Journ

alof

Cell

Scie

nce

Role of Ser129 phosphorylation of a-synuclein inmelanoma cells

Byung Rho Lee1,*, Yasuhiro Matsuo1,2,*, Anil G. Cashikar3 and Tetsu Kamitani1,`

1Department of Medicine, GRU Cancer Center, Georgia Regents University, Augusta, GA 30912, USA2Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan3Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA

*These authors contributed equally to this work.`Author for correspondence (tkamitani@gru.edu)

Accepted 8 November 2012Journal of Cell Science 126, 696–704� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.122093

Summarya-Synuclein, a protein central to Parkinson’s disease, is frequently expressed in melanoma tissues, but not in non-melanocytic cutaneous

carcinoma and normal skin. Thus, a-synuclein is not only related to Parkinson’s disease, but also to melanoma. Recently,epidemiologists reported co-occurrence of melanoma and Parkinson’s disease in patients, suggesting that these diseases could sharecommon pathogenetic components and that a-synuclein might be one of these. In Parkinson’s disease, phosphorylation of a-synuclein atSer129 plays an important role in the pathobiology. However, its role in melanoma is not known. Here, we show the biological relevance

of Ser129 phosphorylation in human melanoma cells. First, we have identified an antibody that reacts with Ser129-unphosphorylated a-synuclein but not with Ser129-phosphorylated a-synuclein. Using this and other antibodies to a-synuclein, we investigated the role ofSer129 phosphorylation in human melanoma SK-MEL28 and SK-MEL5 cells. Our immunofluorescence microscopy showed that the

Ser129-phosphorylated form, but not the Ser129-unphosphorylated form, of a-synuclein localizes to dot-like structures at the cellsurface and the extracellular space. Furthermore, immuno-electron microscopy showed that the melanoma cells release microvesicles inwhich Ser129-phosphorylated a-synuclein localizes to the vesicular membrane. Taken together, our studies suggest that the

phosphorylation of Ser129 leads to the cell surface translocation of a-synuclein along the microtubule network and its subsequentvesicular release in melanoma cells.

Key words: Melanoma, Parkinson’s disease, a-Synuclein, Phosphorylation

Introductiona-Synuclein (a-syn) is an important molecule clinically, since

mutations and copy number variations in its gene have been

linked to familial Parkinson’s disease (PD) (Polymeropoulos

et al., 1997; Kruger et al., 1998; Singleton et al., 2003; Zarranz

et al., 2004). In addition, inclusion bodies containing a-syn,

referred to as Lewy bodies, are pathological hallmarks of familial

and sporadic PD (Fujiwara et al., 2002; Tanji et al., 2006).

Importantly, a-syn is predominantly phosphorylated at Ser129

(S129) in Lewy bodies of the PD brain (Fujiwara et al., 2002;

Anderson et al., 2006). Thus, a-syn is a central molecule in

understanding PD pathobiology, and its phosphorylation at S129

is thought to be involved in PD pathogenesis.

a-Syn is not only related to PD, but also to melanoma, which is

the major cause of skin cancer death worldwide (Matsuo and

Kamitani, 2010). We recently showed that a-syn is highly

expressed in human melanoma cell lines (Matsuo and Kamitani,

2010; Lee and Kamitani, 2011). Moreover, a-syn is frequently

expressed in human melanoma tissues, but it is undetectable in

tissues of non-melanocytic cutaneous carcinoma and normal skin

(Matsuo and Kamitani, 2010). These observations suggest that a-

syn plays a role in the pathobiology of melanoma, as well as that

of PD. Indeed, epidemiological studies have revealed co-

occurrence of melanoma and PD in patients (Olsen et al., 2005;

Olsen et al., 2006; Gao et al., 2009). Specifically, melanoma

patients have an increased risk of subsequently developing PD

(Olsen et al., 2006), and PD patients have an increased risk of

subsequently developing melanoma (Olsen et al., 2005).

Therefore, it is possible that a-syn is shared as a common

pathogenetic component in these two diseases (Matsuo and

Kamitani, 2010).

a-Syn is a small soluble protein containing 140 amino acids

(17 kDa), and predominantly expresses in the brain. a-Syn

localizes to synaptic vesicles on the presynaptic side and the

nucleus – hence the name ‘synuclein’ (Maroteaux et al., 1988;

Auluck et al., 2010). Although the exact function of a-syn is still

unclear, substantial evidence now exists to suggest that this

protein is predominantly natively unfolded in solution but can

bind to phospholipid membranes by adopting an amphipathic

helical conformation in its N-terminal amino acids (Bennett,

2005; McFarland et al., 2008).

a-Syn has at least three experimentally proven phosphorylation

sites, S87, S129 and Y125 (Chau et al., 2009). Among these sites,

S129 has been studied most in the neuropathology field as

described above. With regard to the physiological role of the S129

phosphorylation, there have been several reports. Pronin et al.

reported that the S129 phosphorylation leads to the dissociation of

a-syn from lipids (Pronin et al., 2000). Lou et al. reported that the

S129 phosphorylation reduces the ability of a-syn to regulate

tyrosine hydroxylase (TH) and protein phosphatase 2A (Lou et al.,

696 Research Article

Journ

alof

Cell

Scie

nce

2010). Recently, McFarland et al. showed that the C-terminalpeptide of S129-phosphorylated a-syn interacts preferentially withcytoskeletal proteins and vesicular trafficking proteins (McFarlandet al., 2008), suggesting that the S129 phosphorylation likely has a

profound effect on protein trafficking. Despite these observations,the physiological relevance of S129 phosphorylation is stillunclear.

In this report, we investigated a novel, physiological role of theS129 phosphorylation in human melanoma SK-MEL28 and SK-

MEL5 cells. We first identified an antibody specific to S129-unphosphorylated a-syn. Using this as well as an antibodyspecific to S129-phosphorylated a-syn, we investigated the

subcellular localization of a-syn and found that the S129-phosphorylated form localizes to dot-like structures at the cellsurface and the extracellular space. On the basis of these resultsand others, we have proposed that the phosphorylation of S129

plays a role in the translocation to the cell surface and subsequentvesicular release of a-syn in melanoma cells.

Results4D6 reacts with amino acid residues 124–134 of humana-syn

4D6 is a mouse monoclonal anti-a-syn antibody (Golovko et al.,2005), but its epitope had not been identified. We thereforeperformed epitope mapping. We expressed several deletionmutants of a-syn as fusion proteins in yeast cells and tested for

reactivity with 4D6 using western blotting (Fig. 1A). The resultsmapped the epitope to within 11 amino acid residues (124–134)at the C-terminal region containing the S129 phosphorylation site

(see lane 9). The results are summarized in Fig. 1B,C.

