Prefoldin prevents aggregation of α-synuclein

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Prefoldin prevents aggregation of α-synuclein Mariko Takanoa,b, Erika Tashiroa,b, Akira Kitamurac, Hiroshi Maitaa, Sanae M.M. Iguchi-Arigad, Masataka Kinjoc, Hiroyoshi Arigaa,n a

Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan Graduate School of Life Science, Hokkaido University, Kita-ku, Sapporo 060-0810, Japan c Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Kita-ku, Sapporo 001-0021, Japan d Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo 060-8589, Japan b

art i cle i nfo

ab st rac t

Article history:

Protein aggregation is observed in various neurodegeneration diseases, including Parkin-

Accepted 18 October 2013

son's disease (PD). Alpha-synuclein, a causative gene product of familial PD, is a major component of large aggregates (inclusion bodies) in PD. Prefoldin, a molecular chaperone

Keywords:

comprised of six subunits, PFD1
6, prevents misfolding of newly synthesized nascent

Prefoldin

polypeptides and also prevents aggregation of protein such as a pathogenic form of

Chaperone α-Synuclein

Huntingtin, a causative gene product of Huntington disease. In this study, we first found that aggregation of TagRFP-tagged wild-type α-synuclein and its pathogenic mutants, but

Protein aggregation

not that of GFP-tagged α-synuclein, occurred in transfected Neuro-2a cells. The fluores-

Cell death

cence of GFP is weakened under the condition of pH 4.5–5.0, and TagRFP is a stable red fluorescence protein under an acidic condition. Aggregated TagRFP-wild-type α-synuclein and its pathogenic mutants in Neuro-2a cells were ubiquitinated and were colocalized with the prefoldin complex in the lysosome under this condition. Furthermore, knockdown of PFD2 and PFD5 disrupted prefoldin formation in α-synuclein-expressing cells, resulting in accumulation of aggregates of wild-type and pathogenic α-synuclein and in induction of cell death. The levels of aggregation and cell death in pathogenic α-synuclein-transfected cells tended to be higher than those in wild-type α-synuclein-transfected cells. These results suggest that prefoldin works as a protective factor in aggregated α-synucleininduced cell death. & 2013 Published by Elsevier B.V.

1.

Introduction

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington disease (HD) are caused by neuronal cell death, which is induced by various factors including genetic and environmental factors. In these diseases, proteins with improper conformation are

oligomerized and aggregated into β-sheet-rich fibrils, thereby forming large aggregates (inclusion bodies) in affected neurons, suggesting that dysfunction of repair systems converting improper conformation to normal conformation of proteins leads to the onset of these neurodegenerative diseases (see recent reviews, references therein: Cohen, 2012; Takalo et al., 2013; Chhangani and Mishra, 2013). It is well

n Correspondence to:Hiroyoshi Ariga Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan. Fax: þ81 11 706 4988. E-mail address: [email protected] (H. Ariga).

0006-8993/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.brainres.2013.10.034

