involvement of the cerebral cortex in parkinson disease linked with g2019s lrrk2 mutation without...
TRANSCRIPT
ORIGINAL PAPER
Involvement of the cerebral cortex in Parkinson disease linkedwith G2019S LRRK2 mutation without cognitive impairment
Anna Gomez • Isidre Ferrer
Received: 16 September 2009 / Revised: 5 March 2010 / Accepted: 5 March 2010 / Published online: 16 March 2010
� Springer-Verlag 2010
Abstract Previous studies have shown altered synuclein,
increased oxidative stress damage and increased oxidative
stress responses in patients with sporadic Parkinson’s
disease (PD) without cognitive impairment. Yet no infor-
mation exists about possible molecular alterations in the
cerebral cortex in familial PD. The present study shows
abnormal a-synuclein solubility and aggregation, and
aggregated nitrated a-synuclein, in the cerebral cortex
(area 8) in cases with long-lasting PD linked with the
G2019S LRRK2 mutation, one of them with a few Lewy
bodies (LBs) and the other two without LBs in the cerebral
cortex. Increased expression of the oxidative stress marker
malondialdehyde-lysine (MDAL), together with increased
oxidative stress responses, AGE receptors (RAGE) and
superoxide dismutase 2, occurred in the frontal cortex in
the three LRRK2 cases compared with three controls pro-
cessed in parallel. Bi-dimensional gel electrophoresis,
western blotting, in-gel digestion and mass spectrometry
disclosed glial fibrillary acidic protein as a target of MDAL
adducts. Tubulin b4 and enolase 2 were also identified as
targets of oxidative damage. These results demonstrate
biochemical abnormalities of a-synuclein, and increased
oxidative stress damage and oxidative stress responses in
the frontal cortex in PD linked with G2019S LRRK2
mutation not related with the presence of cortical LBs and
in the absence of apparent cognitive deficits. These findings
show that the cerebral cortex in familial PD linked with
G2019S LRRK2 is affected in a similar way than that seen
in sporadic PD without cognitive impairment.
Keywords Parkinson disease � LRRK2 � a-Synuclein �Oxidative stress � Glial fibrillary acidic protein �Superoxide dismutase
Introduction
Parkinson disease (PD) is a progressive degenerative
disease of the nervous system that is classically charac-
terized by motor symptoms and signs related with the loss
of dopaminergic input of the basal ganglia. Neuropatho-
logically, there is a marked loss of dopaminergic neurons
in the substantia nigra pars compacta accompanied by the
presence of intraneuronal inclusions known as Lewy
bodies (LBs) in many remaining neurons, and aberrant
neurites (LNs). Neuron loss and abnormal inclusions are
not limited to the substantia nigra as many other structures
of the peripheral, autonomic and central nervous system
are also involved, including the olfactory bulb, selected
nuclei of the medulla oblongata and pons, locus ceruleus,
amygdala, nucleus basalis of Meynert, and, in advanced
stages, the striatum and cerebral cortex [7, 15, 21, 32].
LBs and LNs are composed of abnormal protein aggre-
gates [56, 67]. The main component of these aggregates
is a-synuclein, which is abnormally nitrated, phosphory-
lated, and oxidized. It also presents an abnormal
solubility, and is prone to the formation of aggregates and
insoluble fibrils [1–3, 16, 22, 26, 29, 30, 53, 58, 59, 66].
Because of the microscopical visualization of LBs, PD is
considered a Lewy body disease (LBD) [21, 32]. Based
on the main molecular change, PD is categorized as
a-synucleinopathy [25].
A. Gomez � I. Ferrer (&)
Servei Anatomia Patologica, Institut de Neuropatologia,
CIBERNED, IDIBELL-Hospital Universitari de Bellvitge,
Universitat de Barcelona, carrer Feixa LLarga sn,
08907 Hospitalet de LLobregat, Spain
e-mail: [email protected]
123
Acta Neuropathol (2010) 120:155–167
DOI 10.1007/s00401-010-0669-y
In the majority of cases, PD is sporadic, but 5–10% of
cases are due to mutations in selected genes, including
a-synuclein (PARK1) [36, 51, 73], parkin (PARK2) [34],
DJ1 (PARK7) coding for DJ1 [4], PINK1 (PARK6) coding
for PTEN-induced putative kinase 1 [65], and LRRK2
(PARK8) coding for leucine-rich repeat kinase 2 [46, 72].
The roles of HTRA2 (PARK13) coding for HtrA serine
peptidase 2: HtrA2 [60] and UCHL1 (PARK5) coding for
ubiquitin carboxyl-terminal hydrolase L1: UCHL-1 [37]
are still uncertain in PD pathogenesis. PD linked with
LRRK2 mutations is the most common form of familial PD;
LRRK2 mutations are also causative of a number of spo-
radic cases.
About 30 different mutations have been identified
in LRRK2. The most prevalent LRRK2 mutation in the
Mediterranean area is G2019S [18]. Interestingly, the
neuropathology of LRRK2 mutations is not homogeneous;
although neuron loss of dopaminergic neurons is a constant
feature, LBs and LNs are present in some cases but not in
others [55]. Even the same mutations are not always
accompanied by LBs, as in one reported case bearing the
T1699C mutation [69] and another carrying the G2019S
mutation [24]. tau pathology and the absence of LBs
occurred in one case with the G2019S mutation [52]. The
only case examined with the R1441G mutation had loss of
dopaminergic neurons in the substantia nigra, free neuro-
melanin in the neuropil and absence of a-synuclein-,
hyperphosphorylated tau- and ubiquitin-immunoreactive
inclusions [42]. Intriguingly, LRRK2 mutation in one
family was neuropathologically manifested as an LBD in
one member, as a tauopathy with neurofibrillary tangles in
a second affected member, and as loss of neurons with no
intracytoplasmic neuronal inclusions in a third [72]. This
indicates that PD due to LRRK2 mutations is not always an
LBD. Whether a-synuclein is altered in PD with no LBs
and LNs is not known.
