratory Sciences and Technology Research Center and Biochemistry department of Shiraz University of Medical Sciences for technical assistance in this work.
7
r e f e r e nc e s
Banerjee, K., et al., 2014. Dopamine cytotoxicity involves both oxidative and nonoxidative pathways in SH-SY5Y cells: potential role of alpha-synuclein overexpression and proteasomal inhibition in the etiopathogenesis of parkinson’s disease. Park. Dis. 2014, 878935. Bendor, J.T., Logan, T.P., Edwards, R.H., 2013. The function of alpha-synuclein. Neuron 79, 1044–1066. Bertholf, R.L., et al., 1989. A long-term intravenous model of aluminum maltol toxicity in rabbits: tissue distribution, hepatic, renal, and neuronal cytoskeletal changes associated with systemic exposure. Toxicol. Appl. Pharmacol. 98, 58–74. Borza, L.R., 2014. A review on the cause-effect relationship between oxidative stress and toxic proteins in the pathogenesis of neurodegenerative diseases. Rev. Med. Chir. Soc. Med. Nat. Iasi 118, 19–27. Brown, D.R., 2009. Metal binding to alpha-synuclein peptides and its contribution to toxicity. Biochem. Biophys. Res. Commun. 380, 377–381. Campbell, A., Prasad, K., Bondy, S., 1999. Aluminum-induced oxidative events in cell lines: glioma are more responsive than neuroblastoma. Free Radic. Biol. Med. 26, 1166–1171. Chen, T., et al., 2014. delta-Opioid receptor activation reduces alpha-synuclein overexpression and oligomer formation induced by MPP(þ) and/or hypoxia. Exp. Neurol. 255, 127–136. Cheng, F., Vivacqua, G., Yu, S., 2011. The role of alpha-synuclein in neurotransmission and synaptic plasticity. J. Chem Neuroanat. 42, 242–248. Chorfa, A., et al., 2013. Specific pesticide-dependent increases in alpha-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines. Toxicol. Sci. 133, 289–297. Erazi, H., Ahboucha, S., Gamrani, H., 2011. Chronic exposure to aluminum reduces tyrosine hydroxylase expression in the substantia nigra and locomotor performance in rats. Neurosci. Lett. 487, 8–11. Hirsch, E.C., et al., 1991. Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J. Neurochem. 56, 446–451. Johnson, V.J., Kim, S.-H., Sharma, R.P., 2005. Aluminum-maltolate induces apoptosis and necrosis in neuro-2a cells: potential role for p53 signaling. Junn, E., et al., 2009. Repression of α-synuclein expression and toxicity by microRNA-7. Proc. Natl. Acad. Sci. USA 106, 13052–13057. Kay, D.M., et al., 2008. Genetic association between alphasynuclein and idiopathic Parkinson’s disease. Am. J. Med. 147B, 1222–1230. Kumar, V., Bal, A., Gill, K.D., 2009. Aluminium-induced oxidative DNA damage recognition and cell-cycle disruption in different regions of rat brain. Toxicology 264, 137–144. Leng, Y., Chuang, D.M., 2006. Endogenous alpha-synuclein is induced by valproic acid through histone deacetylase inhibition and participates in neuroprotection against glutamateinduced excitotoxicity. J. Neurosci. 26, 7502–7512. Le´vesque, L., et al., 2000. Ligand specific effects on aluminum incorporation and toxicity in neurons and astrocytes. Brain Res. 877, 191–202. Martin, R.B., 1991. Aluminum in biological systems. In aluminum in chemistry. In: Nicolini, P., Zatta, P.F., Corain, B. (Eds.), Biology and Medicine. Raven Press, New York, pp. 3–20. Martin, I., Dawson, V.L., Dawson, T.M., 2011. Recent advances in the genetics of Parkinson’s disease. Annu. Rev. Genom. Hum. Genet. 12, 301–325. Minami, A., et al., 2015. Function of alpha-synuclein and PINK1 in Lewy body dementia (Review). Int. J. Mol. Med. 35, 3–9.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917
BRES : 44692
8
918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953
brain research ] (]]]]) ]]]–]]]
Mizuno, Y., et al., 2008. Progress in the pathogenesis and genetics of Parkinson’s disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 2215–2227. Ohyashiki, T., et al., 2002. Nerve growth factor protects against aluminum-mediated cell death. Toxicology 176, 195–207. Ostrerova-Golts, N., et al., 2000. The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J. Neurosci. 20, 6048–6054. Pan-Montojo, F., Reichmann, H., 2014. Considerations on the role of environmental toxins in idiopathic Parkinson’s disease pathophysiology. Transl. Neurodegener. 3, 10. Peto, M.V., 2010. Aluminium and iron in humans: bioaccumulation, pathology, and removal. Rejuvenation Res. 13, 589–598. Prabhakaran, K., Chapman, G.D., Gunasekar, P.G., 2011. alphaSynuclein overexpression enhances manganese-induced neurotoxicity through the NF-kappaB-mediated pathway. Toxicol. Mech. Method 21, 435–443. Sanchez-Iglesias, S., et al., 2009. Brain oxidative stress and selective behaviour of aluminium in specific areas of rat brain: potential effects in a 6-OHDA-induced model of Parkinson’s disease. J. Neurochem. 109, 879–888. Satoh, E., et al., 2005. Glutathione depletion promotes aluminummediated cell death of PC12 cells. Biol. Pharm. Bull. 28, 941–946. Satoh, E., et al., 2007. Involvement of NO generation in aluminum-induced cell death. Biol. Pharm. Bull. 30, 1390–1394. Savory, J., et al., 1999. Age-related hippocampal changes in Bcl-2: Bax ratio, oxidative stress, redox-active iron and apoptosis associated with aluminum-induced neurodegeneration: increased susceptibility with aging. Neurotoxicology 20, 805–817. Savory, J., Herman, M.M., Ghribi, O., 2006. Mechanisms of aluminum-induced neurodegeneration in animals: Implications for Alzheimer’s disease. J. Alzheimers Dis. 10, 135–144. Seidl, S.E., et al., 2014. The emerging role of nutrition in Parkinson’s disease. Front. Aging Neurosci. 6, 36. Spatola, M., Wider, C., 2014. Genetics of Parkinson’s disease: the yield. Park. Relat. Disord. 20 (Suppl 1), S35–S38.
