J Neuropathol Exp Neurol
Copyright Ó 2012 by the American Association of Neuropathologists, Inc.
Vol. 72, No. 1
January 2013
pp. 53Y60
ORIGINAL ARTICLE
Synaptic Proteins and Choline Acetyltransferase Loss in Visual
Cortex in Dementia With Lewy Bodies
Elizabeta B. Mukaetova-Ladinska, PhD, Alina Andras, PhD, Joan Milne, BA, Zeinab Abdel-All, BA,
Iwo Borr, PhD, Evelyn Jaros, PhD, Robert H. Perry, MD, William G. Honer, MD, Andrea Cleghorn, PhD,
Jeanette Doherty, PhD, Gary McIntosh, PhD, Elaine K. Perry, PhD, Raj N. Kalaria, PhD,
and Ian G. McKeith, MD
Abstract
Functional neuroimaging studies have consistently reported abnormalities in the visual cortex in patients with dementia with Lewy bodies
(DLB), but their neuropathologic substrates are poorly understood. We
analyzed synaptic proteins and choline acetyltransferase (ChAT) in the
primary (BA17) and association (BAs18/19) visual cortex in DLB and
similar aged control and Alzheimer disease (AD) subjects. We found
lower levels of synaptophysin, syntaxin, SNAP-25, and F-synuclein in
DLB subjects versus both aged control (68%Y78% and 27%Y72% for
BA17 and BAs18/19, respectively) and AD cases (54%Y67% and
10%Y56% for BA17 and BAs18/19, respectively). The loss in ChAT
activity in DLB cases was also greater in BA17 (72% and 87% vs AD
and control values, respectively) than in BAs18/19 (52% and 65% vs
AD and control groups, respectively). The observed synaptic and
ChAT changes in the visual cortices were not associated with tau or
A-amyloid pathology in the occipital or the frontal, temporal, and
parietal neocortex. However, the neocortical densities of LBs, particular those in BA17 and BAs18/19, correlated with lower synaptic and
ChAT levels in these brain areas. These findings draw attention to
molecular changes within the primary visual cortex in DLB and correlate with the neuroimaging findings within the occipital lobe in
patients with this disorder.
Key Words: F-Synuclein, ChAT activity, Dementia with Lewy
body, Occipital lobe, SNAP-25, Synaptic proteins, Synaptophysin.
From the Institute for Ageing and Health, Newcastle University (EBM-L, AA, JM,
ZA-A, IB, EJ, RHP, EKP, RNK, IGM); Department of Neuropathology/Cellular Pathology, Royal Victoria Infirmary (EJ, RHP), Newcastle upon Tyne,
UK; Department of Psychiatry, University of British Columbia, Vancouver,
Canada (WGH); and NOVOCASTRA Laboratories Ltd., Newcastle
upon Tyne, UK (AC, JD, GM).
Send correspondence and reprint requests to: Elizabeta B. MukaetovaLadinska, PhD, Institute for Ageing and Health, Wolfson Research Centre,
Campus for Ageing and Vitality, Newcastle University, Newcastle upon
Tyne NE4 5PL UK; E-mail: [email protected]
The study was sponsored by PPP Grant and Alzheimer Society to Elizabeta B.
Mukaetova-Ladinska, Robert H. Perry, and Ian G. McKeith.
This work was supported by the UK NIHR Biomedical Research Centre for
Ageing and Age-Related Disease Award to the Newcastle upon Tyne
Hospitals NHS Foundation Trust. William G. Honer was supported by the
Canadian Institutes of Health Research.
INTRODUCTION
Occipital lobe hypoperfusion (1Y3) and glucose hypometabolism (4Y7) have been consistently reported in both probable and autopsy-confirmed cases of dementia with Lewy bodies
(DLB) (8, 9). Although these findings have low sensitivity and
specificity for routine clinical use (4), they may offer an explanation for the high prevalence of visual symptoms, including
visuoperceptual deficits and visual-related behavioral symptoms (e.g. visual hallucinations) in patients with DLB (10, 11).
The neuroimaging findings are at variance with the limited neuropathology detected in the occipital cortex of DLB.
Thus, the LB density is the lowest in this area (4, 12), with
Brodmann area 17 (BA17) having a significantly lower density
of LBs and Lewy neurites in relation to BA18 and BA19 (13).
Even at the neurochemical level, the tau (paired helical filaments’ accumulation) and amyloid (AA) protein content in the
occipital lobe do not discriminate between older control subjects and those with the LB diseases, including DLB and Parkinson disease (PD) with and without dementia (14). However,
patients with DLB do have significantly lower choline acetyltransferase (ChAT) activity in their visual association cortex
compared with those with AD and/or control subjects (15).
