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The Journal of Neuroscience, July 15, 1999, 19(14):5782-5791
 -Synuclein Shares Physical and Functional Homology with
14-3-3 Proteins
Natalie
Ostrerova1,
Leonard
Petrucelli1,
Matthew
Farrer2,
Nitinkumar
Mehta2,
Peter
Choi1,
John
Hardy2, and
Benjamin
Wolozin1
1 Department of Pharmacology, Loyola University Medical
Center, Maywood, Illinois 60153, and 2 Department of
Pharmacology, Mayo Clinic, Jacksonville, Florida 32224
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ABSTRACT |
 -Synuclein has been implicated in the pathophysiology of many
neurodegenerative diseases, including Parkinson's disease (PD) and
Alzheimer's disease. Mutations in -synuclein cause some cases of
familial PD (Polymeropoulos et al., 1997 ; Kruger et al., 1998). In
addition, many neurodegenerative diseases show accumulation of
-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et
al., 1998). Here, we show that -synuclein shares physical and
functional homology with 14-3-3 proteins, which are a family of
ubiquitous cytoplasmic chaperones. Regions of -synuclein and 14-3-3 proteins share over 40% homology. In addition, -synuclein binds to
14-3-3 proteins, as well as some proteins known to associate with
14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of
-synuclein inhibits protein kinase C activity. The association of
-synuclein with BAD and inhibition of protein kinase C suggests that
increased expression of -synuclein could be harmful. Consistent with
this hypothesis, we observed that overexpression of wild-type -synuclein is toxic, and overexpression of -synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity
and binding profile of -synuclein suggests that it might act as a
protein chaperone and that accumulation of -synuclein could
contribute to cell death in neurodegenerative diseases.
Key words:
14-3-3 proteins; Alzheimer's disease; apoptosis; BAD; extracellular regulated kinase; Parkinson's disease; protein kinase C; synuclein
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INTRODUCTION |
-Synuclein is part of a family of
proteins consisting of -,  -, and  -synuclein (Jakes et al.,
1994; Clayton and George, 1998). The biochemical properties of
-synuclein resemble that of a protein chaperone, capable of binding
other proteins. It is a small 140 amino acid protein, which has an
11-mer repeat that recurs seven times (George et al., 1995). In
solution, -synuclein exists in a random open, rather than globular,
conformation, consistent with a role as a chaperone (Weinreb et al.,
1996). In vitro, the 11-mer repeat has been shown to promote
binding to synthetic acidic phospholipid vesicles (Davidson et al.,
1998). -Synuclein is expressed in a wide variety of autosomal cells,
as well as in neurons (Ueda et al., 1993). In neurons, -synuclein is
enriched in presynaptic terminals in which it is distributed between a soluble pool and a vesicle-bound pool of proteins. The only protein known to interact with (and )-synuclein is phospholipase D2, which is inhibited in vitro by (and )-synuclein with
a KI of 10 nM (Jenco et al.,
1998).
Recent research suggests that -synuclein contributes to the
pathophysiology of many neurodegenerative illnesses. In Parkinson's disease (PD), -synuclein accumulates in Lewy bodies. Dystrophic neurites in PD and amyotrophic lateral sclerosis show accumulations of
-synuclein, as do glia in multiple systems atrophy (Spillantini et
al., 1998; Takeda et al., 1998; Tu et al., 1998). -Synuclein also
accumulates in neuritic plaques in Alzheimer's disease. A number of
studies suggest that -synuclein can be toxic to some cells.
Incubation of -synuclein with the neuronal cells, SK-SY5Y, induces
apoptosis (El-Agnaf et al., 1998a). Molecular genetic studies
have identified two different point mutations in the -synuclein gene, A53T and A30P, that appear to cause familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). The association between
-synuclein and disease suggests that perturbations of -synuclein
biology can be harmful to cells. The current knowledge of the biology of -synuclein, however, is not sufficient to understand how
-synuclein might affect cell viability.
The 14-3-3 proteins constitute a family of protein chaperones that are
particularly abundant in the brain, like -synuclein. The 14-3-3 family of proteins consist of five different isoforms that share
extensive sequence homology, both among the different isoforms and
between similar isoforms in different species (Layfield et al., 1996;
Broadie et al., 1997). 14-3-3 proteins bind to ligands at sites
containing phospho-serine residues. Binding of 14-3-3 to phosphorylated
Raf-1 stabilizes it in an active conformation (Tzivion et al., 1998).
14-3-3 binds to a phosphorylated epitope of protein kinase C
(PKC) and stabilizes PKC in an inactive conformation that is
unable to translocate to the membrane (Meller et al., 1996;
Wheeler-Jones et al., 1996; Matto-Yelin et al., 1997). 14-3-3 also
binds to phosphorylated BAD, which appears to stabilize
maintenance of BAD in a cytoplasmic localization (Zha et al.,
1996).
We now report that -synuclein shares regions of homology with 14-3-3 proteins, binds to 14-3-3 proteins, binds to ligands of 14-3-3, and is
toxic when overexpressed.
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MATERIALS AND METHODS |
Materials. -Synuclein was cloned into
BamHI/NotI sites of pcDNA3. The FLAG sequence was
inserted by PCR into the 5' end of -synuclein cDNA after the
-synuclein ATG start codon and confirmed by DNA sequencing.