Tolleson, C.M., Fang, J.Y., 2013. Advances in the mechanisms of Parkinson’s disease. Discov. Med. 15, 61–66. Trinh, J., Farrer, M., 2013. Advances in the genetics of Parkinson disease. Nat. Rev. Neurol. 9, 445–454. Tsuboi, Y., 2012. Environmental-genetic interactions in the pathogenesis of Parkinson’s disease. Exp. Neurobiol. 21, 123–128. Tsubouchi, R., et al., 2001. Aluminum-induced a poptosis in PC12D cells. Biometals 14, 181–185. Uversky, V.N., Li, J., Fink, A.L., 2001. Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson’s disease and heavy metal exposure. J. Biol. Chem. 276, 44284–44296. Uversky, V.N., et al., 2002. Synergistic effects of pesticides and metals on the fibrillation of alpha-synuclein: implications for Parkinson’s disease. Neurotoxicology 23, 527–536. Vasudevaraju, P., et al., 2008. Molecular toxicity of aluminium in relation to neurodegeneration. Indian J. Med. Res. 128, 545–556. Verstraeten, S.V., Aimo, L., Oteiza, P.I., 2008. Aluminium and lead: molecular mechanisms of brain toxicity. Arch. Toxicol. 82, 789–802. Wan, O.W., Chung, K.K., 2012. The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson’s disease. PLoS One 7 (6), e38545, http://dx. doi.org/10.1371/journal.pone.0038545. Wills, J., et al., 2010. Elevated tauopathy and alpha-synuclein pathology in postmortem Parkinson’s disease brains with and without dementia. Exp. Neurol. 225, 210–218. Zabrocki, P., et al., 2005. Characterization of alpha-synuclein aggregation and synergistic toxicity with protein tau in yeast. Febs J. 272, 1386–1400. Zayed, J., et al., 1990. Parkinson disease and environmental factors. Rev Epidemiol Sante Publique 38, 159–160.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988
pp: 128ðcol:fig: : NILÞ
Model7 brain research ] (]]]]) ]]]–]]]
121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research report
Q2
Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells
Q1
Jamileh Saberzadeh, Rita Arabsolghar, Mohammad Ali Takhshidn Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
art i cle i nfo
ab st rac t
Article history:
Increased expression and aggregation of α-synuclein (α-syn) protein plays a critical role in
Accepted 22 January 2016
mediating the toxic effects of a number of neurodegenerative substances including metals.
Keywords:
Parkinson disease (PD). Aluminum (Al) is a neurotoxic metal that contributes to pathogen-
Thus, knockdown expression of α-syn is proposed as a possible modality for treatment of Alpha synuclein Aluminum maltolate
esis of PD. The aim of this study was to investigate the role of α-syn protein in mediating Al-induced toxicity in PC12 cell. Specific α-syn small interference RNA (siRNA) was applied
Oxidative stress
to knockdown the expression of α-syn protein in PC12 cells. The effects of different
Apoptosis
concentrations of Al-maltolate (Almal) were then evaluated on cell viability and oxidative stress in the α-syn downregulated cells. The results showed that Almal dose dependently induced apoptosis and increased malondialdehyde (MDA) and catalase activity in PC12 cells. Downregulation of α-syn protein significantly increased cell viability and decreased oxidative markers in Almal-treated cells. These findings suggest that α-syn protein may mediate Al-induced apoptosis and oxidative stress in PC12 cells. & 2016 Published by Elsevier B.V.
1.