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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known that molecular chaperones play a role in formation of properly folded proteins through binding to newly synthesized unfolded proteins or through preventing protein aggregations (Morimoto, 2008; Broadley and Hartl, 2009; Clare and Saibil, 2013; Chaari et al., 2013; Kim et al., 2013). It has been reported that Hsp70 prevents aggregation of amyloid β, α-synuclein and pathogenic Huntington in AD, PD and HD, respectively (Warrick et al., 1999; Kazemi-Esfarjani and Benzer, 2000; Muchowski et al., 2000; Evans et al., 2006; Wacker et al., 2004; Klucken et al., 2004) and that chaperonin TriC/CCT prevents aggregation of pathogenic Huntingtin (Behrends et al., 2006; Kitamura et al., 2006; Tam et al., 2006). These findings suggest that elucidation of the molecular mechanisms underlying prevention of protein aggregation by chaperones will lead to an understanding of one of the mechanisms of the onset of neurodegenerative diseases. Prefoldin is a molecular chaperone that assists folding of newly synthesized polypeptide chains such as actin and tubulin (Vainberg et al., 1998; Geissler et al., 1998). Prefoldin binds to proteins that have been synthesized in ribosomes and transports them to chaperonin Tric/CCT in cooperation with Hsp70 and Hsp40 (Vainberg et al., 1998; Geissler et al., 1998; Siegers et al., 1999; Hartl and Hayer-Hartl, 2002). Prefoldin is comprised of six subunits including two α-subunits (PFD3 and PFD5) and four β-subunits (PFD1, PFD2, PFD4 and PFD6). The coiled-coiled regions present in both N- and C-terminal regions in prefoldin form a “jellyfish-like” structure and bind to substrates with their tentacle-like structures (Siegert et al., 2000; Martín-Benito et al., 2007). Furthermore, it has been reported that archaeal prefoldin stimulates formation of soluble amyloid β oligomers to inhibit their fibril formation in vitro (Sakono et al., 2008) and that human prefoldin inhibits Aβ fibrillation to form nontoxic Aβ aggregates (Sörgjerd et al., 2013). We have reported that prefoldin inhibited aggregate and inclusion formation of exogenously added pathogenic Huntingtin in cells (Tashiro et al., 2013) and that knockdown of prefoldin expression and mutation of PFD5 caused accumulation of ubiquitinated protein aggregates in cells and mice, resulting in reduced cell viability (Abe et al., 2013). These findings suggest that prefoldin plays a modifier role against the toxicity of misfolded proteins, including proteins that cause neurodegenerative diseases. PD is a movement disorder that occurs due to dopaminergic cell death in the substantia nigra, resulting in a reduced level of dopamine in the striatum. Although familial PD cases account for 10% of total cases of PD, investigations of the functions of familial PD gene products have provided great insights into the molecular mechanisms of the onset of PD, and familial PD gene products are thought to also play roles in the pathogenesis of sporadic PD (see recent reviews, Sai et al., 2012; Hauser and Hastings, 2013; Ariga et al., 2013). The α-synuclein gene is the familial PD gene park1, and three missense mutations coding for A30P, A53T and E46K α-synuclein have been found (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004). Furthermore, duplication and triplication of the α-synuclein gene are also known as park4 (Zarranz et al., 2004; Chartier-Harlin et al., 2004; Ibáñez et al., 2004). Alpha-synuclein is a major component of the inclusion body termed Lewy body and is ubiquitinated in Lewy bodies of PD patients (Singleton et al., 2003; Liani et al., 2004). The monomer of α-synuclein is assumed to be

misfolded by various stresses and to form fibrils and large inclusions (see recent review: Chaari et al., 2013). Although inclusion bodies containing α-synuclein have long been thought to be a causative factor for PD, accumulating evidence suggests that formation of the inclusion is not correlated with neuronal cell death and that the inclusion body acts as a deposit of aggregated proteins to decrease the risk of neuronal cell death (Zarranz et al., 2004; Chaari et al., 2013). In this study, we investigated the effect of prefoldin on aggregation of TagRFP-tagged α-synuclein and cell death induced by aggregated α-synuclein and we found that knockdown of prefoldin caused accumulation of ubiquitinated α-synuclein aggregates in co-localization with the prefoldin complex and decreased cell viability, suggesting that prefoldin works as a protective factor in aggregated α-synucleininduced cell death.

2.

Results

2.1. Establishment of a monitoring system for detection of α-synuclein aggregation in cells Since wild-type and mutant α-synuclein are known to be prone to aggregation (Polymeropoulos et al., 1997; Krüger et al., 1998; Zarranz et al., 2004; Chaari et al., 2013), GFP-tagged wild-type α-synuclein and three mutants of α-synuclein, A30P, A53T and E46K, were transfected into human SH-SY5Y and mouse Neuro-2a cells and proteins were visualized 48 h after transfection. No or little aggregation of α-synuclein, however, was observed (data not shown). Since it has been reported that ubiquitinated α-synuclein and its aggregates are degraded by the ubiquitin–proteasome system or by lysosome-mediated autophagy in which various proteinases degrade protein aggregates under pH 4.5–5.0 (see a recent review, references therein: Nixon 2013) and since the fluorescence of GFP is weakened under pH conditions of 4.5– 5.0, α-synuclein was tagged with TagRFP (pKa 3.8), which is stable red fluorescence protein under an acidic condition (Merzlyak et al., 2007). Forty-eight hour after transfection of TagRFP-wild-type and mutant α-synuclein into Neuro-2a cells, the expression levels of transfected α-synuclein in cell extracts were examined by Western blotting with antiTagRFP and anti-α-synuclein antibodies and their fluorescence images were examined by using a confocal laser microscope (Fig. 1A and B, respectively). The results showed that the expression levels of wild-type α-synuclein and its three mutants were similar (Fig. 1A). Since a band with a size identical to that of TagRFP was always obtained in Western blotting without any transfection, the band that appeared in a TagRFP-transfected lane is a non-specific band. Stained images and their quantified data showed that dot/aggregation of wild-type α-synuclein and its three mutants, but not that of TagRFP alone, occurred in cells, and that the highest level of aggregation occurred in TagRFP-A53T α-synucleintransfected cells with similar levels of aggregation in wildtype, A30P and E46K α-synuclein-transfected cells, being consistent with results of previous studies (Conway et al., 1998; Narhi et al., 1999; Winner et al., 2011) (Fig. 1B and C).

