Neuropathology of sporadic Parkinson disease before the appearance of parkinsonism: Preclinical Parkinson disease

Abstract

Parkinson disease (PD) is no longer considered a complex motor disorder characterized by parkinsonism but rather a systemic disease with variegated non-motor deficits and neurological symptoms, including impaired olfaction, sleep disorders, gastrointestinal and urinary abnormalities and cardiovascular dysfunction, in addition to other symptoms and signs such as pain, depression and mood disorders. Many of these alterations appear before or in parallel with motor deficits and then worsen with disease progression. Although there is a close relation between motor symptoms and the presence of Lewy bodies (LBs) and neurites filled with abnormal α-synuclein, other neurological alterations are independent of LBs, thereby indicating that different mechanisms probably converge in the degenerative process. This review presents cardinal observations at very early stages of PD and provides personal experience based on the study of a consecutive series of brains with PD-related pathology and without parkinsonism, mainly cases categorized as stages 2-3 of Braak. Alterations in the substantia nigra, striatum and frontal cortex in pPD are here revised in detail. Early modifications in the substantia nigra at pre-motor stages of PD (preclinical PD: pPD) include abnormal small aggregates of α-synuclein which is phosphorylated, nitrated and oxidized, and which exhibits abnormal solubility and truncation. This occurs in association with a plethora of altered molecular events including increased oxidative stress, altered oxidative stress responses, altered balance of L-ferritin and H-ferritin, reduced expression of neuronal globin α and β chains in neurons with α-synuclein deposits, increased expression of endoplasmic reticulum stress markers, increased p62 and ubiquitin immunoreactivity in relation to α-synuclein deposits, and altered distribution of LC3 and other autophagosome/lysosome markers. In spite of the relatively small decrease in the number of dopaminergic neurons in the substantia nigra, which does not reach thresholds causative of parkinsonism, levels of tyrosine hydroxylase and cannabinoid 1 receptor are reduced, whereas levels of adenosine receptor 2A are increased in the caudate in pPD. Moreover, biochemical alterations are also present in the cerebral cortex (at least in the frontal cortex) in pPD including increased oxidative stress and oxidative damage to proteins α-synuclein, β-synuclein, superoxide dismutase 2, aldolase A, enolase 1, and glyceraldehyde dehydrogenase, among others, indicating post-translational modifications of PD-related proteins, and suggesting altered function of pathways involved in glycolysis and energy metabolism in the cerebral cortex in pPD. Current evidence suggests convergence of several altered metabolic pathways leading to chronic neuronal dysfunction, mainly manifested as sub-optimal energy metabolism, altered synaptic function, oxidative and endoplasmic reticulum stress damage and corresponding altered responses, among others. By understanding that these alterations occur at very early stages of PD and that neuronal fatigue and exhaustion may precede, for years, cell death and neuronal loss, we may direct therapeutic strategies towards the prevention and delay of disease progression starting at pre-parkinsonian stages of PD.

Full text

BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - REVIEW ARTICLE
Neuropathology of sporadic Parkinson disease
before the appearance of parkinsonism:
preclinical Parkinson disease
Isidre Ferrer •Anna Martinez •Rosa Blanco •
Ester Dalfo
´•Margarita Carmona
Received: 26 July 2010 / Accepted: 30 August 2010 / Published online: 23 September 2010
ÓSpringer-Verlag 2010
Abstract Parkinson disease (PD) is no longer considered
a complex motor disorder characterized by parkinsonism
but rather a systemic disease with variegated non-motor
deficits and neurological symptoms, including impaired
olfaction, sleep disorders, gastrointestinal and urinary
abnormalities and cardiovascular dysfunction, in addition
to other symptoms and signs such as pain, depression and
mood disorders. Many of these alterations appear before or
in parallel with motor deficits and then worsen with disease
progression. Although there is a close relation between
motor symptoms and the presence of Lewy bodies (LBs)
and neurites filled with abnormal a-synuclein, other neu-
rological alterations are independent of LBs, thereby
indicating that different mechanisms probably converge in
the degenerative process. This review presents cardinal
observations at very early stages of PD and provides per-
sonal experience based on the study of a consecutive series
of brains with PD-related pathology and without parkin-
sonism, mainly cases categorized as stages 2–3 of Braak.
Alterations in the substantia nigra, striatum and frontal
cortex in pPD are here revised in detail. Early modifica-
tions in the substantia nigra at pre-motor stages of PD
(preclinical PD: pPD) include abnormal small aggregates
of a-synuclein which is phosphorylated, nitrated and oxi-
dized, and which exhibits abnormal solubility and trunca-
tion. This occurs in association with a plethora of altered
molecular events including increased oxidative stress,
altered oxidative stress responses, altered balance of
L-ferritin and H-ferritin, reduced expression of neuronal
globin aand bchains in neurons with a-synuclein deposits,
increased expression of endoplasmic reticulum stress
markers, increased p62 and ubiquitin immunoreactivity in
relation to a-synuclein deposits, and altered distribution of
LC3 and other autophagosome/lysosome markers. In spite
of the relatively small decrease in the number of dopami-
nergic neurons in the substantia nigra, which does not reach
thresholds causative of parkinsonism, levels of tyrosine
hydroxylase and cannabinoid 1 receptor are reduced,
whereas levels of adenosine receptor 2A are increased in
the caudate in pPD. Moreover, biochemical alterations are
also present in the cerebral cortex (at least in the frontal
cortex) in pPD including increased oxidative stress and
oxidative damage to proteins a-synuclein, b-synuclein,
superoxide dismutase 2, aldolase A, enolase 1, and glyc-
eraldehyde dehydrogenase, among others, indicating post-
translational modifications of PD-related proteins, and
suggesting altered function of pathways involved in gly-
colysis and energy metabolism in the cerebral cortex in
pPD. Current evidence suggests convergence of several
altered metabolic pathways leading to chronic neuronal
dysfunction, mainly manifested as sub-optimal energy
metabolism, altered synaptic function, oxidative and
endoplasmic reticulum stress damage and corresponding
altered responses, among others. By understanding that
these alterations occur at very early stages of PD and that
neuronal fatigue and exhaustion may precede, for years,
cell death and neuronal loss, we may direct therapeutic
strategies towards the prevention and delay of disease
progression starting at pre-parkinsonian stages of PD.
Keywords Preclinical Parkinson disease 
Incidental Lewy body disease Oxidative stress 
I. Ferrer (&)A. Martinez R. Blanco E. Dalfo
´
M. Carmona
Institut de Neuropatologia, Servei Anatomia Patolo
`gica,
CIBERNED, IDIBELL-Hospital Universitari de Bellvitge,
Universitat de Barcelona, carrer Feixa LLarga sn,
08907 Hospitalet de LLobregat, Spain
e-mail: 8082ifa@gmail.com
123
J Neural Transm (2011) 118:821–839
DOI 10.1007/s00702-010-0482-8

