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-Synuclein Promotes Mitochondrial Deficit and Oxidative Stress
From the Departments of Pathology,*
Neurosciences,
and Reproductive
Medicine,
the University of California at San
Diego, La Jolla, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-synuclein has
recently been implicated in the pathogenesis of Alzheimer’s and
Parkinson’s diseases. Because neurodegeneration in these conditions
might be associated with mitochondrial dysfunction and oxidative
stress, the effects of -synuclein were investigated in a
hypothalamic neuronal cell line (GT1-7). -Synuclein overexpression
in these cells resulted in formation of -synuclein-immunopositive
inclusion-like structures and mitochondrial alterations accompanied by
increased levels of free radicals and decreased secretion of
gonadotropin-releasing hormone. These alterations were ameliorated by
pretreatment with anti-oxidants such as vitamin E. Taken together these
results suggest that abnormal accumulation of -synuclein could lead
to mitochondrial alterations that may result in oxidative stress
and, eventually, cell death.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Synuclein belongs to an expanding family of synuclein proteins
and is homologous to rat and torpedo -synuclein4
and the zebra finch synelfin.5
Other members of the
synuclein family include ß-synuclein6
and 
-synuclein,
or persyn7,8
and a recently identified protein,
synoretin.9
Recent studies have shown that mutations in
the -synuclein gene lead to rare familial forms of Parkinson’s
disease (PD), a disorder characterized by nigral degeneration and
formation of inclusions, denominated Lewy bodies (LBs). In these two
reports, Polymeropoulos et al10
identified a
mutation (A53T) in an Italian and Greek kindred which conferred
susceptibility to early-onset PD. Subsequent analysis by Kruger et
al11
revealed another missense mutation (A30P) found in a
German familial case of PD. Furthermore, in the more frequent forms of
sporadic PD and other forms of LB disease there is significant
-synuclein aggregation in LBs and neurites.12-17
The mechanisms through which
-synuclein might lead to neurodegeneration in LB disease are not yet
completely understood. Recent studies have shown that overexpression of
wild-type and mutant -synuclein leads to synaptic
degeneration18
and cell death19,20
in both
in vivo and in vitro model systems. The role of
abnormal expression of -synuclein in LB disease is currently being
explored. However, in vitro studies have shown that
increasing levels,21
mutations,22
and
oxidative stress23
conditions lead to -synuclein
aggregation. Furthermore, abnormal accumulation of -synuclein might
trigger mitochondrial dysfunction. Supporting this, there is ample
evidence for a central role for mitochondrial dysfunction in the
pathogenesis of PD.24-28
In this context, the main
objective of the present study was to determine whether overexpression
of -synuclein might dysregulate cell function via mitochondrial
alterations. For this purpose, a murine hypothalamic tumor cell line
(GT1-7)29
was transfected with -synuclein and analyzed
for pathological and physiological effects. We present evidence that
overexpression of -synuclein in this cell system leads to
mitochondrial abnormalities and corresponding changes in oxidative
stress markers. These studies support the contention that -synuclein
might play a role in the regulation of mitochondrial function and that
alterations in this pathway may be involved in the pathogenesis of PD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GT1-7 murine hypothalamic tumor cell line (kindly provided by Dr. Pam Mellon, Department of Neurosciences, University of California at San Diego) was selected because of its neuronal origin, expression of many neuronal markers, ability to form synapses in culture, and neurosecretory properties which may represent neurotransmitter-like activity.29 Cells were maintained at 37°C, 5% CO2 in Dulbecco’s modified Eagle’s medium (high glucose), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), and 1% v/v penicillin/streptomycin.
cDNA Subcloning
The murine -synuclein cDNA sequence was obtained as previously
described.30
Briefly, using this plasmid as a template,
the coding region was once again amplified except that a simple
polymerase chain reaction was used with the annealing temperature of
55°C instead of the touchdown program. The amplified product was run
out on a 0.8% agarose gel, purified via the phenol/chloroform
extraction technique, and ligated into a pCR3.1 T-vector (Invitrogen,
Carlsbad, CA). The clones, which contained either the sense or
antisense construct, were selected, verified by sequencing, and
amplified via the Qiagen Maxi-Prep kit (Qiagen, Valencia, CA). These
clones were referred to as #1 and #9B, respectively.
Transfection and Selection of Stable Transfectants
GT1-7 cells were seeded to ~50% confluence in 60-mm tissue culture dishes. The Superfect transfection kit (Qiagen) was used to transfect cells with plasmids #1, #9B, and pCR3.1 (vector alone). Briefly, plasmid DNA (7.5 µg) was diluted with 200 µl of serum-free OPTI-MEM (Life Technologies, Inc., Grand Island, NY) and 40 µl of the Superfect reagent was added to this mix. Cells were incubated for 3 hours at 37°C, washed in phosphate-buffered saline (PBS), and cultured for 12 hours in regular cell media.
