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 -Synuclein in Familial Alzheimer Disease
Epitope Mapping Parallels Dementia With Lewy Bodies and Parkinson Disease
Carol F. Lippa, MD;
M. Luise Schmidt, PhD;
Virginia M.-Y. Lee, PhD;
John Q. Trojanowski, MD, PhD
Arch Neurol. 2001;58:1817-1820.
ABSTRACT
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Background  -Synuclein is a major component of
Lewy bodies (LBs) in Parkinson disease and dementia with LBs and of
glial cytoplasmic inclusions in multiple system atrophy. However,
epitope mapping for -synuclein is distinctive in different
neurodegenerative diseases. The reasons for this are poorly understood
but may reflect fundamental differences in disease mechanisms.
Objective To investigate the -synuclein epitope mapping
properties of LBs in familial Alzheimer disease.
Design and Setting We compared LBs in familial Alzheimer
disease with those in synucleinopathies by probing 6 brains of persons
with familial Alzheimer disease using a panel of antibodies to epitopes
spanning the -synuclein protein. Results were compared with data
from brains of persons with Parkinson disease, dementia with LBs, and
multiple system atrophy.
Results The brains of persons with familial Alzheimer disease
showed consistent staining of LBs with all antibodies, similar
to Parkinson disease and dementia with LBs but different from
-synuclein aggregates that occurred in multiple system atrophy.
Conclusions These data suggest that the epitope profiles
of -synuclein in LBs are similar, regardless of whether the
biological trigger is related to synuclein or a different genetic
pathway. These findings support the hypothesis that the mechanism of
-synuclein aggregation is the same within cell types but distinctive
between cell types.
INTRODUCTION
ALTHOUGH -synuclein
aggregates are traditionally associated with synucleinopathies such as
Parkinson disease (PD) and dementia with Lewy bodies (DLB), they also
occur in Alzheimer disease (AD) and Down syndrome.1, 2, 3 In
particular, most symptomatic patients with mutations of the presenilin
(PS) or amyloid precursor protein (APP) genes have
Lewy bodies (LBs) in the amygdala and adjacent entorhinal
cortex.2 This suggests that the genetic mechanism that
leads to ß-amyloid plaque and tau-rich neurofibrillary tangle
formation in AD with PS and APP mutations also
predisposes individuals to LBs formed by -synuclein filaments.
Questions remain regarding the exact mechanism of -synuclein
aggregation in neurodegenerative diseases. In PD and DLB, full-length
-synuclein is present in filamentous LBs, and they are strongly
immunoreactive for antibodies that recognize epitopes along the entire
protein.4 In multiple system atrophy (MSA), a condition in
which filamentous -synuclein aggregates form glial cytoplasmic
inclusions (GCIs), -synuclein epitope mapping is not
uniform.4 Therefore, structural or conformational
differences in aggregated -synuclein exist in the filamentous
-synuclein lesions of different diseases. This suggests that
mechanisms leading to -synuclein aggregation in LBs vs GCIs are not
identical.
Most epitope-mapping studies of -synuclein have focused on
synucleinopathies. It is unknown whether LB formation in AD occurs
through a distinct mechanism or whether all LBs result from identical
responses to different triggers. Since analysis of the underlying
aggregated protein has the potential to yield clues regarding the
mechanism of inclusion formation, the current study evaluates LB
formation in early-onset familial AD (FAD), in which disease etiology
is not primarily related to an abnormality of the -synuclein
gene.
MATERIALS AND METHODS
TISSUE SAMPLES
We examined affected regions from 6 patients with FAD who were known to
have LBs, including 5 patients with PS-1 mutations and 1
patient with an APP mutation
(Table 1).2 All had
LBs in the amygdala, and 4 (patients 1 and 4-6) also had LBs in the
entorhinal cortex, cingulate gyrus, frontal cortex, nucleus basalis of
Meynert, substantia nigra, and locus coeruleus. Although disease
duration was variable, all patients were in nursing homes, requiring
assistance with all activities of daily living at the time of death.