Endoplasmic reticulum stress Haemoglobin 
Mitochondria Olfaction Cerebral cortex Striatum 
Synuclein Glycolysis
Introduction
Parkinson disease is clinically characterized by a complex
motor disorder known as parkinsonism and is manifested
principally by resting tremor, slowness of initial move-
ment, rigidity and general postural instability. These
symptoms are mainly due to the loss of dopaminergic
neurons in the substantia nigra pars compacta, first the
lateral tier followed by the medial region, leading to
reduced dopaminergic input to the striatum, and accom-
panied by adaptive responses in the internal and external
globus pallidus, subthalamus, thalamus and substantia
nigra pars reticularis. Round, hyaline neuronal cytoplasmic
inclusions called Lewy bodies (LBs), and enlarged aberrant
neurites and threads are found in the parkinsonian sub-
stantia nigra (Forno 1996; Jellinger and Mizuno 2003). In
addition to the substantia nigra, other nuclei are involved
such as the locus coeruleus, reticular nuclei of the brain
stem, and dorsal motor nucleus of the vagus, as well as the
basal nucleus of Meynert, the amygdala and the CA2 area
of the hippocampus. LBs and aberrant neurites as well are
found in these locations (Forno 1996; Braak et al. 1999;
Dickson 2001; Goedert 2001; Jellinger and Mizuno 2003;
Braak and del Tredici 2008).
Cases with LB pathology in the brain stem without
parkinsonism are considered incidental LB disease because
they are unexpectedly discovered following appropriate
post-mortem neuropathological study (Jellinger 2004,
2009; Saito et al. 2004). Whether these cases constitute
pre-parkinsonian PD has been a matter of controversy for
some years, as nobody can ensure that these cases would
have progressed to parkinsonism if they had survived for
longer times. However, the study of consecutive cases in
large series and the recognition of several intermediate
degrees of involvement of the brain stem, limbic structures
and, eventually, the cerebral cortex make it clear that a
prediction of preclinical PD (pPD) as an anterior stage of
PD seems more than reasonable (Jellinger and Mizuno
2003; Braak and del Tredici 2008; Dickson et al. 2008).
LBs and neurites are composed of aggregates of normal,
misfolded and truncated proteins, and ubiquitin, which are
stored in the cytoplasm as non-degraded by-products of the
degenerative process (Schults 2006; Wakabayashi et al.
2007; Leverenz et al. 2007; Xia et al. 2008). The main
component of LBs and aberrant neurites is a-synuclein
which is abnormally phosphorylated, nitrated and oxidized,
has an abnormal crystallographic structure and abnormal
solubility, and is prone to the formation of aggregates and
insoluble fibrils (Spillantini et al. 1997; Wakabayashi et al.
1997; Baba et al. 1998; Hashimoto and Masliah 1999;
Duda et al. 2000; Giasson et al. 2000; Fujiwara et al. 2002;
Anderson et al. 2006).
Systematic study of cases with LB pathology has
prompted a staging classification of PD based on the
putative progression of LB pathology from the medulla
oblongata (and olfactory bulb) to the midbrain, dience-
phalic nuclei, and neocortex (Braak et al. 2002,2003,
2004). Stage 1 is characterized by LBs and neurites in the
dorsal IX/X motor nuclei and/or intermediate reticular
zone; there is also myenteric plexus involvement. Stage 2
affects the medulla oblongata and pontine tegmentum and
covers pathology of stage 1 plus lesions in the caudal raphe
nuclei, gigantocellular reticular nucleus, and coeruleus–
subcoeruleus complex; the olfactory bulb is also involved.
Stage 3 refers to pathology of stage 2 plus midbrain lesions,
particularly in the pars compacta of the substantia nigra.
Stage 4 includes basal prosencephalon and mesocortex
(cortical involvement confined to the transentorhinal region
and allocortex, and CA2 plexus) pathology in addition to
lesions in the midbrain, pons and medulla oblongata. Stage
5 extends to sensory association areas of the neocortex and
prefrontal neocortex. Stage 6 includes, in addition, lesions
in first order sensory association areas of the neocortex and
pre-motor areas; occasionally there are also mild changes
in primary sensory areas and the primary motor field.
This classification shows an acceptable correlation
between pathological findings and clinical data, mainly in a
subgroup of cases with early onset and prolonged duration
in which motor symptoms (parkinsonism) are clearly
dependent on the damage of dopaminergic neurons in the
substantia nigra pars compacta (Braak et al. 2002,2005;
Jellinger 2004; Wolters and Braak 2006). Thus, stages
1 and 2 are not accompanied by parkinsonism and they
fulfil the criteria of pPD. Cases at stage 3 may present with
parkinsonism depending on the amount of cell loss in the
substantia nigra. More strictly, pPD occurs in cases with
LB pathology limited to selected nuclei of the medulla
oblongata and pons, and in cases with a few LBs in the
substantia nigra but without substantial nigral neuron loss
not surpassing a determined threshold, usually considered
as 60% of the total population of dopaminergic neurons
of the pars compacta (Jellinger and Mizuno 2003;
DelleDonne et al. 2008).
Cumulative clinical evidence reveals that olfactory
dysfunction, dysautonomia, sleep fragmentation, rapid eye
movement behaviour disorder, mood and anxiety disorders,
and depression may precede parkinsonian symptoms in a
number of patients with PD clinically characterized by
parkinsonism (Chaudhuri et al. 2005; Postuma et al. 2006;
Iranzo et al. 2006; Siderowf and Stern 2008; Tolosa et al.
2007; Poewe 2007,2008; Ziemssen and Reichmann 2007;
822 I. Ferrer et al.
123