The optimal concentration of G418 sulfate needed for complete cell death in GT1-7 untransfected cells was obtained after a 3-day treatment with various concentrations. G418 (300 µg/ml) was added to the regular media after transfection and replaced every 3 days. After 3 weeks, cloning rings were used to select colonies and cells were screened for expression via the ribonuclease protection assay (RPA), Western blot, and immunocytochemical analysis. After the initial screening, essentially four clones were selected: 1) sense-transfected (overexpressers), 2) antisense-transfected (underexpressers), 3) vector (VEC)-transfected, and 4) nontransfected wild-type controls.
Analysis of -Synuclein mRNA Levels by RPA
Briefly, as previously described, riboprobe templates were
amplified by polymerase chain reaction.30
For the murine
-synuclein template, primers (20 mers) started at nucleotide 145 and
nucleotide 490 of mouse -synuclein (GenBank Accession No. AF044672),
respectively. An actin riboprobe complementary to nucleotides 480 to
559 of mouse ß-actin was used (GenBank Accession No. M18194). RPAs
were carried out with [32P]-labeled antisense
riboprobes, and signals were quantitated with a PhosphorImager and
ImageQuant software, as previously described.31
Actin
signals were used to correct for variations in mRNA content and
loading.
Analysis of -Synuclein Protein Expression by Western Blot
Western blots were analyzed with the affinity-purified rabbit
polyclonal antibody directed against C-terminal -synuclein (amino
acids 131 to 140).32
Briefly, cell pellets were sonicated
and separated into cytosolic and particulate fractions33
and protein levels were determined by the method of Lowry et
al.34
Gels were blotted onto nitrocellulose paper and
incubated with antibodies against -synuclein, (1:1000)
followed by 125I-protein A. Blots were also
stripped and reprobed with a mouse monoclonal antibody against actin
(Chemicon, Temecula, CA) to correct for loading variability. The
specific19-kd signals corresponding to murine -synuclein were
quantified using the ImageQuant software.
Analysis of -Synuclein Immunolocalization by
Double-Immunocytochemistry and Laser Scanning Confocal Microscopy
(LSCM)
Briefly, as previously described35
cells were seeded
onto poly-L-lysine-coated glass coverslips, grown to 60%
confluence, fixed in 4% paraformaldehyde (30 minutes, -4°C),
and pretreated with 0.1%Triton X-100 in PBS (20 minutes,
room temperature). Cells were incubated overnight at 4°C with
antibodies against -synuclein (rabbit polyclonal) and
microtubule-associated protein 2 (mouse monoclonal) (Roche Molecular
Biochemicals, Indianapolis, IN). Cells were then incubated with the
biotinylated anti-rabbit secondary antibody (Vector Laboratories,
Burlingame, CA), followed by tyramide signal amplification-Direct
Red (Dupont-NEN, Boston, MA) and fluorescein
isothiocyanate-conjugated anti-mouse secondary antibody (Vector).
Wild-type and VEC-transfected GT1-7 cells were run in parallel as
negative controls. Coverslips were air-dried overnight, mounted
on slides with antifading media (Vectashield, Vector), and imaged with
the confocal microscope (MRC1024; BioRad).
Gonadotropin-Releasing Hormone (GnRH) Radioimmunoassay
To determine whether levels of -synuclein expression affected
the secretory activity of GT1-7 cells, levels of GnRH release were
investigated by radioimmunoassay, essentially as previously
described.36
Briefly, tubes were incubated overnight at
4°C with 200 µl of 0.1%Triton-PO4 buffer
(0.063 mol/L Na2HPO4, 0.013
mol/L ethylenediaminetetraacetic acid, 0.02%
NaN3, 0.1% Triton X-100, pH 7.4), 1% normal
rabbit serum, and 100 µl of primary SD-7 antibody, (1:30,000)
followed by 100 µl of 125I-GnRH (5000 to 6000
cpm; Dupont-NEN) was added to each tube. On the third day, 100 µl of
goat anti-rabbit globulin secondary antibody (1:25, Calbiochem, San
Diego, CA) in 50 mmol/L ethylenediaminetetraacetic acid buffer (in
0.1% Triton buffer) and 1 ml of 6% polyethylene glycol solution
was added per tube and again incubated overnight at 4°C. Tubes were
then spun down, inverted, dried overnight, and radioactivity was
counted in a counter. A duplicate number of wells and
triplicate numbers of tubes per well were used. Nonspecific binding,
maximum binding, standard curve, and GnRH were added to the assay.