4D6 selectively recognizes the S129-unphosphorylatedform of a-syn

We next determined whether phosphorylation at S129 affects the4D6 immunoreactivity to a-syn. We performed western blotanalysis using both unphosphorylated and phosphorylated formsof a-syn. In brief, a-syn was expressed in bacterial cells and

purified. The unphosphorylated recombinant a-syn protein wastreated with casein kinase-2 (CK2), which is a serine/threonine-selective protein kinase (Fujiwara et al., 2002), to generate a-syn

protein phosphorylated at S129. Both unphosphorylated andphosphorylated forms of a-syn were then analyzed by westernblotting using anti-a-syn monoclonal antibodies LB509 [for total

a-syn (Jakes et al., 1999)], EP1536Y [for S129-phosphorylateda-syn (Fournier et al., 2009; Qing et al., 2009)], and 4D6. Asshown in Fig. 2A, LB509 detected both unphosphorylated and

phosphorylated forms of a-syn. EP1536Y only detected a-synthat was phosphorylated by CK2. 4D6 detected a-syn that wasnot phosphorylated by CK2, but did not detect a-syn that wasphosphorylated by CK2, suggesting that 4D6 selectively reacts

with the S129-unphosphorylated form of a-syn.

We further confirmed the immunoreactivity of 4D6 to S129-

unphosphorylated a-syn using another method. Specifically, a-syn(without tag) was overexpressed in SH-SY5Y cells (Chau et al.,2009) and resolved by two-dimensional gel electrophoresis,

followed by western blot analysis using anti-a-syn antibodiesLB509 (for total a-syn), EP1536Y (for S129-phosphorylated a-syn),and 4D6. As shown in Fig. 2B, LB509 detected at least five species

of a-syn (,17 kDa) at the pH range between 4.4 and 5.0. SinceEP1536Y only detected spot no. 1, we identified spot no. 1 as S129-phosphorylated a-syn and spots no. 2–5 as S129-unphosphorylated

species. 4D6 detected spots no. 2–5, but not spot no. 1 (bottompanel, Fig. 2B), indicating that 4D6 selectively recognizes a-syn

that is not phosphorylated at S129.

Role of S129 phosphorylation in a-syn localization inhuman melanoma SK-MEL28 cells

a-Syn is predominantly expressed in neurons and localizes tosynaptic vesicles and portions of the nucleus (Maroteaux et al.,

1988). Recently, we found that a-syn is expressed more in humanmelanoma tissues and cell lines (Matsuo and Kamitani, 2010; Leeand Kamitani, 2011). However, its subcellular localization in

melanoma cells has not been known. To determine this, weimmunostained human melanoma SK-MEL28 cells using 4D6antibody for a-syn unphosphorylated at S129 (Fig. 3A) and pSyn

Fig. 1. Epitope mapping of 4D6 antibody on a-syn. (A) 4D6

immunoreactivity with deletion mutants of a-syn. To express AD-HA epitope

alone and AD-HA-fused a-syn (full-length and deletion mutants), yeast cells

were transformed and grown in a selection medium. Their total cell lysates

were then prepared and analyzed by western blotting. Anti-HA antibody

16B12 (top panel) and anti-a-syn antibody 4D6 (middle panel) were used to

determine the immunoreactivity with AD-HA (lanes 1 and 6) and AD-HA-

fused a-syn (lanes 2–5 and 7–8). To demonstrate equal loading amounts of

total cell lysates, western blotting using anti-actin antibody was also

performed (bottom panel). The length of wild-type and deletion mutants of a-

syn are indicated by numbers of amino acid residues (e.g. 1–140 for the wild-

type a-syn, 1–50 for the C-terminal deletion mutant and 90–140 for the N-

terminal deletion mutant). Molecular size markers are shown in kilodaltons.

(B) Summary of 4D6 immunoreactivity on full-length a-syn and its deletion

mutants. Amino acid residues 1–60, amphipathic region; amino acid residues

61–95, NAC (non-Ab component of Alzheimer’s disease amyloid) region;

and amino acid residues 96–140, acidic region. S129 residue for

phosphorylation is indicated by an arrowhead. (C) Recognition sites of 4D6

and other anti-a-syn antibodies (EP1536Y, LB509 and pSyn#64) on a-syn.

Recognition sites of antibodies are indicated by arrows.

Release of a-synuclein from melanoma 697

Journ

alof

Cell

Scie

nce

# 64 antibody (Fujiwara et al., 2002) for a-syn phosphorylated at

S129 (Fig. 3B). It should be noted that both 4D6 and pSyn # 64

recognize the same 11 amino acids (residues 124–134) of human

a-syn (see Fig. 1C). For this immunostaining, we counterstained

endogenous NUB1 (Tanji et al., 2006; Tanji et al., 2007) using

rabbit anti-NUB1 antibody, because the counterstaining allowed

us to clearly see the cell shape and cell surface staining (see

below). As shown in Fig. 3, S129-unphosphorylated a-syn

localized to the cytoplasm as small dots, but its nuclear

localization was limited (left panel, Fig. 3A). In contrast, S129-

phosphorylated a-syn localized to the nucleus and cytoplasm (left

panel, Fig. 3B). It should be noted that it also localized to the cell

surface, which was clearly observed at the dendritic processes of

SK-MEL28 cells (see the magnified image on right, Fig. 3B). It

appeared that the small dot-like structures containing S129-

phosphorylated a-syn were released from the cell surface to the

outside (see below for the detail). Importantly, such release of

dot-like structures could not be observed on the cell surface

stained with 4D6 antibody (see the magnified image on right,

Fig. 3A). These results suggest that the phosphorylation of a-syn

at S129 is involved in its cell surface localization as well as its

nuclear localization. In Fig. 3C, we show additional cells

immunostained with 4D6 or pSyn#64 to support the findings

described above.

Dot-like structures containing S129-phosphorylated a-syn

around SK-MEL28 cells

When SK-MEL28 cells were stained with pSyn#64 (Fig. 4B), but

not with 4D6 antibody (Fig. 4A), many dot-like structures were

observed around the cells. Since the staining intensity of each

dot-like structure was weak, longer exposure was needed for the

demonstration in the immunofluorescence microscopy. However,

the dot-like structure with S129-phosphorylated a-syn clearly

existed around the cells (which looked like a starry sky),

suggesting that the phosphorylation of a-syn at S129 is involved

in its cell surface localization and subsequent release.

Immunoreactivity of 4D6 and pSyn#64 antibodies to a-syn-

knockdown SK-MEL28 cells

As described above, we observed differences in 4D6 and

pSyn#64 immunostainings. To confirm that the differences do

Fig. 2. 4D6 immunoreactivity with unphosphorylated and

phosphorylated forms of a-syn. (A) 4D6 immunoreactivity with kinase-

untreated and treated a-syn. Recombinant human a-syn was purified from

bacterial culture and incubated with or without protein kinase CK2.

Unphosphorylated and phosphorylated forms of a-syn were then analyzed by

western blotting using anti-a-syn antibody LB509 (top panel), anti-phospho-

a-syn antibody EP1536Y (middle panel) and anti-a-syn antibody 4D6

(bottom panel). Molecular size markers are shown in kilodaltons. (B) Specific

immunoreactivity of 4D6 with S129-unphosphorylated form of a-syn

expressed in SH-SY5Y cells. a-Syn (without tag) was overexpressed in SH-

SY5Y cells by plasmid transfection. The total cell lysate was resolved by two-

dimensional gel electrophoresis, and western-transferred to a membrane.

Using a stripping method, the membrane was then serially probed with mouse

monoclonal anti-a-syn antibody LB509 (top panel), rabbit monoclonal anti-

phospho-a-syn antibody EP1536Y (middle panel) and mouse monoclonal

anti-a-syn antibody 4D6 (bottom panel) for western blot analysis. Molecular

size markers are shown in kilodaltons.

