J Neuropathol Exp Neurol
Copyright Ó 2007 by the American Association of Neuropathologists, Inc.
Vol. 66, No. 11
November 2007
pp. 965Y974
REVIEW ARTICLE
Protein Aggregation Mechanisms in Synucleinopathies:
Commonalities and Differences
Katrin Beyer, PhD and Aurelio Ariza, MD, PhD
Abstract
Synucleinopathies are characterized by the presence of different
types of >-synuclein (AS)-positive inclusion in the brain. Thus,
whereas Lewy bodies are the hallmark of Parkinson disease and
dementia with Lewy bodies, glial and neuronal cytoplasmic
inclusions are shown by multiple system atrophy. Because the
main component of all these inclusions is conformationally
modified AS, aggregation of the latter is thought to be a key
pathogenic event in these diseases. Although very little information
has been available on AS function and aggregation mechanisms
until 2 years ago, recent investigations have greatly improved our
understanding of the steps involved in the pathogenesis of
synucleinopathies. Additionally, important insights into the specific
molecular events (e.g. differential posttranslational modifications or
isoform expression profiles) underlying each of these conditions
have been gained. The present review summarizes our current
knowledge of the commonalities and differences shown by protein
aggregation mechanisms in the various synucleinopathies.
Key Words: >-Synuclein, Dementia with Lewy bodies, Glial
cytoplasmic inclusion, Inclusion body, Lewy body, Multiple system
atrophy, Parkinson disease.
INTRODUCTION
Parkinson disease (PD), dementia with Lewy bodies
(DLB), and multiple system atrophy (MSA) are considered
to be synucleinopathies because they share >-synuclein (AS)
aggregation into inclusion bodies as their key pathogenic
event. The molecular pathways leading to AS accumulation
in synucleinopathies show interesting commonalities and
differences and gaining an insight into these may significantly help to develop effective diagnostic and therapeutic
approaches to these conditions. After providing a brief
introduction to the various synucleinopathies, in this review
we describe the features of the inclusion bodies found in
these disorders, discuss the mechanisms of inclusion body
From the Department of Pathology, Hospital Universitari Germans Trias i
Pujol, Autonomous University of Barcelona, Badalona, Barcelona, Spain.
Send correspondence and reprint requests to: Aurelio Ariza, MD, PhD,
Department of Pathology, Hospital Universitari Germans Trias i Pujol,
08916 Badalona, Barcelona, Spain; E-mail: [email protected]
This work was supported by the Spanish Ministry of Health FIS Grants PI
030132 and PI 050867 and a Catalonia AGAUR Grant 2005SGR828 and
Marató TV3 Grant 06/1410.
formation, analyze the commonalities and differences
among synucleinopathies in regard to their inclusion body
protein content, and propose working hypotheses and future
research directions.
THE SYNUCLEINOPATHIES
Lewy Body Diseases and Parkinson Disease
First described in 1817, PD is the most common
progressive movement disorder in the elderly and is
characterized by tremor, rigidity, and bradykinesia. There
is increasing evidence that PD is a multisystemic disorder
showing both progressive degeneration of the dopaminergic
nigrostriatal system and widespread extranigral pathology
(1Y3). In PD, Lewy body (LB) pathology first appears in
lower brainstem nuclei such as the dorsal motor nucleus of
the vagus and the olfactory system (stages 1Y2). Afterwards,
ascending progression leads to changes in the coeruleus
complex, substantia nigra pars compacta, basal forebrain
magnocellular nucleus, subthalamic nucleus, and amygdala
(stages 3Y4). Finally, involvement of the neocortex may
supervene (stages 5Y6) (1, 2).
In the last few years advances in the genetics of PD
have revealed that mutations are responsible for a small
proportion of cases, whereas most instances are of sporadic
origin. So far, mutated genes have been found to be
responsible for Mendelian forms of PD. The first PD-related
gene identified was the AS (SNCA) gene, in which
pathogenic point mutations (A30P and A53T) were initially
detected (3, 4). Afterward, a third SNCA mutation (E46K)
was described in a large Spanish pedigree with autosomal
dominant parkinsonism, dementia, and visual hallucinations
(5). SNCA gene duplication and triplication have also been
observed in independent familial PD pedigrees showing a
gene dose-dependent disease course (6, 7). Other familial PD
cases have revealed mutations of the DJ-1 gene (8) and the
phosphatase and tensin homolog (PTEN)-induced putative
kinase 1 (PINK1) gene (9). On the other hand, a synphilin
mutation has been described in patients with sporadic PD
(10). Additionally, the ubiquitin C-terminal hydrolase L1
(UCH-L1) gene I93M mutation has been linked to autosomal
dominant PD (10), whereas the S18Y polymorphism of the
same gene has been associated with a significantly lower risk
of PD (11).
