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-Synuclein Are Not Associated with Aß Amyloid
From the Department of Pathology,*
The University of
Melbourne, Parkville, Victoria, Australia; the Center for Molecular
Biology,
The University of Heidelberg,
Heidelberg, Germany; and the University of Kuopio and University
Hospital of Kuopio,
Kuopio, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-Synuclein (SN), also termed the precursor of the
non-Aß component of Alzheimer's disease (AD) amyloid (NACP),
is a major component of Lewy bodies and Lewy neurites pathognomonic of
Parkinson's disease (PD) and dementia with Lewy bodies (DLB). A
fragment of SN termed the non-Aß component of AD amyloid (NAC) had
previously been identified as a constituent of AD amyloid plaques. To
clarify the relationship of NAC and SN with Aß plaques,
antibodies were raised to three domains of SN. All antibodies
produced punctate labeling of human cortex and strong labeling of Lewy
bodies. Using antibodies to SN(75–91) to label cortical and
hippocampal sections of pathologically proven AD cases, we
found no evidence for NAC in Aß amyloid plaques. Double labeling of
tissue sections in mixed DLB/AD cases revealed SN in dystrophic
neuritic processes, some of which were in close association
with Aß plaques restricted to the CA1 hippocampal region. In brain
homogenates SN was predominantly recovered in the cytosolic fraction
as a 16-kd protein on Western analysis; however, significant
amounts of aggregated and SN fragments were also found in urea
extracts of SDS-insoluble material from DLB and PD cases. NAC
antibodies identified an endogenous fragment of 6 kd in the cytosolic
and urea-soluble brain fractions. This fragment may be produced as a
consequence of SN aggregation or alternatively may accelerate
aggregation of the full-length SN.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-synuclein (SN)
gene are associated with Parkinson's disease (PD),1,2
several reports have shown that SN is a major component of Lewy
bodies and associated Lewy neurites, pathological hallmarks of
Parkinson's disease and dementia with Lewy bodies
(DLB).3-7
SN has also been found in the neuronal and
glial inclusions of multiple system atrophy, Lewy body-like inclusions
in the motor neuron disorder amyotrophic lateral sclerosis, and in
neuronal inclusions in familial Alzheimer's disease
(AD).8-13
Evidence so far indicates all these cytoplasmic
inclusions are filamentous and of similar
composition.14-17
However, SN is not found in
inclusions present in Pick's disease,17
indicating that
SN is selectively deposited in certain neurodegenerative
diseases.
A fragment of SN was earlier shown to co-purify with AD amyloid. Two
peptides isolated after formic acid, CNBr, and protease treatment of AD
brain amyloid are termed the non-amyloid component of AD plaques (NAC).
They were shown to correspond to residues 61–80 and 81–95 of a larger
precursor termed NACP, which was subsequently cloned18
and
found to be a human homolog of the Torpedo ray synuclein,
which had been previously identified in synaptic vesicle
preparations.19
Synucleins constitute a family of proteins
consisting of , ß, and 
SN, now studied in several vertebrates
(reviewed20,21
). - and ß-Synucleins have been shown
to be cytoplasmic proteins associated with presynaptic
junctions22,23
and SN to be the only member associated
with intracellular inclusions in neurodegenerative
conditions.3,5,15,24
Antibodies to peptides in the NAC
region [rabbit antibody (Ab) X1 to SN(61–69) and rabbit Ab Y to
SN(81–87)] were reported to label a large proportion of AD
plaques.16,18,25,26
In the present study, we reexamined
these findings using antibodies raised to SN(75–91) of the NAC
component.
We have also confirmed the immunoreactivity of neuronal inclusions in
dementia with Lewy bodies, and PD using rabbit antibodies to
N-terminal, NAC, and C-terminal domains of SN by
immunocytochemistry. Western blots of brain homogenates from frozen
tissue of these cases were compared with age-matched controls and AD
tissues to examine the expression and solubility of SN and NAC and
their relationship with Aß amyloid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rabbit polyclonal antibodies were raised to the human SN
N-terminal region (1–18) (Ab 97/5), to the C-terminal domain
(116–131) (Ab 97/8), and to the NAC region of SN (75–91) (Ab
42580). Rabbit antibody Ab 98/13 was raised to ß-synuclein (99–113).