Fig. 3. Subcellular localization of S129-unphosphorylated and

phosphorylated forms of endogenous a-syn in human melanoma SK-

MEL28 cells. (A) Double immunostaining of SK-MEL28 cells with anti-a-

syn antibody 4D6 (for S129-unphosphorylated form) and anti-NUB1 antibody

(for counterstaining). (B) Double immunostaining of SK-MEL28 cells with

anti-phospho a-syn antibody pSyn#64 (for S129-phosphorylated form) and

anti-NUB1 antibody (for counterstaining). (C) Comparison between 4D6

immunostaining and pSyn#64 immunostaining. Human melanoma SK-

MEL28 cells were double-immunostained with mouse monoclonal anti-a-syn

antibody (4D6 or pSyn#64) and rabbit polyclonal anti-NUB1 antibody. Cells

were then labeled with fluorescent dye-conjugated secondary antibodies. The

stained cells were analyzed by fluorescence microscopy. In A and B, the

localization of endogenous a-syn is shown by the green fluorescence (left

panels). The localization of endogenous NUB1 is shown by the red

fluorescence. Both images are merged in the middle panels. The framed areas

(middle panels) are magnified in the right panels. Scale bars indicate 20 mm

in the left and middle panels and 5 mm in the right panels. In C, only merged

images are shown with nuclear DAPI staining (the blue fluorescence). Scale

bars indicate 20 mm.

Journal of Cell Science 126 (2)698

Journ

alof

Cell

Scie

nce

not result from non-specific reaction of antibodies, we examinedthe immunoreactivity of 4D6 and pSyn#64 to a-syn-knockdownSK-MEL28 cells. For this purpose, we stably knocked down

endogenous a-syn in SK-MEL28 cells by using lentivirus-mediated RNAi. In brief, SK-MEL28 cells were infected withlentiviruses to express five different sequence-verified shRNAs.We then chose one SK-MEL28 cell line (out of five cell lines)

that was infected with the most efficient lentivirus vectorharboring the shRNA sequence for a-syn RNAi, based onwestern blotting using anti-a-syn antibody 4D6. As shown in the

upper panel of Fig. 5A, the expression of endogenous a-syn wasinterfered in all cell lines expressing a-syn shRNA (no. 1–5).Because endogenous a-syn was strongly knocked down in cell

line no. 3, we chose this cell line for further experiments. Inaddition to the cell line no. 3, we established a cell lineexpressing control shRNA (first lane).

Next, we immunostained these SK-MEL28 cell lines with 4D6

and pSyn#64 antibodies. As shown in Fig. 5B, cells expressingcontrol shRNA were strongly immunostained with bothantibodies (left panels), but cells with knockdown of a-syn

were not (right panels). Thus, we confirmed the specificity of4D6 and pSyn#64 antibodies in a-syn immunostaining.

Ultrastructure of the cell surface of human melanoma SK-MEL28 cells

To further investigate the cell surface of SK-MEL28 cells, we

performed electron microscopy. Fig. 6A,D shows tworepresentative images. In both panels, many small structuresexist at the cell surface. In the magnified images (Fig. 6B,E), we

can clearly observe cross and longitudinal sections of microvillithat contain microfilaments, as reported previously (Rodriguez-

Vicente et al., 1996). In addition, there are microvesicles (MVs),whose average diameter is ,150 nm (arrows, Fig. 6B,E). We

also observed MVs that were budding from or fusing to theplasma membrane (arrowhead, Fig. 6C).

Ultrastructural localization of endogenous a-synphosphorylated at S129 in human melanoma SK-MEL28cells

Next, we performed immuno-electron microscopy to investigate theultrastructural localization of S129-phosphorylated a-syn at the cell

surface and underneath the plasma membrane. As shown in Fig. 7,immuno-electron microscopy revealed that S129-phosphorylated a-

syn localizes to small structures (40 to 60 nm in diameter)underneath the plasma membrane (arrows, Fig. 7A). We speculate

that S129-phosphorylated a-syn is either oligomerized into smallaggregates or accumulated within the 40–60 nm structures. In

addition, S129-phosphorylated a-syn localizes to the membranes ofMVs, whose average diameter is ,150 nm (arrows, Fig. 7B–D).

Thus, our immuno-electron microscopy revealed that SK-MEL28cells release S129-phosphorylated a-syn as MVs.

Role of S129 phosphorylation in a-syn localization invarious melanoma cell lines

In human melanoma SK-MEL28 cells, S129-phosphorylated a-syn localized to the cell surface as well as the nucleus. To

Fig. 4. Dot-like structures with phosphorylated a-syn around SK-MEL28

cells. Cells were immunostained with 4D6 for S129-unphosphorylated a-syn

(A) or with pSyn#64 for S129-phosphorylated a-syn (B). Cells were then

labeled with Alexa Fluor 488-conjugated secondary antibody. The stained

cells were analyzed by fluorescence microscopy with longer exposure to

demonstrate extracellular dot-like structures. The localization of endogenous

a-syn is shown by the green fluorescence of Alexa Fluor 488. The framed

areas (left panels) are magnified in the right panels. Scale bars indicate 20 mm

in the left and 5 mm in the right panels.

Fig. 5. Immunoreactivity of 4D6 and pSyn#64 antibodies to a-syn-

knockdown SK-MEL28 cells. (A) Expression levels of a-syn in SK-MEL28

cells expressing shRNA of a-syn. Total cell lysates were prepared from cells

expressing control shRNA (first lane) and cells expressing a-syn shRNA (no.

1–no. 5). These lysates were analyzed by western blotting using anti-a-syn

antibody 4D6 (upper panel) and anti-actin antibody (lower panel). Molecular

size markers are shown in kilodaltons. (B) Immunostaining of a-syn-

knockdown SK-MEL28 cells. Cells expressing control shRNA (left panels)

and those expressing a-syn shRNA (right panels) were immunostained with

anti-a-syn antibodies 4D6 (upper panels) and pSyn#64 (lower panels). The

localization of a-syn is shown by the green fluorescence. Nuclei are

counterstained blue by DAPI. Scale bars indicate 50 mm.

Release of a-synuclein from melanoma 699

Journ

alof

Cell

Scie

nce

determine whether this is observed in other human melanoma cell

lines, we tested the SK-MEL5, A375, MeWo and WM266-4

melanoma cell lines. First, we examined the expression levels of

endogenous a-syn using different antibodies, including LB509

(for total a-syn), 4D6 (for S129-unphosphorylated a-syn),

pSyn#64 and EP1536Y (for S129-phosphorylated a-syn). As

shown in Fig. 8A, SK-MEL5, MeWo and WM266-4 cells, as

well as SK-MEL28 cells, expressed both S129-unphosphorylated

and phosphorylated forms. Notably, SK-MEL5 cells expressed

them at much higher levels. However, the expression levels were

extremely low in A375 cells as we described previously (Lee and

Kamitani, 2011).

Next, we immunostained HT1080 (as negative control), SK-

MEL5, MeWo and WM266-4 cells using anti-a-syn antibodies 4D6

and pSyn#64. A375 cells were not used because S129-

phosphorylated a-syn was almost undetectable by western blotting.