Recent studies have shown that the cerebral cortex is
affected in sporadic PD. Yet there is no clear relation
between the presence and amount of LBs and LNs in the
cerebral cortex, and cognitive impairment [31, 48, 49].
Therefore, molecular alterations rather than LBs and LNs
are the substrates accounting for the impaired cortical
function. Disturbed mitochondrial function, increased
oxidative damage, impaired energy metabolism, post-
translational a-synuclein modifications, and abnormal
a-synuclein solubility and aggregation, without LBs, are
key abnormalities in sporadic PD cerebral cortex even at
relatively early stages of the disease [19]. No similar
information is available in the cerebral cortex in PD linked
with LRRK2 mutations.
The objective of the present study is to examine bio-
chemical alterations in the cerebral cortex in three
unrelated cases of familial PD linked with the same
G2019S mutation, one of them with classical PD neuro-
pathology, another with LBs in the brain stem and
amygdala but not in the cerebral neocortex, and the third
with neuron loss in the substantia nigra but without LBs or
LNs in any region. None of these cases had evidence of
cognitive deficits during life. These three cases were
selected from a larger series on the basis of the lack of
additional associated cortical pathology that could have
biased molecular observations.
Materials and methods
Tissue samples
Brain samples were obtained from the Institute of Neuro-
pathology and University of Barcelona-Clinic Hospital
Brain Banks following the guidelines of the local ethics
committees.
The cases with PD bearing the G2019S mutation were
three women aged 78, 77 and 83 years (cases 1, 2 and 3,
respectively). Details of clinical and genetic studies (cases
1–3) have been reported elsewhere [23, 24].
Case 1 was a woman with PD starting at the age of
52 years with poor response to medication who was sub-
jected to a left pallidectomy at the age of 70 years, and then
following treatment with Sinemet Plus and Sinemet Retard.
She died at the age of 78 years. No evidence of cognitive
impairment was recorded.
Case 2 was a woman with long-term well-tolerated
parkinsonism, accommodated in a residence for old people
who died at the age of 83 years. She liked to solve cross-
words and sodokus in the company of younger mates and
nurses until a few days before death.
Case 3 was a woman with PD starting at the age of 61,
treated with levodopa (first 300 mg daily, then 600 mg
daily, 10 years later) and pramipexole (0.7 mg three times
a day), and lasting 14 years in whom ‘‘signs of cognitive
impairment, hallucinations, delusions or atypical signs for
PD never developed during the course of the disease’’
[24].
Three controls, all of them women, aged 78, 82 and
83 years were processed in parallel. The post-mortem
delay was between 3 and 7 h.
In all cases, the fresh brain was processed for morpho-
logical and biochemical studies. One cerebral hemisphere
was cut in coronal sections and rapidly frozen and stored at
-80�C until use, while the other hemisphere was fixed in
4% buffered formalin for about 2 months. The brain stem
was cut in transverse sections and consecutive sections
were alternatively frozen or fixed in 4% paraformaldehyde.
The cerebellum was cut on the sagittal plane and selected
samples were frozen or fixed in 4% buffered formalin.
156 Acta Neuropathol (2010) 120:155–167
123
The neuropathological study was carried out on paraffin-
embedded de-waxed sections, 4-lm-thick, of the frontal
(area 8), primary motor, primary sensory, parietal, tem-
poral superior, temporal inferior, anterior cingular, anterior
insular, and primary and associative visual cortices;
entorhinal cortex and hippocampus; caudate, putamen
and globus pallidus; medial and posterior thalamus; sub-
thalamus; nucleus basalis of Meynert; amygdala; midbrain
(two levels), pons and medulla oblongata; cerebellar cor-
tex and dentate nucleus. Tissue sections were stained with
hematoxylin and eosin, and with Kluver-Barrera, or pro-
cessed for immunohistochemistry. After incubation with
methanol and normal serum, the sections were incubated
with one of the primary antibodies at 4�C overnight.
Antibodies to glial fibrillary acidic protein (GFAP, Dako,
Barcelona, Spain), bA4-amyloid (Boehringer-Mannheim,
Barcelona, Spain) and ubiquitin (Dako) were used at
dilutions of 1:250, 1:50, and 1:200, respectively. CD68
(Dako), as a marker of microglia, was diluted at 1:100.
AT8 antibody (Innogenetics, Barcelona, Spain) was used
at a dilution of 1:50. Phospho-specific tau rabbit poly-
clonal antibodies Ser199, Ser202, Ser262, Ser396 and
Ser422 (all of them from Calbiochem, Barcelona, Spain)
were diluted at 1:100. Rabbit polyclonal anti-a-synuclein
antibody (Chemicon, Barcelona, Spain) was used at a
dilution of 1:3,000, and mouse monoclonal anti-phos-
phorylated a-synuclein Ser129 antibody (Wako, USA) at a
dilution of 1:2,000. Anti-nitrated a-synuclein antibody
(Zymed, Madrid, Spain) was used at a dilution of 1:2,000.