Interestingly, mutations of the leucine-rich kinase 2
(LRRK 2) gene encoding dardarin, a multidomain protein of
uncertain function, have been related to both familial PD
J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
965
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Beyer and Ariza
J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
(3%Y10%) and sporadic PD (1%Y8%) in several Europeanderived populations (12). Intriguingly, although the clinical
features of LRRK2-related PD are similar to those of
idiopathic PD, LRRK2-related PD cases show only a small
number of nigral LBs and very occasional cortical LBs (13).
Finally, parkin (PRKN) gene mutations are responsible for
approximately 50% of all autosomal recessive parkinsonism
cases of early onset and for 77% of cases with onset before
21 years of age (14). The vast majority of patients with PD
associated with PRKN mutations lack LBs (15) and
consequently are outside the scope of the present review.
Dementia With Lewy Bodies
DLB is the second most frequent cause of dementia in
the elderly after Alzheimer disease (AD) (16) and is
clinically characterized by progressive dementia, often
accompanied by parkinsonism and psychiatric symptoms
(17). Widespread distribution of LBs in virtually every brain
area is a typical feature of DLB, although the frontal cortex,
pigmented midbrain and brainstem nuclei, dorsal efferent
nucleus of the vagus, basal forebrain nuclei, and limbic
cortical regions are particularly involved (18). DLB cases
may be grouped into types according to their LB distribution:
the brainstem-predominant type, the limbic or transitional
type, and the diffuse neocortical type (17).
Recently revised consensus guidelines (19) for the
pathologic diagnosis of DLB propose a semiquantitative
severity grading of LB-related pathology into mild, moderate,
severe, and very severe cases. This system is based on
semiquantitative assessment of LB density rather than on
previously used counting methods (19). DLB is often
associated with variably extensive AD and is then termed
common Lewy body disease (18). In these cases, the
likelihood that the patient_s clinical syndrome is caused by
LB pathology is directly related to LB density and inversely
related to the severity of concurrent AD-type pathology (19).
An AS gene mutation (E46K) has been described in
a large Spanish pedigree encompassing patients with both
DLB and PD (5). Interestingly, an UCH-L1 gene I93M
mutation has also been detected in another large family with
patients with both PD and DLB (10). Additionally, mutations
(V70M and P123H) of A-synuclein, an AS homolog, have
been found in independent DLB cohorts (20).
Multiple System Atrophy
The term MSA was first proposed by Graham and
Oppenheimer in 1969 for a progressive neurodegenerative
disorder with parkinsonism, ataxia, and autonomic failure
(21). The etiology of this condition remains unknown (21).
Phenotypically, MSA forms with predominantly parkinsonian
symptoms are known as MSA-P and show overall atrophy of
the putamen. On the other hand, MSA forms with predominantly cerebellar symptoms are designated as MSA-C and
show cerebellar atrophy with Purkinje cell depletion, as well
as atrophy of the middle cerebellar peduncles, basis pontis,
and inferior olivary nuclei (22).
Glial cytoplasmic inclusions (GCIs) in oligodendroglial cells, a MSA neuropathologic hallmark, are accompanied by neuronal loss and astrocytosis in the striatonigral
966
system (16). Whereas in stage I MSA there is widespread
distribution of GCIs, neuronal loss is restricted to the
substantia nigra and locus coeruleus, sparing the striatum
(23). These findings suggest that MSA pathogenesis differs
substantially from that of most other neurodegenerative
disorders, in which neurons are primarily targeted (24).
When neurons finally become involved in MSA, they
develop neuronal cytoplasmic inclusions that are particularly
abundant in the anterior central gyrus and supplementary
motor cortex (25). Neuronal cytoplasmic inclusions are
round or ovoid structures that occupy the greater part of
the neuronal cytoplasm and, as shown by immunoelectron
microscopy, contain both granular and filamentous AS (26).
Some MSA cases may also show rare ubiquitin-positive
neuronal nuclear inclusions resembling those seen in motor
neuron disease. A semiquantitative 4-tiered grading system
has recently been proposed for both types of MSA (27).
Although no point mutations have been described in
MSA as yet, cases associated with either minimal GAA1
expansion of the gene frataxin (28) or FMR1 gene
premutation status have been reported (29). These findings
are in accord with MSA phenotypical variability.
INCLUSION BODIES IN SYNUCLEINOPATHIES
Lewy Bodies of PD and DLBs
Morphologically, LBs may be divided into brainstem
and cortical types. Whereas brainstem LBs are found in the
brainstem nuclei and diencephalon, cortical LBs are preferentially seen in the cerebral limbic cortex and amygdala.
Classic brainstem LBs are spherical, intraneuronal
cytoplasmic inclusions that measure 8 to 30 Km in diameter
and are characterized by hyaline eosinophilic cores, concentric lamellar bands, narrow pale halos, and immunoreactivity
for AS and ubiquitin (30). In contrast, cortical LBs typically
lack a halo (31). AS immunolabeling of PD brains has
revealed that AS immunostaining can be detected in approximately 64% of nigral LBs and 31% of cortical LBs (32).