Antibodies used include: polyclonal anti--synuclein (SC1; 1:2000;
against human -synuclein; residues 116-131; sequence, MPVDPDNEAYEMPSEE), monoclonal anti--synuclein-1 (1:1000;
Transduction Laboratories, Lexington, KY), monoclonal anti-FLAG
(1:300; IBI/Kodak, New Haven, CT), polyclonal anti-BAD antibody
(1:1000; Transduction Laboratories), monoclonal anti-PKC-III and
polyclonal pan-PKC (1:1000; Upstate Biotechnology, Lake Placid, NY),
polyclonal 14-3-3, and monoclonal 14-3-3 (1:1000; Transduction Laboratories).
Cell culture. Cells were grown in high glucose DMEM plus
10% FBS supplemented with 500 µg/ml G418, as needed. G418 was used for selection. Transfections used lipofectamine (Life Technologies, Gaithersburg, MD) with 2 µg/ml DNA and 6 µl/ml lipofectamine
in Optimem.
Immunoblotting. Cells were harvested with SDS lysis solution
(2% SDS, 10 mM Tris, pH 7.4, 2 mM -glycerol
phosphate, and 1 µM AEBSF). Protein was determined using
the BCA assay (Pierce, Rockford, IL), and 30 µg/lane was run on 14%
polyacrylamide gels and transferred to nitrocellulose (200 mA, 6 hr). The nitrocellulose was then incubated 1 hr in 5% milk-PBS,
washed, incubated overnight in primary (1°) antibody, washed,
incubated 3 hr in peroxidase-coupled secondary antibody, and developed
with chemiluminescent reagent (DuPont, Billerica, MA).
Immunoprecipitations. Homogenates were extracted with 1%
Triton X-100 in PBS plus protease and phosphatase inhibitors. Some immunoprecipitations of -synuclein were also performed using an
extraction buffer containing 1% Triton X-100-0.2% SDS plus protease
and phosphatase inhibitors. The lysates were spun down (10,000 × g, 15 min), and the supernatants were precleared with protein A Sepharose (Pharmacia, Piscataway, NJ). Agarose-coupled M2
anti-FLAG antibody (25 µl) or 1 µl of antibody was then
added and incubated at 4°C overnight. For the precipitations with
polyclonal SC1, pan-PKC, or 14-3-3 antibodies, the immunocomplexes were
precipitated by incubation with protein A Sepharose at 4°C for 2 hr.
After binding of the solid phase substrate, the samples were washed five times in TBS-1% Triton X-100 and immunoblotted.
PKC assay and cell fractionation. For the PKC assay, cells
were incubated in serum-free conditions overnight, stimulated, washed,
and scraped into 500 µl of 4°C extraction buffer [20
mM Tris, pH 7.4, 2 mM EDTA, 5 mM
EGTA, 0.25 M sucrose, 5 mM -mercaptoethanol, 0.1% Triton X-100, and 20 µg/ml protease inhibitors cocktail (Sigma, St. Louis, MO)]. The samples were then ultrasonicated and centrifuged at 100,000 × g at 4°C for 1 hr. PKC activity in the
supernatant was then determined at 30°C for 10 min using 25 µg of
protein and PKC assay buffer [50 mM Tris-Hcl, pH 7.4, 250 µg/ml BSA, 1 mM EGTA, 10 µM PKC substrate
peptide (Ac-myelin basic protein4-14; Life Technologies),
100 µg/ml phosphatidylcholine/phosphatidylserine (80:20), 50 mM ATP, and 1 µCi -32P-ATP per tube]. For
cell fractionation after harvesting in the PKC assay buffer without
Triton X-100, the cell lysate was sonicated and centrifuged at
100,000 × g at 4°C for 1 hr.
Trypan blue staining. Transfections were performed as
described above using the pGL3 luciferase control plasmid, a luciferase assay kit (Promega), and triplicate points. For the trypan blue viability, assay cells were trypsinized, spun down, and taken up in
HBSS containing 0.4% trypan blue. The total number of cells and the
number of blue-stained cells were then counted using a hemocytometer.
Three different wells are used for each condition (triplicate plating),
and four different fields were counted in each well.
DNA fragmentation. Cells from 100 mm dishes were harvested,
and DNA was isolated by phenol chloroform extraction and analyzed on a
2% agarose gel using Syber Green for visualization.
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RESULTS |
-Synuclein binds to 14-3-3
To explore whether -synuclein might function as a protein
chaperone, we searched the genetic database for homology between -synuclein and other protein chaperones. Using the Multalin
algorithm, we observed regions of sequence homology between members of
the synuclein and 14-3-3 family of proteins (Fig.
1) (Corpet, 1988; Dubois et al., 1997).
This alignment program displays different levels of homology. Exact
homologies are shown as white letters on a black
background (Fig. 1), and homologies between amino acids with
similar chemical properties, such as Ile and Val, are shown in
black letters on a gray background
(Fig. 1). Two regions with 43 and 36% sequence homology were seen
between amino acids 8 and 61 of -synuclein and amino acids between
45 and 102 of 14-3-3 (Fig. 1A). This region contains
domains of 14-3-3 that are thought to be phosphorylated by PKC and
involved in dimerization of 14-3-3 (Dubois et al., 1997; Yaffe et al.,
1997). In contrast, the C terminus of 14-3-3, which has been implicated
in binding to phosphorylated Raf-1, shows no homology to -synuclein
(Ichimura et al., 1997).
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Figure 1.