Herting et al. 2008; Ross et al. 2008; Natale et al. 2008).
Whether these clinical symptoms are associated with LBs
in selected regions of the central, autonomous and
peripheral nervous systems is a matter of study.
a-Synuclein pathology affecting neurons and neurites
occurs in the olfactory bulb and related olfactory nuclei at
very early stages of cases with PD-related pathology
(Daniel and Hawkes 1992; Pearce et al. 1995; Del Tredici
et al. 2002; Braak et al. 2004; Bloch et al. 2006; Hubbard
et al. 2007).
The cardiovascular autonomic system is also affected in
pPD and PD, and alterations implicate both tyrosine
hydroxylase-positive (extrinsic) and negative (intrinsic)
nerves of the cardiac plexus (Iwanaga et al. 1999).
Together, these observations can be summarized as
follows: (a) variegated non-motor deficits and neurological
symptoms, covering impaired olfaction, sleep disorders,
gastrointestinal and urinary abnormalities and cardiovas-
cular dysfunction, in addition to other symptoms and signs
as pain, depression and mood disorders, may occur at early
stages of the disease. (b) A relationship between neuro-
logical symptoms and Lewy pathology exists, but it is not
clear whether the severity of morphological lesions corre-
lates with clinical symptoms. (c) Olfactory tests, poly-
somnographic studies and MIBG myocardial scintigraphy
in combination may be used to discover early signatures of
the disease (Stiasny-Kolster et al. 2005).
Lack of correlation between a-synuclein aggregates and
impaired function is dramatically represented in relation
with cognitive and mental functions. Retrospective clinical
and pathologic studies have shown that there is no rela-
tionship between LB stage and severity of cognitive
impairment in advanced stages of PD (Parkkinen et al.
2005; Weisman et al. 2007; Parkkinen et al. 2008; Jellinger
2008,2009).
These and other studies have led to the visualization of
PD as a systemic disease in which functional deficits are
not exclusively related with LB pathology in neurons and
neurites. This is dramatically manifested in relation with
cerebral cortex involvement in PD in which variegated
pathological molecular events converge to alter neuronal
function (Ferrer 2009a).
Neuropathological examination of pPD in a cohort
of consecutive cases in a general population
Over the last 10 years (2001–2010), 115 cases of LB-rela-
ted pathology were recovered in a consecutive series of
cases subjected to post-mortem study in a general hospital
(Institut de Neuropatologia, Hospital Universitari de
Bellvitge). These were classified as stages 1–2: 24%, stages
3–4: 42%, stages 5–6: 26%, and atypical: 8%. Interestingly,
mixed pathology was very common. AD-related pathology
was found in 81% of cases (40% stages I–II of Braak, 46%
corresponding to stages III–IV, and 14% to stages V and
VI), argyrophilic grain disease and other tauopathies in
24%, vascular pathology in 28%, and other pathological
conditions in 12%. Some cases had multiple pathologies
(mainly LB disease, AD and vascular pathology including
lacunae, multi-infarct encephalopathy, haemorrhages and
demyelination of the centrum semi-ovale reminiscent of
Binswanger encephalopathy). These figures are similar to
those already reported in other series (Jellinger 2004,2008,
2009,2010; Braak et al. 2006; Markesbery et al. 2009;
Beach et al. 2009; Dickson et al. 2010).
The distribution of PD-related pathology in our series
has a gradient with age; cases with stages 5 and 6 are older
as a group than cases at stage 4. However, a number of
cases at stages 1 and 2 were remarkably aged. More than
half were older than 75, indicating that, within the same
age interval, some patients had suffered from fully devel-
oped PD whereas others had remained at pre-motor stages.
These findings are inline with the observation that, at least
some cases of incidental LB disease could represent pre-
clinical PD, arrested PD, or a partial syndrome due to a
lesser burden of causative factors (Frigerio et al. 2009).
For the present purposes, parkinsonism (including slight
motor symptoms, revealed by relatives following an
unbiased post-mortem interview in cases that had not been
diagnosed during life) occurred in 68% of the cases.
Excluding atypical cases, pPD was seen in 28 cases of
stages 1–2, 10 cases of stage 3, and 3 cases of stage 4.
Morphological studies of the substantia nigra were carried
out in this group. In none of these cases did nerve cell loss
in the substantia nigra exceed 50% of the total number of
dopaminergic neurons as revealed by quantitative studies
using tyrosine hydroxylase immunohistochemistry. A sum-
mary of antibodies used for immunohistochemistry, and
single- and double-labelling immunofluorescence and
confocal microscopy, is given in Table 1.
Molecular studies in the frontal cortex were restricted to
20 pPD cases with no associated pathology or with
accompanying AD-related changes stages I–II, to exclude
cases in which cortical pathology could interfere with
changes interpreted to be exclusive of pPD. Control sam-
ples were from cases with no neuropathological changes or
with AD-related pathology stages I–II. Methods employed
were mono- and bi-dimensional gel electrophoresis and
western blotting, immunoprecipitation and functional
assays (Dalfo
´et al. 2004,2005; Dalfo
´and Ferrer 2008;
Muntane
´et al. 2008; Martı
´nez et al. 2010).
The present observations are mainly focused on the
substantia nigra pars compacta, caudate and cerebral cortex
(frontal cortex area 8) in pPD. The olfactory bulb and tract
also deserve a brief comment.
Neuropathology of sporadic Parkinson disease 823
123