TDP-43 was examined using two different antibodies:
a mouse monoclonal antibody (Abnova, Tebu-Bio, Bar-
celona, Spain) raised against a full-length recombinant
human TARDBP, used at a dilution of 1:1,000, and a
rabbit polyclonal antibody (Abcam, Cambridge, UK)
raised against a synthetic peptide corresponding to
C-terminal (aa 350-414) of human TARDBP, used at a
dilution of 1:2,000. Rabbit polyclonal anti-HNE anti-
body (Calbiochem) was used at a dilution of 1:50;
monoclonal anti-SOD1 antibody was used at dilution of
1:50 (Novocastra, Servicios Hospitalarios, Barcelona,
Spain), and rabbit anti-SOD2 was diluted at 1:100
(Stressgen, Bionova, Madrid, Spain).
Sections processed for b-amyloid and a-synuclein
immunohistochemistry were first pre-treated with formic
acid, and incubated with methanol and H2O2 in PBS and
normal serum. Following incubation with the primary
antibody, the sections were incubated with EnVision?
system peroxidase (Dako) for 15 min at room temperature.
The peroxidase reaction was visualized with diam-
inobenzidine and H2O2. Control of the immunostaining
included omission of the primary antibody; no signal was
obtained following incubation with only the secondary
antibody. Sections were lightly counterstained with
hematoxylin. Some sections processed for a-synuclein
immunohistochemistry were pre-treated with proteinase K.
PD-related pathology and AD-related pathology were
graded following Braak stages [5, 6]. Age-matched control
cases were chosen from among a large series on the basis
of the lack of neurological, metabolic and mental disorders,
together with the lack of brain lesions including the
absence of neurofibrillary tangles (even in the entorhinal
cortex), a-synuclein inclusions, TDP-43 abnormalities, and
lack of small vascular disease. A few diffuse b-amyloid
plaques were the only abnormality in the temporal and
orbital cortices in two cases.
Frozen samples of the substantia nigra were only
available in case 1 and, therefore, this region was not the
subject of biochemical studies.
Sample preparation
Frozen samples (0.1 g) of the frontal cortex (area 8) in
LRRK2 cases and controls were homogenized separately in
10 volumes of ice-cold lysis buffer composed of 50 mM
Tris–HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% NaDeox,
0.1% SDS, 50 mM sodium orthovanadate, 1 mM PMSF
and a tablet of protease inhibitors (Roche Molecular Sys-
tems, Alameda, CA, USA), and were incubated by rotation
at 4�C for 1 h. Then, homogenates were centrifuged at
12,000 rpm for 12 min at 4�C. Pellets were discarded.
Protein concentrations were determined by the Bradford
method with bovine serum albumin (BSA) as a standard.
Gel electrophoresis and western blotting
For western blot studies, samples containing 20 lg of pro-
tein were loaded onto 10% acrylamide gels. Proteins were
separated by sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (PAGE) and electrophoretically trans-
ferred to nitrocellulose membranes (200 mA/membrane,
90 min). Immediately afterward, the membranes were
incubated with 5% skimmed milk in TBS-T buffer (100 mM
Tris-buffered saline pH 7.4, 140 mM NaCl and 0.1% Tween
20) for 1 h at room temperature, and then incubated with one
of the primary antibodies in TBS-T containing 3% BSA
(Sigma, Madrid) at 4�C overnight. The following antibodies
were used: rabbit polyclonal anti-a-synuclein (Chemicon),
raised against the 111–131 human a-synuclein residues and
used at a dilution of 1:2,000, nitrated a-synuclein (Zymed,
Madrid, Spain; 1:2,000), superoxide dismutase 1 (SOD1;
Novocastra, UK; 1:10,000), superoxide dismutase 2 (SOD2;
Stressgen, Bionova, Madrid, Spain; 1:8,000), DJ1 (Abcam,
Cambridge, UK; 1:2,000), phospho-a-synuclein Ser129
(a-synuclein-PSser129; Epitomics, Barcelona, Spain; 1:3,000),
malondialdehyde-lysine (MDAL; Calbiochem, Barcelona,
Spain; 1:500), 4-hydroxynonenal (HNE; Calbiochem,
Acta Neuropathol (2010) 120:155–167 157
123
Barcelona, Spain; 1:1,000), advanced glycation end prod-
ucts (AGE; Transgenic, Japan; 1:1,000), and AGE receptors
(RAGE; Serotec, Madrid, Spain; 1:1,000). Anti-GFAP
antibodies (Dako) were used at a dilution of 1:250. Mem-
branes processed for HNE western blotting were first
incubated with a reducer solution of 10 mM NaBH4 in TBS
(100 mM Tris-buffered saline, 140 mM NaCl, pH 7.4) and
then blocked with a solution of 5% skimmed milk in TBST
for 1 h at room temperature.
Subsequently, the membranes were incubated with the
corresponding secondary antibody labeled with horseradish
peroxidase (Dako, Barcelona, Spain) used at a dilution of
1:1,000 for 45 min at room temperature and developed
with chemiluminescence ECL Hyperfilm (Amersham
Biosciences, Barcelona, Spain).
Statistical analysis
The densitometric quantification of western blot bands was
carried out with Total Laboratory v2.01 software (Phar-
macia, Uppsala, Sweden). Densitometric values in each
case were normalized using b-actin. Every case was pro-
cessed in triplicate and patient mean values were obtained.