3-(4,5-Dimethythiazolyl)-2.5-Diphenyl-2H-Tetrazolium-Bromide (MTT) Assay
To assess the effects of -synuclein expression on mitochondrial
activity, the MTT colorimetric assay was used to determine the level of
NADH production within the four cell clones. For vitamin E treatment,
-tocopherol acetate (stock 1:10 in 100% alcohol; Sigma Chemical
Co., St. Louis, MO) was further diluted 1:5 in fetal bovine serum
(Hyclone, Logan, UT) and then mixed 1:5 with regular cell culture
medium. Briefly, after exposure to treatments, the standard medium was
replaced with the MTT dye solution (final concentration, 0.9 mg/ml;
Sigma) and cells were incubated for 4 hours at 37°C. Then, 100 µl
of solubilization solution (50% dimethylformamide/20% sodium dodecyl
sulfate) was added overnight and the blue formazan was detected in a
plate reader at 560 nm.
Lactase Dehydrogenase Activity Assay
To assess cell viability, the CytoTox 96 assay (Promega, Madison, WI) was used. A minimum of 12 replicates per clone was used per treatment and each experiment included a plate of control cells. Briefly, after 3 hours of incubation, media containing various treatments was replaced with 100 µl of normal media and 20 µl of CytoTox reagent was added per well. Cells were incubated for an additional 2 to 3 hours and counted on a 96-well plate reader at 492 nm. Additional analysis of cell viability was performed using the Trypan blue staining method.
2'-7'-Dichlorofluorescein (DCF) Analysis
To determine whether free radical production was associated with
-synuclein expression, DCF loading analysis was performed. Briefly,
cells were cultured on poly-L-lysine (Sigma)-coated glass
coverslips in six-well dishes to 80% confluency. The cell permeable
dye, 2'7'-dichlorodihydrofluorescein diacetate
(H2DCFDA, 75 µmol/L) (Molecular Probes, Eugene,
OR) was added to the media and cells were incubated at 37°C, 5%
CO2 for 15 minutes. Loaded cells were then imaged
with the LSCM.
Glutathione Assay
Because it has been proposed that glutathione is the primary
cellular anti-oxidant response to free radical production, we
determined if levels of glutathione were affected in the four clones
expressing different levels of -synuclein. Briefly, as previously
described,37
cells were collected with 1x pancreatin and
lysed with cold 10% sulfosalicylic acid. Lysates were incubated on ice
for 15 minutes and supernatants were collected by centrifugation. After
neutralization with triethanolamine (diluted 1:4), the concentration of
total glutathione was determined. In addition, a standard curve of
glutathione (0 to 2 nmol/L range; Sigma) was constructed. The
A412 was measured at intervals of 0 seconds, 30
seconds, 60 seconds, and 90 seconds, and the total protein cell pellet
was dissolved in 50 µl of 0.5 mol/L NaOH. Total protein was measured
using the Protein Assay Kit (Pierce, Rockford, IL) and compared to a
standard bovine serum albumin curve. Final concentrations of
glutathione were obtained via linear regression analysis and expressed
as nmol/mg protein.
Electron Microscopy
GT1-7 cells were grown in Lux Permanox culture dishes (Nalgene Nunc International, Naperville, IL) until confluence. Cells were fixed in 1% glutaraldehyde, 2% paraformaldehyde in 0.1 mol/L Na cacodylate buffer (pH 7.4), postfixed in 1% osmium tetroxide, and stained with saturated uranyl acetate in 50% ethanol. The cell layer was dehydrated through a graded series of ethanols to 90% ethanol; 2-hydroxypropyl methacrylate was the intermediate solvent. All infiltrations of 2-hydroxypropyl methacrylate and Scipoxy 812 resin (Energy Beam Sciences, Agawam, MA) were carried out on a shaker at slow speed. After two changes of 100% resin the plates were polymerized in a 65°C oven for 24 hours, the plastic was detached and selected areas were sawed from the plates and glued on dummy blocks. Thin sections (80 nm) were cut on a Reichert Ultracut E Ultramicrotome (Leica, Vienna, Austria), picked up on 200-mesh copper grids, and poststained in ethanolic uranyl acetate followed by bismuth subnitrite. The sections were viewed and photographed with a Zeiss EM10 electron microscope.