Introduction
Numerous studies have indicated the possible role of Al in development of neurodegenerative disease including PD and
PD is a neurodegenerative disorder which is caused by
Alzheimer's disease. The association of Al with increased risk
degeneration of dopaminergic neurons in the substantia
of PD was firstly suggested on the basis of epidemiological
nigra. A complex contribution of several genetic and environ-
studies which revealed higher prevalence of PD in the sub-
mental risk factors is necessary for the initiation and pro-
jects with chronic occupational exposure to Al (Zayed et al.,
gression of PD (Pan-Montojo and Reichmann, 2014; Spatola
1990). The accumulation of Al in neurons of the substantia
and Wider, 2014). Al is a non-essential and toxic metal that
nigra of PD patients (Hirsch et al., 1991), presence of Al in the
can enter the human body through ingestion, inhalation and/
lewy bodies of PD patients (Uversky et al., 2001, 2002),
or during hemodialysis (Peto, 2010; Verstraeten et al., 2008).
decreased locomotor activity in the Al-treated rats (Erazi
n
Corresponding author. Fax: þ98 711 2270301. E-mail address: [email protected] (M.A. Takhshid).
http://dx.doi.org/10.1016/j.brainres.2016.01.037 0006-8993/& 2016 Published by Elsevier B.V.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
BRES : 44692
2
198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
brain research ] (]]]]) ]]]–]]]
et al., 2011), and Al-induced apoptosis and -oxidative stress in the neurons and neural cell lines further indicated the possible role of Al in the pathogenesis of PD (Kumar et al., 2009; Sanchez-Iglesias et al., 2009; Tsubouchi et al., 2001; Satoh et al., 2005). α-syn is a presynaptic protein which is normally expressed in the brain and is proposed to be involved in the regulation of neurotransmitters release, synaptic plasticity, and neuroprotection (Bendor et al., 2013; Cheng et al., 2011). Several lines of evidence have documented the role of α-syn in the pathogenesis of PD (Martin et al., 2011; Seidl et al., 2014; Spatola and Wider, 2014; Tolleson and Fang, 2013; Trinh and Farrer, 2013; Tsuboi, 2012). Firstly, mutations in the gene encoding α-syn are among well-known genetic risk factors for both sporadic and familial PD (Kay et al., 2008). In addition, αsyn protein is a major component of cytoplasmic lewy bodies which is known as a pathological hallmark in the brain of patients with PD (Mizuno et al., 2008). Moreover, it has been demonstrated that overexpression of α-syn can induce progressive neurodegenerative pathological changes in nigrostriatal dopaminergic neurons (Wills et al., 2010). Finally, it has also been reported that toxic effects of several substances like 6-hydroxydopamine (Banerjee et al., 2014), 1-methyl-4phenylpyridinium (Chen et al., 2014), pesticides (Chorfa et al., 2013) and metals such as manganese (Prabhakaran et al., 2011) mediated through increasing expression of α-syn gene. An in vitro study has shown that Al accelerates aggregation of α-syn (Uversky et al., 2001). Nonetheless, the role of α-syn expression in Al-induced neurotoxicity is not well defined. The goal of this study was to investigate the role of cellular α-syn expression in Al-induced cytotoxicity in PC12 cells. In this study, we used Almal to transport Al3þ into the cells. Previous studies have revealed that it can readily transport across the cell membrane and release Al3þ in the cells (Martin, 1991).The findings of the present study revealed that knockdown expression of α-syn using a specific siRNA ameliorated Almal-induced oxidative stress and cell death in PC12 cells.
2.
Results
2.1.
Almal treatment reduced viability of PC12 cells
MTT assay was used to evaluate Almal-induced cytotoxicity in PC12 cells. The cells were treated with increasing concentration of Almal and/or AlCl3 (250–1000 mM) for 48 h and cell viability was determined using MTT assay. The results revealed that the toxic effect of Almal on cell viability was more stronger than AlCl3. Treatment with increasing concentration of AlCl3 for 48 h had no significant effect on cell viability up to 500 mM and reduced cell viability slightly at concentrations higher than 750 mM ,whereas Almal treatment at 250, 500,750 and 1000 μM for 48 h reduced cell viability dose dependently to 75.3374.04%, 4872.5%, 4473.6% ,and 3072% of untreated control values, respectively which showed significant differences (Po0.001) compared to control group (One way ANOVA followed by Tukey's post-hoc test) (Fig. 1).
Fig. 1 – The effect of 48 h treatment with different concentrations (250, 500, 750 and 1000 μM) of AlCl3 (□) and Almal (■) on viability of PC12 cells. Cell viability was determined using MTT assay. The results are the mean7S.D of at least three independent experiments. *Significant difference (# po0.05, * Po0.001) compared to the control group (One way ANOVA followed by Tukey's post-hoc test).
2.2.
Almal treatment induced apoptosis of PC12 cells
Annexin V-FITC/PI flow cytometric analysis was used to explore the effects of Almal on PC12 cells apoptosis. The findings showed that treatment of PC12 cells with Almal resulted in a dose-dependent increase in the percentage of apoptotic cells. 48 h after treatment of PC12 cells with 250, 500, 750 and 1000 μM of Almal, the percentage of apoptotic cells were 23.0171.4%, 27.870.81%, 55.0770.66% and 6171.03%, respectively which showed significant differences compared to the control group (Fig. 2) (Po0.001 , One way ANOVA followed by Tukey's post-hoc test).