After this aggregation only one data value per case was
obtained, resulting in three independent values per group.
Data were analyzed by Mann–Whitney’s U test with sta-
tistical software SPSS (PASW). All data are given as
mean ± SEM. Comparisons between control and LRRK2
group were considered statistically significant when the
level of significance was \0.05. The error bars correspond
to the standard error of the mean (SEM). All statistical
analyses were performed in an exploratory way and do not
provide confirmatory statistical evidence.
Solubility and aggregation of a-synuclein
a-Synuclein solubility and aggregation were analyzed as
detailed elsewhere [13]. In short, brain samples (0.2 g)
from the frontal cortex (area 8) were homogenized in a
glass homogenizer in 1 ml of ice-cold PBS (sodium
phosphate buffer, pH 7.0, plus protease inhibitors), soni-
cated and centrifuged at 2,744 rpm at 4�C for 5 min.
The pellet was discarded and the resulting supernatant
was ultra-centrifuged at 54,000 rpm at 4�C for 1 h. The
supernatant (S2) was kept as the PBS-soluble or cytosolic
fraction. The resulting pellet was re-suspended in a solution
of PBS, pH 7.0, containing 0.5% sodium deoxycholate, 1%
Triton and 0.1% SDS, and then ultra-centrifuged at
54,000 rpm at 4�C for 1 h. The resulting supernatant (S3)
was kept as the deoxycholate-soluble fraction. The corre-
sponding pellet was re-suspended in a solution of 2% SDS
and maintained at room temperature for 1 h. Immediately
afterward, the samples were centrifuged at 54,000 rpm at
25�C for 1 h, and the resulting supernatant (S4) was the
SDS-soluble fraction.
Equal amounts of each fraction were mixed with
reducing sample buffer and processed for 10% SDS–PAGE
electrophoresis and western blot analysis. The membranes
were incubated with rabbit polyclonal anti-a-synuclein
(Chemicon), produced against residues 111–131, used at a
dilution of 1:2,000 or guinea-pig anti-a-synuclein (Cal-
biochem), raised against residues 123–140, diluted at
1:500, or anti-nitrated a-synuclein (Zymed) used at
1:2,000, or anti-phosphorylated a-synuclein Ser129 anti-
body (Wako) at a dilution of 1:2,000. Protein bands were
visualized with the ECL method.
2D gel electrophoresis, western blotting
and identification of lipoxidized proteins
by mass spectrometry
Samples of the frontal cortex (area 8) of control and dis-
eased cases were processed in parallel. A total of 200 lg of
protein was mixed with 2D lysis buffer composed of
40 mM Tris pH 7.5 containing 7 M urea, 2 M thiourea plus
0.2% Byolites (v/v), 4% CHAPS (Bio-Rad, Barcelona,
Spain), 2 mM TBP, and 0.1% bromophenol blue in a final
volume of 150 ll. For first dimension electrophoresis,
200 lg of protein was applied onto 7 cm pH 3–10 non-
linear gradient ReadyStrip IPG strips (Bio-Rad). After
rehydration of the strips for 24 h, proteins were focused at
500 V for 1 h, 1,000 V for 8 h, 10,000 V for 4 h, and 50 V
for 5 h. Strips were incubated in equilibration buffer
composed of 50 mM Tris–HCl pH 6.8, 6 M urea, 1% SDS,
30% glycerol, and 2% dithiothreitol (Sigma). A second
equilibration step was performed incubating the strips for
10 min in equilibration buffer containing 2.5% iodoaceta-
mide (Bio-Rad). All strips were placed onto 10%
polyacrylamide gels, and, for the second dimension, gels
were run at 0.02 A per gel. For gel staining, a mass spec-
trometry-modified silver staining method was used as
described by the supplier (Amersham Biosciences). Con-
trol and diseased samples were run in parallel. In every
case, one gel was stained with silver while the other gel
was transferred to a nitrocellulose membrane at 200 mA
per membrane for 90 min. The anti-MDAL and anti-HNE
antibodies (Calbiochem) were used at a dilution of 1:500
and 1:1,000, respectively, in TBST containing 3% BSA.
Anti-GFAP antibodies (Dako) were used at a dilution of
1:250. After incubation with the primary antibody, the
membranes were washed for 5 min in TBST three times.
The horseradish peroxidase-linked secondary antibody
(Dako) diluted at 1:1,000 in 5% skimmed milk in TBST
was then added for 1 h at room temperature. Membranes
were washed and developed by exposure to ECL Hyperfilm
(Amersham Biosciences).
158 Acta Neuropathol (2010) 120:155–167
123
In-gel digestion
Proteins were in-gel digested with trypsin (Sequencing
grade modified, Promega, Barcelona, Spain) in the auto-
matic Investigator ProGest robot of Genomic Solutions.
Briefly, excised gel spots were washed sequentially with
ammonium bicarbonate buffer and acetonitrile. Proteins
were reduced with 10 mM dithiothreitol solution for 30 min
and alkylated with 100 mM solution of iodine acetamide for
15 min. After sequential washings with buffer and aceto-
nitrile, proteins were digested overnight at 37�C with
trypsin 0.27 nM. Tryptic peptides were extracted from the
gel matrix with 10% formic acid and acetonitrile. The
extracts were pooled and dried in a vacuum centrifuge.