Because components of the cholinergic neurotransmitter
system (e.g. ChAT activity) correlate with a loss of synaptophysin in the midYfrontal neocortex in DLB (16), we undertook
a biochemical analysis of synaptic proteins (synaptophysin,
syntaxin, SNAP-25, and F-synucleins) of autopsy-proven DLB,
AD, and age-matched control subjects with no neurologic disorder. The aim of the study was to explore whether the primary and association visual cortices in DLB subjects exhibit
changes in the level of these synaptic proteins, and if so, how
they are related to the ChAT changes seen in these areas.
MATERIALS AND METHODS
Cases
Brain tissues were obtained from the Newcastle Brain
Tissue Resource. We acquired 22 prospectively assessed cases
with clinical and neuropathologic diagnoses of DLB, 11 with
AD, and 16 cases with no cognitive impairment and no neurologic and psychiatric disorders, which were used as a similarage control group. The demographic and neuropathologic
characteristics of the groups (sex, age at death, disease duration,
J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
53
J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
Mukaetova-Ladinska et al
postmortem delay, cortical LB score, LB disease diagnostic category, and Braak stage) are provided in Table 1. Use of autopsy
brain tissues was approved by the Local Research Ethics Committee of Newcastle and North Tyneside Health Authority.
Clinical data of DLB, AD, and control subjects were
obtained from the Institute for Ageing and Health database. The
review of the clinical histories for the control subjects showed
no presence of cognitive impairment or psychiatric and/or neurologic disorders. Clinical information for the cognitive functioning of the AD subjects was recorded at their baseline and
contained Mini-Mental State Examination and Cambridge
Assessment for Mental Disorders in the Elderly information.
The clinical diagnosis of DLB was based on the consensus
clinical criteria (17, 18). Clinical assessments were repeated
annually until death and included a standardized psychiatric
history (History and Aetiology Schedule) (19), assessment of
cognitive function using the CAMDEX (20), a standardized
physical examination including the modified Unified Parkinson’s Disease Rating Scale (21), and assessment of psychosis
using the Columbia University Scale for Psychopathology in
Alzheimer’s disease (22). The presence of psychotic features,
including visual hallucinations, auditory hallucinations, and
delusional phenomena (including delusional misidentification),
was estimated according to Burns et al (23).
in paraffin, and 6-Km-thick sections were examined using
standard neurohistopathological stains, including hematoxylin
and eosin for general histology, cresyl fast violet for neuronal
population density, and Loyez method for myelin staining.
Further neuropathologic assessment was done on paraffinembedded tissue sections immunostained for ubiquitin (DAKO
Glostrup, Denmark), Tau2 (Sigma, St. Louis, MO), AT8
(Endogen, Pierce, Rockford, IL), A-amyloid (DAKO), and
>-synuclein (Novocastra Laboratories, Ltd, Newcastle upon
Tyne, UK). The densities of amyloid plaques, neurofibrillary
tangles, and LBs from frontal (BA9), temporal (BA20Y22),
parietal (BA40), and occipital lobe (BA17 and BAs18/19)
were recorded. The >-synucleinYimmunoreactive Lewy neurites
in each region were examined at a magnification of 250,
and their density was expressed on a semiquantitative scale
of 0 to 3 (0, absent; 1, sparse; 2, moderate; and 3, frequent
[24]). Concomitant neurofibrillary pathology was assessed
using the Braak staging (25). The neuropathologic diagnosis
of DLB and LB staging was established according to criteria
recommended by the First and Third Report of the DLB
Consortium (17, 18), whereas the neuropathologic diagnosis
of AD was consistent with CERAD (Consortium to Establish
a Registry for Alzheimer’s Disease) (26) and NIA-Reagan
(27) neuropathologic criteria.