2.3.
Almal caused DNA fragmentation
To investigate the effect of Almal on cellular DNA fragmentation, PC12 cells were treated with 500 μM Almal for 48 h. As can be seen in Fig. 3, Almal induced DNA fragmentation in PC12 cells.
2.4. The role of α-syn in Almal-induced PC12 cell cytotoxicity To assess the possible role of α-syn in Almal-induced cell cytotoxicity, PC12 cells were treated with Almal (500 μM) for 48 h and the cellular level of α-syn were then evaluated using western blotting analysis. As can be seen in Fig. 4, western blot analysis showed that exposure with Almal had no significant effect on α-syn accumulation and the level of αsyn in Almal-treated PC12 cells was similar to the basal levels observed in untreated control cells. In a separate experiment, the effect of knocking down expression of α-syn on Almal cytotoxicity was investigated. Thus, PC12 cells were firstly treated with siRNA against α-syn to knocking down expression of α-syn and then treated with Almal. Western blotting analysis showed a significant downregulation of α-syn opon using siRNA (Fig. 5A and B). After treatment with 500 μM of Almal for 48 h, cell viability in α-syn-knocking down cells was
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317
BRES : 44692 brain research ] (]]]]) ]]]–]]]
318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377
3
Fig. 2 – The effect of Almal on apoptosis of PC12 cells. PC12 cells were treated with 0(A), 250(B), 500(C), 750(D), and 1000(E) μM of Almal for 48 h and the percentage of apoptotic cells was measured using AnnexinV-FITC/PI flocytometric method. Data were expressed as percentage of apoptotic cells in 105 events per sample and were mean7S.D of three experiments (F). Asterisks (*) show significant difference (po0.001) compared to the control group (One way ANOVA followed with Tukey's post-hoc test).
significantly higher than siRNA-untreated cells (62.0372.5% and 46.6773.6% compared to the control, respectively) (Fig. 5B). Knocking down expression of α-syn also reduced Almal-induced apoptosis in α-syn-knocking down cells compare to siRNA-untreated cells (Fig. 5A).
by specific siRNA significantly inhibited the effect of Almal on increasing catalase activity (Fig. 7A) and MDA levels (Fig. 7B). These results suggest that α-syn may mediate Almal-induced oxidative stress.
2.5.
3.
Almal induced oxidative stress in PC12 cells
To investigate the role of oxidative stress in Almal-induced cell death, PC12 cells were treated with different concentrations of Almal for 48 h and markers of oxidative stress including MDA levels and catalase activity were then determined. The results showed that treatment with Almal increased MDA levels (Fig. 6A) and catalase activity (Fig. 6B) dose dependently. In addition, silencing of α-syn expression
Discussion
The critical role of α-syn aggregation in the pathogenesis of PD has been clearly documented (Martin et al., 2011; Seidl et al., 2014; Spatola and Wider, 2014; Tolleson and Fang, 2013; Trinh and Farrer, 2013). Increased intracellular accumulation and structural modifications of α-syn protein are two main causes of α-syn aggregation. Accumulation of α-syn is determined by the level of α-syn gene expression and protein
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
BRES : 44692
4
438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497
brain research ] (]]]]) ]]]–]]]
Fig. 3 – Agarose gel electrophoresis of Almal-induced genomic DNA fragmentation of PC12 cell. PC12 cells were treated with 500 lM of Almal (A) or vehicle (B) for 48 h. Cellular DNA were then extracted, analyzed, by a 1% agarose gel electrophoresis stained with ethidium bromide, and viewed under UV light. Lane C displays 50 bp DNA ladder.
Fig. 4 – The effect of Almal on α-syn accumulation in PC12 cells. Almal treatment had no significant effect on α-syn accumulation in PC12 cells. PC12 cells were treated with Almal (500 μM) for 48 h and the level of α-syn was then evaluated by western blotting analysis. β actin was probed as a protein loading control.