Acquisition of mass spectrometry and MS/MS spectra
Proteins manually excised from the 2D gels were digested
and analyzed by CapLCnano-ESI-MS-MS mass spectrome-
try. The tryptic-digested peptide samples were analyzed
using on-line liquid chromatography (CapLC, Micromass-
Waters, Manchester, UK) coupled with tandem mass spec-
trometry (Q-TOF Global, Micromass-Waters). Samples were
re-suspended in 12 ll of 10% formic acid solution and 4 ll
was injected for chromatographic separation into a reverse-
phase capillary C18 column (75 lm internal diameter and
15 cm in length, PepMap column, LC Packings, Amster-
dam). The eluted peptides were ionized via coated nano-ES
needles (PicoTipTM, New Objective, Woburn, MA, USA). A
capillary voltage of 1,800–2,200 V was applied together with
a cone voltage of 80 V. The collision in the collision-induced
dissociation was 25–35 eV and argon was employed as the
collision gas. Data were generated in PKL file format and
submitted for database searching in MASCOT server (Matrix
Science, Boston, MA, USA). NCBI database was used with
the following parameters: trypsin enzyme, 1 missed cleav-
age, carbamidomethyl (C) as fixed modification, and
oxidized (M) as variable modification, and mass tolerance of
150–250 ppm. A probability-based MOWSE score was used
to determine the level of confidence in the identification from
the mass spectra. MOWSE scores greater than 50 were
considered to indicate a high confidence of identification. All
the experiments were carried out in triplicate.
Results
Neuropathological findings in LRRK2 cases
Case 1
The brain weight was 1,100 g. The macroscopic exami-
nation revealed atherosclerosis grade I/II and loss of
pigmentation of the substantia nigra. The microscopical
study demonstrated loss of neurons in the substantia nigra
pars compacta together with free neuromelanin in the
neuropil and slight astrocytic gliosis and microgliosis. LBs
and a-synuclein-immunoreactive neurites were present in
the substantia nigra, locus ceruleus, dorsal nucleus of the
vagus nerve and hippoglossus, reticular formation, nucleus
basalis of Meynert, hypothalamus, nucleus subthalamicus,
hippocampus, amygdala, transentorhinal cortex, anterior
gyrus cinguli and spinal cord. LBS and LNs were also
stained with anti-phosphorylated a-synuclein Ser129 and
anti-nitrated a-synuclein antibodies. The olfactory bulb
was not available for study. Diffuse slight astrocytic gliosis
occurred in the cerebral cortex and striatum. In addition, a
few neurofibrillary tangles were observed in the entorhinal
and transentorhinal cortices. b-Amyloid plaques were
absent. Main neuropathological changes were consistent
with PD-associated pathology stage 5 of Braak, and AD-
related pathology stage I–II/0.
Case 2
The brain weight was 1,120 g. The macroscopic exami-
nation revealed atherosclerosis grade II, an old infarct
(22912920 mm) in the right occipital lobe and depig-
mentation of the substantia nigra. The microscopical study
revealed LBs and a-synuclein-immunoreactive neurites in
the substantia nigra, locus ceruleus, dorsal nucleus of the
vagus nerve and hippoglossus, reticular formation, nucleus
basalis of Meynert, amygdala and olfactory bulb. LBS and
LNs were also stained with anti-phosphorylated a-synuc-
lein Ser129 and anti-nitrated a-synuclein antibodies. In
addition, neurofibrillary tangles and threads were present in
the entorhinal and perirhinal cortices, subiculum, hippo-
campus, nucleus basalis of Meynert, amygdala and locus
ceruleus. Diffuse plaques were present in the temporal
cortex. Moderate astrocytic gliosis was seen in the deep
layers of cerebral cortex and white matter. Main neuro-
pathological changes were consistent with PD-associated
pathology stage 4 of Braak, AD-related pathology stage III/
A, and old right occipital infarction.
Case 3
The brain weight was 1,160. The macroscopic examination
disclosed atherosclerosis grade II/III, a lacunar infarct in
the head of the right caudate and slight depigmentation of
the substantia nigra. The microscopic study revealed loss of
neurons in the substantia nigra pars compacta together with
free neuromelanin in the neuropil and astrocytic gliosis.
Remaining neurons of the substantia nigra were unre-
markable excepting for the presence of Marinesco bodies.
LBs and neurites, and a-synuclein-immunoreactive or
Acta Neuropathol (2010) 120:155–167 159
123
ubiquitin-positive inclusions were absent in the substantia
nigra and other nuclei of the brain stem. In fact, the use of
frozen samples which were fixed in formalin for morpho-
logical studies, in addition to the initial formalin-fixed
samples, excluded any possibility of biochemical analysis
of the substantia nigra in this case. Moreover, a-synuclein-
immunoreactive inclusions were not seen in any brain
region, including the amygdala, nucleus basalis of Meyn-
ert, hippocampus, spinal cord, temporal cortex, insular
cortex and frontal cortex. In the same line, anti-phosphor-
ylated a-synuclein Ser129 and anti-nitrated a-synuclein
antibodies revealed no morphological alterations. A few
neurofibrillary tangles were found in the entorhinal and
perirhinal cortex but not in other regions; more specifically
hyperphosphorylated tau deposits were absent in the sub-
stantia nigra. b-Amyloid plaques were not seen in any
region. Moderate astrocytic gliosis was seen in the striatum
and cerebral cortex. The main neuropathological diagnoses
were non-Lewy type PD, AD-related pathology stage II/0,
small blood vessel disease and lacunar infarct in the right
caudate.