Statistical Analysis
To ascertain differences among the various cell lines and treatment groups, one-way analysis of variance (ANOVA) with post hoc Scheffé test was performed using the SuperANOVA program (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Synuclein-Transfected Cell Lines
RPA analysis showed that cells transfected with the
sense construct (ST) expressed eightfold to ninefold in- creased
levels of murine mRNA -synuclein compared to VEC-transfected and
nontransfected control cells. In contrast, cells transfected with
antisense (AST) showed a 50% reduction in -synuclein mRNA levels
(Figure 1
, A and B). To
determine whether the increase in mRNA levels was accompanied by
similar changes at the protein level, Western blot analysis was
performed. Consistent with previous studies32
-synuclein immunoreactivity in GT1-7 cells was identified as an
18-kd band both in cytosolic (Figure 1C)
and particulate fractions (not
shown). Semiquantitative Western blot analysis by PhosphorImager of
-synuclein signals showed that ST cells expressed twofold over
control, whereas AST cells expressed 50% of control levels (Figure 1D)
.
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-Synuclein
Overexpressing Cells
LSCM of double-immunolabeled control and VEC-transfected GT1-7
cells showed that with the mouse-specific -synuclein antibody there
was a moderate granular cytoplasmic immunoreactivity (Figure 2A)
. In contrast, ST cells showed intense
immunoreactivity associated with granular structures (Figure 2
; B, C,
and E) and dense cytoplasmic aggregates (Figure 2, F–G)
. Control
experiments where the primary antibodies were inactivated show no
immunoreactivity (Figure 2D)
. AST cells showed microtubule-associated
protein 2 immunoreactivity, but mild or no -synuclein immunostaining
(Figure 2H)
. Analysis of the ST cells by transmitted electron
microscopy showed that, compared to nontransfected cells (Figure 3, A and B)
, some of the mitochondria
were enlarged (Figure 3, C and D)
and displayed abnormal vacuolized
cristae (Figure 3, E and F)
. In addition, some of the ST cells
presented aggregates of electrodense material localized mainly in the
perinuclear region (Figure 4, C and D)
.
These inclusion-like structures had a diameter of ~5 to 7 µm and
were usually surrounded by laminated bodies. These alterations were not
observed in VEC or control cells (Figure 4, A and B)
. Furthermore, some
ST cells displayed numerous secondary lysosomes and laminated
bodies in their cytoplasm and a relative lack of neuritic processes
(Figure 4, E and F)
.
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-Synuclein
Overexpressing Cells
To determine whether structural alterations observed in the
mitochondria were associated with functional deficits in the ST cells,
the MTT assay (a measure of mitochondrial activity) was performed. In
ST cells, this study showed a 20% reduction compared to VEC and
control cells (Figure 5)
. Because
previous studies have shown that mitochondrial dysfunction might lead
to oxidative stress via disruption of the respiratory
chain,27,33,38,39
it is possible that mitochondrial
deficiencies might be normalized by treatment with anti-oxidants such
as vitamin E. Consistent with this, vitamin E-treated ST cells showed
levels similar to VEC and control cells (Figure 5)
. Because it is
possible that reduction in mitochondrial activity might be secondary to
cell death rather than primarily associated with -synuclein
overexpression, the lactate dehydrogenase assay was performed. No
significant differences in cell viability were observed among the
clones (data not shown).
|
-Synuclein Overexpressing Cells
Because vitamin E pretreatment ameliorated the mitochondrial
deficiencies observed in ST cells, we used the membrane permeable dye,
DCF to determine whether the cells were under oxidative stress. For
this purpose, DCF-loaded GT1-7 cells were imaged with the LSCM. This
study showed a mild fluorescent signal in control cells (Figure 6A)
and an increased fluorescent signal
in the ST cells (Figure 6C)
. AST cells showed no significant increase
in the levels of DCF reactivity (not shown). Experiments where control
cells were treated with hydrogen peroxide showed similar increased
levels of the fluorescent signal as in the ST cells (Figure 6B)
. To
further corroborate that the increased DCF signal observed in the ST
cells was associated with generation of oxidative stress, cells were
pretreated with vitamin E. This anti-oxidant treatment resulted in a
reduction in the signal in ST cells (Figure 6D)
to levels similar to
those found in nontransfected control and VEC cells (Figure 6A)
.
|
. AST cells showed no
significant alterations in the levels of glutathione expression (Figure 7A)
.
|
-Synuclein Overexpressing Cells
To further confirm that the mitochondrial alterations and
oxidative stress conditions affected the function of ST cells, levels
of GnRH release were determined by radioimmunoassay. This study showed
that in ST cells there was a 60% reduction in the levels of GnRH
secretion compared to VEC and control cells and treatment of ST cells
with the anti-oxidant vitamin E resulted in normalization of GnRH
secretion (Figure 7B)
. GnRH release was within normal limits in AST
cells (not shown).