As shown in Fig. 8B, S129-unphosphorylated a-syn localized

to the cytoplasm as small dots, but its nuclear localization was

limited in three melanoma cell lines (top panels). In contrast,

S129-phosphorylated a-syn localized to the nucleus and

cytoplasm in these cell lines (middle and bottom panels). It

should be noted that it also localized to the cell surface in SK-

MEL5 cells, but not in MeWo and WM266-4 cells (see the

magnified images at the bottom). In supplementary material Fig.

S1, we showed additional SK-MEL5 cells immunostained with

4D6 or pSyn#64 to support these findings. Thus, we found that

S129-phosphorylation plays a role in localization of a-syn to the

cell surface in two (SK-MEL5 and SK-MEL28) out of five

human melanoma cell lines tested in this study.

Moreover, we showed that the dot-like structures with S129-

phosphorylated a-syn existed around SK-MEL5 cells (data not

shown), suggesting that the phosphorylation of a-syn at S129 isinvolved in its cell surface localization and subsequent release inSK-MEL5 as well as SK-MEL28 cells (also see Fig. 4).

Translocation of a-syn-positive structures along themicrotubule network

Previously, tubulin was revealed to be an a-syn-binding protein(Alim et al., 2004). In neurons, a-syn was shown to be transportedby both kinesin and dynein motor proteins along microtubules

(Utton et al., 2005). These observations suggested that a transportsystem along microtubules is involved in translocation of a-syn inmelanoma cells. To test this possibility, we compared thedistributions of dot-like structures of a-syn and the microtubule

network in melanoma cells. Specifically, we double-immunostainedendogenous a-syn (unphosphorylated or phosphorylated at S129)and endogenous a-tubulin of the microtubule network in flat

extended SK-MEL5 cells to determine their distributions byfluorescence microscopy. As shown in the upper panels of Fig. 9,a-tubulin formed a fine network structure of microtubules, while

S129-unphosphorylated a-syn was observed as dot-like structures.When the images were merged, the microtubule network wasabundantly decorated with dot-like structures of a-syn (Fig. 9A).

Importantly, the magnified image clearly showed that the dot-likestructures are located along the microtubules (Fig. 9B). In contrast,S129-phosphorylated a-syn localized to the nucleus and cell surface

Fig. 6. Electron microscopy for ultrastructure of SK-MEL28 cells. Cells

were postfixed in osmium tetroxide, stained en bloc with uranyl acetate, and

embedded in epon-araldite resin. Ultrathin sections were cut and sequentially

stained with uranyl acetate and lead citrate. The sections were observed by a

transmission electron microscope. The framed areas in A are magnified

in B and C. The framed area in D is magnified in (E). Scale bars indicate

2 mm in A and D and 400 nm in B, C, and E. Arrows in B and E indicate MVs

on the cell surface. An arrowhead in C indicates the vesicular ‘budding’ or

‘fusion’ on the cell surface.

Fig. 7. Ultrastructural localization of S129-phosphorylated, endogenous

a-syn in SK-MEL28 cells. Immuno-colloidal gold electron microscopy was

performed using an antibody to S129-phosphorylated a-syn (pSyn#64). Thin

sections of SK-MEL28 cells were sequentially incubated with pSyn#64 and

secondary antibody conjugated with colloidal gold particles (15 nm in

diameter). After staining with uranyl acetate, the sections were observed by a

transmission electron microscope. (A) Vertical section showing a-syn clusters

or oligomers in the cytoplasm. (B–D) Sections showing MVs containing a-

syn. Scale bars indicate 200 nm. Arrowheads indicate microvilli on the cell

surface. Arrows indicate the localization of a-syn labeled with colloidal

gold particles.

Journal of Cell Science 126 (2)700

Journ

alof

Cell

Scie

nce

predominantly (Fig. 9C), as described above. The magnified image

showed that the dot-like structures were located along the

microtubules (Fig. 9D). These results suggest that dot-like

structures with unphosphorylated a-syn locate on the microtubule

network and, upon phosphorylation at S129, they are transported to

the nucleus and cell surface.

Discussiona-Syn has at least three experimentally proven phosphorylation

sites (Chau et al., 2009), of which the S129 residue is thought to

be the major site (Wang et al., 2012). The phosphorylation of

S129 plays an important role in the pathobiology of

neurodegenerative a-synucleinopathy, because it is selectively

and extensively phosphorylated in a-synucleinopathy lesions

such as the Lewy bodies and Lewy neurites found in the PD brain

(Fujiwara et al., 2002; Anderson et al., 2006). However, little is

currently known of the physiological (not pathological) relevance

of this phosphorylation, partly due to the technological

difficulties of the required experiments. Specifically, to clearly

determine the physiological role of S129 phosphorylation, we

have to examine S129-unphosphorylated a-syn as well as S129-

phosphorylated a-syn. For this purpose, two different antibodies

to a-syn are needed: one antibody for a-syn unphosphorylated at

S129 and another antibody for a-syn phosphorylated at S129.

However, although the latter antibody is commercially available,

no antibody for S129-unphosphorylated a-syn had previously

been identified, making it difficult to study the physiological role

of S129 phosphorylation.

In our present study, we identified 4D6 as an antibody to S129-

unphosphorylated a-syn. Importantly, 4D6 recognizes the same

C-terminal region as pSyn#64 does. Unlike 4D6, however,

pSyn#64 is an antibody to a-syn phosphorylated at S129 and has

commonly been used for immunostaining to determine the

localization of S129-phosphorylated a-syn (Fujiwara et al., 2002;

Tanji et al., 2006; Tanji et al., 2011). Therefore, we expected that

immunostaining using both 4D6 and pSyn#64 antibodies would

allow us to more clearly and exactly uncover the role of S129

phosphorylation in the subcellular localization of a-syn. Using

these antibodies, we immunostained human melanoma SK-

MEL28 and SK-MEL5 cells that highly express endogenous a-

syn (Matsuo and Kamitani, 2010; Hansen et al., 2011; Lee and

Fig. 8. Localization of S129-unphosphorylated and phosphorylated forms

of endogenous a-syn in various human melanoma cell lines.

(A) Expression levels of S129-unphosphorylated and phosphorylated a-syn.

Total cell lysates were prepared from human melanoma cell lines, SK-

MEL28, SK-MEL5, A375, MeWo and WM266-4, and human lung

fibrosarcoma cell line HT1080 (negative control). The lysates were analyzed

by western blotting using anti-a-syn antibodies LB509 (for total a-syn), 4D6

(for S129-unphosphorylated form), pSyn#64 (for S129-phosphorylated form)

and EP1536Y (for S129-phosphorylated form). In addition, expression levels

of NUB1 and actin were examined. Molecular size markers are shown in

kilodaltons. (B) Double immunostaining of HT1080 and three melanoma cell

lines with anti-a-syn antibody (4D6 or pSyn#64) and anti-NUB1 antibody (for

counterstaining). The localization of endogenous a-syn is shown by the green

fluorescence. The localization of endogenous NUB1 is shown by the red

fluorescence. Nuclear counterstaining is shown by the blue fluorescence of

DAPI. These three-color images are merged in all panels. The framed areas

(middle panels) are magnified in the bottom panels. Scale bars indicate 20 mm

in the top and middle panels and 5 mm in the bottom panels.