The substantia nigra in pre-motor stages of PD
It is classically considered that the substantia nigra in pre-
motor stages of PD is not morphologically affected or
damaged to an extent not surpassing a threshold currently
accepted as involving less than 60% of the total of dopa-
minergic neurons of the substantia nigra pars compacta.
Using the staging nomenclature of PD-related pathology in
our series, pre-motor or incidental PD may correspond to
stages 1–3. Surprisingly, parkinsonian symptoms have
been reported in stage 2 of Braak (Jellinger 2009). This is
not our experience: parkinsonian symptoms and signs
appear at stage 3 in our series and stage 3 may be also
encountered in pPD cases in which decline of substantia
nigra dopaminergic neurons does not exceed 50% of the
total. Since vascular pathology is not rare in ours and in
other series, we cannot rule out the possibility of primary
vascular lesions in the striatum may account for motor
deficits in cases with PD-related pathology stage 2 in other
series.
a-Synuclein in the substantia nigra
An important issue is the relative amount of a-synuclein in
the substantia nigra pars compacta when compared with
other brain regions. Studies in normal human brain are
puzzling since it is not clear whether the substantia nigra
and striatum had the lowest (Rockenstein et al. 2001)or
the highest levels (Solano et al. 2000)ofa-synuclein.
Regarding PD, comparative studies of control and diseased
Table 1 Antibodies, species,
source and dilution used for
immunohistochemistry and
immunofluorescence in the
present study
Tissue sections were pre-treated
with formic acid for 3 min and
then with 10 mM sodium citrate
buffer pH 6 for 20 min at 95°C
Antibody Species Manufacturer Dilution
ATP-synthase aMouse BD Biosciences (Madrid, Spain) 1:1,000
Casein kinase II Rabbit Stressgen (bioNova, Madrid, Spain) 1:100
COX7A2 Mouse Abnova (Taipei, Taiwan) 1:50
eif2aRabbit Abcam (Cambridge, UK) 1:100
eif2a-P Rabbit Cell signalling (Izasa, Barcelona, Spain) 1:100
Ferritin Goat Affinity Bioreagents (bioNova, Madrid, Spain) 1:500
Haemoglobin (bchain) Mouse Abcam (Cambridge, UK) 1:200
8-Hydroxy-2
deoxyguanosine
Mouse Abcam (Cambridge, UK) 1:200
LAMP-1 Rabbit Sta. Cruz (Quimigen, Barcelona) 1:100
LAMP-2 Rabbit Sta. Cruz (Quimigen, Barcelona) 1:500
LC-3 Rabbit Cell signalling (Izasa, Barcelona, Spain) 1:100
Neuroketals Goat Chemicon (Millipore, Madrid, Spain) 1:200
Oxphos Mouse Mitosciences (Eugene, Oregon, USA) 1:1,000
Phospho-p38 Rabbit Cell signalling (Izasa, Barcelona, Spain) 1:100
p62 Rabbit BIOMOL (Quimigen, Madrid, Spain) 1:500
Peroxiredoxin-2 Rabbit Abcam (Cambridge, UK) 1:1,000
VDAC, porin Mouse Calbiochem (bioNova, Madrid, Spain) 1:300
SOD-1 (Cu/Zn) Mouse Novocastra (Newcastle, UK) 1:50
SOD-2 (Mn) Rabbit Stressgen (bioNova, Madrid, Spain) 1:100
a-Synuclein Mouse Novocastra (Newcastle, UK) 1:100
a-Synuclein Rabbit Chemicon (Millipore, Madrid, Spain), epitope
111–131
1:500
a-Synuclein Rabbit Abcam (Cambridge, UK), epitope 11–26 1:100
a-Synuclein Guinea
pig
Calbiochem (bioNova, Madrid, Spain), epitope
123–140
1:50
a-Synuclein Mouse ATGen (Montevideo, Uruguay), epitope 61–90 1:100
Nitrated a-synuclein Mouse Zymed (San Francisco, CA, USA) 1:300
Phospho-a-synuclein Mouse WAKO (Rafer, Madrid, Spain) 1:500
Transferrin Rabbit Affinity Bioreagents (bioNova, Madrid, Spain) 1:300
Ubiquitin Rabbit DAKO (Barcelona, Spain) 1:500
8-dOHG Mouse Abcam 1:50
Tyrosine hydroxylase Mouse Chemicon 1:1,000
CB-1 receptor Rabbit Frontier Science 1:200
824 I. Ferrer et al.
123