A, Alignment of -synuclein and
the 14-3-3 family of proteins. The alignment was performed using the
Multalin program (http://www.toulouse.inra.fr/multalin.html). To
observe homology, we excluded the first 30 amino acids of 14-3-3 proteins while performing the alignment algorithm. Exact matches are
shown as white letters on a black
background, and matches of proteins with similar properties are
shown as black letters on a gray
background. In addition, we have noted aligned serines and
threonines in black because both amino acids can be
phosphorylated by serine/threonine kinases. B,
Association of -synuclein with 14-3-3. Rat brain tissue was
fractionated into cytoplasmic (C) and membrane
(M) components, taken up in
immunoprecipitation buffer, and treated as shown. The
left shows an immunoblot of a 14-3-3
immunoprecipitate with anti--synuclein antibody, and the
right shows an immunoblot of the lysates. The omit lane
is labeled Ø and refers to immunoprecipitations done
using protein A but no 1° antibody. C, The
left shows an immunoblot of -synuclein
immunoprecipitates with anti-14-3-3 antibody, and the
right shows an immunoblot of the lysates. The omit lane
is labeled Ø and refers to an immunoprecipitation done
using protein A but no 1° antibody.
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Because the homology between -synuclein and 14-3-3 covers a region
known to mediate dimerization of 14-3-3 proteins, we investigated whether -synuclein associates with 14-3-3. Rat brain homogenates were fractionated into membrane and cytoplasmic components, and 14-3-3 protein was immunoprecipitated. The resulting precipitates were immunoblotted using anti--synuclein antibody. As shown in Figure 1B, -synuclein and 14-3-3
coimmunoprecipitated. Comparison of the exposure times for the
immunoprecipitate and the lysate indicates that immunoprecipitation of
14-3-3 concentrated -synuclein ~40-fold because -synuclein
could be observed in the immunoprecipitates after an exposure of 0.5 sec but required a 20 sec exposure to be observed in the lysates (Fig.
1B). -Synuclein was most abundant in the 14-3-3
immunoprecipitates obtained from the cytoplasmic samples (Fig.
1B). The association between -synuclein and 14-3-3 could also be observed by immunoprecipitating -synuclein and probing
the resulting immunoblots with anti-14-3-3 antibody (Fig. 1C). We used an antibody to the 14-3-3 isoform because of
the availability of a monoclonal mouse antibody (which can be
distinguished from the polyclonal rabbit anti-synuclein antibody used
for the immunoprecipitation). These data show that -synuclein
associates with 14-3-3.
-Synuclein binds and inhibits PKC
To examine the significance of this association, we examined
whether -synuclein binds to other proteins that associate with 14-3-3 proteins. We first examined the binding to PKC. Rat brain tissue
was homogenized in PKC assay buffer and stimulated for 30 min with 1 µM phorbol 12,13-myristate acetate (PMA). The homogenate was then separated into membrane and cytoplasmic components, the PKC
was immunoprecipitated with pan-PKC antibody, and the precipitates were
probed with monoclonal anti--synuclein-1 antibody. The presence of
-synuclein in the PKC immunoprecipitates was readily apparent, and
immunoprecipitation of -synuclein concentrated the PKC approximately fivefold based on exposure times (4 sec exposure for the
immunoprecipitate and 20 sec exposure for the lysate) (Fig.
2). Association between -synuclein and
PKC was also evident by immunoprecipitating -synuclein and probing
with antibodies specific to PKC, , ,  , or (Fig. 2). The
pattern of association and amount of association with PKC was
isoform-specific. Based on the intensity of the immunoprecipitations, -synuclein appeared to give the following rank order of association with PKC isoforms: = > > = . Complexes of -synuclein with PKC were apparent in all
samples, whereas complexes with PKC, , or were present only
in the membrane fraction under basal or stimulated conditions, and
complexes with PKC were apparent in the membrane fractions only
after stimulation (data not shown).
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Figure 2.
Association of -synuclein with PKC
isoforms in rat brain tissue. Left panels show
immunoblots of -synuclein immunoprecipitates with isoform-specific
PKC antibodies. Right panels show immunoblots of each
PKC isoform in the membrane and cytoplasmic fractions. Before each
immunoprecipitation, half of the brain homogenates were treated with 1 µM PMA plus 1 mM Ca2+ at
30°C for 30 min. The homogenates were then fractionated into membrane
and cytoplasmic components taken up in immunoprecipitation buffer, and
proteins were isolated as shown. Cytoplasmic, C;
membrane, M; omit, Ø, which is an
immunoprecipitation done using protein A but no 1° antibody.
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The ability of -synuclein to associate with PKC suggests that it
might regulate PKC. To examine this issue, we generated lines of 293 human embryonic kidney (HEK) cells overexpressing wild-type or A53T
-synuclein, as well as a vector transfected control line (Fig.
3A). First, we examined
whether complexes of -synuclein and PKC could be immunoprecipitated.
Lysates were prepared from cells grown under basal conditions or
conditions known to stimulate PKC. We then immunoprecipitated the
-synuclein PKC complex by precipitating with anti--synuclein
antibody and probing with anti-PKC (Fig. 3B) or by
precipitating with anti pan-PKC antibody and probing with
anti--synuclein (Fig. 3C). As with the brain tissue, we
observed that -synuclein in these cell lines also binds PKC. The
association of -synuclein with PKC was most apparent after
membrane translocation (Fig. 3B). Next, we determined
whether overexpression of -synuclein affected PKC activity. Control,
wild-type, and A53T--synuclein 293 HEK cell lines were treated with
1 µM PMA for 30 min, and PKC activity in the lysates was
analyzed in vitro. The cell lines overexpressing wild-type
or A53T -synuclein did not show any increases in PKC activity after
PMA treatment, despite strong stimulation observed in the control cell
lines and despite robust translocation of PKC observed in each line
after PMA treatment (Fig. 3D,E).