For preabsorption experiments 10 µg of immunizing peptide was
preincubated with 1 to 2 µl of antisera for 16 hours at 4°C in 0.1
ml of PBS, pH 7.4, for immunohistochemistry or in 0.5 ml of TBS-T (50
mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl with 0.2% Tween 20) for
Western blot, before appropriate dilution for use. Mouse monoclonal
antibodies (MAb) 1E8 and WO2 recognize Aß(17–24)27
and
Aß(5–8),28
respectively. Rabbit antiserum to human tau
was from Dako (Glostrup, Denmark). Synaptophysin mouse MAb SY38 was
from Boehringer Mannheim (Mannheim, Germany).
Tissue Collection
Brain tissue was collected at autopsy. Tissue from clinically and pathologically confirmed cases of five DLB, four PD, seven sporadic AD, five DLB/AD, and seven age-matched controls were used in the study. The pathological diagnosis was made according to standard criteria. AD was diagnosed using CERAD criteria.29 DLB cases were diagnosed using the consensus guidelines30 when cortical Lewy bodies were seen using ubiquitin immunohistochemistry on the initial screen. A pathological diagnosis of PD was made in conjunction with clinical PD and pathology predominating in the midbrain. For histochemistry, tissues were fixed in 10% formalin in PBS. For Western blot analysis tissues were frozen and stored at -70°C.
Immunocytochemistry
Formalin-fixed tissue from the substantia nigra, hippocampus, and cortex was embedded in paraffin. Sections were treated with 80% formic acid for 5 minutes, treated with 3% hydrogen peroxide for 5 minutes, and incubated in blocking buffer (50 mmol/L Tris-HCl, 175 mmol/L NaCl, pH 7.4, with 20% serum corresponding to species for secondary Ab) before incubation with primary antibody. Ab 97/8 was used at 1:2000, Ab 97/5 at 1:500, Ab 42580 at 1:100, Ab 98/13 1:200, MAb 1E8 was undiluted hybridoma culture supernatant, and rabbit anti-human tau was used at 1:400. Secondary reagents linked to horseradish peroxidase were used and visualized with diaminobenzidine. For double labeling, sections were reacted with an additional secondary antibody conjugated to alkaline phosphatase and developed with 5-bromo-4-chloro-3-indoxyl phosphate and nitro blue tetrazolium chloride (blue) from Dako (K598) or new fuchsin (Dako, K0596) (red). Sections were counterstained with hematoxylin.
Immunofluorescence Labeling
Hippocampal and cortical primary neurons were cultured from embryonic day 15 rats on poly(L)-lysine-coated glass coverslips for 15 days, as previously described.31 Cells were washed twice with PBS containing 1 mmol/L CaCl2 and 1 mmol/L MgCl2 and fixed with 4% formaldehyde in PBS, pH 7.4, for 15 minutes at room temperature (RT) or for 30 seconds in acetone at -20°C. Cells were then washed twice in PBS. Formaldehyde-fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at RT. Cells were treated with 20% sheep serum in PBS for 10 minutes at RT before incubation with primary Ab diluted in 1% bovine serum albumin in PBS. After washing, cells were reacted with FITC-conjugated sheep anti-rabbit Ig (Amrad, Boronia, Victoria, Australia) or Texas Red conjugated sheep anti-mouse Ig (Amersham, Little Chalfont, England). Double labeling steps were performed sequentially. Coverslips were mounted in 2.6% DABCO (Sigma) in 90% glycerol/10% PBS and imaged with a BioRad 1024 confocal system.
Western Blotting
Brain homogenates were prepared from tissue stored at -70°C by
sonication in 1:10 (g/vol) in TBS buffer containing 50 mmol/L Tris-HCl,
pH 7.4, 175 mmol/L NaCl, 5 mmol/L EDTA, and the protease inhibitors
PMSF (2 mmol/L), aprotinin (2 µg/ml), leupeptin (2 µg/ml), antipain
(2 µg/ml), and pepstatin (2 µg/ml). After 5 minutes of
centrifugation at 1000 x g, the supernatants were
centrifuged at 150,000 x g for 1 hour at 4°C. This
high speed supernatant fraction is termed TBS-soluble. The pellets were
rinsed twice in TBS before solubilizing in 5% SDS in TBS and further
centrifugation at 150,000 x g for 30 minutes. This
supernatant was termed the SDS-soluble fraction. The SDS-insoluble
pellet was solubilized in 8 mol/L urea/5% SDS in TBS and termed the
urea-soluble fraction. Protein concentration was determined in the TBS
and SDS soluble fractions using a BCA assay (Pierce, Rockford, IL).