Ultrastructurally, brainstem LBs are composed of
radially arranged 7- to 20-nm intermediate filaments associated with granular electron-dense material and vesicular
structures. Their core shows tightly packed filaments and
dense granular material, whereas radiating fibers occupy
their periphery (30). As expected from their uniform density
on light microscopy, cortical LBs are composed almost
entirely of circular or oval fibrillary material (33).
Although a number of proteins have been identified in
LBs, their precise biochemical composition has not yet been
elucidated (Table 1). Granulofilamentous accumulation of
AS has recently been shown in Purkinje cell axons in PD
and DLB brains, albeit if cerebellar pathology has not been
unequivocally demonstrated in LB diseases (34).
GCIs of Multiple System Atrophy
GCIs are faintly eosinophilic, sickle-shaped, oval or
conical inclusions that displace the nucleus eccentrically.
Their localization to microglia has been established by
double staining techniques (21). Ultrastructurally, GCIs
consist of loosely aggregated filaments with cross-sectional
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TABLE 1. Main Protein Components Found in Lewy Bodies
(18, 35) and Glial Cytoplasmic Inclusions (18, 39)
Lewy Bodies
AS
Ubiquitin
>-, A-Crystallin
Complement proteins (C3d, C4d, C7, C9)
Cyclin-dependent-kinase 5 (cdk-5)
Cytochrome c
Dorfin
Heat shock protein 90 (Hsp90)
Microtubule associated proteins
(MAP-2, MAP-5/MAP-1b)
Multicatalytic protease
Neural precursor cell expressed,
developmentally downregulated
8 (NEDD8)
Neurofilaments
Nuclear factor-JB
p25
Sphingomyelin
Sept4
Synaptic vesicle-specific protein
Synaptophysin
Torsin A
Tubulin
Tyrosine hydroxylase
Ubiquitin C-terminal hydroxylase
Glial Cytoplasmic
Inclusions
AS
Ubiquitin
>-, A-Tubulin
bcl-2
Carbonic anhydrase
isoenzyme II
cdk-5
Dorfin
Heat shock protein 90
(Hsp90)
MAP-5
Mitogen-activated protein
kinase (MAPK)
NEDD8
Phosphatidylinositol
3-kinase (PI3K)
p25
Tau 2 protein
Sept4
diameters of 20 to 40 nm, in addition to granulated material,
that often entrap cytoplasmic organelles such as mitochondria and secretory vesicles (21). Further immunoelectron
microscopic analysis of GCIs has revealed the presence of
different types of AS filaments whose width may either be
uniform or show periodic variations (35). It is suspected that
other cytoskeletal proteins, such as >-tubulin and A-tubulin,
may be involved in AS filament formation (36).
AS is the main component of GCIs, but the latter
contain many other proteins such as ubiquitin and E3
ubiquitin ligases. The main GCI protein components
detected by immunocytochemistry are listed in Table 1.
MECHANISMS OF INCLUSION BODY
FORMATION IN SYNUCLEINOPATHIES
Brainstem LBs
AS aggregation is now accepted as the key step
preceding LB formation. Although the precise events
responsible for AS aggregation remain unknown, accruing
evidence suggests a complex mechanism encompassing
phenomena as diverse as posttranslational modification and
alternative splicing (37). Furthermore, mutations affecting
AS intrinsic structure are also responsible for aggregation
enhancement, as suggested by the occurrence of accelerated
Ó 2007 American Association of Neuropathologists, Inc.
Protein Aggregation in Synucleinopathies
fibril formation in the presence of A30P, E46K, and A53T
mutated AS (38, 39). In LB diseases there is altered
solubility of aggregated AS as well as substantial accumulation of detergent-soluble and detergent-insoluble AS
species of various molecular weights, particularly in the
grey matter (30).
An immunoelectron microscopic study of AS in PD
and DLB showed AS filaments in LBs, pale bodies, and
perikaryal threads (40). In LBs, AS material was largely
detected in radially arranged peripheral filaments, whereas
the cores showed just a small number of grains. Pale
bodies showed AS labeling to be related to loosely aggregated filaments, whereas perikaryal threads revealed a
loose meshwork of small filament bundles that were
immunoreactive for AS but not for ubiquitin. The ultrastructural similarities among filaments in LBs, pale bodies,
and perikaryal threads prompted the hypothesis that AS
perikaryal threads are an early stage of filament assembly
that may then progress to pale bodies and, finally, to
classic LBs (40). Other studies confirmed these data and
showed that pale bodies occur after incorporation of p62
and assimilation of less aggregated forms of AS (31, 41).
Afterwards, the appearance of LBs would be coincidental
with increasing ubiquitin immunoreactivity and further AS
incorporation (41).