Neuropathology
Protein Extraction and Quantitative Assays
At autopsy, the right cerebral hemisphere, cerebellum,
and brainstem were fixed in formalin; samples were embedded
Left cerebral hemispheres were coronally sliced at 1-cm
intervals and snap-frozen for neurochemical studies at the time
TABLE 1. Demographic and Neuropathologic Characteristics of Cases
Parameters
Sex, F/M*
Age at death, years
Duration of disease, years
Cognitive measures at baseline
MMSE
CAMCOG
PM delay, hours
Total cLB score
Occipital cLB score
BA17
BAs18/19
LBD diagnostic categories
BST
Occipital neurofibrillary tangle density*
BA17
BAs18/19
Occipital plaque density (AA)
BA17
BAs18/19
Occipital vascular pathology*†
DLB
AD
Control
12/10
82.14 T 1.36
5.68 T 0.67
4/7
80.46 T 2.71
6.45 T 0.47
8/8
77.31 T 3.68
NA
13.25 T 1.91
38.17 T 8.09
42.41 T 3.84
14.36 T 1.05 (4Y20)
23.03 T 3.01
85.51 T 13.52
19.83 T 3.55
0.00 T 0.00
NA
NA
30.07 T 8.45
0.00 T 0.00
0.1665 T 0.07698
0.3382 T 0.16816
2.86 T 0.25
3.54 T 0.27 (IIYV)
0.00 T 0.00
0.00 T 0.00
0.00 T 0.00
5.00 T 0.00 (V)
p
0.613
0.573
0.969
0.273
0.207
0.137
G0.0001‡§
0.00 T 0.00
0.00 T 0.00
0.00 T 0.00
1.86 T 0.63 (0YIV)
0.013
0.003‡§
G0.0001‡§
G0.0001‡§k
0.069 T 0.053
0.152 T 0.119
0.410 T 0.205
1.560 T 0.463
0.00 T 0.0
0.00 T 0.000
0.011k
G0.0001§k
5.43 T 1.533
6.02 T 1.402
2/20
10.55 T 2.543
7.90 T 2.793
4/7
0.03 T 0.065
0.09 T 0.085
0/16
0.008‡k
0.05‡k
0.0151k
Total cLB score represents a sum of semiquantitative scores from the transentorhinal (TE), anterior cingulate (AC), temporal (T), frontal (F), and parietal (P) cortex using the
scoring scheme recommended by the Third Report of the DLB Consortium, where presence of LBs is scored as: 0, none; 1, few; 2, moderate; 3, high; 4, very high. The LBD diagnostic
categories are defined by a total cLB score as follows: L (Limbic) = 2 to 9 (2 to 6 in TE and AC and 0 to 3 in T and F and P); DN (diffuse neocortical) = 7 to 20 (4Y8 in TE and AC;
+3Y12 in T and F and P) (18). Lewy neurites were not detected in neocortical areas in AD or control cases, whereas in DLB cases, BA17 and BAs18/19 were devoid of Lewy neurites.
Values represent mean T SEM (range). *W2 tests; †the identified vascular pathology consisted of microinfarcts, small vessel disease, and ischemic changes.
Significance values p G 0.05: ‡DLB versus control; §DLB versus AD; kAD versus control.
BST, Braak stage; CAMCOG, Cambridge Assessment for Mental Disorders in the Elderly; cLB, cortical LB; F/M, female/male; LBD, LB disease; MMSE, Mini-Mental State
Examination; NA, not applicable; PM, postmortem.
54
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Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
DLB: Synaptic and ChAT Loss in Visual Cortex
TABLE 2. Levels of Synaptic Proteins and ChAT in Primary and Association Visual Cortex of Control and DLB Cases
BA17
Protein Measures
Synaptophysin
Syntaxin
SNAP-25
F-Synuclein
ChAT activity
BAs18/19
DLB
AD
Control
p
DLB
AD
Control
p
5.78 T 0.99
4.67 T 3.24
7.58 T 1.02
0.66 T 0.08
0.31 T 0.10
12.62 T 2.52
11.13 T 1.53
18.68 T 2.51
2.01 T 0.33
1.10 T 0.80*
21.89 T 3.85
21.14 T 3.38
23.80 T 4.18
2.72 T 0.32
2.24 T 0.29
G0.0001‡§
G0.0001‡§k
G0.0001 ‡§
G0.0001‡§
G0.0001‡k
10.62 T 1.92
8.60 T 1.43
12.09 T 2.03
0.78 T 0.12
0.80 T 0.18
13.78 T 2.18
11.42 T 1.75
18.51 T 3.10
1.79 T 0.29
1.68 T 0.19†
14.55 T 1.87
12.65 T 11.37
19.81 T 2.57
2.83 T 0.29
2.26 T 0.18
0.001‡§
0.001‡§
G0.0001‡§
0.002‡§
0.0006‡§
*BA17 ChAT data from (36) and †BAs18/19 data from (37). The AD ChAT activity data were obtained using the same methods as for the DLB and control subjects.
p G 0.001: ‡DLB versus control; §DLB versus AD; kAD versus control.
Values represent mean T SEM of relative arbitrary units of immunoreactivity as determined by ELISA for the synaptic proteins and nanomoles per hour per milligram for ChAT
activity.
of autopsy. For biochemistry, brain tissue from primary (BA17)
and association (BAs18 and 19) visual cortex (0.5 g wet weight
gray matter) was subdissected from the frozen slices (R.H. Perry
et al, unpublished data) and used for the protein extraction and
quantitative assays. The extraction of synaptic (synaptophysin,
syntaxin, and SNAP-25) and heat-stable proteins (F-synuclein)
was done according to a previously published protocol (28).
Briefly, the brain tissue was homogenized in 0.32 mol/L
sucrose, and 100 KL of this brain homogenate was retained and
used for F-synuclein analysis. The rest of the brain homogenate
was subdivided into 2 portions and used for extraction of
synaptic proteins (precipitated with 1 mol/L NaCl) and heatstable proteins (precipitated with 60% ammonium sulfate).