Tissue samples for the current analysis were derived from the amygdala
and adjacent entorhinal cortex in all cases, and also included the
other involved brain regions in patient 5. These data were compared
with those acquired using the same method from brains of persons with
PD, DLB, and MSA.4
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Clinical Features and Epitope Mapping in the Amygdala of Patients With Familial Alzheimer Disease*
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IMMUNOHISTOCHEMICAL ANALYSIS AND ANTI–--SYNUCLEIN ANTIBODIES
Tissue blocks were formalin fixed, paraffin embedded, and serially cut
at 6 µm. Tissues were then stained with a panel of
monoclonal antibodies (Mabs) (SNL-4, SYN204, LB509, 211, 202)
(Figure 1), described
previously,4 that recognize identified epitopes spanning
the -synuclein protein. Formic acid pretreatment optimized
staining,4 and the topographically distinct -synuclein
epitopes were detected using avidin-biotin complex kits
(Vector Laboratories, Burlingame, Calif) and
3,3'-diaminobenzidine.4 Positive control specimens
consisted of LB-rich brain sections from the amygdala of a patient with
DLB. Consecutive sections stained with the supernatant from unfused
SP2/0-Ag14 mouse myeloma cells in place of primary antibodies were used
as negative controls. A previously described2
semiquantitative scale was used to document the degree of LB staining
in the amygdala, where 0 indicates no LBs; 1, 1 to 5 LBs; 2, 6 to 20
LBs; and 3, more than 20 LBs. Since LBs were numerous in several of
these cases, we added an additional category (grade 4) where there were
more than 20 LBs per microscopic field (magnified
x20).
RESULTS
Using the same LB509 Mab as in prior studies,2, 3, 4 we
confirmed that there is robust staining of LBs and Lewy neurites in
sections of amygdala and the adjacent entorhinal cortex in FAD. Using
the other Mabs that recognize epitopes spanning the
protein (Figure 2A-E), we
observed a similar pattern of LB staining in FAD. In particular, the
SNL-4 Mab stained equivalent numbers of LBs as LB509 and the other
Mabs, although the intensity of staining was slightly less with SNL-4
than with the other Mabs. The staining pattern was indistinguishable
between our patients with DLB (Figure 2F-J) and FAD, except that the
LBs in patients with FAD (Figure 2A-E) more often had a sharply
irregular, eccentric shape due to the co-occurrence of neurofibrillary
tangles in some neurons with LBs. Identical, uniform epitope staining
was seen in LBs in other cortical regions. Nigral LBs had a staining
pattern similar to those of the substantia nigra in PD.4
However, the -synuclein epitope profile of LBs in FAD differed from
that of GCIs in MSA, in which a reduced number of cells were
immunoreactive for the SNL-4 Mab.4 We noted no differential
patterns of immunoreactivity when comparing FAD with PS and
APP mutations, or FAD with different PS mutations,
and LB staining was graded 4 with all antibodies for all of our
patients with FAD.
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Figure 2.
Photomicrographs of -synuclein epitope recognition in Lewy bodies
(LBs) in the amygdala comparing patient 2 with familial Alzheimer
disease (A-E) with a patient with dementia with LBs (F-J).
The panels show immunostaining for the SNL-4 (A and F), SYN204 (B and
G), LB509 (C and H), 211 (D and I), and 202 (E and J) antibodies.
Numerous LBs are appreciated using all of the antibodies in both
conditions. Similar numbers of LBs stain for SNL-4 compared with the
other antibodies, although the intensity of immunoreactivity of SNL-4
is reduced in both patients. Scale bar indicates 20 µm for all
images.
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COMMENT
We extended previous data on LB formation from filamentous
-synuclein aggregates in FAD by demonstrating that LBs in FAD
contain the full-length -synuclein protein. We showed that the
-synuclein epitope profile was indistinguishable between the LBs in
FAD and the LBs in DLB and PD, but that the profile differed from that
seen in the GCIs in MSA. Moreover, we found no differential staining
pattern for -synuclein in LBs between different mutations in FAD.