Interestingly, initial perikaryal AS accumulation seems
to be specifically constituted by AS112, a major AS isoform
resulting from alternative splicing. AS112 lacks part of the
unfolded protein C terminus but conserves intact membraneprotein and protein-protein binding domains. Therefore,
AS112 seems to be an aggregation-prone isoform with
enhanced membrane-binding properties (37, 42). Additionally, the fact that aggregation of AS is preceded by its
interaction with membranes (43) and that the small fraction
of membrane-bound AS seeds the accumulation of the far
more abundant cytosolic form (44) point to the AS112
membrane-protein interaction domain as a crucial element in
early aggregation steps (37).
Cortical LBs
The development of cortical LBs has been divided into
stages based on morphologic and immunoelectron microscopic observations (45). In Stage 1 there is cytoplasmic
granular AS accumulation in the neuronal cell body.
Subsequent addition of filamentous AS leads to stage 2. In
stage 3, AS accumulation gives rise to typical round LBs
composed of dense granulofilamentous material. Stage 4 is
characterized by involvement of dendrites. In stage 5 LBs
are distorted and acquire a loose periphery. Finally, in stage
6 LBs undergo degradation, become extracellular, and show
loose filamentous components, decreased AS immunoreactivity, and astroglial process involvement (45). Brains with
mild LB pathology show stages 1 to 2 LBs in the neocortex,
whereas brains with severe LB pathology show stages 5 to 6
LBs in the limbic cortex.
Glial Cytoplasmic Inclusions
As happens in LBs, profound solubility modifications
and pathologic accumulation of AS precedes GCI formation
967
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Beyer and Ariza
J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
(46). However, as suggested by the absence of detergentinsoluble AS, properties of aggregated AS are different in
MSA and LB diseases (30). A reverse correlation between
buffered saline-soluble and detergent-soluble levels of AS in
individual MSA cases indicates a transition toward insolubility with disease progression (30). As in LB diseases,
altered AS accumulation precedes AS ubiquitination, GCI
development, and neuronal loss (35, 47). The diameter of
GCI filaments matches the diameter of in vitro AS
aggregates (48) and, although full-length AS has been
detected in GCIs, the predominant form of the aggregated
protein seems to be C-terminally truncated (49).
Two main hypotheses have been proposed to explain
the presence of aggregated AS in oligodendroglia. The first
hypothesis is based on the observations that AS is transiently
expressed in cultured rat oligodendrocytes (50) and that
glia show low AS levels in vivo (51). Impaired ability to
degrade AS or selective upregulation of AS expression in
glial cells would lead to its accumulation and aggregation in
these cells (52). The second hypothesis claims that oligodendrocytes actively uptake AS previously released by dying
neurons (25, 53). In agreement with this, a patient with
early-stage MSA presented a number of neuronal inclusions
that drastically exceeded the number of GCIs (54). Because
later MSA stages are characterized by the presence of
numerous GCIs, translocation of aggregated AS from dying
neurons to oligodendroglial cells can be invoked as a possible mechanism of GCI formation.
It has been suggested that GCI evolution is different
according to brain region and that GCI composition
changes with disease duration. Thus, albeit the total number
of MSA GCIs does not vary with disease duration, it
has been shown that the number of AS-positive, ubiquitinpositive GCIs decreases and the number of AS-negative,
ubiquitin-positive GCIs increases as the disease evolves.
This finding indicates that MSA progression is marked by
incorporation of additional proteins into GCIs and their
further compaction (55).
THE PATHOGENESIS OF
SYNUCLEIONOPATHIES: COMMONALITIES
An important commonality of synucleinopathies, also
shared by most neurodegenerative conditions, is the genetic
and neuropathologic heterogeneity shown by each of these
disorders. Whereas the ever-increasing number of gene
mutations associated with synucleinopathies underlines their
genetic heterogeneity, the variety of protein components
found in inclusion bodies bears witness to their neuropathologic heterogeneity. Of these proteins, some are present
in all inclusion bodies discussed in this review, whereas
others are specific markers of certain inclusion forms.
Elevated phosphorylated AS levels have been detected
in all synucleinopathies. AS is constitutively phosphorylated
at serine residue 129 (56), and repeated phosphorylation and
dephosphorylation within the AS C-terminal domain, mediated by casein kinases CK-1 and CK-2, has been observed
in vivo (57). Extensive AS phosphorylation may be a significant pathogenic event and, probably, a necessary step in
LB formation. This hypothesis is suggested by the fact that
968
more that 90% of insoluble AS is phosphorylated in brains
with synucleinopathies, whereas phosphorylation is a feature
of only about 4% of normal AS (58).
Another common early event of inclusion body
formation in synucleinopathies is the occurrence of
AS/rab3a binding. This phenomenon, absent in the normal brain, is characterized by indirect binding of rab3a to
AS with the aid of rabphilin intermediation (59). In MSA,
AS/rab3a binding has been shown in both GCI-rich
cerebellum and pons and GCI-free cerebral cortex, pointing
to a specific role for rab3a in AS aggregation preceding
GCI formation (60).