degradation. Point mutations, oxidative stress and metal ions are among the factors that induced structural modifications in the α-syn protein (Minami et al., 2015). The associations between α-syn overexpression and cell death and conversely, down regulation of α-syn gene with cell protection and
survival against neurotoxin have been indicated in several studies (Banerjee et al., 2014; Chen et al., 2014; Chorfa et al., 2013; Prabhakaran et al., 2011). The findings of the present study showed that knocking down of α-syn gene expression using a specific siRNA increased the cell viability and ameliorated the oxidative stress in Almal-treated PC12 cells. To the best of our knowledge, this was the first study which showed the role of α-syn protein in Al-induced cell death and oxidative stress. Consistent with the results of previous studies (Satoh et al., 2005; Satoh et al., 2007), the findings of the current study showed that Almal dose dependently increased cell death in the PC12 cells. However, AlCl3 up to concentration of 500 mM had no significant effect on viability of PC12 cell and at higher concentrations slightly reduced PC12 cell viability which is in accordance with the results of previous studies (Johnson et al., 2005; Ohyashiki et al., 2002; Tsubouchi et al., 2001). Higher toxicity of Almal may be due to higher stability and solubility of Almal than AlCl3 in aqueous solutions (Vasudevaraju et al., 2008; Campbell et al., 1999; Savory et al., 2006). In addition, Almal is an electro-neutral and lipophilic compound, mediating its more efficient transport across the cell membrane and releasing of Al3þ into the cells (Lévesque et al., 2000; Ohyashiki et al., 2002; Savory et al., 1999). Flow cytometric evaluation and DNA fragmentation assay indicated that apoptosis was the main cause of Al-induced cell death. A number of studies have suggested the critical role of α-syn in metals-induced cytotoxicity (Brown, 2009; Ostrerova-Golts et al., 2000; Uversky et al., 2001). As increased intracellular accumulation of α-syn, arising from increased αsyn gene expression or reduced proteosomal degradation of the protein can be lead to formation of aggregated forms of αsyn which is the only forms of the protein with cytotoxic effects (Junn et al., 2009; Zabrocki et al., 2005; Wan and Chung, 2012). Thus, to elucidate the role of α-syn in Alinduced cell death, we first investigated the effect of Almal treatment on intracellular accumulation of α-syn. However, findings from western blot analysis showed that Almal treatment had no significant effect on the accumulation of α-syn in PC12 cells. These data indicate that increase accumulation of α-syn may not contribute to Almal-induced cytotoxicity in PC12 cells. We then applied specific α-syn siRNA to evaluate the effect of α-syn downregulation on Alinduced cell death. The results revealed that knockdown of αsyn expression increased viability and decreased Al-induced apoptosis in PC12 cells. These findings imply that α-syn at its basal cellular level, similar to that present in the untreated cells, may mediate Al-induced cell toxicity. The exact mechanism linking α-syn to Al-induced cell death is not clear. However, oxidative stress can be a possible mechanism. Our data showed that Almal treatment increased levels of markers of oxidative stress, including MDA level and activity of catalase in PC12 cells. It has been suggested that a cause-effect relationship and vicious cycle may exist between reactive oxygen species production and αsyn aggregation so that increased oxidative stress can lead to aggregation of α-syn in the cells and vice versa α-syn can induce oxidative stress (Borza, 2014). In this study, the knockdown of α-syn expression reduced the level of MDA and
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557
BRES : 44692 brain research ] (]]]]) ]]]–]]]
558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617
5
Fig. 5 – The effects of α-syn knocking down by α-syn siRNA on Almal-induced cell cytotoxicity. Western blotting analysis showed a significant downregulation of α-syn opon using α-syn siRNA. α-syn downregulation ameliorated Almal-induced apoptosis (A) and protected against Almal-mediated cell death (B). Data are mean7SD of 3 independent experiments. Asterisks indicate significant statistical difference between group (***Po 0.001, ** Po0.01, and *Po0.05, one way ANOVA followed by Tukey's post-hoc test).
Fig. 6 – The effect of Almal on MDA levels (A) and catalase activity (B) in PC12 cells. PC12 cells were treated with 0 (control), 250, 500. 750 and 1000 μM Almal for 48 h . MDA levels and catalase activity were then measured. *Significant difference (po0.001) compare to control group.
catalase activity to 50 and 45% of Almal-treated cells. This implies that α-syn lies upstream of oxidative stress induced by Almal exposure and is required for mediating the Almalinduced oxidative stress. Uversky et al. (2001) have reported that polyvalent metal ions, such as Al3þ, can directly accelerate aggregation of α-syn in vitro. Hence, Al-induced oxidative stress may arise in part from the effect of Al on α-syn aggregation.
3.1.
Conclusions
In conclusion, our finding showed that knocking down expression of α-syn ameliorated Al-induced cell apoptosis and oxidative stress in PC12 cells. Although further studies are needed to understand the exact potential mechanisms by
which α-syn modulates Al-induced cytotoxicity, our data suggest that oxidative stress may link α-syn to Al-induced neurotoxicity.
4.
Experimental procedure
4.1.
Cell culture
PC12 cell lines were obtained from Pasture institute (pasture institute, Iran) and cultured in DMEM (Gibco) with 10% Horse serum, 5% fetal bovine serum (Gibco) and 1% penicillin– streptomycin (Cinagen, Iran) at 37 1C and 5% CO2. Cells were seeded at 104 cells/ml for each experiment and allowed to grow for 24 h prior to treatment with Almal.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677
BRES : 44692
6
678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737
brain research ] (]]]]) ]]]–]]]
Fig. 7 – Effects of α-syn downregulation on Almal-induced oxidative stress in PC12 cells. MDA level (A) and catalase activity (B) were measured in PC12 cells after 48 h treatment with 500 μM of Almal in the presence or absence of α-syn gene silencing using α-syn siRNA. Data are mean7SD of three experiments. Asterisks (*) and hash symbols (#) show significant difference compared to control and Almal-treated cells, respectively (Po0.05, one way ANOVA followed by Tukey's post-hoc test).