Samples were mixed with 2X Laemmli sample buffer containing 10%
ß-mercaptoethanol and boiled for 5 minutes. 5 µg of protein of TBS
fractions, 10 µg of protein of SDS fractions, and 30 µl of
urea-soluble material from approximately 30 µg equivalent of frozen
tissue were electrophoresed on 10% SDS Tris-tricine polyacrylamide
gels and analyzed by Western blot developing with chemiluminescence
(ECL, Amersham).32,33
Western blot analysis of the
1000 x g pellet revealed Aß and SN but not the
6-kd NAC fragment in all of the samples. The expression levels of SN
in this pellet follows the pattern of the urea fraction, ie, high in
DLB/PD cases and minimal amounts in control/AD cases (data not shown).
We chose to use 8 mol/L urea/5% SDS to solubilize SN/NAC
aggregates, because in our laboratory we have found that 8 mol/L urea
solubilizes amyloid plaques from human AD brain homogenate more
efficiently and less variably than formic acid.34
Under
our conditions, materials have been completely solubilized with 8 mol/L
urea/5% SDS, as no pellet was left after this step. Furthermore,
SDS-insoluble Aß was also detected with this solubilization method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SN in Brain by Immunocytochemistry and
Western Blot
To investigate SN expression, rabbit antibodies were raised to
three SN regions: N-terminal (1–18) Ab (97/5), anti-NAC (75–91) Ab
42580, and the C-terminal domain (116–131) Ab 97/8. This last peptide
is most specific for SN, a region of least homology with other
members of the synuclein family.21
Immunocytochemistry
showed that all three antibodies to SN reacted with a fine punctate
pattern for human brain regions rich in synapses and strongly labeled
Lewy bodies and Lewy neurites of DLB (n = 5) and
PD (n = 4). Representative labeling of Lewy
bodies and a Lewy neurite is shown for cingulate gyrus cortex (Figure 1,A and C)
and substantia nigra (Figure 1B)
from a DLB case. Antisera to ß-synuclein (Ab 98/13) also produced
a punctate synaptic type pattern by histochemistry but did not label
Lewy body or neurite inclusions (not shown). Preabsorption with
immunizing peptides abolished reactivity and the preimmune sera were
negative (not shown).
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SN reacted with a major band with apparent
mobility of 16 kd (Figure 2,A–C)
, a
result consistent with earlier studies.22,23,35
The
ß-synuclein Ab 98/13 reacted with a band migrating close to that for
SN as detected by Ab 97/8 (Figure 2D)
. Preabsorption of antibodies
with the immunizing peptides removed reactivity as detected by Western
analysis (Figure 2)
. Preimmune sera were also negative on Western
blots.
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SN reactivity by immunofluorescence in comparison with
the integral synaptic membrane protein synaptophysin. After fixation
with formaldehyde, double labeling for the two proteins showed
reactivity for all of the SN Abs and considerable co-localization
with synaptophysin as shown for mature hippocampal neurons (Figure 3, A–F)
similar to the results reported
by Withers et al36
using a C-terminal SN MAb. However,
not all synaptophysin punctate reactivity was also positive for SN
as indicated in Figure 3 (E and F)
. It is also of interest that most
SN reactivity was lost after acetone-alone fixation, indicative of a
loose association with cellular structures, as shown for SN labeling
with Ab 97/8 (Figure 3H)
, in contrast to the strong labeling of the
integral membrane protein synaptophysin which was retained after
acetone fixation (Figure 3G)
. Cell body labeling was also found for the
anti-NAC antibody which was used at a lower dilution and is considered
nonspecific, since it was not observed for the other two -SN
antibodies and could not be removed by preabsorption (data not shown).
Withers et al36
also observed nonspecific cell body
labeling of cultured hippocampal neurons with SN antibodies using
similar labeling conditions.