Fig. 9. Double immunostaining of microtubules and a-syn in human

melanoma SK-MEL5 cells. Cells were fixed in cold methanol,

permeabilized with 0.1% Triton X-100, and immunostained with both rat

monoclonal anti-a-tubulin antibody and mouse monoclonal anti-a-syn

antibody (4D6 or pSyn#64). After washing, the cells were labeled with both

Alexa Fluor 488-conjugated anti-rat IgG antibody and Alexa Fluor 555-

conjugated anti-mouse IgG antibody. The cells were then stained with DAPI

and analyzed by fluorescence microscopy. The localization of microtubules is

shown by the green fluorescence of Alexa Fluor 488, and the localization of

endogenous a-syn is shown by the red fluorescence of Alexa Fluor 555.

Nuclear counterstaining is shown by the blue fluorescence of DAPI.

(A,B) double immunostaining with 4D6 and anti-a-tubulin antibody.

(C,D) double immunostaining with pSyn#64 and anti-a-tubulin antibody.

Panels A and C are magnified in panels B and D, respectively. Scale bars

indicate 20 nm (A,C) and 5 nm (B,D).

Release of a-synuclein from melanoma 701

Journ

alof

Cell

Scie

nce

Kamitani, 2011). The immunostaining showed that S129-

phosphorylated a-syn localizes to the cell surface as small dots,but S129-unphosphorylated a-syn does not. Importantly, we alsodetected the released dot-like structures containing S129-

phosphorylated a-syn by using immunofluorescence microscopy.Furthermore, immuno-electron microscopy revealed that SK-MEL28 melanoma cells release MVs in which S129-phosphorylated a-syn localizes to the vesicular membrane. These

results suggest that the S129 phosphorylation plays a role in thecell surface localization and subsequent vesicular release of a-synin some of the human melanoma cells.

a-Syn release has intensively been studied using neuronal, butnot melanoma, cells. These studies revealed that a-syn is releasedfrom neuronal cells in a constitutive manner (Lee et al., 2005; Leeet al., 2008). This is supported by a previous clinical finding that a-

syn is present in human cerebrospinal fluid at low nanomolarconcentrations (Borghi et al., 2000; El-Agnaf et al., 2003). Sinceboth neuronal cells and melanocytes developmentally originate

from the neural crest and share several features, the a-syn releasemay be a common feature in both cell types.

How is a-syn release regulated in melanoma cells? As describedabove, our immunofluorescence microscopy revealed that the dot-

like structures are immunostained by pSyn#64 antibody, but not by4D6 antibody, at the cell surface and outside of melanoma cells.These results suggest that S129 phosphorylation regulates the a-

syn release in melanoma cells. Indeed, McFarland et al.demonstrated that S129 phosphorylation enables a-syn tointeract with vesicular trafficking proteins of mouse brain

(McFarland et al., 2008). Most recently, Wang et al. detected theselective elevation of S129-phosphorylated a-syn in thecerebrospinal fluid of PD patients (Wang et al., 2012). On the

basis of these observations and our results, S129 phosphorylationmight regulate the release of a-syn through the vesiculartrafficking proteins in melanoma cells as well as neuronal cells.Importantly, our double immunostaining of a-syn and a-tubulin

suggested that, upon phosphorylation at S129, a-syn is traffickedalong the microtubule network.

What is the biological and clinical relevance of the a-syn release

in melanoma? Although not known for melanoma, this has beenwell investigated in neuronal cells, and an interesting hypothesishas been proposed for a-syn release in the pathogenesis of PD. Inthis hypothesis, molecules of a-syn with an abnormal structure are

released from one neuron and taken up by another, leading toneurodegeneration by prion-like mechanisms (Frost and Diamond,2010; Olanow and McNaught, 2011; Steiner et al., 2011). In

melanoma, epidemiological studies have revealed co-occurrenceof melanoma with PD (Olsen et al., 2005; Olsen et al., 2006; Gaoet al., 2009). Specifically, melanoma patients have an increased

risk of subsequently developing PD (Olsen et al., 2006). Therefore,it is possible that a-syn release from melanoma cells plays a role inthe pathogenesis or progression of PD, if it is propagated into

neuronal cells. Indeed, this possibility is partially supported by arecent observation that a-syn is propagated from human melanomaSK-MEL5 cells to mouse neuroblastoma N2a cells in co-cultureconditions (Hansen et al., 2011).

In conclusion, we have reported that the S129-phosphorylatedform, but not the S129-unphosphorylated form, of a-syn localizesto dot-like structures at the cell surface and the extracellular

space of melanoma cells. Our studies suggest that thephosphorylation of S129 leads to the cell surface translocationof a-syn along the microtubule network and subsequent vesicular

release in melanoma cells. Further studies will be required to

determine the clinical relevance of this finding.

Materials and MethodsCell lines and culture conditions

The following human cell lines were purchased from American Type CultureCollection (Manassas, VA): SH-SY5Y (human neuroblastoma), SK-MEL28(human malignant melanoma), SK-MEL5 (human malignant melanoma), A375(human malignant melanoma), MeWo (human malignant melanoma), WM266-4(human malignant melanoma), and HT1080 (human lung fibrosarcoma). All cellswere maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% fetal calf serum (FCS) and antibiotics.

Antibodies

Rat monoclonal anti-a-tubulin (YL1/2) antibody and mouse monoclonal anti-a-synantibodies LB509 (specific for amino acid residues 115–122) and 4D6 werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonalanti-phospho a-syn antibody pSyn#64 (specific for amino acid residues 124–134)was purchased from Wako Chemicals USA (Richmond, VA). Rabbit monoclonalanti-phospho a-syn antibody EP1536Y (specific for amino acid residues 120–140)was purchased from Epitomics (Burlingame, CA). Mouse monoclonal anti-HAantibody 16B12 was purchased from Covance (Richmond, CA). Rabbit polyclonalanti-NUB1 antibody was generated by immunization with a GST fusion protein ofNUB1, corresponding to amino acid residues 432–601 (Kito et al., 2001).

Yeast transformation and cell lysate preparation

For epitope mapping of mouse monoclonal anti-a-syn antibody 4D6, severaltruncated fragments of a-syn were expressed in yeast cells and detected by westernblotting using 4D6. In brief, the cDNAs of truncated a-syn were amplified bypolymerase chain reaction (PCR) from pcDNA3/a-syn (see Two-dimensional gelelectrophoresis below) using appropriate primers. These cDNAs were inserted intothe multi-cloning site of yeast expression vector pGADT7 (Clontech, MountainView, CA). Since the multi-cloning site was located downstream of the cDNAencoding Gal4-activating domain (AD) and HA tag, the plasmids allow us toexpress AD-HA-fused a-syn fragments in yeast cells. For the expression, AH109yeast cells were transformed with the plasmids using the lithium acetate method(Okura et al., 1996). Transformed cells were grown on a Leu2 synthetic agar platefor 3 days at 30 C. The yeast cells were then grown in a Leu2 synthetic medium.Afterwards, using the boiling-SDS-glass bead method (Matsuo et al., 2004), thetotal cell lysate was prepared for western blotting.