Abnormalities of aggregated -synuclein are implicated in the
pathogenesis of PD, DLB, and MSA. The current data are interesting
because they reinforce the notion that the morphologic features of the
-synuclein filaments that form in neurons differ from those of
-synuclein filamentous aggregates in glia. Duda et al4
determined that the C-terminal epitopes are strongly immunoreactive for
-synuclein in GCIs, whereas N-terminal epitopes show less consistent
immunoreactivity. In comparison, LBs in PD and DLB are uniformly
immunoreactive for all -synuclein antibodies. The
reasons for these differences are unclear. The
differences between LBs and GCIs might be related to conformational
differences in the aggregated protein in different cell types, leading
to differential exposure of the N-terminal epitopes. The similarity
between LBs in FAD, DLB, and PD supports the notion that neuronal LB
formation occurs through a single mechanism. Our data suggest that in
cases of PS-1 and APP mutation, the formation of LBs
is also identical. Although we had only 1 case with an APP
mutation in the present study, these data build on those of previous
work documenting the common occurrence of LBs in these
kindreds.2 We argue that filamentous -synuclein
aggregates in neurons are similar, whether or not the primary disease
is a synucleinopathy. Thus, LB formation appears to be a common end
point of different genetic pathways.
It could be argued that LB formation is not linked to neuronal
degeneration in our patients with FAD, but rather that LBs are a
nonspecific manifestation of end-stage neuronal degeneration. All of
our subjects had severe dementia with advanced AD pathologic features
(Braak stage V or VI).5 Indeed, LBs are not a
feature of preclinical AD related to Down syndrome or PS
mutations.2, 3 However, the consistently high densities of
these inclusions indicate that their occurrence was meaningful. In
addition, in patient 5, symptoms of DLB, including marked clinical
fluctuations, spontaneous parkinsonism, and visual hallucinations, were
present. Patient 2 also had early signs of parkinsonism. Although
retrospective examination of medical records showed no definite signs
of DLB in the other cases, clinical symptoms or signs of amygdaloid
dysfunction were not specifically tracked.
Although -synuclein, ubiquitin, and neurofilaments are all present
in LBs, increasing evidence suggests that -synuclein is the major
protein component of LBs, and mutations of the -synuclein gene have
been shown to be pathogenic for familial PD,6 whereas
-synuclein has been shown to be incorporated into LBs, pale bodies,
and Lewy neurites more consistently and at an earlier point than is
ubiquitin.7 -Synuclein is a presynaptic protein that is
abundantly expressed in neurons throughout the brain, and it is thought
to play a role in synaptic plasticity. Furthermore, Richter-Landsberg
et al8 recently showed that during development,
oligodendroglial cells express -synuclein, indicating that
-synuclein is also normally present in these glial cells, thereby
implicating -synuclein in glial functions.
It is unclear why limbic regions are vulnerable to LB formation in FAD.
Kosaka9 noted the amygdala's susceptibility to LB
formation when he described a DLB variant with LBs restricted to the
cerebral cortex and
amygdala. The amygdala also is susceptible to
accumulations of LBs in PD10 and DLB, and Schmidt et
al11 described susceptibility of the amygdala to LB
formation in sporadic AD. In that study, they showed that LBs in the
amygdala frequently co-occur with tau-rich neurofibrillary tangles.
Thus, amygdaloid neurons appear to be susceptible to LB formation
in response to a variety of neurodegenerative disease–initiating
events. Studies of the amygdala in other neurodegenerative diseases
will help determine whether the propensity to LB formation is
restricted to synucleinopathies and amyloidopathies, or
whether it is a more universal phenomenon in the degenerating
brain.
CONCLUSIONS
In the present study, we compared cases in which disease etiology is
linked directly to genetic defects that primarily influence ß-amyloid
plaque or PS metabolism (or processing) with diseases primarily
involving -synuclein. Although there is no known interaction between
APP or PS proteins and -synuclein, the present study shows that
full-length -synuclein is expressed in LBs in cases in which disease
due to genetic mutations is unrelated to the -synuclein gene. Thus,
our data suggest that the mechanisms of LB formation in neurons are
identical regardless of the biological trigger, including mutations
that cause FAD, but the different -synuclein epitope profile seen in
GCIs in MSA indicates that the mechanism of aggregation of these
filamentous inclusions may differ in different cell types.