Similar results were observed in different monoclonal lines of 293 HEK cells stably transfected with wild-type or A53T -synuclein (data not
shown). These data show that -synuclein binds to and inhibits PKC.
Most of the PKC isoforms show strong association with membrane-bound -synuclein (Figs. 2, 3B). This suggests that
membrane-bound -synuclein has a stronger affinity for PKC, which is
consistent with observations that membrane-bound -synuclein has a
more stable secondary structure (Davidson et al., 1998). Inhibition of
PKC by -synuclein, however, does not appear to affect membrane
translocation of PKC. 14-3-3 also binds to and inhibits PKC but does so
in the cytoplasm.
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Figure 3.
A, An immunoblot of 293 HEK cell
lines stably transfected with empty vector (Vec),
wild-type (WT), or A53T -synuclein using
-synuclein antibody SC1. Control 293 HEK cells do endogenously
express low levels of -synuclein (arrow), whereas the
transfected cells show increased expression of the 19 kDa -synuclein
proteins. B, Immunoblot of PKC in -synuclein
immunoprecipitates. 293 HEK cell lines stably transfected with empty
vector (Vec), wild-type (WT), or
A53T -synuclein were grown under basal conditions or treated with 20 nM bradykinin for 30 min, and the lysates were
immunoprecipitated using agarose-coupled anti-FLAG resin.
Immunoblotting of the resulting samples with anti-PKC type III antibody
showed coassociation of -synuclein with PKC only in the
bradykinin-treated samples (top panel). The
bottom panel shows an immunoblot of PKC in the
corresponding total cell lysates. C, Immunoblot of
-synuclein in PKC immunoprecipitates. Lane 1,
Lysates were treated with 1 µM PMA for 30 min, and PKC
was immunoprecipitated using anti-pan-PKC antibody (lane
2) (this antibody recognizes PKC, , and ;
Upstate Biotechnology) and immunoblotted with anti--synuclein SC1
antibody. Lane 2 shows an immunoprecipitation with the
anti-pan-PKC antibody omitted. Lane 3
(Lys) shows a parallel anti-synuclein immunoblot of the
lysate (30 µg of lysate). No reactivity was seen in absence of PMA
stimulation. D, PKC activity does not increase in 293 HEK cell lines overexpressing wild-type or A53T -synuclein after
stimulation with PMA (1 µM, 30 min).
*p < 0.001. E, Immunoblots of
PKC in fractionated cell lysates (cytoplasm to membrane) after
treatment with PMA (1 µM, 30 min). All cell lines
(vector, wild-type -synuclein, and A53T -synuclein) showed robust
translocation of PKC after PMA treatment. C,
Cytoplasm; M, membrane.
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-Synuclein binds to BAD and extracellular
regulated kinase
14-3-3 is also known to associate with BAD, which is a Bcl-2
homolog that regulates cell death (Yang et al., 1995). To examine whether -synuclein also associates with BAD, we
immunoprecipitated -synuclein from rat cortex (Fig.
4B). BAD was readily
detected after immunoprecipitation of -synuclein, and -synuclein
was readily detected after immunoprecipitation of BAD. The BAD was concentrated approximately ninefold in the -synuclein
immunoprecipitates (10 sec exposure for the immunoprecipitate vs 1.5 min exposure for the lysates) (Fig. 4A). We were also
able to show an association between BAD and -synuclein in cell
lines. Coimmunoprecipitation of BAD and -synuclein was apparent in
lysates from 293 HEK cells transfected with -synuclein (Fig.
4B). We also observed coimmunoprecipitation of BAD
and -synuclein in HeLa cells stably transfected with wild-type or
A53T -synuclein (Fig. 4C). In these immunoprecipitations, we investigated the phosphorylation state of the immunoprecipitated BAD
and found that the immunoprecipitated -synuclein was positive for
BAD but negative for phospho-BAD112 or
phospho-BAD136 (Fig. 4C). Finally, we examined
the regulation of -synuclein binding to BAD and found a pattern
opposite to that of PKC. Stimulation of 293 HEK cells with either
carbachol or bradykinin reduced -synuclein-BAD complex
formation (Fig. 4B). Similar results were observed in brain in which treatment of brain homogenates with 1 µM
PMA decreased the association of BAD with -synuclein (Fig.
4A). Thus, binding of BAD and PKC to -synuclein
are inversely correlated. Binding of 14-3-3 and -synuclein to BAD
are also inversely correlated, because 14-3-3 has been shown to bind
phospho-BAD, whereas -synuclein binds dephospho-BAD (Zha et al.,
1996).
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Figure 4.
A, Coimmunoprecipitation of
-synuclein and BAD from brain lysate. Homogenates from rat cortex
were fractionated into membrane and cytoplasmic components and taken up
in immunoprecipitation buffer. BAD protein was then immunoprecipitated,
and precipitates were immunoblotted with monoclonal anti--synuclein.
Cytoplasmic, C; membrane, M; omit,
Ø, which is an immunoprecipitation done using protein A
but no 1° antibody. B, Association of
-synuclein with BAD in 293 HEK cells and regulation by agents that
stimulate PKC. 293 HEK cells (which express endogenous -synuclein)
were treated with carbachol (1 mM, 30 min) or bradykinin
(20 nM, 30 min). -Synuclein was then immunoprecipitated
from total cellular lysates with anti-synuclein SC1 antibody, and the
resulting immunoblots were probed with anti-BAD antibody.