4.2.
Almal preparation
Almal was prepared from Aluminum chloride hexahydrate (AlCl3 6H2O) and Maltol (3-hydroxy-2-methyl-4-pyrone) (Sigma) as described previously by Bertholf et al. (1989). Almal stock solution was prepared in double distilled water and sterilized using a 0.2 mm filter.
4.3.
Cell viability assay
The effect of Almal on cell viability was measured by MTT assay. In brief, 103 PC12 cells/100 ml of culture medium were seeded into each well of a 96-well plate and cultured for 24 h. The cells were then treated with various concentrations of Almal (final concentration 250, 500, 750 and 1000 μM) and or AlCl3 for 48 h. Next, 20 ml of MTT (5 g/L) was added to each well and incubated for 4 h at 37 1C. The culture medium was then replaced with 150 ml of dimethyl sulfoxide (DMSO). The absorbance rate at 570 nm was determined using ELISA reader. The rate of cell growth inhibition was calculated as the percentage of the control group.
4.4.
Flow cytometric apoptosis assay
Flow cytometric evaluation using AnnexinV–FITC/PI kit (Dako,cat no: k235011) was performed to identify the role of apoptosis in Almal cytotoxicity. Briefly, PC12 cells were cultured on T75 culture flasks for 24 h. The cells were then treated with various concentrations of Almal (final concentration 250, 500, 750 and 1000 μM) for 48 h. The cells were washed in cold PBS and suspended in 1 ml of ice cold binding buffer. 1 ml of Annexin V-FITC and 2.5 ml of PI were added to 96 ml of cell suspension and gently mixed, followed by keeping the tubes on ice and incubating for 10 min in dark. The cells were acquired within 5 min using a BD flow cytometer. The fluorochromes was excited using the 488 nm line of argon-ion laser, and annexinV and PI emissions were monitored at 519 and 620 nm, respectively. A total of 10,000 events were acquired for each sample.
4.5.
DNA fragmentation assay
PC12 cells were treated with 500 mM of Almal for 48 h. The cells were then harvested and lysed in lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM EDTA and 1% Triton X-100). Cellular DNA was then extracted using DNA extraction kit (Cinagen, Iran) according to manufacturer's instruction. The extracted DNA was separated on 1% of agarose gel. The gel was then stained with ethidium bromide and visualized on an UVtrans illuminator and photographed. Fragmented DNA, shown as DNA ladder in the gel, indicates apoptotic cell death.
4.6.
Western blot
In order to identify the effect of Almal on expression-α synuclein, western blot analysis was conducted. PC12 cells were plated on T75 culture flasks for 24 h. They were then treated with 500 mM of Almal for 48 h. After treatments, the cells were harvested and frozen in micro-tubes containing PBS and stored at 801c until used. Almal-treated and nontreated PC12 cells were washed with PBS and lysed in 10 mM Tris–HCl pH 7.4, containing 0.825 M NaCl and 1% NP-40. For completing the lysis, sonication was used. Protein concentration was determined using Bradford method. Equal amounts of cell lysate were separated on 16% SDS–PAGE gel and transferred to a nitrocellulose membrane following the standard protocol. Immunoblotting was performed with anti-αsyn and anti-actin (ab1801; Abcam, Canada). A horseradish peroxidase-conjugated anti-rabbit secondary antibody (Sigma, USA) and chemiluminescence substrates (ECL; Amersham Bioscience AB) were used to detect the immunolabeled bands.
4.7.
siRNA transfection
Knocking down of α-syn gene expression was performed with a specific siRNA molecule against α-syn mRNA. siRNA were selected based on the Genbank rat α-syn sequence NM019169 and the candidate sequence was blasted and confirmed for
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797
BRES : 44692 brain research ] (]]]]) ]]]–]]]
798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857
specificity (Leng and Chuang, 2006). The siRNA sense and antisense sequence were 50 GGGAGUCGUUCAUGGAGUG 30 and 50 CACUCCAUGAACGACUCCC 30 , respectively (Synthesized by Eurofins MWG operon, Germany). The sense and antisense oligonucleotides were annealed to make doublestranded siRNA. Electroporation was used for α-syn siRNA transfection. Briefly, 5 105 PC12 cells were added to a mixture of 50 μl of serum-free medium and 50 μl of sterile distilled water (DW) with (10 nM) or without annealed siRNA. The RNA-cells mixtures were transferred into 4 mm Biorad Gene Pulser cuvettes, and electroporated (960 μF and 250 V in 4 mm cuvettes) by Gene Pulser X cell Electroporation system (BioRad, US). After electreporation, the cells were re-plated onto T25 flask. 48 h after treatment, siRNA transfected cells were harvested to evaluate the α-syn protein using western blot analysis as previously described.
4.8.