Total levels of protein in the extract were determined using
the Lowry protein assay, based on the reaction of Cu+ produced
by the oxidation of peptide bonds, with Folin-Ciocalteu reagent
(29). There were no significant differences in the total protein
concentrations between DLB, AD, and control groups (BA17:
4.63 T 1.75 Kg/mL vs 3.59 T 1.21 Kg/mL vs 4.13 T 1.22 Kg/mL
for DLB, AD, and control groups, respectively, F = 0.575, p =
0.567; BAs18/19: 5.20 T 1.19 Kg/mL vs 5.49 T 1.00 Kg/mL vs
5.74 T 1.08 Kg/mL for DLB, AD, and control groups, respectively, F = 0.057, p = 0.945). All measurements were done
blind to clinical and neuropathologic diagnoses.
Description of Immunoprobes
The characteristics of the synaptic immunoprobes against
synaptophysin, syntaxin, and SNAP-25 (mAbs EP10, SP8,
and SP12, respectively) have been previously published (30).
The level of F-synuclein was estimated with mAb 21H12
(antiYF-synuclein antibody), the epitope of which remains un-
known. This immunoprobe was raised against a 65-AA sequence
of F-synuclein and was screened against >- and A-synuclein
peptides to identify specific reactivity to the F-synuclein protein (31). None of the biochemical measures were influenced
by postmortem delay, fixation, or agonal state, similarly to
previous studies (32, 33).
Immunoassays
Indirect ELISA was used to determine the protein level
of synaptophysin (1:10 dilution), syntaxin, SNAP-25, and
F-synuclein (all used in 1:100 dilution). Briefly, triplicates of
double dilutions of the antigen over 6 wells were coated overnight at 4-C using carbonate-bicarbonate buffer, washed in
0.05% Tween, blocked with 1% dried skim milk, and incubated
for 1 hour at 37-C. Plates were washed again and incubated for
1 hour at 37-C with primary antibodies to the antigen of interest
and diluted in 0.05% Tween in PBS (pH 6.8). After another
wash in 0.05% Tween in PBS, plates were incubated with secondary antibodies conjugated to horseradish peroxidase and
incubated again for 1 hour at 37-C. Colorimetric analysis of
reaction with trimethyl benzadine was performed, and the reaction was quenched after 10 minutes with 2N H2SO4. Plates
were read with Vmax plate reader (Molecular Devices, Sunnyvale, CA), and assay curves were plotted using SOFTmaxPro
(Version 4.7.1; Molecular Devices). All values were normalized
for 0.3-mL fraction from 0.3 to 0.5 g of brain tissue and
expressed as relative arbitrary units of immunoreactivity, similar to a previous report (28).
To determine ChAT activity, brain homogenates were
prepared from 20- to 30-mg frozen brain tissue from both analyzed area. The ChAT activity was measured using the modified Fonnum radiochemical method (34, 35), which includes
both zero time (0 minutes) and 90 minutes of incubation. The
TABLE 3. Correlations Between ChAT Activity and Synaptic Markers
Parameters
Brain Area
Groups
Synaptophysin
Syntaxin
SNAP-25
F- Synuclein
BA17
DLB
Control
DLB
Control
r = 0.775, p = 0.0001*
r = 0.097, p = 0.836
r = j0.068, p = 0.387
r = 0.538, p = 0.088
r = 0.762, p = 0.0001*
r = 0.038, p = 0.936
r = j0.168, p = 0.239
r = 0.439, p = 0.177
r = 0.737, p = 0.0001*
r = j0.33, p = 0.943
r = j0.187, p = 0.215
r = 0.433, p = 0.183
r = 0.517, p = 0.003*
r = j0.650, p = 0.890
r = j0.252, p = 0.142
r = 0.081, p = 0.814
BAs18/19
*Significant correlation, p e 0.003.
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55
J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
Mukaetova-Ladinska et al
ChAT activity was expressed as nanomoles of acetylcholine
produced per hour per milligram protein.
variables was determined by the Spearman rank correlation.
Significance of analysis was set at p = 0.05.
RESULTS
Statistical Analysis
Statistical analysis was performed using SPSS v.19.
Because data were not normally distributed (Shapiro-Wilk test),
group differences were tested with the Kruskal-Wallis one-way
analysis of variance test, whereas the specific sample pairs for
significant differences were addressed with the Mann-Whitney
U test with a Bonferroni correction for multiple comparisons.