The incorporation of ubiquitin into all inclusion
bodies in LB diseases and MSA is a further commonality
of synucleinopathies. Nevertheless, the deposition of
mono- or diubiquitinated phosphorylated AS in LB diseases and MSA indicates that AS ubiquitination occurs
after AS aggregation (61). Consequently, AS ubiquitination
seems to be an advanced step in LB as well as GCI
formation (62).
One of the common components of LBs and GCIs is
oxidative stress-related, ubiquitin-binding protein p62,
whose immunostaining patterns are markedly similar to
those of ubiquitin (63). It has been shown that the
association of p62 with polyubiquitinated proteins enhances
aggregation and plays an important role in protecting cells
against the toxicity of misfolded proteins, particularly in
advanced disease stages (64).
Synphilin-1 was initially identified as an AS-binding
protein and, in association with AS, constitutes a major
component of LBs (65). As shown by immunocytochemical
and ultrastructural studies, most PD brainstem LBs show
intense central core positivity for synphilin-1. On immunoelectron microscopy, the reaction product is seen to be
located in filamentous and circular structures. In contrast,
synphilin-1 is not present in pale bodies and only a small
fraction of DLB cortical LBs contains synphilin-1. Similarly
to brainstem LBs, numerous GCIs are positive for synphilin1, suggesting that the role of abnormal synphilin-1 accumulation is not restricted to brainstem LB formation (66).
Interestingly, dorfin colocalizes with ubiquitin in LBs
and GCIs. This E3 ubiquitin ligase participates in the
formation of ubiquitinated AS-positive inclusions in PD,
DLB, and MSA (67). Because dorfin physically binds and
ubiquitinates synphilin-1 through its central portion but does
not ubiquitinate AS (68), it may be surmised that the
synphilin-1 central domain plays an important role in
inclusion body formation.
Intensive investigations on AS aggregation and inclusion body formation have recently identified additional
proteins in LBs and GCIs. One of them is neural precursor
cell expressed, developmentally downregulated 8 (NEDD8),
an ubiquitin-like protein that controls vital biologic events
through its conjugation to culling ubiquitin E3 ligases (69).
Its presence in LBs and GCIs indicates that NEDD8 is
involved in the formation of various ubiquitinated inclusions
via the ubiquitin-proteasome system. Studies of heat shock
proteins (Hsp), which facilitate refolding of denatured
polypeptides, revealed that Hsp90 and AS colocalize in
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TABLE 2. Proteins Variably Found in Brainstem and Cortical
Lewy Bodies and in Glial Cytoplasmic Inclusions
Lewy Bodies
Proteins
Partially detected
14-3-3
Tau
Clusterin
Brainstem
Cortical
GCIs
++/j
+++/j
j
++/j
+++/j
+/j
+/j
+/Y Y Y
+/Y Y Y Y
Y, negative; +++/j, most inclusion bodies are positive; ++/j, many inclusion
bodies are positive; +/j, 50% of inclusion bodies are positive; +/j j, some inclusion
bodies are positive; +/j j j, a few inclusion bodies are positive; +/j j j j, very
few inclusion bodies are positive.
GCI, glial cytoplasmic inclusions.
LBs and GCIs. These findings suggest a role for Hsp90 in
AS inclusion body formation (70).
Sept4, a member of the septin protein family, is
consistently found in LBs and GCIs (71). Because tagged
Sept4 and AS synergistically accelerate cell death induced
by the proteasome inhibitor and this effect is further
enhanced by LB-associated synphilin-1 expression, another
LB-associated protein, it may be postulated that Sept4 is a
potential cofactor in inclusion body formation.
Finally, investigation of other AS proaggregation
proteins identified brain-specific p25> as a candidate that
preferentially binds to AS in its aggregated state. In vitro
stimulation of AS aggregation by purified recombinant
human p25> and detection of p25> in LBs and GCIs
prompted the suggestion that this protein plays a proaggregation role in neurodegeneration associated with AS aggregates (72).
THE PATHOGENESIS OF SYNUCLEINOPATHIES:
DIFFERENCES
Protein Aggregation in Synucleinopathies
mediated ubiquitination of proteins in LB-like inclusions
coexpressing synphilin-1, AS, and parkin occurs predominantly
via K63 linkages and formation of these inclusions is enhanced
by K63-linked ubiquitination (78).
LBs may contain tau as well. In fact, about 80% of
PD cases show tau-immunoreactive LBs. Tau positivity is
detected at the periphery of LBs, regardless of Braak
stage. LBs and neurofibrillary tangles (NFTs) keep an
intriguing relationship inasmuch as the proportion of tauimmunoreactive LBs is greater in NFT-vulnerable neurons
and lesser in NFT-resistant neurons (Table 2). These
observations suggest that tau may coaggregate with AS in
LBs, especially in neuronal populations that are vulnerable to both NFTs and LBs (79).