Ø represents an immunoprecipitation performed without
1° anti--synuclein antibody. Immunoblots of the lysates
showed that equal amounts of protein were loaded in each lane (data not
shown). C, Immunoblot of BAD (arrow,
top) or phospho-BAD136
(bottom, arrow points to absent band;
1:200; New England Biolabs, Beverly, MA) after immunoprecipitation of
FLAG-tagged -synuclein from HeLa cell lysates. -Synuclein was
immunoprecipitated with agarose-coupled anti-FLAG resin, and the
immunoblots were probed with anti-BAD antibody (top) or
anti-phospho-BAD136 antibody (bottom). No
specific staining for phospho-BAD136 was observed. The
bands in the phospho-BAD136 immunoblot were present in the
control cell line that was not transfected with FLAG-tagged
-synuclein, and therefore these bands represent nonspecific binding.
An immunoblot of the lysates showed equal expression of FLAG-tagged
wild-type and A53T -synuclein (data not shown).
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We also examined binding of -synuclein to Raf-1 and extracellular
regulated kinase (ERK), which are key kinases in the ERK cascade.
14-3-3 has been shown to bind to phosphorylated Raf-1 protein. First,
we immunoprecipitated -synuclein from rat brain lysate and blotted
with antibody to Raf-1. -Synuclein did not bind to Raf-1 protein (5 sec exposure for the immunoprecipitate and lysate) (Fig.
5). We investigated this issue further by
examining whether -synuclein might bind to another member of the ERK
cascade. We next blotted the -synuclein immunoprecipitate with
anti-ERK 1 2 antibodies and observed that strong anti-ERK reactivity
(Fig. 5). Based on exposure times, immunoprecipitation of -synuclein concentrated ERK 20-fold (0.5 sec was used for exposure of the immunoprecipitate, and 10 sec was used for exposure of the lysate) (Fig. 5). This suggests that -synuclein associates with ERK
and might also play a role in regulating the ERK cascade. Because 14-3-3 is known to both Raf-1 and phospho-BAD, the inability of -synuclein to bind Raf-1 or phospho-BAD suggests that the
association of -synuclein with its protein partners (PKC, BAD, and
ERK) can occur independently of the association of -synuclein with
14-3-3.
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Figure 5.
Association of -synuclein with
members of the ERK cascade. Left panels show immunoblots
of -synuclein immunoprecipitates with antibodies to either Raf-1 or
ERK. No association was seen with Raf-1, whereas strong binding was
seen with ERK. Right panels show immunoblots with the
Raf-1 and ERK antibodies in the membrane and cytoplasmic fractions.
Before each immunoprecipitation, half of the rat brain homogenates were
treated with 1 µM PMA plus 1 mM
Ca2+ at 30°C for 30 min. The homogenates were then
fractionated into membrane and cytoplasmic components and taken up in
immunoprecipitation buffer, and proteins were isolated as shown.
Cytoplasmic, C; membrane, M, and omit,
Ø, which is an immunoprecipitation done using protein A
but no 1° antibody.
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Overexpression of -synuclein is toxic
The correlation between mutations in -synuclein and familial PD
and the presence of -synuclein pathology in multiple
neurodegenerative diseases suggests that -synuclein could be harmful
to cells. Our observations that -synuclein binds to proteins that
are known to affect cell viability, such as BAD, suggests biochemical
mechanisms that might underlie the toxicity of -synuclein.
Determining whether -synuclein is indeed toxic is of fundamental
importance to neuropathology. Expression of -synuclein in cells has
not yet been shown to be toxic to cells. The only study performed to
date addressing this issue has shown that extracellular administration
of aggregates of -synuclein is toxic (El-Agnaf et al.,
1998a). The conditions used in this study differ from
physiological conditions because -synuclein is an intracellular
protein and not an extracellular protein. Because -synuclein
interacts with three proteins known to affect cell viability, BAD, PKC,
and ERK, we sought to address directly whether increases in
intracellular -synuclein were toxic.
We first examined whether the regulation of -synuclein levels was
responsive to cell stress. To examine the regulation of -synuclein,
we measured the amount of -synuclein that was endogenously expressed
in 293 HEK cells under basal conditions and after serum deprivation for
24 or 48 hr. We observed that serum deprivation increased expression of
-synuclein after 24 hr (Fig.
6A). The level of
-synuclein remained elevated at the 48 hr time point as well. Thus,
cell stress can increase -synuclein expression.
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Figure 6.
Toxicity of -synuclein. A,
-Synuclein expression increased during incubation of 293 HEK cells
in serum-free medium. The left shows an immunoblot of
-synuclein with SC1 antibody, and the right shows the
same immunoblot reprobed with anti-actin antibody (Sigma).
B, Transfection of 293 HEK cells with an antisense
-synuclein construct (AS; 2 µg) reduced the amount
of endogenous -synuclein expressed in the cells
(left). The control lane
(Ctrl) shows lysates from cells transfected with
an empty pcDNA3 plasmid under the same conditions at the same time. The
immunoblot was then reprobed with anti-actin antibody to show that
equal amounts of protein were loaded in each lane
(right). C, Transient transfection of 293 HEK (left) or SK-N-SH cells (middle) with
a pGL3 luciferase plasmid and vector, wild-type, A53T, or A30P
-synuclein constructs induces a dose-dependent decrease in
luciferase activity. In contrast, transfection with antisense
-synuclein increased luciferase expression (right).