Assay of catalase acticity and MDA concentration
Almal-treated cells and non-treated control cells were lysed by sonication in ice-cold 50 mM sodium phosphate buffer (pH 7.0). The cell lysates were then centrifuged at 13,000g for 10 min at 4 1C, and the supernatants were used for assay of catalase activity and MDA concentration. Catalase activity was measured based on the rate of decomposition of H2O2 at 25 1C, which was monitored by the decrease in absorbance at 240 nm. MDA concentration was estimated according to the method of thiobarbituric acid. The amount of protein in the supernatants was determined using method of Bradford. Catalase activities and MDA concentration are expressed as units/mg protein and nmol/ mg protein, respectively.
4.9.
Statistical Analysis
All experiments were performed for a minimum of three times and data have been expressed as mean7SD. Statistical analysis was performed using SPSS 15.0 statistical package (SPSS Inc., Chicago, IL, USA). Statistical significance of the results was calculated using one-way ANOVA followed by Tukey's multiple comparison tests. Differences were considered statistically significant at Po0.05.
Conflict of Interest None declared.
Acknowledgment This manuscript was extracted from the M.Sc. thesis of Jamileh Saberzadeh and was supported by Grant number 905970 from the Vice-Chancellor for Research Affairs of Shiraz University of Medical Sciences, Shiraz, Iran. We are also grateful to all staff of Diagnostic Laboratory Sciences and Technology Research Center and Biochemistry department of Shiraz University of Medical Sciences for technical assistance in this work.
7
r e f e r e nc e s
Banerjee, K., et al., 2014. Dopamine cytotoxicity involves both oxidative and nonoxidative pathways in SH-SY5Y cells: potential role of alpha-synuclein overexpression and proteasomal inhibition in the etiopathogenesis of parkinson’s disease. Park. Dis. 2014, 878935. Bendor, J.T., Logan, T.P., Edwards, R.H., 2013. The function of alpha-synuclein. Neuron 79, 1044–1066. Bertholf, R.L., et al., 1989. A long-term intravenous model of aluminum maltol toxicity in rabbits: tissue distribution, hepatic, renal, and neuronal cytoskeletal changes associated with systemic exposure. Toxicol. Appl. Pharmacol. 98, 58–74. Borza, L.R., 2014. A review on the cause-effect relationship between oxidative stress and toxic proteins in the pathogenesis of neurodegenerative diseases. Rev. Med. Chir. Soc. Med. Nat. Iasi 118, 19–27. Brown, D.R., 2009. Metal binding to alpha-synuclein peptides and its contribution to toxicity. Biochem. Biophys. Res. Commun. 380, 377–381. Campbell, A., Prasad, K., Bondy, S., 1999. Aluminum-induced oxidative events in cell lines: glioma are more responsive than neuroblastoma. Free Radic. Biol. Med. 26, 1166–1171. Chen, T., et al., 2014. delta-Opioid receptor activation reduces alpha-synuclein overexpression and oligomer formation induced by MPP(þ) and/or hypoxia. Exp. Neurol. 255, 127–136. Cheng, F., Vivacqua, G., Yu, S., 2011. The role of alpha-synuclein in neurotransmission and synaptic plasticity. J. Chem Neuroanat. 42, 242–248. Chorfa, A., et al., 2013. Specific pesticide-dependent increases in alpha-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines. Toxicol. Sci. 133, 289–297. Erazi, H., Ahboucha, S., Gamrani, H., 2011. Chronic exposure to aluminum reduces tyrosine hydroxylase expression in the substantia nigra and locomotor performance in rats. Neurosci. Lett. 487, 8–11. Hirsch, E.C., et al., 1991. Iron and aluminum increase in the substantia nigra of patients with Parkinson’s disease: an X-ray microanalysis. J. Neurochem. 56, 446–451. Johnson, V.J., Kim, S.-H., Sharma, R.P., 2005. Aluminum-maltolate induces apoptosis and necrosis in neuro-2a cells: potential role for p53 signaling. Junn, E., et al., 2009. Repression of α-synuclein expression and toxicity by microRNA-7. Proc. Natl. Acad. Sci. USA 106, 13052–13057. Kay, D.M., et al., 2008. Genetic association between alphasynuclein and idiopathic Parkinson’s disease. Am. J. Med. 147B, 1222–1230. Kumar, V., Bal, A., Gill, K.D., 2009. Aluminium-induced oxidative DNA damage recognition and cell-cycle disruption in different regions of rat brain. Toxicology 264, 137–144. Leng, Y., Chuang, D.M., 2006. Endogenous alpha-synuclein is induced by valproic acid through histone deacetylase inhibition and participates in neuroprotection against glutamateinduced excitotoxicity. J. Neurosci. 26, 7502–7512. Le´vesque, L., et al., 2000. Ligand specific effects on aluminum incorporation and toxicity in neurons and astrocytes. Brain Res. 877, 191–202. Martin, R.B., 1991. Aluminum in biological systems. In aluminum in chemistry. In: Nicolini, P., Zatta, P.F., Corain, B. (Eds.), Biology and Medicine. Raven Press, New York, pp. 3–20. Martin, I., Dawson, V.L., Dawson, T.M., 2011. Recent advances in the genetics of Parkinson’s disease. Annu. Rev. Genom. Hum. Genet. 12, 301–325. Minami, A., et al., 2015. Function of alpha-synuclein and PINK1 in Lewy body dementia (Review). Int. J. Mol. Med. 35, 3–9.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917