Finally, it is worth noting that AS shares physical
and functional homology with 14-3-3 proteins, to which it
binds. In agreement with this finding, 14-3-3 proteins have
been detected in LBs (Table 2) (80), although it is not
clear which 14-3-3 proteins are specifically involved in
LB formation.
Brainstem LBs
Because certain proteins are present only in brainstem
LBs and not in the other inclusion types discussed in this
review (Table 3), it may be assumed that specific mechanisms are responsible for brainstem LB formation (Fig. 1).
Synphilin-1, an AS interacting protein, accumulates in
brainstem LBs but its presence in cortical LBs is only
occasional. Through its interaction with AS, synphilin-1
promotes AS aggregation and ubiquitination. Evidence of
localization of Pael-R, a parkin substrate, in LB cores
suggests that Pael-R is involved in the early phases of LB
formation (81). PINK1, a protein with both mitochondrial
targeting and serine/threonine kinase domains, is detected in
a proportion of brainstem LBs in both sporadic PD and
PINK1 gene mutation-associated PD. The presence of
PINK1 in brainstem LBs in addition to its mitochondrial
Lewy Bodies
Oxidative stress is a strong inducer of cell protein
alterations. Mainly affected are mitochondria, as shown by
mitochondrial complex I decreased activity and increased
reactive oxygen species production in PD substantia nigra
(73). An important protein target of oxidative damage is
Cu,Zn-superoxide dismutase, a key antioxidant enzyme
whose function is altered by oxidative modifications (74).
Because AS seems to be a further target of oxidative stress
in PD presymptomatic stages, AS oxidation may be a
significant enhancer of AS aggregation properties (75).
Colocalization of parkin and AS in both brainstem and
cortical LBs indicates that functional parkin protein may be
required for LB formation (76). Parkin, in common with both
dorfin and 7 in absentia homolog (SIAH), acts as an E3
ubiquitin ligase and is involved in protein turnover via the
ubiquitin-proteasome system. Substrates ubiquitinated by parkin are thought to be destined for proteasomal degradation.
Besides, it is currently known that parkin interacts with
synphilin-1 and ubiquitinates it in a nonclassic, proteasomalindependent manner that involves lysine 63 (K63)-linked
polyubiquitin chain formation (77). Furthermore, parkinÓ 2007 American Association of Neuropathologists, Inc.
TABLE 3. Proteins Specifically Found in Brainstem and
Cortical Lewy Bodies and in Glial Cytoplasmic Inclusions
Lewy Bodies
Proteins
Partially specific
Synphilin-1
Parkin
SOD1
SOD2
Very specific
Pael-R
Pink-1
Clusterin
DJ-1
Elk-1
Midkine
Brainstem
Cortical
GCIs
+
+
+
+
Y
+
+
+
+
Y
Y
Y
+
+
Y
Y
Y
Y
Y
Y
+
Y
Y
Y
Y
Y
Y
+
+
+
+, present; Y, absent
GCI, glial cytoplasmic inclusions; SOD, superoxide dismutase.
969
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Beyer and Ariza
J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
FIGURE 1. Putative steps of progressive protein aggregation and accumulation in brainstem Lewy body (LB) formation. In
accordance with previous reports (31, 40, 41), 4 main steps are represented. Whether these steps can be further subdivided
remains to be clarified by careful study of neuropathologically well-characterized cases. AS, >-synuclein.
localization, underpins the significance of mitochondrial
dysfunction in the pathogenesis of nigral cell degeneration
in PD (82). Further evidence in favor of the existence of
distinctive production mechanisms for each of the two LB
types is the absence of clusterin in brainstem LBs but not in
cortical LBs (83).
In addition to dorfin, synphilin-1 interacts with E3
ubiquitin ligases SIAH-1 and SIAH-2, which ubiquitinate
synphilin-1 and promote its degradation by means of the
ubiquitin-proteasome system. Inability of the proteasome
to degrade the synphilin-1/SIAH complex leads to robust
formation of ubiquitinated cytosolic inclusions. AS also
associates with SIAH (mainly SIAH-2) and is monoubiquitinated by it. Detection of SIAH immunoreactivity in
PD LBs has implicated this protein in inclusion body
formation (84).
Cortical LBs
Several alterations have been described to be specific
for cortical LBs in DLB (Table 3; Fig. 2). First, it has been
seen that AS112 is overexpressed in DLB brains but not in
control or AD brains. This suggests that AS112 is related to
LB formation and probably constitutes a primary step in
DLB pathogenesis. Additionally, UCHL-1 is selectively
downregulated in the frontal cortices of DLB brains (85).
Because synphilin-1 is present only in a small fraction of
cortical LBs (66), it may be proposed that LB formation in
DLB (or at least in a DLB subset) is promoted by
FIGURE 2. Putative steps of progressive protein aggregation and accumulation in cortical Lewy body (LB) formation. The fact
that clusterin is present in only about 50% of cortical LBs, in conjunction with their morphologic heterogeneity, strongly supports
the existence of different cortical LB types (32). Question marks (???) indicate a hypothetical event. AS, >-synuclein.