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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Ta gR FP W T A 30 P A 53 T E4 6K

TagRFP -α-synuclein

TagRFP -α-synuclein

α-TagRFP

TagRFP TagRFP -α-synuclein

α-α-synuclein

TagRFP-α-synuclein TagRFP

WT

A30P

**

25

**

20

A53T

E46K

** **

**

19.8

15.7 12.6

15

12.3

10 5 0.7

6K

**p<0.01 n=3

E4

53

T

P A

30 A

W T

0

Ta gR FP

Aggregation-positive cell (%)

227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286

TagRFP-α-synuclein Fig. 1 – Aggregation of wild-type and mutant α-synuclein in Neuro-2a cells. (A) Neuro-2a cells were transfected with pTag RFP-N1, pTagRFP-α-synuclein and its mutant versions. Forty-eight hour after transfection, proteins were extracted from cells and analyzed by Western blotting with anti-TagRFP and anti-α-synuclein antibodies as described in Section 4. (B) Neuro-2a cells were transfected with pTagRFP-N1, pTagRFP-α-synuclein and its mutant versions. Forty-eight hour after transfection, cell images were obtained using a confocal microscope. Arrows indicate dots/aggregates of α-synuclein. Aggregated dots in this study were defined as visible large spots of TagRFP signals in cells. (C) Aggregate-containing cells in B were quantified. About 300 cells were counted in each figure. Ratios of aggregate-containing cells/total cells counted are shown as mean7S.D. of the three experiments. Significance: nnpo0.01.

Wild-type and A53T α-synuclein were therefore used for further study.

2.2. Co-localization of aggregated α-synuclein with the prefoldin complex Since aggregation of TagRFP-α-synuclein, but not that of GFPα-synuclein, was observed in Neuro-2a cells, it was thought

that aggregation of TagRFP-α-synuclein occurred in acidic organelles such as lysosomes. To examine this, Neuro-2a cells were transfected with wild-type and A53T α-synuclein and stained with Lysotracker at 48 h after transfection, and cell images were obtained under a confocal laser microscope. As shown in merged figures of Fig. 2A, all of the dots/ aggregates of TagRFP-α-synuclein were co-localized with those of LysoTracker signals. TagRFP-wild-type and TagRFP-A53T

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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TagRFP

LysoTracker

Merge

TagRPF

α -Ubiquitin

Merge

TagRFP

α -Prefoldin

α -synuclein WT

A53T

α -synuclein WT

A53T

Merge

TagRFP -WT α -synuclein

TagRFP -A53T

α -synuclein

Fig. 2 – Co-localization of α-synuclein with lysosome and the prefoldin complex. (A)–(C) Neuro-2a cells were transfected with pTagRFP-wild-type α-synuclein and pTagRFP-A53T α-synuclein. Forty-eight hour after transfection, cells were stained with Lysotracker Green DND-26 (A), an anti-ubiquitin antibody (B) and anti-prefoldin antibody (C), and cell images were visualized by using a confocal laser microscope.