brains are not illuminating. a-Synuclein mRNA levels in
the PD substantia nigra have been reported to be unmodi-
fied, increased, or decreased when compared with age-
matched controls (Rockenstein et al. 2001; Wirdefeldt et al.
2001; Kingsbury et al. 2004; Chiba-Falek et al. 2006).
Cleaved (truncated) fragments of a-synuclein are prone to
aggregate in vitro and have been considered prime com-
ponents in LBs (Murray et al. 2003; Li et al. 2005).
Our recent studies have shown consistently lower levels
of a-synuclein protein expression in the human substantia
nigra and nucleus basalis of Meynert when compared with
other brain regions independent of age and pathology.
Phosphorylated a-synuclein at Ser129 is increased in the
same regions, thus correlating with the total amount of
a-synuclein. Additionally, truncated a-synuclein is natu-
rally observed in control and diseased brains, correlating
with the total amount of a-synuclein (Muntane
´et al., in
preparation).
a-Synuclein pathology and Lewy inclusions
in the substantia nigra in pPD
Phosphorylated a-synuclein at Ser129 is considered a key
step in the development of a-synuclein aggregates
(Anderson et al. 2006). Phosphorylated a-synuclein at
Ser129 co-localizes with a-synuclein in punctuate Lewy-
type aggregates in the substantia nigra at stage 3 of Braak
(Fig. 1a–c). It is difficult to ascertain the amount of phos-
phorylated a-synuclein of the total a-synuclein on the basis
of immunohistochemistry using antibodies that may have
different binding capacities to their corresponding sub-
strates. However, double-labelling studies suggest that
phosphorylated a-synuclein at Ser129 predominates in LB
lesions in pPD. Similar pattern was seen in affected nuclei
of the medulla oblongata and affected neurons in the sub-
stantia nigra in pPD. Casein kinase II, one of the enzymes
that phosphorylates a-synuclein at Ser129 (Ishii et al. 2007;
Waxman and Giasson 2008), is currently found in LB in
the substantia nigra in advanced stages of PD (Ryu et al.
2008). Yet casein kinase II is also observed in neurons with
early a-synuclein inclusions not sequestered by LBs in pPD
(Fig. 1d–f), indicating that increased casein kinase II
expression is associated with increased a-synuclein phos-
phorylation at early stages of PD-related pathology. Other
activated kinases, the functions of which have not been
clearly identified in PD, have also been reported in asso-
ciation with LBs. One of them, active p38 (p38-P), is
accumulated in cytoplasmic granules in the vicinity of
LBs or in association with irregular-shaped or diffuse
a-synuclein deposits in a percentage of substantia nigra
pars compacta neurons in PD (Ferrer et al. 2001) and in a
few neurons in pPD at stage 3 (Fig. 1g–i). In addition,
nitrated a-synuclein is also found in small granules in
dopaminergic neurons before the appearance of LBs in
pPD. Finally, oxidative damage of a-synuclein has also
been found using biochemical methods in the substantia
nigra in pPD (Dalfo
´and Ferrer 2008). These observations
indicate that modifications of a-synuclein, including oxi-
dation, nitration and phosphorylation at Ser129, are early
events in the substantia nigra at stage 3 of PD and occur in
neurons with punctuate a-synuclein aggregates.
Truncated a-synuclein variants, detected with antibodies
raised against different C- and N-terminal epitopes of
a-synuclein, are components of punctate a-synuclein
aggregates and LBs (Fig. 2).
These observations indicate that altered expression of
proteins is not restricted to LBs, at least at early stages of
LB formation. Rather, altered expression of proteins is seen
in the vicinity but not within LBs in pPD. On the other
hand, the structure of LBs is not homogeneous but different
forms of a-synuclein are differentially incorporated into
LBs.
Mitochondria dysfunction
Deficiencies in complex I subunits of the respiratory chain
and impaired activity of the electron transport chain are
well-known abnormalities in the substantia nigra in spo-
radic PD with parkinsonism (Mizuno et al. 1989; Parker
et al. 1989; Schapira 2008; Onyango 2008).
Early studies showed no modifications in the activity of
mitochondrial complexes II, III and IV in pPD (Jenner
et al. 1992; Dexter et al. 1994). More especially, complex I
activity in pPD was normal or reduced to intermediate
levels among controls and cases with PD (Jenner et al.
1992; Dexter et al. 1994). However, studies in total
homogenates do not permit the detection of modifications
restricted to individual neurons. The use of immunofluo-
rescence with appropriate markers offers limited informa-
tion, as oxphos, porin, cytochrome Coxidase subunits and
ATP synthase immunoreactivities are preserved in neurons
of the substantia nigra pars compacta in stage 3 of Braak,
independent of the presence or absence of a-synuclein
inclusions (data not shown). Whether subunits of mito-
chondrial complex I are oxidatively damaged, functionally
impaired and misassembled in pPD substantia nigra as
reported in PD (Keeney et al. 2006) is not known.
Oxidative stress
Reduced glutathione levels are decreased in the substantia
nigra in pPD and they are considered one of the first
alterations in the substantia nigra in PD (Jenner et al. 1992;
Jenner 1993,1998; Dexter et al. 1994; Zeevalk et al. 2008).
Oxidative damage in the substantia nigra is already
present in pPD at stages 2 and 3 of Braak (Dalfo
´et al.
Neuropathology of sporadic Parkinson disease 825
123

Fig. 1 Double-labelling immunofluorescence and confocal micros-
copy of a-synuclein (green) and a-synuclein PSer129 (red)(a–c),
casein kinase II (green) and a-synuclein (red)(d–f), and p38 kinase-P
(green) and a-synuclein (red)(g–i) in the substantia nigra pars
compacta in pPD stage 3. a-Synuclein co-localizes with a-synuclein
PSer129 in several but not all punctiform, neuritic and cytoplasmic
aggregates. Casein kinase II and p38 kinase-P are expressed in the
cytoplasm of neurons with a-synuclein aggregates. p38-P also co-
localizes with synuclein in LBs. j–lNegative controls without
primary antibodies
826 I. Ferrer et al.
123

2005). This is further documented by increased neuroketal
(NKT) immunofluorescence in neurons with a-synuclein
deposits and by the presence of NKTs in LBs (Fig. 3a–c).
Neuroketals are a class of compounds that result from the
oxidation of docosahexaenoic acid (DHA), a membrane
polyunsaturated fatty acid especially vulnerable to free
radical attack, which is present in large amounts in the
brain. Therefore, the presence of neuroketals indicates
oxidation of DHA in substantia nigra dopaminergic neu-
rons at early stages of PD.
Oxidative stress responses are also augmented in neurons
with a-synuclein pathology. Expression of peroxiredoxin 2,
superoxide dismutase 1 (SOD1) and SOD2 is increased in
neurons with a-synuclein inclusions (Fig. 3d–l). In addition
to cytoplasmic increase in SOD1 and SOD2, these proteins
can also be sequestered in LBs (Fig. 3j–l). It is interesting to
have in mind that SOD2 has been reported oxidatively
damaged in PD (Choi et al. 2005; Dalfo
´et al. 2005). Thus,
proteins produced to counteract oxidative stress are, in turn,
oxidatively damaged and sequestered into LBs.
In the line evidencing early oxidative damage in the
substantia nigra in pPD is the observation of lipoxidative
damage of a-synuclein in pPD (Dalfo
´and Ferrer 2008).
Therefore, available observations show that oxidative
Fig. 2 Triple-labelling immunofluorescence and confocal micros-
copy of a-synuclein inclusions in the substantia nigra in pPD. Triple
labelling with antibodies directed to the NAC region (AB9, green),
N-terminal (AB2, red) and C-terminal (AB8, blue)ofa-synuclein
show slight differences in the staining of a-synuclein inclusions and
LBs in the substantia in pPD. Several punctiform inclusions are
differentially stained with the different anti-a-synuclein antibodies
whereas co-localization of all three antibodies is found in LB (white
in the merge construction)
Neuropathology of sporadic Parkinson disease 827
123