Mono-dimensional gel electrophoresis
and western blotting
Gel electrophoresis of total homogenates of the cerebral
cortex and western blotting for a-synuclein showed a band
of about 17 kDa in control and diseased cases. No differ-
ences between LRRK2 cases and controls were seen in the
total levels of a-synuclein of 17 kDa. Levels of nitrated
a-synuclein of 17 kDa and a-synuclein-PSer129 were higher,
although not statistically significant, in LRRK2 cases when
compared with controls (Fig. 1). No differences in the
expression levels of DJ1 and SOD1 were detected when
comparing LRRK2 cases and controls (Fig. 1). Cortical
SOD2 levels were significantly higher in LRRK2 cases
Fig. 1 Gel electrophoresis and western blotting for nitrated
a-synuclein, phosphorylated a-synuclein Ser129, SOD1, SOD2 and
DJ1 in the frontal cortex (area 8) of three cases of PD linked with the
G2019S mutation, and three controls. b-Actin is used as a control of
protein loading. Accompanying diagrams show the mean val-
ues ± SEM of the three controls and the three LRRK2 cases.
Significant differences were found regarding SOD2 in LRRK2 cases
when compared with controls (Mann–Whitney’s U test, *p \ 0.05)
160 Acta Neuropathol (2010) 120:155–167
123
when compared with controls (Fig. 1). Images selected for
Figs. 1, 2, 3, 4, and 5 are representative of one of the three
replicas carried out per sample.
Solubility and aggregation of a-synuclein
a-Synuclein (about 17 kDa) was recovered in the PBS-
soluble (cytosolic) and SDS-soluble fractions in control and
diseased cases. In addition, a-synuclein-immunoreactive
bands of high molecular weight of about 40 kDa were
recovered in the cytosolic and SDS fractions in LRRK2
cases 1 and 3 using the Chemicon antibody (residues
111–131); a faint band of about of 25 kDa was seen in the
PBS fraction in LRRK2 case 1; finally, one band of about
38 kDa occurred in case 2 in deoxycholate fraction. These
modifications in synuclein solubility and aggregation were
not seen in control cases (Fig. 2). A different pattern was
found using the Calbiochem antibody (residues 123–140).
A band of about 25 kDa was present in the PBS-soluble
fractions in G2019S cases, particularly in case 2, but not in
controls, whereas the bands of 40 kDa were only barely
stained. The band of 17 kDa was clearly identified in con-
trol and diseased cases (Fig. 2). Anti-nitrated a-synuclein
antibodies showed weak bands of about 40 kDa in LRRK2
cases 1 and 3 (Fig. 2). Anti-phosphorylated a-synuclein
antibodies disclosed faint bands of 25 kDa in the PBS
fraction of LRRK2 cases 1 and 2. No phosphorylated
bands were recovered in the deoxycholate and SDS frac-
tions (Fig. 2). Together, these findings show aggregated
a-synuclein of about 40 kDa partially nitrated but not
phosphorylated in cases 1 and 3. A different pattern of
aggregation was seen in case 2 with aggregates of synuclein
Fig. 2 Solubility and aggregation of a-synuclein in the frontal cortex
(area 8) in PD cases linked with the G2019S mutation and two
controls processed in parallel. In addition to the band of about 17 kDa
in all the samples, bands of higher molecular weight of about 40 kDa
(arrows) are seen in the cytosolic- (PBS-) and SDS-soluble fractions
in G2019S cases 1 and 3 using the Chemicon antibody raised against
111–131 of human a-synuclein. These bands were also recognized
with anti-nitrated synuclein antibodies, but barely with Calbiochem
antibodies raised against residues 123–140. In contrast, a band of
25 kDa (arrowhead) was seen in case 2 with the Calbiochem but not
with the Chemicon antibodies in the PBS fraction in case 2. This band
also appeared to be nitrated as seen with anti-nitrated synuclein
antibodies. Anti-phosphorylated synuclein antibodies disclose faint
bands of 25 kDa in the PBS fraction in LRRK2 cases 1 and 2
(arrowhead). Such band in case 1 seems to correspond to the faint
band of 25 kDa disclosed with the Chemicon antibody (arrowhead).
No phosphorylated synuclein aggregates are recovered in deoxycho-
late and SDS fractions
Acta Neuropathol (2010) 120:155–167 161
123
of 25 kDa in PBS fraction recognized with antibodies
directed to the C-terminal. Phosphorylated a-synuclein in
aggregates was very limited to light bands of about 25 kDa
in PBS fractions in LRRK2 cases 1 and 2.
Gel electrophoresis, western blotting and mass
spectrometry identification of GFAP as a target
of increased oxidative damage in LRRK2 cases
Several MDAL- and HNE-immunoreactive bands were
found in control and diseased cases. Increased MDAL and
HNE immunoreactivity of bands between 38 and 45 kDa
occurred in LRRK2 cases, mainly in cases 1 and 3 (Fig. 3).
Increased AGE immunoreactivity in the three diseased
cases was predominant in bands of about 70 kDa (Fig. 3),
thus indicating different substrates of MDAL, HNE and
AGE adducts. In line with these observations, increased
immunoreactivity to RAGE was present in LRRK2 cases
when compared with controls (Fig. 3). Significant differ-
ences (Mann–Whitney’s U test; *p \ 0.05) were found
between control and LRRK2 cases regarding total levels of
MDAL and RAGE.