Purification of recombinant human a-syn from bacterial culture

The cDNA of wild-type a-syn was subcloned into pT7-7 expression plasmid vector(USB, Cleveland, OH) for inducible expression in Escherichia coli. Rosetta (DE3)expression host cells (Novagen, Madison, WI) were then transformed with theplasmid and grown in 1l of LB medium containing ampicillin and chloramphenicolat 37 C with shaking. When the optical density of the culture medium reached 0.6at 600 nm, a-syn was induced by adding 1 mM isopropyl thiogalactoside,followed by an additional culture for 3 h. Cells were harvested by centrifugationand the pellet was subjected to purification essentially as described previously(Volles and Lansbury, 2007). Briefly, cells were lysed by boiling in the buffer[50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 150 mM NaCl]. Nucleic acids wereremoved by precipitation with streptomycin sulfate and acetic acid. a-Syn wassubsequently purified by ammonium sulfate precipitation. The pellet wasresuspended in ammonium acetate, followed by ethanol precipitation threetimes. The final pellet was resuspended in ammonium acetate, aliquoted intoappropriate amounts, subjected to lyophilization, and stored until use for ‘‘in vitro

phosphorylation assay’’.

In vitro phosphorylation of a-syn

Recombinant human a-syn (3.5 mg) was incubated in 10 ml of in vitro

phosphorylation buffer [20 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2,5 mM ATP] (Fujiwara et al., 2002) with or without 500 units of purified caseinkinase-2 (CK2; New England Biolabs, Ipswich, MA) for 18 h at 30 C. After thekinase reaction, protein samples were heated for 1 h in 2% SDS treating solutionand analyzed by western blotting.

Western blot analysis

Protein samples were heated for 30 min in a sample-treating solution containing2% SDS and 5% b-mercaptoethanol. After SDS-PAGE, proteins were transferredto a polyvinylidene difluoride (PVDF) membrane, Immobilon P (Millipore,Bedford, MA). Western blotting was then performed as described previously(Matsuo and Kamitani, 2010; Lee and Kamitani, 2011). Horseradish peroxidase(HRP)-conjugated antibodies against mouse IgG or rabbit IgG (Santa CruzBiotechnology) were used as secondary antibodies.

Journal of Cell Science 126 (2)702

Journ

alof

Cell

Scie

nce

Two-dimensional gel electrophoresis

The cDNA of human a-syn (wild-type) was amplified using PCR with appropriateprimers from a human brain cDNA library (Invitrogen, Carlsbad, CA). The cDNAsequence was confirmed by automated DNA sequencing. The cDNA was insertedinto pcDNA3 (Invitrogen) to generate the plasmid pcDNA3/a-syn. The plasmidwas then transfected into SH-SY5Y cells using Lipofectamine 2000 (Invitrogen) toexpress the wild-type a-syn without a tag. Twenty hours after transfection, thecells were collected by centrifugation, lysed in 200 ml of lysis buffer [20 mM Tris-HCl (pH 7.5), 400 mM NaCl, 0.5% NP-40, 10 mM NaF, 1 mM Na3VO4, 1 mMEDTA, protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)] andcentrifuged at 18,000 g for 10 min at 4 C. The supernatant was collected anddesalted by Sephadex G-25 spin column equilibrated with gel filtration buffer[5 mM Tris-HCl (pH 7.5), 5 mM NaF, 1 mM Na3VO4, 1 mM EDTA, proteaseinhibitor cocktail]. Using the desalted supernatant, two-dimensional gelelectrophoresis was performed according to the method reported by Alberio et al.(Alberio et al., 2010), with minor modifications. Briefly, the supernatant (50 ml)was diluted to 320 ml with a sample buffer [8 M urea, 2% CHAPS, 0.5% IPGbuffer 3.5–5.0 (GE Healthcare Biosciences), 30 mM dithiothreitol, and traces ofbromophenol blue], and loaded on an 18 cm IPG Dry Strip (GE HealthcareBiosciences) with a 3.0–5.6 pH gradient for in-gel rehydration (13 h at 20 V).Isoelectrofocussing was performed at 20 C on Ettan IPGphor3 (GE HealthcareBiosciences) with the schedule: 1 h at 200 V, 1 h linear gradient to 500 V, 2 hlinear gradient to 1000 V, 3 h linear gradient to 8000 V, and 3 h at 8000 V. TheIPG strip was then equilibrated for 15 min in the SDS treating buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, 1% dithiothreitol, and traces ofbromophenol blue]. The IPG strip was placed on a 15%, 1.0 mm-thick separatingpolyacrylamide gel. SDS-PAGE as a second dimension was carried out at 15 mA(current constant) using Mini-PROTEAN Tetra system (Bio-Rad, Hercules, CA).

Double immunostaining for endogenous a-syn and NUB1

Human cell lines were cultured on a coverslip in a 3.5-cm dish. After 24 h, thecells were fixed in a 4% paraformaldehyde solution for 20 min, stained with0.1 mg/ml of 49,6-diamidine-2-phenylindole dihydrochloride (DAPI; RocheDiagnostics, Indianapolis, IN) for 10 min, and permeabilized with 0.1% TritonX-100 for 10 min at room temperature. The cells were first labeled with rabbitanti-NUB1 antibody (dilution 1:5000) and mouse monoclonal anti-a-syn antibody4D6 (IgG1 isotype; dilution 1:2000) or mouse monoclonal anti-phospho a-synantibody pSyn#64 (IgG1 isotype; dilution 1:3000) overnight at 4 C. After washing,the cells were labeled with Alexa Fluor 594-conjugated anti-rabbit IgG (dilution1:1000) for NUB1 and Alexa Fluor 488-conjugated anti-mouse IgG (dilution1:1000) for a-syn or phospho a-syn for 1 h at room temperature. These secondaryantibodies were purchased from Molecular Probes (Eugene, OR). Finally, the cellswere analyzed by an Axio Imager M1 microscope (Zeiss, Thornwood, NY). Thelocalization of a-syn or phospho a-syn was shown by the green fluorescence ofAlexa Fluor 488. The localization of NUB1 was shown by the red fluorescence ofAlexa Fluor 594. Their co-localization was examined by the merging of bothfluorescent images.

Double immunostaining for a-tubulin and a-syn

SK-MEL5 cells were cultured on a coverslip, fixed in methanol at 220 C,permeabilized in PBS containing 0.1% Triton X-100, blocked with 5% horseserum, and then immunostained with rat monoclonal anti-a-tubulin (YL1/2)antibody and mouse monoclonal anti-a-syn antibody (4D6 or pSyn#64) as wedescribed previously (Tanaka et al., 2010). The localization of endogenous a-tubulin was shown by the green fluorescence of Alexa Fluor 488. The localizationof endogenous a-syn was shown by the red fluorescence of Alexa Fluor 555.Nuclear counterstaining is shown by the blue fluorescence of DAPI. Theircolocalization was examined by the merging of fluorescent images.