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Earlier studies have indicated that antibodies to the NAC fragment
of SN label AD amyloid plaques.16,18,25,26
Since these
reports have not been confirmed with independently prepared antibodies,
we reexamined this question using antibodies raised to the central NAC
domain using Ab 42580 on AD cortical and hippocampal sections. We found
no evidence of NAC labeling in plaques of cortical or hippocampal
tissue sections from AD only cases (Figure 4, B and D)
. Serial sections labeled in
parallel for Aß revealed numerous plaques (Figure 4, A and C)
. Our
other SN antibodies which strongly labeled synapses and Lewy body
inclusions (Ab 97/5 and Ab 97/8) also showed no plaque labeling in AD
only cases (not shown).
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SN Deposits Are Occasionally Associated with the Periphery of
Aß Plaques and Tau-Positive Neurofibrillary Tangles
In cases with mixed AD and DLB pathology the distribution of Lewy
bodies was similar to that of DLB cases. In the cortex SN
immunoreactivity in and around Aß plaques was not seen; however, in
sections from the hippocampus of DLB/AD double staining for SN (Ab
97/8) and Aß (MAb 1E8) showed strong SN labeling in the CA1 region
in dystrophic globular neurites (Figure 5A)
at the periphery of Aß-positive
plaques. There were also clusters of SN-positive globular neuritic
structures not in direct association with Aß plaques (Figure 5B)
.
These neuritic structures were also labeled with the NAC antibody (not
shown) which indicated that full-length SN and possibly also the NAC
fragment may be aggregating as neuritic inclusions that may be
associated only occasionally with Aß plaques. These neuritic
SN-positive clusters are similar to those described in AD
hippocampus by Munoz and Wang.37
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SN and tau in the same cases revealed occasional
association of SN inclusions within cells that were also strongly
reactive for intraneuronal tau (Figure 5C)
as well as close association
of SN-positive dystrophic neuritic structures with tau-positive
deposits (Figure 5D)
. Close expression of tau immunoreactive
neurofibrillary tangles and SN reactivity was also shown in DLB
hippocampus by Iseki et al.38
Expression and Solubility of SN and NAC in Human Brain
Homogenate Fractions
To investigate the solubility of SN and its fragments
differential extraction procedures were carried out on brain
homogenates. TBS homogenates were centrifuged at 150,000 x
g to generate a cytosolic TBS-soluble fraction and
particulate membrane fraction (TBS-pellet). The pellets were then
solubilized with 5% SDS and centrifuged at 150,000 x
g to generate an SDS-soluble fraction. The SDS-insoluble
pellet was subsequently solubilized with urea to generate the
urea-soluble fraction. Western blot analysis of the SN expression in
TBS-soluble extracts of human brain cingulate gyrus cortex by Western
blotting showed similar reactivity from cases with DLB, PD, AD, and
controls (Figure 6A)
using C-terminal
domain Ab 97/8. AD cases (Figure 6A
, lanes 7 and 8) had apparently less
SN which maybe indicative of neuronal synaptic loss. Antibody to the
central hydrophobic domain of SN(75–91), NAC Ab 42580, also
detected full-length SN and a less abundant putative NAC fragment of
about 6 kd. The presence of the soluble 6-kd NAC was not apparently
related to the disease process. Another fragment of about 12 kd was
also detected (Figure 6B)
.
|
SN (Figure 6, D and E)
but no
detection of the 6-kd NAC fragment (Figure 6E)
. Analysis of the
SDS-insoluble pellet which was solubilized by urea showed marked
differences between brain samples. DLB and PD cases showed the most
SN reactivity (Figure 6G)
with low amounts in control brains and
with variable amounts from low to an intermediate amount in AD cortex
samples. Immunoreaction with the NAC antibody again detected
full-length SN (Figure 6H)
as well as detection of the NAC fragment
in the DLB cases and some PD cases (Figure 6H)
. Significant amounts of
apparently aggregated SN and/or NAC were detected in urea-soluble
fractions, especially with the NAC antibody as evidenced by bands of
slower mobility (ie, higher molecular weights) in the DLB and some PD
cases (Figure 6H)
. The 6-kd band detected by the NAC antibody in both
the TBS (soluble fraction) and the insoluble urea fractions (Figure 6, B and H)
are similar as they both run just below the 6.5-kd molecular
weight marker and ran with the same electrophoretic mobility when these
fractions were run on the same gel (data not shown). Ab 98/13 staining
of the urea-soluble fractions revealed no reactivity indicating that
ß-synuclein is not aggregating like the SN (not shown).