AUTHOR INFORMATION
Accepted for publication May 23, 2001.
This research was supported in part by grants from the National
Alzheimer's Association, Chicago, Ill, and by grants AG09215 and
AG10124 from the National Institute on Aging of the National Institutes
of Health, Bethesda, Md.
We acknowledge Terry Schuck for assistance with technical aspects of
the study. We also wish to thank the families of the patients and the
patients who donated tissue for research.
From the Department of Neurology, Medical College
of Pennsylvania–Hahnemann University (Dr Lippa),
and the Center for Neurodegenerative Disease Research, Department
of Pathology and Laboratory Medicine, University of Pennsylvania School of
Medicine (Drs Schmidt, Lee, and Trojanowski), Philadelphia.
Corresponding author and reprints: Carol F. Lippa, MD, Department of
Neurology, Medical College of Pennsylvania–Hahnemann University, 3300
Henry Ave, Philadelphia, PA 19129 (e-mail: carol.lippa{at}drexel.edu).
REFERENCES
| |
1. Hamilton RL. Lewy bodies in Alzheimer's disease: a neuropathological
review of 145 cases using -synuclein immunohistochemistry. Brain Pathol. 2000;10:378-384.
ISI
| PUBMED
2. Lippa CF, Fujiwara H, Mann DM, et al. Lewy bodies contain altered
-synuclein in brains of many familial Alzheimer's disease patients
with mutations in presenilin and amyloid precursor protein genes. Am J Pathol. 1998;153:1365-1370.
FREE FULL TEXT
3. Lippa CF, Schmidt ML, Lee VM, Trojanowski JQ. Antibodies to
-synuclein detect Lewy bodies in many Down's syndrome brains with
Alzheimer's disease. Ann Neurol. 1999;45:353-357.
FULL TEXT
|
ISI
| PUBMED
4. Duda JE, Giasson BI, Gur TL, et al. Immunohistochemical and biochemical
studies demonstrate a distinct profile of -synuclein permutations in
multiple system atrophy. J Neuropathol Exp Neurol. 2000;59:830-841.
ISI
| PUBMED
5. Braak H, Braak E. Diagnostic criteria for neuropathologic assessment of
Alzheimer's disease. Neurobiol Aging. 1997;18(suppl):S85-S88.
6. Polymeropoulos MH, Lavedan C, Leroy E, et al. Mutation in the
alpha-synuclein gene identified in families with Parkinson's disease. Science. 1997;276:2045-2047.
FREE FULL TEXT
7. Irizarry MC, Growdon W, Gomez-Isla T, et al. Nigral and cortical Lewy
bodies and dystrophic nigral neurites in Parkinson's disease and
cortical Lewy body disease contain -synuclein immunoreactivity. J Neuropathol Exp Neurol. 1998;57:334-337.
ISI
| PUBMED
8. Richter-Landsberg C, Gorath M, Trojanowski JQ, Lee VM. -Synuclein is
developmentally expressed in cultured rat brain oligodendrocytes. J Neurosci Res. 2000;62:9-14.
FULL TEXT
|
ISI
| PUBMED
9. Kosaka K. Diffuse Lewy body disease. Rinsho Shinkeigaku. 1995;35:1455-1456.
PUBMED
10. Braak H, Braak E, Yilmazer D, de Vos RA, Jansen EN, Bohl J. Pattern of
brain destruction in Parkinson's and Alzheimer's diseases. J
Neural Transm. 1996;103:455-490.
FULL TEXT
|
ISI
| PUBMED
11. Schmidt ML, Martin JA, Lee VM, Trojanowski JQ. Convergence of Lewy
bodies and neurofibrillary tangles in amygdala neurons of Alzheimer's
disease and Lewy body disorders. Acta Neuropathol (Berl). 1996;91:475-481.
FULL TEXT
| PUBMED
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