Bi-dimensional gel electrophoresis and western blotting
to anti-MDAL and anti-HNE showed increased immuno-
reactivity of spots between 35 and 45 kDa in LRRK2 cases
compared with controls (Fig. 4). Parallel silver stained gels
were used to identify the oxidized spots. In-gel digestion
and mass spectrometry identified GFAP as the main oxi-
dized protein. Tubulin b4 and enolase 2 were also
identified as targets of oxidative damage (Table 1).
Fig. 3 Gel electrophoresis and western blotting for MDAL, HNE,
AGE and RAGE in the frontal cortex (area 8) of the three cases of PD
linked with the G2019S mutation, and three controls. Proteins were
separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) at 12 or 10%. b-Actin is used as a control
of protein loading. Increased immunoreactivity is found in LRRK2
cases when compared with controls. Anti-MDAL recognizes different
expression levels of bands of about 40 kDa and below (arrows) in
LRRK2 cases. Anti-HNE discloses two differential bands of about
40 kDa in cases 1 and 3 (arrows). Anti-AGE antibodies recognize
increased immunoreactivity of bands of about 70 kDa (arrow) in the
three LRRK2. Anti-RAGE antibodies recognize several bands the
intensity of which is increased in PD cases bearing the G2019S
mutation (arrows). Significant differences were found regarding
MDAL and RAGE in LRRK2 cases when compared with controls
(Mann–Whitney’s U test, *p \ 0.05)
162 Acta Neuropathol (2010) 120:155–167
123
GFAP oxidative damage is not a mere result
of the total amount of GFAP
In order to learn whether oxidative damage to GFAP in
LRRK2 cases was a mere reflection of the total amount of
GFAP, mono-dimensional gel electrophoresis and western
blotting disclosed similar GFAP immunoreactivity in
G2019S cases 2 and 3, and control 3, whereas lower levels
were seen in G2019S case 1 and in controls 1 and 2. Bi-
dimensional gel electrophoresis and western blotting further
confirmed high levels of GFAP in control 3 and G2019S
cases 2 and 3 when compared with G2019S case 1 (Fig. 5).
These results did not match with the predicted amount of
oxidized GFAP when compared with control versus patho-
logic cases (see control 3 and G2019S 3 in Fig. 4 for
comparison), thus indicating that the amount of oxidized
GFAP is not merely dependent on the total amount of GFAP.
Discussion
The present study has shown increased oxidative damage,
increased oxidative stress responses, abnormal a-synuclein
solubility and aggregation, and increased a-synuclein
nitration in the frontal cortex (area 8) in cases of PD
associated with the G2019S LRRK2 mutation in whom
parkinsonian symptoms were not accompanied by apparent
cognitive deficits. The limited number of cases selected for
study was due to the fact that other cases were excluded
because of accompanying neuropathological changes that
may bias molecular observations (i.e. advanced stages of
AD, multi-infarct encephalopathy and Binswanger dis-
ease), and post-mortem delays longer than 12 h that may
modify the expression of oxidative stress markers [20].
Similar changes in a-synuclein solubility and aggrega-
tion are seen in sporadic PD, other a-synucleinopathies and
related transgenic models [3, 13, 17, 30, 33, 38]. Yet these
biochemical abnormalities are not accompanied by the
presence of LBs in the cerebral cortex in the present series.
In the same line, changes of a-synuclein solubility and
aggregation are found independently of the presence of
intracytoplasmic a-synuclein inclusions in transgenic mice
models [13]. Together these findings suggest that bio-
chemical aggregates of a-synuclein and a-synuclein
nitration are not necessarily manifested as LBs. The rea-
sons for abnormal a-synuclein solubility, aggregation and
nitration in LRRK2 cases are not known, but oxidative
stress favors a-synuclein aggregation in vitro [28, 45, 50,
64, 70]. Furthermore, two cases had aggregates of synuc-
lein of about 40 kDa, whereas another case has aggregates
of 25 kDa which are recognized with antibodies directed to
the C-terminal, thus probably indicating that aggregates are
composed of different synuclein variants.
Phosphorylation of a-synuclein at Ser129 is another
dominant pathological modification in human a-synuc-
leinopathies [1, 22, 53]. Phosphorylated a-synuclein at
Ser129 also accumulates in the nervous system in a-syn-
uclein transgenic mice and flies, partly associated with
inclusions in a limited number of neurons [44, 62, 68].
Phosphorylation of a-synuclein of 17 kDa was not
Fig. 4 Bi-dimensional gel electrophoresis and western blotting for
HNE and MDAL in control case 3 and in the three PD cases linked
with the G2019S mutation. Spots of about 40 kDa and below are
strongly expressed in PD cases when compared with the control.
In-gel digestion and mass spectrometry of the corresponding spots in
parallel gels disclosed GFAP, tubulin and enolase as the MDAL-
modified protein. Spot numbers correspond to those identified in
Table 1
Acta Neuropathol (2010) 120:155–167 163
123
significantly increased in the present cases when compared
with age-matched controls. Yet, faint aggregates of 25 kDa
in PBS fractions were observed in cases 1 and 2.
The functional consequences of a-synuclein modifica-
tions are still not clear. In vitro, a-synuclein phosphory-
lation, among other factors, facilitates fibril formation,
whereas nitration of a-synuclein facilitates folding and
aggregation into octamers but prevents fibrillation [49, 62,
63, 69]. Although phosphorylated and nitrated a-synuclein
accumulate in LBs and LNs in a-synucleinopathies [1, 2,
16, 22, 26], the present findings suggest that nitrated
a-synuclein is not restricted to aberrant inclusions. There-
fore, the present findings, in line with observations in
animal models, support the notion that alterations in vivo
are more complex than those predicted by in vitro models.