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-synuclein in
GT1-7 cells leads to mitochondrial dysfunction, as reflected by the
presence of giant mitochondria, formation of laminated bodies and
autophagozomes, and decreased MTT levels. Consistent with this
possibility, a recent study has shown that -synuclein exhibits
homology with 14-3-3 proteins and is capable of interacting with
BAD, a Bcl-2 homologue that controls mitochondrial function and
promotes cell death.40
In this regard, BAD interacts with
Bcl-x1 to form a heterodimer which displaces Bcl-2 leading to release
of cytochrome c from mitochondria, which is thought to be one of the
earliest steps involved in apoptotic cell death.41
Because
recent studies have also shown that cytochrome c interacts with
-synuclein in LBs,21,42
then release of this
mitochondrial molecule might not only trigger cell death, but also
promote -synuclein aggregation.
As to other potentially important consequences of the
-synuclein-mediated mitochondrial dysfunction, the present study
showed that overexpression of -synuclein in GT1-7 cells resulted in
decreased GnRH release and cellular changes consistent with oxidative
stress. Regarding the reduced vesicular secretion of GnRH in ST cells,
it has been demonstrated that cellular energy stores affect
functioning of vesicular transport proteins such as
N-ethylmaleimide-sensitive fusion protein.43
Vesicle activity correlates well with synaptic function or neural
plasticity and levels of -synuclein seem to follow those of other
proteins important for synaptogenesis and development.30
In addition, it has been shown that in the zebra finch model,
levels of -synuclein homologue, synelfin, are developmentally
regulated in the brain during critical periods of song
learning.5
Furthermore, -synuclein has been shown
to be capable of binding to synthetic membranes of acidic phospholipids
and preferentially to vesicles of smaller diameter suggestive of
selective regulation at the nerve terminal.44
-Synuclein has also been shown to inhibit the activity of
phospholipase D2,45
an enzyme that has been implicated in
the vesicular activity pathway.46
Taken together these
studies suggest that decreased neurosecretory activity in transfected
GT1-7 cells may be indirectly associated with mitochondrial
dysfunction. Alternatively, the neurosecretory alterations might
also be associated with other toxic changes such as generation of
radical oxygen species. In this regard, consistent with previous
reports showing that mitochondrial dysfunction leads to oxidative
stress,27,47
the present study indicated that
-synuclein overexpression results in mitochondrial alterations,
accompanied by increased DCF fluorescence and altered glutathione
levels. Furthermore, these alterations were reversible by treatment
with the anti-oxidant, vitamin E. It should be noted that whereas we
observe an increase in cellular glutathione levels, this is in contrast
to the observed decrease in glutathione levels in the brains of
patients with PD.48
It is possible that this difference
can be accounted for by the selection process of viable cell clones and
corroborates the hypothesis that the clones have altered their cellular
anti-oxidant defenses to counter the increased oxidative load.
These results are of significant importance for the understanding of PD
pathogenesis, because in this disorder mitochondrial function is
significantly compromised as reflected by alterations, among others, in
mitochondrial complex I levels23,25,49-54
and oxidative
stress has been postulated to play a central role in the
neurodegenerative process in PD.27
In this context, it is
possible that overexpression of -synuclein leads to mitochondrial
alterations which, in turn, lead to generation of oxygen radicals. This
is notable in view of recent studies showing that oxidative stress
leads to -synuclein aggregation23
and that cytochrome
c, which is centrally involved in the mitochondrial-associated
apoptotic pathway, is closely associated with -synuclein in LBs and
triggers -synuclein aggregation.42
These results
suggest that oxidative stress might further accelerate -synuclein
aggregation which, in turn, may result in LB formation.
In summary, the present study supports the contention that
-synuclein might be involved in regulating mitochondrial activity
and alterations in -synuclein expression may lead to mitochondrial
dysfunction and generation of radical oxygen species, that eventually
results in neurodegeneration in conditions associated with LBs such as
PD.
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Footnotes |
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Supported by National Institutes of Health grants AG5131 and AG10869.
Accepted for publication April 20, 2000.
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-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease. Brain Res 1998, 799:301-306[Medline]
-synuclein in vitro. NeuroReport 1999, 10:717-721[Medline]
-Synuclein shares physical and functional homology with 14-3-3 proteins. J Neurosci 1999, 19:5782-5791-synuclein aggregation in Lewy body disease. J Biol Chem 1999, 274:28849-28852This article has been cited by other articles:
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