Fig. 3 Double-labelling immunofluorescence and confocal micros-
copy of neuroketal (green) and a-synuclein (red)(a–c), peroxiredoxin
(green) and a-synuclein (red)(d–f), superoxide dismutase 2 (green)
and a-synuclein (red)(g–i), and superoxide dismutase 1 (green) and
a-synuclein (red)(j–l) in the substantia nigra pars compacta in pPD
stage 3. Markers of oxidative damage and markers of oxidative stress
responses are expressed in neurons of the substantia nigra. In addition,
neuroketal, SOD1 and SOD2, but rarely peroxiredoxin accumulate in
LBs
828 I. Ferrer et al.
123

damage is an early event in PD and that oxidative damage
in the substantia nigra in stage 2 precedes the formation of
LBs in pPD substantia nigra.
Further biochemical studies have shown increased levels
of neuroketals in the substantia nigra in cases with PD-
related pathology stages 1 and 2 (Fig. 4), thus further
supporting that oxidative damage to specific lipids in the
substantia nigra occurs at very early stages of PD before
the appearance of regional a-synuclein aggregates. These
observations indicate early oxidative damage to lipids and
certain proteins. In addition, evidence of DNA damage in
the substantia nigra in pPD is shown in Fig. 4.
Iron
Iron plays an important role in the regulation of several
enzymatic activities and in redox modulation. Iron dysho-
meostasis has been observed in PD (Andersen 2004;
Salazar et al. 2006; Hirsch 2006; Barnham and Bush 2008;
Youdim 2008). Neuromelanin has a protective function
under physiological conditions contributing to iron
homeostasis (Double 2006). However, neuromelanin
lipoxidation, altered a-synuclein binding to neuromelanin
and increased levels of L-ferritin in neuromelanin have
harmful effects on dopaminergic cell fate (Halliday et al.
2005; Double and Halliday 2006; Zecca et al. 2006,2008;
Tribl et al. 2009). Iron is increased in the substantia nigra in
PD and it is stored in glial cells and macrophages at the
time that neuromelanin decreases in vulnerable neurons
(Gerlach et al. 2006; Fasano et al. 2006). In addition,
increased iron levels have been found in individual neurons
in PD (Oakley et al. 2007). As a result, increased iron
levels, and particularly increased levels of labile iron, may
increase iron-mediated oxidative damage and contribute to
redox imbalance in PD (Wang et al. 2008; Chinta and
Andersen 2008; Wypijewska et al. 2010).
No evidence of alteration in iron, copper, manganese or
zinc in the pPD substantia nigra was noted in early studies
(Jenner et al. 1992). However, recent observations have
shown that L-ferritin concentration in the substantia nigra
is lower in pPD (and PD) when compared with controls,
whereas H-ferritin in PD is higher than in pPD and con-
trols, thus indicating fine abnormalities in iron metabolism
in the substantia nigra at early stages of PD (Koziorowski
et al. 2007).
Neuronal globin aand bchain in the substantia
nigra in pPD
Recent studies have demonstrated the presence of globin a
and bin neurons in the mouse, rat and human brains, using
robust combined methods including transcriptome analysis
(cDNA microarrays and nanoCAGE) of laser-captured
micro-dissected neurons, quantitative transcriptase-poly-
merase chain reaction (RT-PCR), in situ hybridization and
immunohistochemistry with a panel of different specific
antibodies (Biagioli et al. 2009; Richter et al. 2009). Nerve
cells also express erythropoietin receptor, erythropoietin
and hypoxia-inducible factor Hif1a(Matsuda et al. 1994;
Digicaylioglu et al. 1995).
Globin aand bis present in dopaminergic neurons of the
substantia nigra, and its expression is reduced in vulnerable
neurons in parallel with abnormal a-synuclein deposition
(Fig. 5). In contrast, the expression of neuroglobin and
erythropoietin receptor is preserved in the same cells.
These preliminary data point to the possibility that
decreased globin aand bmay play a role in the patho-
genesis of PD.
Endoplasmic reticulum stress
Accumulation of misfolded proteins can trigger a reticu-
lum stress response which is manifested by the activation
of certain markers such as pancreatic endoplasmic retic-
ulum kinase (PERK) and eukaryotic initiation factor 2 a
(eIF2a). Phosphorylated PERK and phosphorylated eIF2a
expression is increased in dopaminergic neurons of the
substantia nigra in advanced stages of PD (Hoozemans
et al. 2007). Increased eIF2aand phosphorylated eIF2aare
also found in neurons of the substantia nigra pars com-
pacta, they partly co-localize with a-synuclein inclusions
in pPD (Fig. 6). Together, these observations support the
idea that endoplasmic reticulum stress plays a role in the
pathogenesis of early stages of PD (Wang and Takahashi
2007).
Protein degradation: the ubiquitin–proteasome system
and autophagy in iPD
LBs and neurites are manifestations of impaired protein
degradation in vulnerable cells. Since protein degradation
putatively occurs via cytosolic proteases, chaperone-
mediated autophagy (CMA), ubiquitin–proteasome system
(UPS) and micro-, macroautophagy, all these mechanisms
have been analysed in experimental models of PD. How-
ever, much less is known about what happens in PD.
Several in vitro studies have shown impaired UPS
function in PD (Olanow and McNaught 2006). In favour of
impaired UPS function is the cardinal early observation
that ubiquitin occurs in Lewy inclusions and that p62
immunoreactivity appears in association with abnormal
a-synuclein inclusions at early stages of LB (Kuusisto et al.
2003). However, the sequence of events is obscure. Mor-
phological evidence in PD neurons shows fine a-synuclein
deposition in the cytoplasm of selected neurons. Ubiquitin
and p62 decorates some of but not all these inclusions
Neuropathology of sporadic Parkinson disease 829
123