Additional factors, such as autophagy and the ubiquitin
proteasomal system of protein degradation, play key roles
in the formation of aberrant intracytoplasmic and neuritic
inclusions in LBDs.
Increased oxidative stress and oxidative damage are
major contributory factors in the pathogenesis of PD.
Increased expression of oxidative markers, increased oxi-
dative damage to selected proteins, and increased oxidative
responses have been observed in the cerebral cortex in
sporadic PD even at relatively early stages of the disease
[9–12, 14, 27, 71]. Significant increased expression of the
oxidative stress marker MDAL has been found in the frontal
cortex in the three PD cases associated with G2019S LRRK2
mutation. This is accompanied by increased expression
levels of RAGE and SOD2. Moreover, bi-dimensional gel
electrophoresis, western blotting and mass spectrometry
have identified GFAP as a target of protein oxidation in the
cerebral cortex of G2019S LRRK2 mutation. Tubulin b4 and
enolase 2 were also identified as targets of oxidative dam-
age. This finding does not exclude the presence of further
oxidized proteins, as only a few spots stained with anti-
MDAL antibodies were selected for study. HNE-modified
proteins and putative proteins with AGE adducts were not
processed for bi-dimensional gel electrophoresis, western
blotting and mass spectrometry; and total homogenates
rather than enriched fractions were analyzed. This is an
important point as the different band patterns obtained with
the different markers of lipoxidative and glycoxidative
stress were different thus suggesting that particular oxida-
tive modifications affect different proteins. Enolase was
also identified as a target of oxidation in the frontal cortex in
sporadic PD [27], and tubulins are common targets of oxi-
dative damage in the cerebral cortex in AD [40, 61].
Increased oxidative stress and oxidative damage to pro-
teins are not specific to PD as they are a common feature in
Fig. 5 GFAP expression in the frontal cortex in LRRK2 cases [1–3]
linked to the G2019S mutation and three controls. GFAP immuno-
reactivity is similar in G2019S cases 2 and 3, and control 3, whereas
lower levels are seen in G2019S and controls 1 and 2. Bi-dimensional
gel electrophoresis and western blotting further confirm high levels of
GFAP in control 3 and cases G2019S 2 and 3 when compared with
G2019S. These marks are in contrast with the spots corresponding to
oxidized GFAP (spots 1, 2 and 3) in Fig. 4, thus indicating that GFAP
oxidative damage is not a mere expression of the total level of GFAP
Table 1 Identification of
oxidized proteins, as revealed
with MDAL, excised from 2D
gels
Spot Protein Molecular
weight (kDa)
pI MOWSE
score
Peptides
matched
ID number
1 GFAP 49,907 5.42 1,029 28 gi 4503979
2 GFAP 49,907 5.42 403 8 gi 4503979
3 GFAP 49,907 5.42 340 7 gi 4503979
4 Enolase 2 47,581 4.91 1,046 21 gi 5803011
5 Tubulin beta, 4 50,010 4.78 569 15 gi 21361322
164 Acta Neuropathol (2010) 120:155–167
123
several degenerative diseases of the nervous system [8, 40,
61]. Increased oxidative damage to GFAP occurs in AD,
tauopathies (Pick’s disease, progressive supranuclear palsy,
frontotemporal lobar degeneration associated with mutation
in the tau gene, and argyrophilic grain disease) and Hun-
tington’s disease [35, 41, 43, 47, 54, 57]. In this line, the
present findings indicate that (1) the cerebral cortex is
affected by increased oxidative stress and increased oxi-
dative damage to proteins, (2) GFAP is one of the targets of
oxidative damage, and (3) other proteins are also targets of
oxidative stress in the cerebral cortex in LRRK2 cases.
Interestingly, no mention of a-synuclein inclusions is
found in the description of brain lesions in LRRK2 trans-
genic mice [39]. Unfortunately, biochemical studies of a-
synuclein are not yet available in this model. Evidence of
altered a-synuclein solubility and aggregation, and post-
translational modifications of a-synuclein in this model, will
help make clear whether observations in the cerebral cortex
in individuals with PD due to G2019S LRRK2 mutation are
reproducible in corresponding transgenic models.
In summary, the present observations, although limited
to three cases, have shown involvement of the cerebral
cortex in PD linked with the G2019S LRRK2 mutation in
the absence of overt cognitive deficits. Since these changes
are similar to those encountered in sporadic PD cases with
no clinical symptoms, and not subjected to any treatment,
the present observations are likely primary events in the
natural course of PD rather than consequences of long-term
anti-parkinsonian drug administration. Involvement of the
cerebral cortex in PD linked to G2019S LRRK2 mutation
without apparent cognitive deficits stresses the necessity
for refining clinical methods to detect early impairment of
cortical function in PD, and further supports the concept
that PD linked to G2019S LRRK2 mutation is a systemic
disease not restricted to the motor system.
Acknowledgments This work was funded by grants from the
Spanish Ministry of Health, Instituto de Salud Carlos III PI08/0582,
and supported by the European Commission under the Sixth Frame-
work Programme BrainNet Europe II, LSHM-CT-2004-503039 and
INDABIP FP6-2005-IFESCIHEALTH-7 Molecular Diagnostics.
Thanks to T. Yohannan for editorial help.
Conflict of interest statement There is no conflict of interest
including any financial, personal or other relationships with other people
or organizations within the 3 years from the beginning of the work.
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