BRES : 44692
8
918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951 952 953
brain research ] (]]]]) ]]]–]]]
Mizuno, Y., et al., 2008. Progress in the pathogenesis and genetics of Parkinson’s disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363, 2215–2227. Ohyashiki, T., et al., 2002. Nerve growth factor protects against aluminum-mediated cell death. Toxicology 176, 195–207. Ostrerova-Golts, N., et al., 2000. The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity. J. Neurosci. 20, 6048–6054. Pan-Montojo, F., Reichmann, H., 2014. Considerations on the role of environmental toxins in idiopathic Parkinson’s disease pathophysiology. Transl. Neurodegener. 3, 10. Peto, M.V., 2010. Aluminium and iron in humans: bioaccumulation, pathology, and removal. Rejuvenation Res. 13, 589–598. Prabhakaran, K., Chapman, G.D., Gunasekar, P.G., 2011. alphaSynuclein overexpression enhances manganese-induced neurotoxicity through the NF-kappaB-mediated pathway. Toxicol. Mech. Method 21, 435–443. Sanchez-Iglesias, S., et al., 2009. Brain oxidative stress and selective behaviour of aluminium in specific areas of rat brain: potential effects in a 6-OHDA-induced model of Parkinson’s disease. J. Neurochem. 109, 879–888. Satoh, E., et al., 2005. Glutathione depletion promotes aluminummediated cell death of PC12 cells. Biol. Pharm. Bull. 28, 941–946. Satoh, E., et al., 2007. Involvement of NO generation in aluminum-induced cell death. Biol. Pharm. Bull. 30, 1390–1394. Savory, J., et al., 1999. Age-related hippocampal changes in Bcl-2: Bax ratio, oxidative stress, redox-active iron and apoptosis associated with aluminum-induced neurodegeneration: increased susceptibility with aging. Neurotoxicology 20, 805–817. Savory, J., Herman, M.M., Ghribi, O., 2006. Mechanisms of aluminum-induced neurodegeneration in animals: Implications for Alzheimer’s disease. J. Alzheimers Dis. 10, 135–144. Seidl, S.E., et al., 2014. The emerging role of nutrition in Parkinson’s disease. Front. Aging Neurosci. 6, 36. Spatola, M., Wider, C., 2014. Genetics of Parkinson’s disease: the yield. Park. Relat. Disord. 20 (Suppl 1), S35–S38.
Tolleson, C.M., Fang, J.Y., 2013. Advances in the mechanisms of Parkinson’s disease. Discov. Med. 15, 61–66. Trinh, J., Farrer, M., 2013. Advances in the genetics of Parkinson disease. Nat. Rev. Neurol. 9, 445–454. Tsuboi, Y., 2012. Environmental-genetic interactions in the pathogenesis of Parkinson’s disease. Exp. Neurobiol. 21, 123–128. Tsubouchi, R., et al., 2001. Aluminum-induced a poptosis in PC12D cells. Biometals 14, 181–185. Uversky, V.N., Li, J., Fink, A.L., 2001. Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson’s disease and heavy metal exposure. J. Biol. Chem. 276, 44284–44296. Uversky, V.N., et al., 2002. Synergistic effects of pesticides and metals on the fibrillation of alpha-synuclein: implications for Parkinson’s disease. Neurotoxicology 23, 527–536. Vasudevaraju, P., et al., 2008. Molecular toxicity of aluminium in relation to neurodegeneration. Indian J. Med. Res. 128, 545–556. Verstraeten, S.V., Aimo, L., Oteiza, P.I., 2008. Aluminium and lead: molecular mechanisms of brain toxicity. Arch. Toxicol. 82, 789–802. Wan, O.W., Chung, K.K., 2012. The role of alpha-synuclein oligomerization and aggregation in cellular and animal models of Parkinson’s disease. PLoS One 7 (6), e38545, http://dx. doi.org/10.1371/journal.pone.0038545. Wills, J., et al., 2010. Elevated tauopathy and alpha-synuclein pathology in postmortem Parkinson’s disease brains with and without dementia. Exp. Neurol. 225, 210–218. Zabrocki, P., et al., 2005. Characterization of alpha-synuclein aggregation and synergistic toxicity with protein tau in yeast. Febs J. 272, 1386–1400. Zayed, J., et al., 1990. Parkinson disease and environmental factors. Rev Epidemiol Sante Publique 38, 159–160.
Please cite this article as: Saberzadeh, J., et al., Alpha synuclein protein is involved in Aluminum-induced cell death and oxidative stress in PC12 cells. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.01.037
954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988