The strength of association (monotonic relationship) between
Neuropathologic Features in Visual Areas of the
DLB Group
The total cortical LB score was significantly higher in the
DLB cases (p G 0.0001; Table 1). As expected, AD subjects
had the highest density of both neurofibrillary tangles and
amyloid plaques in occipital areas, whereas the control group
had only an insignificant amount of plaques. The DLB cases
TABLE 4. Correlation of Synaptic and Pathologic Markers in DLB and AD
Measurements
F-NFT
T-NFT
P-NFT
BA17-NFT
BAs18/19-NFT
F-AA SP
T-AA SP
P-AA SP
BA17-AA SP
BAs18/19-AA SP
F-LB
T-LB
P-LB
BA17-LB
BAs18/19-LB
F-LN
T-LN
P-LN
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
DLB
AD
BA17 Synaptophysin
BA17 Syntaxin
BA17 SNAP-25
BA17 F-Synuclein
0.707; 0.182
j0.238; 0.570
0.224; 0.818
j0.657; 0.156
0.707; 0.182
0.180; 0.670
j0.215; 0.362
j0.49; 0.909
j0.020; 0.936
0.119; 0.779
j0.300; 0.624
0.2; 0.800
j0.600; 0.285
j0.009, 0.912
j0.300; 0.624
j0.008; 0.932
j0.308; 0.199
0.262; 0.531
j0.372; 0.117
0.381; 0.352
j0.700; 0.188
NA
j0.900; 0.037*
NA
j0.667; 0.219
NA
j0.546; 0.029*
NA
j0.539; 0.026*
NA
j0.144; 0.502
NA
j0.077; 0.719
NA
j0.024; 0.913
NA
0.354; 0.559
0.095; 0.823
j0.112; 0.858
j0.543; 0.266
0.354; 0.559
0.695; 0.056
j0.123; 0.604
j0.122; 0.774
0.105; 0.668
0.167; 0.693
j0.5; 0.391
0.8; 0.2
j0.7; 0.188
0.008; 0.932
j0.5; 0.391
0.5; 0.667
j0.319; 0.184
j0.071; 0.867
j0.352; 0.139
0.071; 0.867
j0.4; 0.505
NA
j0.7; 0.188
NA
j0.410; 0.493
NA
j0.449; 0.081
NA
j0.449; 0.071
NA
j0.122; 0.569
NA
j0.059; 0.784
NA
0.113; 0.600
NA
0.354; 0.559
j0.024; 0.955
j0.112; 0.858
j0.543; 0.266
0.354; 0.559
0.611; 0.108
j0.145; 0.542
j0.244; 0.560
0.072; 0.768
0.238; 0.570
j0.086; 0.780
j0.8; 0.2
j0.7; 0.188
0.008; 0.932
j0.242; 0.425
0.5; 0.667
j0.347; 0.145
j0.429; 0.289
j0.404; 0.087
j0.024; 0.955
j0.4; 0.505
NA
j0.7; 0.188
NA
j0.410; 0.493
NA
j0.465; 0.070
NA
j0.523; 0.031*
NA
j0.097; 0.652
NA
j0.092; 0.668
NA
0.120; 0.575
NA
0.008; 0.932
j0.786; 0.036*
j0.224; 0.718
0.5; 0.391
0.009, 0.912
j0.054; 0.908
j0.60; 0.800
0.317; 0.444
j0.148; 0.545
0.524; 0.183
0.006; 0.986
0.009, 0.912
0.1; 0.873
0.087; 0.821
j0263; 0.386
0.009, 0.912
j0.28; 0.908
0.786; 0.021*
j0.041; 0.869
0.960; 0.0001*
0.2; 0.747
NA
0.1; 0.873
NA
j0.308; 0.614
NA
j0.191; 0.479
NA
j0.318; 0.213
NA
j216; 0.310
NA
j0.297; 0.159
NA
j0.355; 0.089
NA
Correlations and p values are shown. AD cases were devoid of LBs and LNs in any of the neocortical areas analyzed. In the DLB subjects, no LNs were detected in BA17 or
Ba18/19. Significant correlations and p values are underlined. *p G 0.05.
AD ChAT activities were determined in another cohort of patients.
AA SP, A-amyloid senile plaques; BA17, Brodmann area 17; BAs18/19, Bradman areas 18 and 19; F, frontal lobe; LN, Lewy neurite; NA, not applicable; NFT, neurofibrillary
tangle; NP, not performed; P, parietal lobe; T, temporal lobe.
56
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J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
had a lower density of neurofibrillary tangles and amyloid
plaques in BAs18/19 versus AD (p G 0.0001) but a significantly
higher density of LBs compared with both AD and control
groups (p G 0.0001). Vascular abnormalities in the occipital
lobes were not detected in any of the control subjects, but they
were present in 30% of the AD group and in only 2 DLB cases
(Table 1).
Synaptic Protein Levels in Visual Areas of DLB
Subjects
The DLB subjects had significantly lower levels of synaptic (synaptophysin, syntaxin, SNAP-25, and F-synuclein)
proteins in both the primary and association visual cortices
DLB: Synaptic and ChAT Loss in Visual Cortex
compared with AD and control subjects (Table 2). The reduction of these proteins was more pronounced in BA17 (3- to
5-fold and 2- to 3-fold lower level of synaptophysin, syntaxin,
SNAP-25, and F-synuclein in comparison with those in the
controls and AD subjects) than in BAs18/19 (1.5- to 3-fold
lower). Similar significant reductions, with more pronounced
changes in BA17 than in BAs18/19, were apparent in ChAT
activities for the DLB group (Table 2).