970
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J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
overrepresentation of aggregation-prone AS112 in the
absence of AS-interacting synphilin-1. Moreover, about
50% of cortical LBs are immunoreactive for clusterin, a
molecular chaperon with a role in the refolding of misfolded
proteins. In agreement with this function, clusterin colocalizes with AS, as indicated by the almost complete overlap
of immunoreactivity of both proteins. Identification of some
LBs showing high clusterin but low AS levels suggests that
clusterin may influence LB formation (83).
Glial Cytoplasmic Inclusions
In contrast to cortical LBs, clusterin is detected in only
approximately 10% of GCIs (Table 2) (83). On the other hand,
only a subset of GCIs (or none in some cases) contain
hypophosphorylated tau and 14-3-3 proteins (Table 2) (86).
When present, 14-3-3 proteins associate with AS in GCIs. A
negative correlation between the degree of tissue degeneration
and the density of 14-3-3 proteins suggests that the latter are AS
cofactors in GCI formation (87), at least in some MSA cases.
The absence of Pael-R and parkin in MSA GCIs points to the
existence of different formation pathways for LBs and GCIs.
DJ-1, one of the GCI-specific components detected to
date (Table 3), is present as an insoluble, modified protein
and seems to incorporate into GCIs after undergoing
upregulation in reactive astrocytes and neurons (88).
Another GCI-specific protein is Elk-1, which does not
directly bind to AS but shares with AS its ability to bind
to extracellular signal-related kinase (ERK)-2, a mitogenactivated protein (MAP) kinase. Interaction of AS with the
MAP kinase pathway might cause dysfunction of neurons
and oligodendrocytes and lead to neurodegeneration (89).
Immunohistochemical detection of midkine in GCIs and the
subsequent immunoelectron microscopic identification of
midkine-positive, granule-coated fibrils indicated that this
new neurotrophic factor could be an essential, specific
constituent of GCIs (90).
A further specific feature of MSA seems to be
extensive accumulation of A-synuclein, an AS aggregation
inhibitor, in Purkinje cells. The latter cells show no
abnormal AS accumulation in MSA, in contrast to the
increased AS levels shown in PD and DLB (54).
WORKING HYPOTHESES
Brainstem and Cortical LBs
The occurrence of significant differences between
brainstem and cortical LB components suggests that their
formation mechanisms are markedly disparate (Figs. 1, 2).
Brainstem LBs contain the AS-interacting protein synphilin1, whose core location would indicate that interaction
between AS and synphilin-1 is an early event. Subsequent
failure of protein refolding agents would result in protein
sequestration within LBs. E3 ubiquitin ligases would
ubiquitinate synphilin-1 and AS for their targeting for
proteasome degradation, and parkin would carry out K63mediated ubiquitination, thus enhancing protein aggregation
and LB formation. Intervention of additional E3 ubiquitin
ligases, such as dorfin and SIAH, would result in their
sequestering within LBs. As cell stress increases, mitochonÓ 2007 American Association of Neuropathologists, Inc.
Protein Aggregation in Synucleinopathies
dria would be recruited to participate in the dissociation of
aggregates, and mitochondrial proteins such as PINK1
would also be sequestered. The outcome of this series of
misfoldings and subsequent refolding attempts would be the
accumulation and further insolubilization of protein aggregates and eventual LB formation. PD subtypes are still to be
well characterized, but probably each of them will feature
subtype-specific LB formation mechanisms whose elucidation is necessary for the development of effective treatments.
The main morphologic difference between brainstem
and cortical LBs is the core-halo structure observed in
brainstem LBs but not in cortical LBs. On the other hand,
cortical LBs lack synphilin-1, which is abnormally ubiquitinated by parkin. This absence of synphilin-1 would be
responsible for the more uniform distribution of aggregated
material in cortical LBs.
Figure 2 shows the possible molecular pathways of
cortical LB formation, although at present the steps involved
are well known only for the late stages. AS112 overexpression, in addition to AS phosphorylation and oxidation,
would result in the primary AS aggregates sequestering
soluble AS. Different PD and DLB subgroups could be
characterized by specific primary AS modifications. For
example, in some cases AS112 overexpression could be
more important than AS phosphorylation, whereas in other
instances AS oxidation could play a primary role. In
contrast, other PD and DLB subgroups would develop the
disease as a consequence of combined AS alterations. In
subsequent stages, E3 ubiquitin ligases (parkin, dorfin, and
SIAH) would ubiquitinate AS and other proteins for their
proteasome-mediated degradation. Additionally, the attempts
by clusterin to refold AS would result in its sequestration
in less dense LBs devoid of cores and halos. In the
absence of clusterin, 14-3-3 proteins acting as molecular
chaperones would attempt AS refolding just to undergo
sequestration and integration into LBs as well. In contrast
to the 4 steps of brainstem LB formation, cortical LBs
would be the result of the initial 3 steps in the absence of
the final compaction phase. Confirmation of this hypothesis makes detailed immunohistochemical study of the
cortical LB formation process mandatory.