Examination of Aß in the brain fractions revealed weak reactivity in
the TBS-soluble fraction (Figure 6C)
. There were significant amounts of
Aß associated with the particulate pellet which was solubilized by
SDS (SDS-soluble Aß) (Figure 6F)
, and considerable SDS-insoluble but
urea-soluble Aß (Figure 6I)
in DLB, one PD, both AD, and negligible
amounts in one PD and the controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SN all reacted with
Lewy body inclusions in DLB brain sections, reacted by Western blotting
in brain homogenates with a major product of 16 kd, and also by
immunofluorescence at synaptic sites indicating that all epitopes for
these antibodies and most likely full-length SN are expressed at
these sites.
These results confirm previous studies which primarily used N- and
C-terminal region antibodies, indicating that SN is a major
component of Lewy bodies.3-7
The relative mobility on the
10% Tris-tricine gel system for SN of apparent 16 kd is closer to
the predicted molecular weight of the 140 amino acid protein than 19 kd
reported previously for samples resolved on Tris-glycine gel
systems.18,22,24
We have used the Tris-tricine system for
better resolution of smaller peptides such as Aß32,33
and used it in this study for improved detection of the NAC peptide.
These gels did not resolve a clear difference between the mobility of
- and ß-synuclein (134 amino acids) as shown
previously.24
Although Uéda et al first described isolation of NAC peptide
associated with AD brain amyloid in 199318
there has been
little further information to confirm or characterize this fragment in
human brain. Western blot analysis in Figure 2B
and Figure 6, B and H
,
shows that the NAC Ab 42580 detected the full-length SN as well as a
6-kd fragment. This size for the putative NAC is larger than
anticipated for a fragment of 35 amino acids. Since NAC was originally
isolated using CNBr and protease digestion, the size predicted for the
putative NAC may underestimate the size of an endogenous fragment found
in the brain. The 6-kd fragment detected in our system appears to be
longer than the suggested SN(61–95) residues.
By immunocytochemistry the NAC antibody failed to show labeling of Aß
plaques in brain sections from patients with pathologically confirmed
AD or of the cores of plaques from mixed DLB/AD cases. In mixed DLB/AD
cases SN immunoreactivity was found at the periphery of some Aß
plaques and as clusters of bulbous neuritic processes in only a limited
region of the hippocampus, the CA1 region. We also noted dense SN
immunoreactive Lewy neurites localized to the CA2 hippocampal region of
all DLB and mixed DLB/AD cases as has been reported
previously.15,17
Lewy neurites in this region have been
noted as a distinguishing feature of DLB and PD.39,40
Thus
all SN immunoreactivity appeared to be intracellular in contrast to
the extracellular deposition of Aß amyloid plaques, which is
suggestive of independent insoluble accumulation of these proteins.
Western blot analysis also showed lack of correlation between
expression of Aß and NAC or between Aß and SN detection in
TBS-soluble fractions or urea-soluble fractions. Especially in the AD
cases studied, urea-soluble NAC or SN did not accumulate with
urea-soluble Aß. However, in the Lewy body disease cases there was
significant expression of SN, NAC, and Aß in the urea-soluble
fractions. Cases 1 to 3 did not have sufficient neuritic Aß plaque
morphology to be classified as AD pathology; however, they had moderate
numbers of diffuse plaques and cases 2 and 3 also had a congophilic
angiopathy. One PD case examined had very few diffuse plaques and low
co-expression of SN, NAC, and Aß in the urea-soluble fraction
(case 4).
It is only relatively recently that DLB has been distinguished as a
major cause of dementia. When DLB and AD co-exist, the incidence of
Lewy bodies is difficult to distinguish from neurofibrillary tangles in
cortical regions since both label with ubiquitin antibody which has
been used to date to detect Lewy bodies.40,41
An improved
assessment of the incidence and relationship of DLB and mixed DLB/AD
will now be possible with the advent of SN antibodies to identify
more accurately Lewy bodies in routine pathological assessment.