Control subjects had higher levels of synaptophysin
(F = 2.930, p = 0.097) and syntaxin (F = 5.416, p = 0.027)
but not of SNAP-25 (F = 0.658, p = 0.424) or F-synuclein
(F = 0.061, p = 0.807) in BA17 compared with BAs18/19
(Table 2). In contrast, in DLB, this relationship was reversed
with BAs18/19, showing higher levels of synaptic proteins
.
BA17 ChAT
j0.027; 0.949
NP
j0.006; 0.988
NP
j0.027; 0.949
NP
j0.142; 0.562
NP
j0.52; 0.836
NP
0.378; 0.403
NP
0.685; 0.090
NP
0.523; 0.229
NP
j0.311; 0.195
NP
j0.318; 0.185
NP
0.185; 0.661
NA
0.026; 0.952
NP
j0.203; 0.630
NP
j0.523; 0.038*
NP
j0.609; 0.009*
NP
j0.235; 0.365
NP
0.182; 0.485
NP
j0.165; 0.526
NP
BAs18/19 Synaptophysin
j0.354; 0.559
0.119; 0.779
0.112; 0.858
j0.429; 0.397
0.354; 0.559
0.084; 0.844
j0.007; 0.976
j0.537; 0.170
j0.224; 0.357
j0.429; 0.289
0.255; 0.400
0.2; 0.8
0479; 0.098
j0.008; 0.932
0.105; 0.668
j0.008; 0.932
0.321; 0.180
0; 1
0.366; 0.123
0.095; 0.823
0.5; 0.391
NP
0.3; 0.624
NA
0.872; 0.054
NA
0.257; 0.337
NA
0.393; 0.119
NA
0.137; 0.524
NA
0.116; 0.591
NA
j0.005; 0.980
NA
BAs18/19 Syntaxin
j0.354; 0.559
0.452; 0.260
0.112; 0.858
j0.543; 0.266
j0.354; 0.559
0012; 0.902
j0.109; 0.648
j0.732; 0.039*
j0.309; 0.197
j0.643; 0.086
j0.072; 0.815
j0.6; 0.4
0.237; 0.436
0.071; 0.867
0.1; 0.873
0.009, 0.912
0.285; 0.237
j0.381; 0.352
0.373; 0.115
j0.286; 0.493
0.5; 0.391
NA
0.3; 0.624
NA
0.872; 0.054
NA
0.431; 0.096
NA
0.597; 0.011*
NA
0.211; 0.323
NA
0.035; 0.872
NA
0.000; 0.999
NA
BAs18/19 SNAP-25
j0.707; 0.182
0.214; 0.610
0.224; 0.718
j0.314; 0.544
j0.707; 0.182
j0.216; 0.608
j0.157; 0.508
j0.537; 0.170
j0.316; 0.187
j0.619; 0.102
0.161; 0.600
j0.8; 0.2
0.1; 0.873
j0.009; 0.912
0.768; 0.002*
j0.008; 0.932
0.297; 0.218
0; 1
0.340; 0.155
j0.24; 0.955
0.5; 0.391
NA
0.600; 0.285
NA
0.975; 0.005*
NA
0.426; 0.1
NA
0.536; 0.027*
NA
0.225; 0.290
NA
j0.001; 0.995
NA
0.004; 0.985
NA
BAs18/19 F-Synuclein
j0.354; 0.559
0.214; 0.610
0.112; 0.858
j0.314; 0.544
0.354; 0.559
j0.216; 0.608
0.077; 0.746
j0.048; 0.911
0.040; 0.872
0.014; 0.912
0.1; 0.873
j0.8; 0.2
0.460; 0.114
j0.041; 0.869
0.1; 0.873
j0.008; 0.932
0.372; 0.117
j0.537; 0.170
0.359; 0.131
j0.5; 0.207
0.5; 0.391
NA
0.3; 0.624
NA
0.872; 0.054
NA
0.228; 0.395
NA
0.329; 0.197
NA
0.167; 0.437
NA
j0.066; 0.760
NA
j0.148; 0.491
NA
BAs18/19 ChAT
j0.082; 0.846
NP
0.134; 0.752
NP
j0.082;0.846
NP
0.343; 0.193
NP
0.26;2 0.346
NP
0.613; 0.144
NP
0.667; 0.102
NP
0.685; 0.090
NP
j0.114 0.673
NP
j0.091;0.737
NP
0.247; 0.555
NP
0.012; 0.952
NP
0.266; 0.525
NP
j0.772;0.002*
NP
j0.701;0.005*
NP
j0.481; 0.082
NP
j0.299; 0.299
NP
j0.161; 0.583
NP
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57
Mukaetova-Ladinska et al
compared with BA17 (synaptophysin, F = 5.009, p = 0.031;
syntaxin, F = 6.142, p = 0.017; SNAP-25, F = 3.949, p = 0.053;
but not F-synuclein, F = 0.482, p = 0.633). There were no
differences with respect to synaptic content between BA17
and BAs18/19 in the AD group (synaptophysin, F = 1.739,
p = 0.098; syntaxin, F = 0.466, p = 0.647; SNAP-25, F = 0.228,
p = 0.822; F-synuclein, F = 0.513, p = 0.614). The ChAT
activities were similar between BA17 and BAs18/19 in control
(F = 0.247, p = 0.625) and AD subjects (F = 1.236, p = 0.231),
whereas DLB subjects had a higher level of ChAT in BAs18/
19 in comparison with that in BA17 (F = 6.333, p = 0.016).