suggesting that abnormal synuclein deposition is a first step
followed by ubiquitination of a-synuclein and association
of p62 to polyubiquitinated proteins (Kuusisto et al. 2003;
Nakaso et al. 2004). More recent observations have pro-
vided evidence of altered expression of components of the
UPS in PD (Chu et al. 2009). Moreover, some of them
appear to be modified by oxidation thus resulting in lost of
function (Choi et al. 2004; Chung et al. 2004; see later).
Therefore, altered degradation by the UPS of abnormal
a-synuclein and several hundred proteins associated with
LBs does probably depend on multiple factors (Shamoto-
Nagai et al. 2007; Chu et al. 2009).
The role of autophagy, including CMA and macro-
autophagy in PD, is postulated mainly on the basis of
observations in cell models (Stefanis et al. 2001; Cuervo
et al. 2004; Engelender 2008; Martı
´nez-Vicente et al.
2008; Xilouri et al. 2008, Vogiatzi et al. 2008; Mak et al.
2010). Little is known, however, about the role of
autophagy in human PD. Several morphological studies
have shown that neuromelanin in the substantia nigra is
associated with the autophagosome/lysosome system
(Anglade et al. 1997; Tribl et al. 2006) thus providing a
possible subtle link between a-synuclein and lysosomes in
the substantia nigra under normal conditions and in PD.
Certain autophagy markers are altered in the substantia
nigra in PD (Pan et al. 2008). More straightforward in
the present context is the observation that the expression
and localization of proteins linked with autophagy and
Fig. 4 a Increased neuroketal expression in total homogenates of the
substantia nigra in pPD (iPD) compared with controls (CTL).
Accompanying diagram illustrtes significant differences P\0.001
(Student’s ttest). bIncreased oxidative damage to nucleic acids of
glial cells in the substantia nigra of pPD at stage 3. Double-labelling
immunofluorescence and confocal microscopy to 8-hydroxyguanine
(8-dOHG) (green) and a-synuclein (red) shows green staining in the
nuclei of several glial cels but not in neurons with a-synuclein
inclusions. Nuclei (blue) are stained with TO-PRO
830 I. Ferrer et al.
123

Fig. 5 Double-labelling immunofluorescence and confocal micros-
copy of ferritin (green) and a-synuclein (red)(a–f), a-synuclein
(green) and globin a-chain (red) (G-L) in the substantia nigra pars
compacta in pPD stage 3. Ferritin is decreased in neurons with
a-synuclein inclusions (compare a–cwith d–f). Similarly, globin
a-chain expression is reduced in neurons with a-synuclein inclusions
(compare g–iwith j–l)
Neuropathology of sporadic Parkinson disease 831
123

lysosomes as microtubule-associated protein 1 light chain
3 alpha (LC3), lysosomal-associated protein 1 (LAMP1)
and LAMP2 (Klionsky et al. 2008) are altered in pPD
(Fig. 7). In the same line, recent findings suggest that
CMA activity is reduced in PD brain (Alvarez-Erviti et al.
2010).
Fig. 6 Double-labelling immunofluorescence and confocal micros-
copy of eif2a(green) and a-synuclein (red)(a–f), and eif2a(green)
and eif2a-P (red)(g–l) in the substantia nigra pars compacta in pPD
stage 3. eif2aco-localizes with a-synuclein inclusions (a–f). The
majority of eif2ais phosphorylated (g–l) thus suggesting activation of
the reticulum stress responses
832 I. Ferrer et al.
123

In summary, experiments in vitro strongly support the
idea of altered UPS and autophagosome/lysosome function
in models of altered a-synuclein turnover and clearance,
but precise information about the amount of a-synuclein
cleared by the UPS and the CMA systems is not known in
human disease.
Fig. 7 Double-labelling immunofluorescence and confocal micros-
copy of microtubule-associated protein 1 light chain 3 alpha (LC3)
(green) and a-synuclein (red)(a–c), lysosomal associated protein 2
(LAMP2) (green) and a-synuclein (red)(d–f), LAMP1 (green)
and a-synuclein (red)(g–h) in substantia nigra pars compacta in
pPD stage 3. Markers of autophagy are expressed in association with
a-synuclein aggregates. j–lNegative controls processed without
primary antibodies
Neuropathology of sporadic Parkinson disease 833
123

The basal ganglia in pPD
Even in the absence of motor symptoms, loss of neurons in
the substantia nigra translates into loss of dopaminergic
innervation and consequent remodelling of the putamen.
This is clearly illustrated by the loss of tyrosine hydroxy-
lase (TH) immunoreactivity and loss of vesicular mono-
amine transporter 2 (VMAT2) in the putamen in pPD, with
values that are intermediate between those in normal
individuals and in parkinsonian stages of PD (DelleDonne
et al. 2008; Dickson et al. 2008) (Fig. 8). These morpho-
logical aspects are also sustained by observations using
serial metabolic imaging with [(18)F]-fluorodeoxyglucose
positron emission tomography (PET) in sporadic cases
(Tang et al. 2010). Multitracer PET scans are, therefore,
useful tools to unveil dopaminergic dysfunction in patients
with suspected pPD.
The balance of other molecules is also modified in the
striatum in pPD. Leu-encephalin levels are reduced in the
putamen and undetectable in the substantia nigra in PD, but
Leu-encephalin levels are only barely reduced in the
putamen in pPD (Fernandez et al. 1996).
Adenosine receptors 2A (A
2A
Rs) in the striatum are
practically restricted to GABAergic neurons projecting
from the caudate and putamen to the external globus pal-
lidus, which also expresses dopamine D
2
receptors (D
2
Rs)
(Fuxe et al. 2007). A
2A
Rs expression levels in the striatum
are increased in PD and correlate with motor symptoms
(Varani et al. 2010). Recently, we have seen increased
A
2A
R expression levels in the caudate in cases with pPD
stage 3 of Braak, thus indicating that this is an early
abnormality of the denervated striatum (Buira et al. 2010).
Finally, early and pre-symptomatic stages of PD are
associated with desensitization/downregulation of type 1
cannabinoid (CB1) receptors, whereas advanced stages are
characterized by up-regulatory responses of CB1 receptors
(Garcı
´a-Arencibia et al. 2009). Whether down-regulation
of CB1 receptors at early stages is pathogenic remains to be
elucidated; this may constitute an important putative target
for therapeutic intervention. Decreased CB1 receptor
expression is also observed in the caudate in pPD, showing
that this is a very early alteration in the course of PD
(Fig. 8).
The olfactory bulb and tract in pPD
In the present series, the morphological study of the
olfactory bulb and tract revealed the presence of small
numbers of neurons and neurites bearing a-synuclein
aggregates which are also recognized with antibodies
against nitrated a-synuclein and against phospho-specific
a-synuclein Ser129 antibodies (Fig. 9). However, it is hard
to assume that such a small number of structural anomalies
may account for the sophisticated olfactory deficits that
include not only loss of olfaction but also altered dis-
crimination of odours with increased perception in some
circumstances. It may be hypothesized that, as in other
regions, olfactory alterations in pPD and PD are the result
of more complicated settings resulting from several
molecular deficits.
The cerebral cortex in pPD
Altered behaviour, apathy and depression may occur in
patients with pPD (Poewe 2008; McKinlay et al. 2009;
Pedersen et al. 2009). The molecular substrates of such
alterations are scarcely known but pieces of knowledge are
rapidly growing (Ferrer 2009a).
Brain cortex and mitochondrial O
2
uptake and complex I
activity are significantly lower in PD, whereas mtNOS
activity, cytochrome content, expression of SOD2, mito-
chondrial mass, and oxidative damage are significantly
higher in the frontal cortex in PD. The decreases in tissue
and mitochondrial O
2
uptake and in complex I activity are
considered the consequences of mitochondrial oxidative
damage in the cerebral cortex in PD (Navarro et al. 2009).
Unfortunately, no similar data are available in pPD.
Recent observations have shown abnormal lipid com-
position in the frontal cortex in pPD which is manifested
Fig. 8 Expression levels of tyrosine hydroxylase (TH) and cannab-
inoid 1 receptor (CB-1 receptor) in the caudate nucleus in controls
and in pPD (iPD Braak 3). Reduced expression of TH (62 kDa) and
CB-1 receptor (52 kDa) is observed in diseased cases when compared
with controls (P\0.01, Student’s ttest)
834 I. Ferrer et al.
123