We found immunocytochemical labeling patterns with the NAC antibody
indistinguishable from the other SN antibodies as also reported for
another recently generated NAC antibody tested on ischemic gerbil
hippocampus.42
Examination of double labeling for SN
and synaptophysin reactivity on cultured hippocampal rat neurons
confirmed a synaptic localization for SN that was easily disrupted
by acetone fixation, suggesting a loose association with cellular
structures. This was consistent with SN solubility properties and
earlier localization studies.35,36
Recent in
vitro studies indicate synthetic SN associates with small lipid
vesicles by electrostatic interaction.43
Hsu et
al44
showed that SN is expressed later in murine
development than synaptophysin and progressively moves from being
solely cytosolic to more particulate throughout development, consistent
with our Western blot data that show that a pool of SN is found in
the membrane particulate fraction (Figure 6, D and E)
.
Comparison of immunoreactivity by Western blotting of extracts of human
brain homogenates indicated that most SN is expressed in the
TBS-soluble cytosolic fraction generated from the 150,000 x
g centrifugation step in different disease states. Some
reduction may occur in AD with loss of neuronal synapses. The 6-kd
putative NAC is also present in low amounts in all of the TBS-soluble
samples. The levels of both soluble SN and soluble 6-kd fragment
show no correlation with disease state. The presence of significant
amounts of SN in the SDS fraction is consistent with our
immunofluorescence data and other reports that SN can associate with
small lipid vesicular structures, since the SDS fraction is derived
from the TBS-insoluble fraction and comprises membranous structures.
Since there is no detectable 6-kd NAC fragment in the SDS fraction,
this indicates that the fragment is probably a normal soluble breakdown
product of SN and not associated with membrane/vesicle structures.
Marked differences were seen in the urea-soluble brain fractions, with
most SN and 6-kd immunoreactivity in the Lewy body disorder cases of
DLB and some of the PD cases, indicating that less soluble and
aggregated SN and the putative NAC accumulates in these disorders.
The NAC fragment of 6 kd and higher molecular weight aggregates
particularly detected with the NAC Ab 42580 indicate the SDS-insoluble
material forms aggregates which could form the Lewy bodies and Lewy
neurites and is disease-related. These aggregates are not recognized by
the ß-synuclein antibody consistent with the negative
immunoreactivity of ß-synuclein antibody for Lewy bodies in brain
sections by immunocytochemistry. Fractions containing SN of reduced
solubility have also been reported for neurodegenerative cases of
multiple system atrophy24
and familial AD cases that
contained many Lewy body inclusions.10
Truncated SN
polypeptides of 14 to 16 kd as well as full-length SN and aggregates
were detected using a monoclonal antibody by Baba et al in formic acid
extracts of purified Lewy bodies.3
The results presented here indicate the urea-soluble fraction may
represent aggregated SN deposits from the Lewy bodies and neurites
of these cases and may include aggregated NAC. NAC may therefore be a
comparatively protease-resistant core of the protein which is
particularly prone to aggregation, as shown previously with NAC
synthetic peptide.25,45-47
NAC may accumulate as a
natural breakdown product of SN aggregation or NAC aggregates may
contribute to further aggregation of the full-length SN, rather than
seeding aggregation of the Aß amyloid peptide as had been proposed
earlier. The mechanism underlying SN aggregation will be important
for understanding the role of SN in neurodegeneration.
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Acknowledgements |
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Footnotes |
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Supported by the National Health and Medical Research Council of Australia. KB and TH are supported by the AFI, Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie. PJ is supported by the Academy of Finland and Alexander von Humboldt Foundation.