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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α-synuclein-transfected Neuro-2a cells were then stained with an anti-ubiquitin antibody followed by an FITC-conjugated secondary antibody, and the results showed that aggregates of both wild-type and A53T α-synuclein were colocalized with ubiquitin, indicating that TagRFP-wild-type and TagRFP-A53T α-synuclein were ubiquitinated (Fig. 2B). We have established an anti-prefoldin antibody that recognizes the prefoldin complex but not its 6 subunits (Abe et al., 2013). By using this antibody, aggregates of both TagRFP-wild-type and TagRFP-A53T α-synuclein were found to be co-localized with the prefoldin complex (Fig. 2C). These results indicate that ubiquitinated TagRFPα-synuclein aggregates were co-localized with the prefoldin complex in the lysosome.

2.3. Stimulation of α-synuclein aggregation and cell death in prefoldin-knockdowned Neuro-2a cells To examine the role of prefoldin in α-synuclein aggregation, knockdown of the expression of prefoldin subunits in cells was carried out using siRNAs targeting prefoldin subunits. Since the prefoldin complex contains two α-type subunits (PFD3 and PFD5) and four β-type subunits (PFD1, PFD2, PFD4 and PFD6), siRNAs targeting PFD5 (α-type subunit) and PFD2 (β-type subunit) were chosen for knockdown. As previously reported (Miyazawa et al., 2011; Tashiro et al., 2013; Abe et al., 2013), knockdown of either PFD2 or PFD5 reduced the expression levels of other prefoldin subunits (Fig. 3A) and knockdown of PFD2 and PFD5 disrupted the prefoldin complex detected by a glycerol density gradient centrifugation (data not shown). Neuro-2a cells were transfected with PFD2 siRNA, PFD5 siRNA or non-specific siRNA as a control. Twenty-four hour after transfection, cells were transfected with expression vectors for TagRFP, TagRFP-wild-type α-synuclein and TagRFP-A53T α-synuclein. Forty-eight hour after transfection of TagRFP proteins, proteins in cells were first examined by Western blotting with anti-TagRFP, anti-α-synuclein, antiPFD2 and anti-PFD5 antibodies. As shown in Fig. 3A, expression levels of PFD2 and PFD5 were reduced, but those of TagRFP proteins were not changed after transfection of siRNAs into Neuro-2a cells. TagRFP signals in cells were then analyzed using a fluorescence microscope at 48 h after transfection of TagRFP proteins. As shown in Fig. 3B–D, both PFD2 and PFD5 knockdown increased the number of dots/ aggregates in TagRFP-wild-type α-synuclein and TagRFPA53T α-synuclein-expressing cells but not in cells expressing TagRFP. The number of cells containing more than 2 aggregates/cell was greater in TagRFP-A53T α-synuclein-expressing cells than in TagRFP-wild-type α-synuclein-expressing cells. Neuro-2a cells were transfected with PFD2 siRNA and PFD5 siRNA and then transfected with expression vectors for TagRFP, TagRFP-wild-type α-synuclein and TagRFP-A53T α-synuclein as described above. Forty-eight hour after transfection of TagRFP proteins, cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. As shown in Fig. 4A, knockdown of both PFD2 and PFD5 significantly decreased the viability of TagRFP-wildtype α-synuclein and TagRFP-A53T α-synuclein-transfected cells but not that of TagRFP-transfected cells. LDL assays were also carried out to examine the effect of prefoldinknockdown on viability of TagRFP-α-synuclein-transfected

5

cells. As shown in Fig. 4B, prefoldin-knockdown using PFD2 siRNA and PFD5 siRNA specifically increased cell toxicity. These results suggest prefoldin-dependent protective reactions against toxicity of α-synuclein aggregation in Neuro-2a cells.

3.