For the antisense experiments in the right, cells were
transfected and then serum-deprived for 24 hr, after which luciferase
activity was measured. Cells transfected with antisense -synuclein
showed less toxicity than cells transfected with vector.
*p < 0.05; **p < 0.01;
n = 4 for each point. D, Similar
experiments showed a dose-dependent increase in toxicity as shown by
trypan blue staining after serum deprivation for 0, 24, or 48 hr.
Parallel experiments with a -galactosidase vector showed a 40%
transfection rate in 293 HEK cells. Based on 20% of the cells being
positive for trypan blue after transfection, we estimate that
transfection of 1 µg of A53T -synuclein induced ~50% cell death
in 293 HEK cells. +p < 0.05;
*p < 0.01; **p < 0.001. E, Effects of -synuclein on DNA fragmentation. No
fragmentation was seen under basal growth conditions in the control
cell line (Vec, lane 1), wild-type
-synuclein overexpresser (WT, lane 2),
or A53T -synuclein expresser (A53T, lane
3). After 24 hr incubation in serum-free medium,
oligomeric DNA fragmentation was strong in the control cell line
(lane 4), moderate in the wild-type -synuclein
cell line (lane 5), and absent in the A53T -synuclein
cell line (lane 6). A slight increase in highly
degraded DNA is apparent in lane 6 above the dye front,
suggesting increased necrotic DNA. F, Wild-type
-synuclein increases BAD toxicity, but mutant -synuclein (A53T
and A30P) does not increase BAD toxicity. 293 HEK cells were
cotransfected with the constitutively active 1 µg of pGL3 luciferase
plasmid with or without 100 ng of BAD, and with or without 500 ng of
-synuclein (wild-type, A53T, or A30P). A -galactosidase
vector was used as a ballast to maintain the DNA amount at 2 µg; this
vector does not affect luciferase activity. *p < 0.0001; n = 4; comparing samples with or without
BAD. The A53T and A30P transfections alone were also significantly
different from vector at p < 0.0001;
n = 4.
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To examine whether increased expression of -synuclein was harmful to
cells, we analyzed the expression of a constitutively active luciferase
reporter vector after transient transfection of -synuclein or
control constructs. Apoptosis or necrosis reduces the amount of
luciferase expressed because protein synthesis decreases during cell
death. 293 HEK cells or SK-N-SH cells, a human neuroblastoma cell line,
were transiently transfected with constructs containing vector,
wild-type, A53T, or A30P -synuclein, as well as with a pGL3 plasmid
that constitutively expresses luciferase. Transfection of wild-type,
A53T, and A30P -synuclein induced dose-dependent increases in
toxicity in both the 293 HEK cells and the SK-N-SH cells (Fig.
6C). The A53T -synuclein construct was more toxic than
the wild-type construct in both cell lines at each dose tested. The
A30P -synuclein was more toxic than wild type up to 500 ng of DNA,
after which the toxicity reached a plateau.
In separate experiments, we observed that transfection with antisense
-synuclein was cytoprotective (Fig.
6B,C). 293 HEK cells were
transfected with the constitutively active pGL3 luciferase vector and
either antisense -synuclein or empty pcDNA3. Immunoblots of the
cells with anti--synuclein antibody showed that the antisense construct reduced the expression of endogenous -synuclein (Fig. 6B). -Synuclein reactivity was not completely
eliminated because, in transient transfections, some of the cells do
not receive the antisense construct. The cells were then serum-deprived
for 24 hr, and luciferase activity was measured. Serum deprivation
caused a 25% decrease in luciferase activity in control cells that had been transfected with pGL3 and pcDNA3 (Fig. 6C, right
panel). In contrast, cells transfected with pGL3 and
antisense -synuclein showed a 26% increase in luciferase activity
after serum deprivation (Fig. 6C, right
panel). The ability of the antisense -synuclein construct to protect against toxicity suggests that -synuclein participates in cell death processes and that reducing -synuclein interferes with cell death processes.
We confirmed the link between -synuclein and toxicity using trypan
blue staining. 293 HEK cells were transiently transfected with empty
vector, wild-type, or A53T -synuclein and subsequently serum-starved
for 0, 24, or 48 hr. The percentage of dead cells, as determined using
trypan blue staining, was much greater in cells transfected with A53T
-synuclein than either the wild-type -synuclein or the vector
(Fig. 6D). Because the trypan blue assay is better at
detecting necrosis than apoptosis, the increased cell death induced by
A53T -synuclein suggests that A53T -synuclein induces more
necrosis than apoptosis.
To analyze the mechanism of cell death further, we examined DNA
fragmentation in the 293 HEK cell lines stably transfected with vector,
wild-type, or A53T -synuclein. 293 HEK cells were subjected to serum
withdrawal for 12, 18, 24, 36, or 48 hr to induce apoptosis, and the
DNA was analyzed by agarose gel electrophoresis. Maximal amount of DNA
fragmentation was observed at 24 hr. The same experiment was then
repeated using 293 HEK cells expressing vector, wild-type, or A53T
-synuclein. After 24 hr of serum deprivation, the fragmented DNA ran
as a ladder in the serum-deprived control cell line, which is
characteristic of apoptosis, but was less apparent and more smeared in
the cell line expressing wild-type -synuclein (Fig.
6E). In the A53T -synuclein cell line, the DNA
was highly degraded and appeared mainly as smear at the dye front (Fig.
6E). This suggests that overexpression of wild-type -synuclein allows some apoptosis, but expression of A53T
-synuclein potentiates necrosis.