Lentivirus-mediated RNAi

We stably knocked down endogenous a-syn by RNAi and established an a-syn-knockdown cell line. We used lentivirus-mediated RNAi, called MissionH shorthairpin RNA (shRNA) system (Sigma-Aldrich). Five sequence-verified shRNAlentiviral plasmids (pLKO.1-puro) for a-syn knockdown were purchased fromSigma-Aldrich. Lentiviruses were generated by triple transfection of HEK293Tcells with pLKO.1-puro plasmid (for shRNA), pMDG-2 plasmid (for envelope),and psPAX2 plasmid (for gag-pol) using FuGENE6 (Roche). After 24 h,lentivirus-containing medium was harvested. SK-MEL28 cells were theninfected using the lentivirus-containing medium and selected by puromycin.Finally, we chose one SK-MEL28 cell line (out of five cell lines) that was infectedwith the most efficient lentivirus vector harboring the shRNA sequence for a-synRNAi, based on western blotting using anti-a-syn antibody 4D6.

Electron microscopy

SK-MEL28 cells were fixed with 0.1 M sodium cacodylate (NaCac) buffer(pH 7.4) containing 2% glutaraldehyde for 3 min on a culture dish, scraped with acell scraper, and pelleted by centrifugation. After 1 h, the cells were postfixed in

0.1 M NaCac buffer (pH 7.4) containing 2% osmium tetroxide for 1 h, stained enbloc with 2% uranyl acetate, dehydrated with a graded ethanol series, andembedded in epon-araldite resin. Thin sections were cut with a diamond knife onan EM UC6 ultramicrotome (Leica Microsystems, Inc., Bannockburn, IL),collected on copper grids, and sequentially stained with 2% uranyl acetate for5 min and Reynolds lead citrate for 3 min. The sections were observed by a JEM1230 transmission electron microscope (JEOL USA Inc., Peabody, MA) at 110 kVand imaged with UltraScan 4000 CCD Camera and First Light Digital CameraController (Gatan Inc., Pleasanton, CA).

Immuno-electron microscopy

SK-MEL28 cells were fixed on a culture dish for 3 min in 0.1 M NaCac buffer(pH 7.4) containing 4% paraformaldehyde and 0.2% glutaraldehyde, scraped witha cell scraper, and pelleted by centrifugation. After 1 h, the cells were dehydratedwith a graded ethanol series and embedded in LR White resin (ElectronMicroscopy Sciences, Hatfield, PA). Thin sections were cut with a diamond knifeon an EM UC6 ultramicrotome and collected on nickel grids. Sections wereincubated in the blocking buffer [50 mM Tris-HCl (pH 7.4), 118 mM NaCl,10 mM NaF, 5% BSA, 3% normal donkey serum, 0.05% Tween-20] at roomtemperature for 2 h, followed by incubation with mouse monoclonal anti-phosphoa-syn antibody pSyn#64 (dilution 1:75) overnight at 4 C. The sections werewashed and stained for 2 h at room temperature with donkey anti-mouse IgG(Abcam, Cambridge, MA) that was conjugated with colloidal gold particles(15 nm in diameter) as described previously (Slot and Geuze, 1985). Afterwashing, the sections were stained with 2% uranyl acetate alone for the cytoplasm(Fig. 7A) or with 2% uranyl acetate and 0.04% bismuth subnitrate sequentially forcell surface structures (Ainsworth et al., 1972) (Fig. 7B–D). The stained sectionswere then observed by a JEM 1230 transmission electron microscope as describedabove.

AcknowledgementsWe thank Mr Robert Smith (Georgia Regents University) forexcellent technical assistance.

FundingThis work was supported in part by National Institutes of HealthGrant [grant number R01AG024497] to T.K. Deposited in PMC forrelease after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122093/-/DC1

ReferencesAinsworth, S. K., Ito, S. and Karnovsky, M. J. (1972). Alkaline bismuth reagent for

high resolution ultrastructural demonstration of periodate-reactive sites. J. Histochem.

Cytochem. 20, 995-1005.

Alberio, T., Bossi, A. M., Milli, A., Parma, E., Gariboldi, M. B., Tosi, G., Lopiano,

L. and Fasano, M. (2010). Proteomic analysis of dopamine and a-synuclein interplayin a cellular model of Parkinson’s disease pathogenesis. FEBS J. 277, 4909-4919.

Alim, M. A., Ma, Q. L., Takeda, K., Aizawa, T., Matsubara, M., Nakamura, M.,

Asada, A., Saito, T., Kaji, H., Yoshii, M. et al. (2004). Demonstration of a role fora-synuclein as a functional microtubule-associated protein. J. Alzheimers Dis. 6, 435-442.

Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K.,

Caccavello, R. J., Barbour, R., Huang, J., Kling, K., Lee, M. et al. (2006).Phosphorylation of Ser-129 is the dominant pathological modification of a-synucleinin familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752.

Auluck, P. K., Caraveo, G. and Lindquist, S. (2010). a-Synuclein: membraneinteractions and toxicity in Parkinson’s disease. Annu. Rev. Cell Dev. Biol. 26, 211-233.

Bennett, M. C. (2005). The role of a-synuclein in neurodegenerative diseases.Pharmacol. Ther. 105, 311-331.

Borghi, R., Marchese, R., Negro, A., Marinelli, L., Forloni, G., Zaccheo, D.,Abbruzzese, G. and Tabaton, M. (2000). Full length a-synuclein is present incerebrospinal fluid from Parkinson’s disease and normal subjects. Neurosci. Lett. 287,65-67.

Chau, K. Y., Ching, H. L., Schapira, A. H. and Cooper, J. M. (2009). Relationshipbetween a synuclein phosphorylation, proteasomal inhibition and cell death:relevance to Parkinson’s disease pathogenesis. J. Neurochem. 110, 1005-1013.

El-Agnaf, O. M., Salem, S. A., Paleologou, K. E., Cooper, L. J., Fullwood, N. J.,Gibson, M. J., Curran, M. D., Court, J. A., Mann, D. M., Ikeda, S. et al. (2003). a-synuclein implicated in Parkinson’s disease is present in extracellular biologicalfluids, including human plasma. FASEB J. 17, 1945-1947.

Fournier, M., Vitte, J., Garrigue, J., Langui, D., Dullin, J. P., Saurini, F., Hanoun,

N., Perez-Diaz, F., Cornilleau, F., Joubert, C. et al. (2009). Parkin deficiencydelays motor decline and disease manifestation in a mouse model of synucleinopathy.PLoS ONE 4, e6629.

Release of a-synuclein from melanoma 703

Journ

alof

Cell

Scie

nce

Frost, B. and Diamond, M. I. (2010). Prion-like mechanisms in neurodegenerativediseases. Nat. Rev. Neurosci. 11, 155-159.

Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg,M. S., Shen, J., Takio, K. and Iwatsubo, T. (2002). a-Synuclein is phosphorylatedin synucleinopathy lesions. Nat. Cell Biol. 4, 160-164.

Gao, X., Simon, K. C., Han, J., Schwarzschild, M. A. and Ascherio, A. (2009).Family history of melanoma and Parkinson disease risk. Neurology 73, 1286-1291.

Golovko, M. Y., Faergeman, N. J., Cole, N. B., Castagnet, P. I., Nussbaum, R. L.and Murphy, E. J. (2005). a-synuclein gene deletion decreases brain palmitateuptake and alters the palmitate metabolism in the absence of a-synuclein palmitatebinding. Biochemistry 44, 8251-8259.