Discussion

In this study, we first found that TagRFP-wild-type α-synuclein and pathogenic mutants of α-synuclein were ubiquitinated and aggregated in co-localization with the prefoldin complex in the lysosome (Figs. 1 and 2). We then found that knockdown of prefoldin expression increased the level of aggregated α-synuclein and reduced viability of transfected Neuro-2a cells (Figs. 3 and 4). These results suggest that prefoldin plays a role in preventing αsynuclein-induced cell toxicity. It is known that α-synuclein is oligomerized and aggregated into β-sheet-rich fibrils in the cytoplasm, thereby forming large aggregates/inclusion termed Lewy bodies (Zarranz et al., 2004; Chaari et al., 2013). Alpha-synuclein in Lewy bodies was shown to be ubiquitinated (Singleton et al., 2003; Liani et al., 2004). It has been suggested that after disruption of the lysosome-mediated degradation system due to accumulation of some α-synuclein aggregates in the lysosome, formation of α-synuclein aggregates in the cytoplasm is further accelerated and then a Lewy body is formed (Zarranz et al., 2004). Since TagRFP-tagged α-synuclein used in this study was aggregated and ubiquitinated (Figs. 1 and 2B), it is thought that aggregates of TagRFP-α-synuclein are aggregates before or during Lewy body formation. Aggregates of TagRFP-wild-type α-synuclein and TagRFP-mutants of α-synuclein were colocalized with the prefoldin complex in the lysosome, though most of the prefoldin was localized in the cytoplasm (Fig. 2A and C). This conclusion is consistent of the results in our previous report, in which the prefoldin complex was co-localized with ubiquitinated protein (Abe et al., 2013). Furthermore, the number of α-synuclein aggregate-containing cells and ratio of cell death were increased in prefoldin-knockdown cells (Figs. 3 and 4). Small aggregates of α-synuclein are thought to be more toxic than large aggregates (Chaari et al., 2013). Since prefoldin assists folding of newly synthesized unfolded proteins (Vainberg et al., 1998; Geissler et al., 1998) and since prefoldin reduced aggregation of α-synuclein in this study, it is thought that prefoldin inhibits early stages of the aggregation of α-synuclein such as formation of small oligomers. Indeed, we have shown that prefoldin inhibits formation of pathogenic Huntingtin oligomers that are larger than a dimer and trimer (Tashiro et al., 2013). Alternatively, it is also possible that the effects of prefoldin knockdown on synuclein aggregation and toxicity are indirect, perhaps through the accumulation of unrelated misfolded nascent proteins that then compromise the overall folding capacity of the cell. Many of the α-synuclein aggregates are known to be degraded by autolysosomes through macroautophagy (Yu et al., 2009; Spencer et al., 2009; Ebrahimi-Fakhari et al., 2011). Although prefoldin prevents aggregation of ubiquitinated proteins even in the absence of autophagy (Abe et al., 2013), the results in the present study also suggest that prefoldin assists autophagy-dependent

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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6

si-PFD2

TagRFP -α-synuclein

A

53

T

53 T Ta gR FP W T

53 T Ta gR FP W T

TagRFP -α-synuclein

A

Ta gR FP W T

TagRFP -α-synuclein

si-PFD5

A

si-contrrol

TagRFP -α-synuclein α-TagRFP

TagRFP TagRFP -α-synuclein

α-α-synuclein

PFD2

α-PFD2

PFD5

α-PFD5

si-contrrol

si-PFD5

si-PFD2

TagRFP

WT

TagRFP -α-synuclein

A53T

TagRFP -α-synuclein

TagRFP -WT α-synuclein

TagRFP -A53T α-synuclein

5

2 D

-P FD si

-P F si

5 D

-c on tr r

nt

ol

0

si

rr ol -P FD 2 si -P FD 5 si -c on tr ro l si -P FD 2 si -P FD 5 si

-c o

si TagRFP

20

nt

5

2

-P FD si

FD

-P si

si

-c

on tr

ro l

0

40

2

5.3

4.5

-c o

4.9

si

10

-P F

15.7

**

**

*

60

D

18.0

20

** 80

si

25.5

TagRFP -A53T α-synuclein

100

-P F

26.1

l

29.1

25.2

rr o

**

Aggregate-containing cell (%)

**

30

TagRFP -WT α-synuclein

** **

si

40

Aggregate-containing cell (%)

587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646

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1 aggregate/cell *p<0.05, **p<0.01 >2 aggregates/cell n=5

Fig. 3 – Stimulation of aggregation of α-synuclein in prefoldin-knockdown cells. (A) Neuro-2a cells were transfected with PFD2 siRNA, PFD5 siRNA or non-specific siRNA (si-control). Twenty-four hour after transfection, cells were transfected with pTagRFP, pTagRFPwild-type α-synuclein and pTagRFP-A53T α-synuclein. Proteins were extracted 48 h after transfection and analyzed by Western blotting with antibodies against PFD2, PFD5, TagRFP and α-synuclein. (B) Neuro-2a cells were transfected with PFD2 siRNA, PFD5 siRNA or non-specific siRNA (si-control) and then with pTagRFP, pTagRFP-wild-type α-synuclein and pTagRFP-A53T α-synuclein as described in the legend for (A). Cell images of TagRFP fluorescence were visualized by using a confocal laser microscope. Cells containing a dot or dots were marked. (C) Aggregate-containing cells were counted in cell images of (B), and data are shown as mean7S.D. of the three experiments. Significance: nnpo0.01. (D) Numbers of cells containing one aggregate and more than two aggregates were counted in cell images of (B), and data are shown as mean7S.D. of the three experiments. Significance: npo0.05 and nn po0.01.