Accepted for publication June 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-synuclein in Parkinson's disease. Nat Genet 1998, 18:106-108[Medline]
-synuclein gene identified in families with Parkinson's disease. Science 1997, 276:2045-2047-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies. Am J Pathol 1998, 152:879-884[Abstract]
-synuclein immunoreactivity. J Neuropathol Exp Neurol 1998, 57:334-337[Medline]
-synuclein in neurodegenerative disorders. Am J Pathol 1998, 152:367-372[Abstract]
-synuclein immunoreactivity in fibrillary components of neuronal, and oligodendroglial cytoplasmic inclusions in the pontine nuclei in multiple system atrophy. Acta Neuropathol 1998, 96:439-444[Medline]
-synuclein disease? Lancet 1998, 352:547-548[Medline]
-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes. Am J Pathol 1998, 153:1365-1370-synuclein inclusions link multiple system atrophy with Parkinson's disease, and dementia with Lewy bodies. Neurosci Lett 1998, 251:205-208[Medline]
-Synuclein immunoreactivity in glial cytoplasmic inclusions in multiple system atrophy. Neurosci Lett 1998, 249:180-182[Medline]
-synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 1998, 95:6469-6473-synuclein in Lewy body disease as revealed by proteinase K and formic acid pretreatment. Lab Invest 1998, 78:1169-1177[Medline]
-synuclein/NACP is a cytopathological feature common to Lewy body disease and multiple system atrophy. Acta Neuropathol 1998, 96:445-452[Medline]
-synuclein. Ann Neurol 1998, 44:415-422[Medline]
-synuclein-immunoreactive in the hippocampus of patients with diffuse Lewy body disease. Neurosci Lett 1998, 258:81-84[Medline]
-synuclein in an ischemic gerbil hippocampus. Brain Res 1998, 788:311-314[Medline]
-synuclein secondary structure upon binding to synthetic membrane. J Biol Chem 1998, 273:9443-9449-synuclein) during murine brain development. J Neurochem 1998, 338–344
-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of ß-sheet and amyloid-like filaments. FEBS Lett 1998, 440:71-75[Medline]
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 Y.-H. Suh and F. Checler Amyloid Precursor Protein, Presenilins, and alpha -Synuclein: Molecular Pathogenesis and Pharmacological Applications in Alzheimer's Disease Pharmacol. Rev., September 1, 2002; 54(3): 469 - 525. [Abstract] [Full Text] [PDF]
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H. Miake, H. Mizusawa, T. Iwatsubo, and M. Hasegawa Biochemical Characterization of the Core Structure of alpha -Synuclein Filaments J. Biol. Chem., May 17, 2002; 277(21): 19213 - 19219. [Abstract] [Full Text] [PDF]
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 P. J. Kahle, M. Neumann, L. Ozmen, V. Muller, S. Odoy, N. Okamoto, H. Jacobsen, T. Iwatsubo, J. Q. Trojanowski, H. Takahashi, et al. Selective Insolubility of {alpha}-Synuclein in Human Lewy Body Diseases Is Recapitulated in a Transgenic Mouse Model Am. J. Pathol., December 1, 2001; 159(6): 2215 - 2225. [Abstract] [Full Text] [PDF]
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R. J. Perrin, W. S. Woods, D. F. Clayton, and J. M. George Exposure to Long Chain Polyunsaturated Fatty Acids Triggers Rapid Multimerization of Synucleins J. Biol. Chem., November 2, 2001; 276(45): 41958 - 41962. [Abstract] [Full Text] [PDF]
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 J. E. Galvin, V. M.-Y. Lee, and J. Q. Trojanowski Synucleinopathies: Clinical and Pathological Implications Arch Neurol, February 1, 2001; 58(2): 186 - 190. [Abstract] [Full Text] [PDF]
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P. J. Kahle, M. Neumann, L. Ozmen, V. Muller, H. Jacobsen, A. Schindzielorz, M. Okochi, U. Leimer, H. van der Putten, A. Probst, et al. Subcellular Localization of Wild-Type and Parkinson's Disease-Associated Mutant alpha -Synuclein in Human and Transgenic Mouse Brain J. Neurosci., September 1, 2000; 20(17): 6365 - 6373. [Abstract] [Full Text] [PDF]
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P. H. Jensen, K. Islam, J. Kenney, M. S. Nielsen, J. Power, and W. P. Gai Microtubule-associated Protein 1B Is a Component of Cortical Lewy Bodies and Binds alpha -Synuclein Filaments J. Biol. Chem., July 7, 2000; 275(28): 21500 - 21507. [Abstract] [Full Text] [PDF]
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B. I. Giasson, I. V. J. Murray, J. Q. Trojanowski, and V. M.-Y. Lee A Hydrophobic Stretch of 12 Amino Acid Residues in the Middle of alpha -Synuclein Is Essential for Filament Assembly J. Biol. Chem., January 19, 2001; 276(4): 2380 - 2386. [Abstract] [Full Text] [PDF]
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