In the primary visual cortex in DLB subjects, previously
reported ChAT activities correlated with the expression of
synaptophysin, syntaxin, SNAP-25, and F-synuclein, whereas
these correlations did not reach significance for the secondary
visual cortex (Table 3). There was no correlation between
ChAT activities and levels of synaptic proteins in both BA17
and BAs18/19 in the control group.
Correlative Biochemical and Neuropathologic
Analysis
We conducted a correlative biochemical-neuropathologic
analysis to address whether the changes in occipital lobe synaptic content in DLB and AD groups were associated with local
or more remote neuropathology (Table 4). Neurofibrillary and
amyloid pathology in the frontal, temporal, parietal, and occipital lobes (BA17 and BAs18/19 analyzed separately) did not
play a major role in the decline of synaptic proteins and ChAT
in DLB and AD subjects, with the exception of the density of
neurofibrillary tangles in the frontal lobe (negatively correlated with BA17 F-synuclein levels; r = j0.786, p = 0.036)
and BA17 (negatively correlated with BAs18/19 syntaxin levels; r = j0.732, p = 0.039). Interestingly, the amyloid load of
BA17 and BAs18/19 was positively correlated with the BA17
F-synuclein in AD subjects only. However, the presence of
LBs in the temporal, parietal, and occipital lobes was negatively correlated with synaptic proteins and ChAT activity in
BA17 but not in the same manner in BAs18/19 (syntaxin and
SNAP-25) in DLB subjects. In contrast, the presence of Lewy
neurites did not seem to influence the level of synaptic proteins
and ChAT activity in the occipital lobe (Table 4).
DISCUSSION
We demonstrate significant changes in synaptic proteins
and ChAT activities in the primary and association visual cortices in DLB. This loss occurs in the presence of minor histopathologic changes detected in these areas, suggesting that low
levels of LB pathology (consisting of >-synuclein aggregates)
might instigate or result in these changes either on their own or
with amyloid-A enhancing the >-synucleinYinduced synapse
damage, as recently demonstrated in vitro (38).
There are some limitations to the present study. We focus
here on the occipital lobe rather than including a broader comparison with other neocortical brain regions. Therefore, the
results reported herein will necessarily have to be placed into
context with results of comparable analyses to be obtained from
other brain regions. Moreover, because ChAT data on cases of
PD with dementia were not available for the present study, such
58
J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
cases were not included. Thus, it may also be difficult at this
time to know how to incorporate the present results into the
broader range of diseases with LB pathology. Moreover, we did
not perform biochemical measurements of A-amyloid in the
cortical samples. Nevertheless, the present data suggest possible correlates of the neuroimaging findings of hypoperfusion
and molecular substrates of dysfunction that are characteristic
of the occipital lobes in subjects with DLB.
Synaptic loss (assessed using synaptophysin and SNAP-25
immunoprobes) in the medial temporal lobe and the frontal
neocortex has been previously reported in DLB with and without
concomitant AD neuropathology (39Y47). Some of these studies
have related the substantial depletion of synaptophysin and
SNAP-25 in DLB to the presence of AD pathology, with the
synaptic loss being similar to that observed in AD (40, 42). A
larger clinicobiochemical and neuropathologic correlative study
in AD also reported a significant negative correlation between
neurofibrillary tangles and synaptophysin expression (48). In
contrast, individuals with pure DLB and those with LBs
without dementia have either been reported to have unchanged
levels of synaptophysin versus control subjects (43) or somewhat lower levels of synaptic proteins but not to the extent
observed in AD (40, 45, 46). We demonstrate that synaptic
changes in DLB occur in the presence of low levels of LB
pathology but not of AD pathology or Lewy neurites. Thus,
our results contrast with those in previous reports. Nevertheless, we show that in DLB, in particular, the loss of synaptic
proteins might reflect cortico-cortical disconnectivity, similar
to that described in AD (49, 50). In support of this are recent
findings of reduced fractional anisotropy restricted predominantly to the parieto-occipital white matter tracts in DLB subjects only (51), highlighting the potential importance of white
matter tract changes as a possible mode for >-synuclein propagation in DLB.
Our findings of differential synaptic protein expression in
DLB visual areas point toward distinct neurobiologic mechanisms underlying the synaptic changes in BA17 and BAs18/19.