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706

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120

*

N.S.

*

*

*

Cell viability (%)

100 80 60 40 20

tr ol FD 2 si -P D F5 si -c on tr ol si -P FD 2 si -P D F 5 si -c on tr ol si -P FD 2 si -P D F5 si -P

on

si -c

C on

tr

ol

0

TagRFP

*p<0.05 n=15

TagRFP TagRFP -WT α-synuclein -A53Tα-synuclein

140

*

120

N.S.

*

100 80 60 40 20

F5

2

D -P si

ol

FD

tr on

-P si

-c si

2

F5 D -P

si

ol tr

FD

-P

on -c si

si

2

F5

si

-P

D

FD

-P si

si

-c

on

tr

ol

0

TagRFP

TGTGACAACAGTGGCTGAGA-3′; α-synuclein-A53Tantisense; 5′-TCTCAGCCACTGTTGTCACACCATGCACCA-3′; α-synucleinE46Ksense: 5′-GCTCCAAAACCAAGAAGGGAGTGGTGCATG-3′ and α-synuclein-E46Kantisense: 5′-CATGCACCACTCCCTTCTTGGTTTTGGAGC-3′. pTagRFP-N1 was obtained from Evrogen (Moscow, Russia). For pTagRFP-α-synuclein, PCR was carried out using two primers (α-synuclein5′-EcoRI and α-synuclein3′SalI) and pHM5-α-synuclein as a template. The resultant PCR product was then digested with EcoRI and SalI and inserted into EcoRI/SalI sites of pTagRFP-N1. For pTagRFP-A30P α-synuclein, pTagRFP-A53T α-synuclein and pTagRFP-E46K α-synuclein, PCR was carried out using sense and antisense primers corresponding to their mutations described above and using pTagRFP-αsynuclein as a template. The resultant PCR product was then digested with DpnI and used for transformation into E. coli.

4.2.

**

130

Cytotoxicity (%)

707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766

TagRFP TagRFP -WT α-synuclein -A53Tα-synuclein

*p<0.05 **p<0.01 n=4

Fig. 4 – Stimulation of aggregated α-synuclein-induced cell death in prefoldin-knockdown cells. Neuro-2a cells were transfected with PFD2 siRNA, PFD5 siRNA or non-specific siRNA (si-control). Twenty-four hour after transfection, cells were transfected with pTagRFP, pTagRFP-wild-type α-synuclein and pTagRFP-A53T α-synuclein. Forty-eight hour after transfection, cell viability was assessed by MTT assays (A) and LDL assays (B). Data are shown as mean7S.E. of 15 experiments (A) and of 4 experiments (B). Significance: n po0.05 and nnpo0.01.

degradation of α-synuclein aggregates by delivering them into the lysosome.

4.

Experimental procedures

4.1.