Finally, as an initial step in examining whether interaction with BAD
contributes to the mechanism of cell death induced by overexpressed
wild-type or mutant -synuclein, we investigated whether
overexpression of -synuclein affects the toxicity of BAD. 293 HEK
cells were cotransfected with 1 µg of the constitutively active pGL3
luciferase vector, with 500 ng of vector, wild-type, A53T, or A30P
-synuclein, and 100 ng of BAD. The amount of luciferase reactivity
quantitates cell viability. The total amount of plasmid DNA used for
transfections was kept constant by use of a -galactosidase ballast
plasmid, which we have shown does not affect luciferase activity.
Transfection with wild-type -synuclein added to the toxicity of
BAD (Fig. 6F). Transfection with BAD alone produced 56% (p < 0.0001; n = 4)
toxicity, whereas cotransfection with BAD plus wild-type -synuclein
produced 65% (p < 0.0001; n = 4) toxicity. In contrast, cotransfection of mutant -synuclein (A53T or A30P) with BAD did not show any increase in toxicity over
transfection with BAD alone (Fig. 6F). Transfection
with BAD alone produced 56% toxicity, whereas transfection with BAD
plus A53T or A30P -synuclein produced only 49 and 51%
(p < 0.0001; n = 4) toxicity. The inability of mutant -synuclein constructs to add to toxicity when cotransfected with BAD suggests that A53T and A30P mutant -synucleins cause toxicity through a mechanism utilizing BAD, although further experiments will need to be done to prove this point.
| |
DISCUSSION |
Our data show that -synuclein binds to the protein chaperone
14-3-3 and shares a small region of homology with 14-3-3. By examining
proteins known to associate with 14-3-3, we were able to identify three
groups of proteins that also associate with -synuclein. The proteins
that bind both 14-3-3 and -synuclein include five different isoforms
of PKC, ERK, and BAD (Meller et al., 1996; Broadie et al., 1997). The
ability of -synuclein to concentrate ligands during
immunoprecipitation appears to follow a rank order association of
14-3-3 > ERK > BAD > PKC isoforms. Based on this rank
order, the association between -synuclein and 14-3-3, ERK, or BAD
might be stronger than that for PKC. However, the ability of
overexpressed -synuclein to inhibit PKC activity indicates that
there is a significant functional association between the two proteins
and suggests that the limited ability of -synuclein to concentrate
PKC during immunoprecipitations reflects technical rather than
functional issues.
-Synuclein shares a region of homology with 14-3-3 proteins, binds
to many of the same proteins as 14-3-3, and modifies the activity of
these proteins. Based on this, we propose that -synuclein functions
like 14-3-3 proteins and could be considered as part of a 14-3-3 superfamily. Interestingly, the same amino acids that are homologous
between -synuclein and 14-3-3 are also present in - and
-synuclein, which suggests that these two proteins might also be
part of this superfamily. In addition, - and -synuclein might
also bind 14-3-3, PKC, BAD, and ERK, like -synuclein. The 14-3-3 family of proteins are thought to be protein chaperones that bind to
kinases and stabilize their activity (Tzivion et al., 1998).
Examination of the mechanism of action of 14-3-3 suggests potential
models for how -synuclein might act. In the case of PKC, 14-3-3 binds to a phosphorylated epitope on PKC and holds the protein in an
inactive conformation that prevents it from translocating to the
membrane, despite the presence of diacylglycerol and calcium (Aitken et
al., 1995; Meller et al., 1996; Reurther and Pendergast, 1996;
Matto-Yelin et al., 1997). For Raf-1, this interaction is understood in
even more detail (Muslin et al., 1996; Yaffe et al., 1997). After
activation of Raf-1 by Ras, dimeric 14-3-3 binds to Raf-1 and
stabilizes it in a conformation that remains active even after Ras has
dissociated (Tzivion et al., 1998). Thus, 14-3-3 prolongs the duration
of Raf-1 activation. For both PKC and Raf-1, the essential function of
14-3-3 is to stabilize the protein in a particular conformation, either
active, as with Raf-1, or inactive, as with PKC. Based on the
functional and physical homology to 14-3-3, we hypothesize that
-synuclein has a similar chaperone function. Previous reports have
suggested that -synuclein is a chaperone because of its open,
unstructured character in solution (Weinreb et al., 1996). Our data now
provide biochemical support for this hypothesis.
14-3-3 might therefore be a useful model for studying -synuclein.
The specific binding patterns of 14-3-3 and -synuclein, however,
appear to differ. Raf-1, which is a protein known to bind to 14-3-3, does not bind to -synuclein (Tzivion et al., 1998). In addition,
whereas 14-3-3 binds to phosphorylated BAD, -synuclein binds to
dephospho-BAD (Zha et al., 1996). If -synuclein binds to proteins as
part of a complex with 14-3-3, we would expect -synuclein to
associate with all proteins that associate with 14-3-3, but this is not
what we observe. The lack of binding to Raf-1 and the binding to
dephospho-BAD suggest that -synuclein binds to proteins
independently of 14-3-3 rather than as part of a larger
14-3-3--synuclein complex. Raf-1 has been shown to bind to the C
terminus of 14-3-3, which is a region of the protein that shares no
homology with -synuclein (Tzivion et al., 1998). Thus, the inability
of -synuclein to bind to Raf-1 is consistent with the lack of
homology between -synuclein and the C-terminal domain of 14-3-3.