Glial Cytoplasmic Inclusions
GCIs have been much less extensively studied than
LBs and, consequently, few data from which to infer
possible GCI formation pathways are available. Perhaps
the most interesting feature regarding GCI protein content is
the combined presence of synphilin-1 and absence of parkin,
in which case abnormal protein ubiquitination is not to be
expected. Moreover, because AS-positive material is less
dense in GCIs than in brainstem LBs, it can be proposed that
K63-mediated synphilin-1 ubiquitination by parkin is
responsible for the increased compaction of LB cores, which
are mainly constituted by interacting AS and synphilin-1.
Additionally, a primary involvement of mitochondria in GCI formation has been proposed, especially in
relation to oxidative stress (54). In concordance with this
proposal, GCIs have been seen to contain DJ-1, which is
involved in oxidation damage repair, and Elk-1, which is
971
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J Neuropathol Exp Neurol Volume 66, Number 11, November 2007
Beyer and Ariza
instrumental in neuronal differentiation, cell proliferation,
and apoptosis (91). Widespread detection of midkine in
GCI granule-coated fibrils suggests that this neurotrophic
factor, initially associated with fetal morphogenesis,
participates in GCI formation in a probable attempt to
repair AS accumulation damage.
WHERE TO FROM HERE?
FUTURE RESEARCH DIRECTIONS
Whereas synucleinopathies show an unexpected
homology in regard to their neuropathologic findings, these
disorders demonstrate an equally unexpected phenotypic
heterogeneity, mainly due to genetic variations. Thus, PD,
DLB, and MSA are each constituted by different subgroups
that seem to be the result of idiosyncratic molecular
mechanisms, as suggested by the heterogeneous brain
findings of these conditions (Table 2).
Although AS aggregation is a common event preceding inclusion body formation in synucleinopathies, increasing evidence strongly suggests that different factors trigger
and/or enhance this aggregation in different settings (Fig. 2).
Accurate characterization of all PD, DLB, and MSA
subgroups is necessary for the development of efficient
preventive treatments. This would initially require comprehensive neuropathologic studies for accurate determination
of protein content variations in LBs and GCIs in different
settings. These studies should be carried out on brain areas
that are differentially involved in the earlier and later stages
of these conditions, as well as on uninvolved brain regions. In
this regard, a very recent work analyzed the brain distribution
of AS pathology independently of clinical phenotype.
Interestingly, the authors identified cases showing a high AS
pathology burden in both brainstem and cortical areas in the
absence of clinical symptoms (92). The question then arises
as to why some individuals become symptomatic and others
do not while showing similar neuropathologic changes. The
aforesaid observations provide further evidence in favor of
the heterogeneity of factors conditioning the development of
synucleinopathies (92). Delineating the differences in protein
distribution and contents in inclusion bodies would help to
elucidate the mechanisms of formation for each subgroup. If
inclusion body protein distribution and contents can be
correlated with the disease genotype or phenotype, the choice
of therapeutic strategies in each patient could be guided by
knowledge of the operating mechanism as deduced from the
individual genotype or phenotype.
CONCLUSIONS
The basic feature of neurodegenerative disorders is the
accumulation of misfolded proteins that subsequently
undergo sequestration and aggregation as proteinaceous
inclusions. It is thought that these inclusions protect the cell
from the toxicity of soluble misfolded proteins, although
conflicting evidence suggests that the presence of inclusion
bodies may also lead to cell dysfunction and death. Therefore, it is of paramount importance to determine which
proteins are misfolded and which are sequestered as they
attempt to refold altered proteins.
972
Synucleinopathies share AS aggregation as the main
pathogenic event preceding inclusion body formation, but
different molecular mechanisms seem to be responsible for
the development of LBs in PD and DLB and of GCIs in
MSA. It thus seems mandatory to accurately delineate
different subsets of these diseases and their molecular
mechanisms. Appropriate diagnosis and treatment of individual patients will not be possible until the complete
characterization of the genotypic and phenotypic alterations
specifically associated with each synucleinopathy subgroup
has been achieved. Only elimination of primary causes
through either enhancement of AS solubility or inhibition of
AS aggregation will bring about effective disease control. It
should be emphasized, however, that the precise pathogenetic factors underlying each synucleinopathy subtype must
be fully unraveled before we are able to influence AS
solubility or aggregation.
ACKNOWLEDGMENTS
We thank Dr. Isidro Ferrer, University of Barcelona,
for his invaluable suggestions and comments on the manuscript. We thank Dr. Sarah Hunt for the critical reading of
the manuscript.
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