Plasmids

Analysis of aggregate-containing cells

Neuroblastoma Neuro-2a cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Cells were cultured on a 3.5-cm glass-bottom dish (Iwaki, Tokyo, Japan) coated with type IV collagen (Cellmatrix, Nitta Gelatin, Osaka, Japan) and transfected with 0.05 mg of pTagRFP-N1, pTagRFP-α-synuclein and its mutant versions using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Forty-eight hour after transfection, cells were fixed and stained with 100 nM Lysotracker Green DND-26 (Molecular Probes, Eugene, OR, USA) or with anti-multi-ubiquitin (1/50 dilution, DAKO, Glostrup, Denmark) and anti-prefoldin (1/50 dilution) antibodies followed by respective secondary antibodies. Cell images were visualized by using a confocal laser microscope (LSM510, Zeiss, Jena, Germany). An anti-prefoldin antibody was established by us as described previously (Abe et al., 2013). Aggregated dots in this study were defined as visible large spots of TagRFP signals in cells. About 300 cells were counted in each figure. To examine the role of prefoldin in aggregation of α-synuclein, knockdown of α-synuclein expression was carried out using specific siRNAs. Nucleotide sequences of the upper and lower strands for siRNA were as follows: 5′GGAGCAUGUGCUUAUUGAUGU-3′ and 5′-AUCAAUAAGCACAUGCUCCAC-3′ for PFD2, and 5′-GGAGCGGACUGUCAAAGAATT-3′ and 5′-UUCUUUGACAGUCCGCUCCTT-3′ for PFD5. Neuro-2a cells were first transfected with PFD2 siRNA and PFD5 siRNA using Lipofectamine 2000 reagent. Allstars Negative control siRNA (Qiagen, Hilden, Germany) was used as a non-specific negative control (Qiagen). Twenty-four hour after transfection, cells were transfected with pTagRFP-N1, pTagRFP-α-synuclein and its mutant versions as described above. Forty-eight hour after transfection, cell images were obtained as described above.

4.3. Nucleotide sequences used for PCR primers are as follows: α-synuclein5′-EcoRI: 5′-GGGAATTCATGGATGTATTCATGAAAGGA-3′; α-synuclein3′-SalI: 5′-GTCGACGGGGCTTCAGGTTCGTA-3′; α-synuclein-A30Psense; 5′-GTGTGGCAGAAGCACAGGAAAGACAAAAG-3′; α-synuclein-A30Pantisense: 5′-CTTTTGTCTTTCCTGGTGCTTCTGCCACAC-3′; α-synuclein-A53Tsense: 5′-TGGTGCATGG

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Analysis of cell death

Neuro-2a cells in 96-well plates (3.3  103 cells/dish) were first transfected with 1.33 mm PFD2 siRNA, PFD5 siRNA and control siRNA using Lipofectamine 2000 reagent. Twenty-four hour after transfection, cells were transfected with 1.67  10  3 mg of pTagRFP-N1, pTagRFP-α-synuclein and its mutant versions.

Please cite this article as: Takano, M., et al., Prefoldin prevents aggregation of α-synuclein. Brain Research (2013), http://dx.doi. org/10.1016/j.brainres.2013.10.034

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Forty-eight hour after transfection, cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. To assess cell viability, LDH assays were carried out using cell extracts from cultured Neuro-2a cells as described above. LDH assays were carried out using an LDL assay kit (CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit, Promega) according to supplier's protocol. Briefly, 300 ml of cell extract was mixed with 300 ml of CytoTox-ONE reagent and incubated at 2 1C for 10 min, and its fluorescence intensity (Ex¼ 560 nm, Em¼590 nm) was measured using a fluorescent spectrophotometer (F-2500, Hitachi).

4.4.

Western blotting

Neuro-2a cells in 6-well dishes (1.25  10  5 cells/well) were transfected with 0.05 mg of pTagRFP-N1, pTagRFP-α-synuclein and its mutant versions using Lipofectamine 2000 reagent. Forty-eight hour after transfection, proteins were extracted from cells after incubation of cells with a Hepes buffer containing 40 mM Hepes–NaOH (pH 7.4), 120 mM NaCl, 1 mM EDTA, 0.5% NP-40 and protease inhibitors and subjected to Western blot analysis with anti-α-synuclein (1/5000 dilution, BD, Franklin Lakes, NJ, USA), anti-TagRFP (1/5000 dilution, Evrogen), anti-PFD2 (1/1000 dilution) and anti-PFD5 (1/1000 dilution, S-20, Santa Cruz, Santa Cruz, CA, USA) antibodies. Proteins were then reacted with an IRDye800- or Alexa Fluor 680-conjugated secondary antibody and visualized by using an infrared imaging system (Odyssey, LI-COR, Lincoln, NE, USA). An anti-PFD2 antibody was established by us as described previously (Tashiro et al., 2013).

4.5.

Statistical analyses

Data are expressed as mean7S.D. or 7S.E. Statistical analyses were performed using analysis of variance (one-way ANOVA) followed by the Tukey–Kramer test.

Acknowledgment This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), from New Energy and Industrial Technology Development Organization (NEDO) and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) in Japan.

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