Hansen, C., Angot, E., Bergstrom, A. L., Steiner, J. A., Pieri, L., Paul, G., Outeiro,

T. F., Melki, R., Kallunki, P., Fog, K. et al. (2011). a-Synuclein propagates frommouse brain to grafted dopaminergic neurons and seeds aggregation in culturedhuman cells. J. Clin. Invest. 121, 715-725.

Jakes, R., Crowther, R. A., Lee, V. M., Trojanowski, J. Q., Iwatsubo, T. and

Goedert, M. (1999). Epitope mapping of LB509, a monoclonal antibody directedagainst human a-synuclein. Neurosci. Lett. 269, 13-16.

Kito, K., Yeh, E. T. H. and Kamitani, T. (2001). NUB1, a NEDD8-interacting protein,is induced by interferon and down-regulates the NEDD8 expression. J. Biol. Chem.

276, 20603-20609.Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek,

H., Epplen, J. T., Schols, L. and Riess, O. (1998). Ala30Pro mutation in the geneencoding a-synuclein in Parkinson’s disease. Nat. Genet. 18, 106-108.

Lee, B. R. and Kamitani, T. (2011). Improved immunodetection of endogenous a-synuclein. PLoS ONE 6, e23939.

Lee, H. J., Patel, S. and Lee, S. J. (2005). Intravesicular localization and exocytosis ofa-synuclein and its aggregates. J. Neurosci. 25, 6016-6024.

Lee, S. J., Jeon, H. and Kandror, K. V. (2008). a-synuclein is localized in asubpopulation of rat brain synaptic vesicles. Acta Neurobiol. Exp. (Warsz.) 68, 509-515.

Lou, H., Montoya, S. E., Alerte, T. N., Wang, J., Wu, J., Peng, X., Hong, C. S.,Friedrich, E. E., Mader, S. A., Pedersen, C. J. et al. (2010). Serine 129phosphorylation reduces the ability of a-synuclein to regulate tyrosine hydroxylaseand protein phosphatase 2A in vitro and in vivo. J. Biol. Chem. 285, 17648-17661.

Maroteaux, L., Campanelli, J. T. and Scheller, R. H. (1988). Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal. J. Neurosci.

8, 2804-2815.Matsuo, Y. and Kamitani, T. (2010). Parkinson’s disease-related protein, a-synuclein,

in malignant melanoma. PLoS ONE 5, e10481.Matsuo, Y., Tanaka, K., Nakagawa, T., Matsuda, H. and Kawamukai, M. (2004).

Genetic analysis of chs1+ and chs2+ encoding chitin synthases fromSchizosaccharomyces pombe. Biosci. Biotechnol. Biochem. 68, 1489-1499.

McFarland, M. A., Ellis, C. E., Markey, S. P. and Nussbaum, R. L. (2008).Proteomics analysis identifies phosphorylation-dependent a-synuclein protein inter-actions. Mol. Cell. Proteomics 7, 2123-2137.

Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C.-F., Chang, H.-M.and Yeh, E. T. H. (1996). Protection against Fas/APO-1- and tumor necrosis factor-mediated cell death by a novel protein, sentrin. J. Immunol. 157, 4277-4281.

Olanow, C. W. and McNaught, K. (2011). Parkinson’s disease, proteins, and prions:milestones. Mov. Disord. 26, 1056-1071.

Olsen, J. H., Friis, S., Frederiksen, K., McLaughlin, J. K., Mellemkjaer, L. andMøller, H. (2005). Atypical cancer pattern in patients with Parkinson’s disease. Br. J.

Cancer 92, 201-205.Olsen, J. H., Friis, S. and Frederiksen, K. (2006). Malignant melanoma and other

types of cancer preceding Parkinson disease. Epidemiology 17, 582-587.Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A.,

Pike, B., Root, H., Rubenstein, J., Boyer, R. et al. (1997). Mutation in the a-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045-2047.

Pronin, A. N., Morris, A. J., Surguchov, A. and Benovic, J. L. (2000). Synucleins area novel class of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275,26515-26522.

Qing, H., Wong, W., McGeer, E. G. and McGeer, P. L. (2009). Lrrk2 phosphorylatesa synuclein at serine 129: Parkinson disease implications. Biochem. Biophys. Res.

Commun. 387, 149-152.Rodriguez-Vicente, J., Vicente-Ortega, V. and Canteras-Jordana, (1996). Azelaic

acid was sensitizing effect in the chemotherapeutic treatment of several melanomacell lines. Pigment Cell Res. 9, 317-325.

Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J.,

Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R. et al. (2003). a-Synucleinlocus triplication causes Parkinson’s disease. Science 302, 841.

Slot, J. W. and Geuze, H. J. (1985). A new method of preparing gold probes formultiple-labeling cytochemistry. Eur. J. Cell Biol. 38, 87-93.

Steiner, J. A., Angot, E. and Brundin, P. (2011). A deadly spread: cellular mechanismsof a-synuclein transfer. Cell Death Differ. 18, 1425-1433.

Tanaka, M., Tanji, K., Niida, M. and Kamitani, T. (2010). Dynamic movements ofRo52 cytoplasmic bodies along microtubules. Histochem. Cell Biol. 133, 273-284.

Tanji, K., Tanaka, T., Mori, F., Kito, K., Takahashi, H., Wakabayashi, K. and

Kamitani, T. (2006). NUB1 suppresses the formation of Lewy body-like inclusionsby proteasomal degradation of synphilin-1. Am. J. Pathol. 169, 553-565.

Tanji, K., Mori, F., Kakita, A., Zhang, H., Kito, K., Kamitani, T., Takahashi,

H. and Wakabayashi, K. (2007). Immunohistochemical localization of NUB1, asynphilin-1-binding protein, in neurodegenerative disorders. Acta Neuropathol. 114,365-371.

Tanji, K., Mori, F., Kito, K., Kakita, A., Mimura, J., Itoh, K., Takahashi, H.,Kamitani, T. and Wakabayashi, K. (2011). Synphilin-1-binding protein NUB1 iscolocalized with nonfibrillar, proteinase K-resistant a-synuclein in presynapses inLewy body disease. J. Neuropathol. Exp. Neurol. 70, 879-889.

Utton, M. A., Noble, W. J., Hill, J. E., Anderton, B. H. and Hanger, D. P. (2005).Molecular motors implicated in the axonal transport of tau and a-synuclein.J. Cell Sci. 118, 4645-4654.

Volles, M. J. and Lansbury, P. T., Jr (2007). Relationships between the sequence of a-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity. J.

Mol. Biol. 366, 1510-1522.Wang, Y., Shi, M., Chung, K. A., Zabetian, C. P., Leverenz, J. B., Berg, D., Srulijes,

K., Trojanowski, J. Q., Lee, V. M., Siderowf, A. D. et al. (2012). Phosphorylated a-Synuclein in Parkinson’s Disease. Sci. Transl. Med. 4, 121.

Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lezcano, E., Ros, R., Ampuero, I.,

Vidal, L., Hoenicka, J., Rodriguez, O., Atares, B. et al. (2004). The new mutation,E46K, of a-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55,164-173.

Journal of Cell Science 126 (2)704