with significantly increased expression levels of the highly
peroxidizable DHA and increased peroxidability index
(Dalfo
´et al. 2005). More dramatically, several key proteins
are targets of oxidative damage in the frontal cortex, as
revealed by bi-dimensional gel electrophoresis, redox
proteomics and mass spectrometry, in pPD including
a-synuclein, b-synuclein and SOD2 (Dalfo
´et al. 2005;
Dalfo
´and Ferrer 2008). In addition, increased oxidative
damage of aldolase A, enolase 1 and glyceraldehyde
dehydrogenase (GAPDH), all of them involved in glycol-
ysis and energy metabolism, is found in the frontal cortex
in pPD (and PD as well) (Go
´mez and Ferrer 2009). Other
oxidatively damaged proteins are phosphoprotein enriched
in astrocytes 15, SH3 domain binding glutamic acid-rich
protein like, ubiquitin-conjugating enzyme E2N-like, pro-
hibitin, proteasome subunit Y and thioredoxin. Finally,
cortical synapses are abnormal in pPD (and PD), as tau
phosphorylation and a-synuclein phosphorylation are
increased in synaptic-enriched fractions of frontal cortex
homogenates (Muntane
´et al. 2008).
Concluding comments
Until recently, PD was considered to be a movement dis-
ease mainly characterized by the loss of dopaminergic
neurons and occurrence of LBs in the remaining cells in the
substantia nigra. This point of view has served to drive
major advances in the treatment of patients with PD,
including pharmacological and neurosurgical therapeutic
interventions geared to balancing dopamine deficits and
related neurotransmitters and modulators in the nigrostri-
atal system. However, PD is a systemic disease affecting
several distinct neuronal populations of the central and
peripheral nervous system, with the appearance of motor
symptoms in a substantial number of patients as the top of a
plethora of non-motor signs and symptoms affecting the
heart, gastrointestinal tract, olfaction, sleep and mental
functions. Moreover, neuropathological and clinical studies
have shown only a partial correlation between regional
a-synuclein inclusions and corresponding neurological
deficits at early stages of the disease, and no clear relation
between LBs, and motor and cognitive impairment.
Cumulative evidence in recent years indicates that mito-
chondrial dysfunction, oxidative stress and oxidative
damage to crucial proteins, post-translational modifications
of proteins, and endoplasmic reticulum stress converge in
the pathogenesis of PD. This is further supported by pro-
teomic studies showing dysregulation of proteins involved
in energy metabolism, oxidative stress, signal transduction,
electron transport and detoxification pathways in PD
(Srivastava et al. 2010). Furthermore, the use of redox
proteomics has revealed that protein altered by oxidative
damage may have an impact on protein function in spite of
apparently normal total levels of the protein (Martı
´nez
et al. 2010). Importantly, some of these changes occur at
very early stages of PD-related pathology in areas with no
accompanying Lewy-like inclusions, such as the substantia
Fig. 9 Images of the olfactory bulb at stages 2 and 3 of Braak. Scattered neurites and cell bodies are immunoreactive with anti-a-synuclein
antibodies. Paraffin sections slightly counterstained with haematoxylin
Neuropathology of sporadic Parkinson disease 835
123

nigra in stage 2, and the striatum and cerebral frontal cortex
in stages 2 and 3 of Braak. Furthermore, although still
patchy, several pieces of information suggest that failure in
energy metabolism may have an additive deleterious effect
on neuronal function. The term ‘exhausted neuron’ was
applied in the context of Alzheimer disease to describe the
particular situation of neurons working under suboptimal
conditions of reduced energy production, increased energy
demands and oxidative damage (Ferrer 2009b). This term
can also be applied to pPD and PD in which neurons suffer
from a convergence of intrinsic and external deficiencies
that may impair neuronal function and eventually result in
cell death. Recognition of this scenario helps us understand
day-to-day variations in neurological deficits, fluctuations,
apparent transient recoveries and subtle mining of neuronal
integrity in neurodegenerative disease. More importantly,
many of these alterations may be targets of therapeutic
intervention geared to delaying disease progression.
Acknowledgments Work carried out in the Institute of Neuropa-
thology was partially funded by grants from the Spanish Ministry of
Health, Instituto de Salud Carlos III PI05/1570, PI05/2214 and PI08/
0582, and supported by the European Commission: BrainNet Europe
II, LSHM-CT-2004-503039 and INDABIP FP6-2005-LIFESCI-
HEALTH-7 Molecular Diagnostics. Thanks to T. Yohannan for
editorial help.
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