Thus, whereas the decline in synaptic proteins in the primary
visual cortex might be explained by LB pathology alone, it
seems that the slightly higher LB score in BAs18/19 may have
resulted in elevated synaptic protein levels in these brain areas.
A biochemical study reported that >-synuclein aggregates (considered by some to be early stages of LBs [52]) were accompanied by an increase in syntaxin and synaptophysin levels
(53). This would be in agreement with an increase in the Nterminal end of >-synuclein in this brain area (Elizabeta B.
Mukaetova-Ladinska, unpublished data). These findings have
now been further confirmed in a transgenic >-synuclein animal
model in which >-synuclein aggregates contribute to the redistribution of t-SNARE synaptic proteins in the absence of major
quantitative synaptic changes, as seen on immunoblots (54). We
have previously reported upregulation of neocortical synaptic
proteins (synaptophysin, syntaxin, and SNAP-25) accompanied
by MAP2 and >-synuclein increase at the time of emerging
neurofibrillary pathology in AD (28). These findings provide
further support for the concept that the initial stages of neurodegenerative pathology, including LB and neurofibrillary
pathology, are accompanied by reorganization of presynaptic
end terminals.
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J Neuropathol Exp Neurol Volume 72, Number 1, January 2013
Our results showing depleted syntaxin levels in DLB
individuals are in contrast to a previous report of upregulation
of the syntaxin gene (syntaxin 8) in nigral dopaminergic neurons in vitro (55) and unchanged striatum syntaxin levels in a
transgenic animal model of PD (54). Because oxidative stress
is a primary pathogenic mechanism of nigral dopaminergic cell
death in PD and other LB-bearing diseases (56), this raises the
possibility of highly selective changes occurring in brain areas
harboring dopaminergic neurons rather than uniformly in all
brain areas. However, we cannot exclude the possibility that
expression patterns of synaptic proteins may vary in different
systems (e.g. tissue culture and transgenic animal models vs
human cerebral tissue). The loss of syntaxin within the occipital lobe parallels the loss of another SNARE protein, SNAP25, and indicates that the t-SNARE complex is substantially
impaired in DLB, a similar finding to our previous report of
t-SNARE loss in the LB variant of AD but not pure AD (57).
This may thus underlie the characteristic profound neurotransmitter deficit in DLB. In support of this is our finding of
significant ChAT depletion within the same areas.
A decrease of F-synuclein in the occipital lobe suggests
the possibility of a generalized decrease in synaptic proteins in
DLB subjects. This is supported by correlations between the
level of F-synucleins with the level of synaptophysin, syntaxin,
and SNAP-25 in both the primary and secondary visual cortex
in DLB. Our results are in contrast to those of a previous study
that reported unchanged level of F-synuclein mRNA in DLB
neocortex (58). This decrease in F-synuclein in DLB appears in
areas that have minimal >-synuclein and neurofibrillary pathology in contrast to AD subjects who had a substantially higher
density of neurofibrillary tangles in both analyzed visual areas.
This is notable because F-synuclein has recently been implicated in tubulin polymerization and inducing microtubule
bundling (59). Thus, the observed decrease in AD subjects (all
having Braak stage V at the time of their death) may indicate
early microtubule disassembly, preceding the overt neurofibrillary pathology occurring in the primary and secondary
visual cortex in Braak stage VI.
The truncation and hyperphosphorylation of tau protein
alone, however, cannot be the explanation for the F-synuclein
decline in DLB because our group had a lower Braak stage.
Interestingly, a previous study failed to detect F-synuclein in
DLB brain lysates (60); thus, our findings of up to 86% lower
F-synuclein levels would be in keeping with the results of
the latter study. Another explanation for the lower level of
F-synuclein may be attributed to the greater social submissiveness of DLB subjects compared with that of AD patients
because F-synuclein mRNA can be regulated via behavioral
patterns, with higher levels found widespread in all neocortical
areas in dominant subjects only (61).
In summary, we describe the presence of substantial depletion of synaptic proteins in the occipital lobes of subjects
with DLB that, except for a relatively minor LB pathology,
is not associated with overt neuropathology. Further studies will
need to concentrate on questions examining whether the depletion of synaptic proteins and ChAT reflect pathologic changes
(including amyloid-A and >-synuclein oligomers) in the afferent input and/or an arrest in synaptic remodeling in the primary
and association visual areas of DLB patients. Our finding of a
DLB: Synaptic and ChAT Loss in Visual Cortex
greater synaptic protein depletion in the primary than in the
association visual cortex in DLB needs to be explored further
to determine its clinical usefulness in terms of developing
novel diagnostic tools and/or pharmacologic treatments for
DLB patients, particularly those with prominent visual spatial
deficits and persistent visual hallucinations.
ACKNOWLEDGMENTS
The authors thank all participants and their families
who have taken part in the longitudinal clinical studies and
participated in the autopsy tissue program and Mrs Caroline
Kirk and Mrs Alyson Goldwater for secretarial help and
editing the text.
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