Based on protein binding patterns of -synuclein, we were also
able to identify functional consequences of increased expression of
-synuclein. Overexpression of -synuclein inhibits PKC activity. Interestingly, despite inhibiting PKC activity, -synuclein does not
inhibit PKC membrane translocation. This suggests that -synuclein acts by blocking the catalytic site of PKC but does not prevent the
conformational change associated with membrane translocation. Multiple
studies have documented the dissociation between PKC translocation and
PKC activation (Lu et al., 1994; Mochly-Rosen and Kauvlar, 1998). The
interaction of -synuclein with PKC provides a mechanism through
which the a dissociation of PKC translocation and activation could occur.
Another functional consequence of -synuclein expression that we
investigated was toxicity. The ability of -synuclein to inhibit PKC
and to bind to BAD suggested to us that overexpression of -synuclein
might be toxic. Consistent with this hypothesis, we observed that
-synuclein levels increase during conditions promoting cell stress,
overexpression of -synuclein induces toxicity, apparently by
increasing necrosis, and reduced expression of -synuclein protects
against toxicity. Because -synuclein appears to associate with
multiple proteins, the mechanism of -synuclein toxicity may be
multifactorial. Our preliminary data, however, suggest that the added
toxicity associated with overexpression of A53T and A30P -synuclein
is caused by the actions of BAD. Although the mutant
-synuclein constructs A53T and A30P are toxic when transfected
alone, neither mutant form of -synuclein induces significant
toxicity when cotransfected with BAD. The inability of mutant
-synuclein to increase toxicity in cells that have been
cotransfected with BAD is consistent with a hypothesis that the two
proteins (mutant -synuclein and BAD) use the same mechanism of
toxicity and that the additional toxicity seen with both mutant -synucleins is caused by activation of BAD. Proof of this point, however, awaits a demonstration that inhibiting BAD activity prevents the added toxicity associated with the mutant -synucleins.
Unlike mutant -synucleins, wild-type -synuclein causes similar
toxicity in control or BAD transfected cells. This suggests that
wild-type -synuclein does not activate BAD. The binding of wild-type
-synuclein to BAD contrasts with its apparent inability to affect
BAD toxicity. This discrepancy between binding and activity is also
seen with 14-3-3 protein, which binds to BAD but has not been shown to
affect BAD function (Zha et al., 1996). The inability to detect
regulation of BAD can occur because protein chaperones, such as 14-3-3, sometimes regulate the trafficking of their ligands rather than
directly affecting their activity. In such cases, measurements of
activity fail to detect regulation by the chaperone, despite a
clear interaction between the chaperone and its ligand (Zha et al.,
1996). Because of the physical and functional homology between
-synuclein and 14-3-3, we believe that the inability to detect
regulation of BAD by wild-type -synuclein might result from its
function as a chaperone.
The unstructured character of -synuclein may contribute to its
tendency to aggregate in neurodegenerative diseases. Several studies
have noted that -synuclein has a tendency to aggregate in solution
and that the A53T -synuclein, but not the A30P -synuclein, has an
increased tendency to aggregate in vitro (Conway et al., 1998; Hashimoto et al., 1998; Paik et al., 1998). The A30P
-synuclein mutant might have a reduced tendency to associate with
membranes, which could also increase its tendency to aggregate
(El-Agnaf et al., 1998b; Jensen et al., 1998). The propensity of
-synuclein to aggregate may underlie its tendency to accumulate as
focal accumulations in dystrophic neurites in neurodegenerative
diseases such as PD, Alzheimer's disease, multiple systems atrophy, or amyotrophic lateral sclerosis (Spillantini et al., 1998; Takeda et al.,
1998).
The significance of accumulations of -synuclein is unknown,
but our study suggests that -synuclein can be toxic. Our findings are supported by a recent study showing that -synuclein is toxic when incubated with cells (El-Agnaf et al., 1998a). The ability of -synuclein to cause toxicity suggests that accumulation of -synuclein might contribute to the synaptic loss and cell death that
underlies neurodegenerative diseases. The mechanism of toxicity is
unknown. In the case of other protein aggregates, such as A, the
aggregated protein induces production of free radicals by binding to
proteins that stimulate free radical production or by binding metals
and stimulating free radical production through Fenton reactions (Behl
et al., 1994; Yan et al., 1996; Atwood et al., 1998). Free radical
production is also thought to be important in PD because the neurons of
the substantia nigra contain dopamine and iron, both of which generate
free radicals. In addition, the neuroprotective transcription factor
NF- B has been shown to be activated in the substantia nigra in PD
(Hunot et al., 1997). In our studies we observed that -synuclein
binds to the neurotoxic protein BAD, and the cell death induced by A53T
and the A30P -synuclein in cell lines appears to involve BAD. The
linkage between -synuclein and BAD raises the possibility that BAD
contributes to neurodegeneration in familial PD. The putative
contribution of BAD to neurodegeneration in the CNS needs to be investigated.
| |
FOOTNOTES |
Received March 8, 1999; revised May 7, 1999; accepted May 7, 1999.
This research was supported in part by grants from the Neuroscience
Research and Education Fund and the National Parkinson Foundation. We
thank Michael Comb (New England Biolabs) for providing anti-phospho-BAD
antibodies and Yahong Zhang for her technical assistance.
N. Ostrerova and L. Petrucelli contributed equally to this work.
Correspondence should be addressed to Dr. Benjamin Wolozin, Department
of Pharmacology, Loyola University Medical Center, Building 102, Room
4644, 2160 South 1st Avenue, Maywood, IL 60154.
| |
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TOP
ABSTRACT
INTRODUCTION
