Supplementary Information
Table S1. Characteristics of subjects used in array tomography studies
Case
Age (y)
Gender
Diagnosis
Braak stage
AD1
77
male
AD
V/VI
APOE
genotype
3/4
AD2
90
male
AD
IV/VI
3/3
AD3
83
female
AD
VI/VI
3/4
AD4
74
female
AD
VI/VI
3/4
AD5
61
male
AD
V/VI
3/4
AD6
84
female
AD
VI/VI
4/4
AD7
89
male
AD
VI/VI
3/3
AD8
80
female
AD
VI/VI
4/4
AD9
84
male
AD
V/VI
3/4
AD10
81
male
AD
VI/VI
3/3
AD11
90
male
AD
VI/VI
4/4
HC1
66
male
N/A
3/3
HC2
69
male
left hemisphere
stroke*
NNP
N/A
3/3
HC3
70
male
NNP
N/A
3/3
HC4
75
male
NNP
N/A
3/3
HC5
62
male
NNP
N/A
3/3
NNP – no neurological phenotype, AD – Alzheimer’s disease, *tissue taken from right
hemisphere, postmortem intervals approximately 24hrs. N/A- tissue not available for staging.
Table S2. Characteristic of subjects used in biochemical studies
Braak stage
APOE
genotype
control
I/VI
3/3
male
control
I/VI
3/4
80
female
control
II/VI
2/3
HC9
88
female
control
II/VI
3/3
HC10
85
male
control
II/VI
3/3
HC11
57
female
control
N/A
3/3
AD12
84
female
AD
VI/VI
3/3
AD13
92
female
AD
VI/VI
3/3
AD14
65
male
AD
V/VI
3/3
AD15
74
male
AD
V/VI
3/3
AD16
75
female
AD
VI/VI
3/3
AD17
93
male
AD
V/VI
3/3
AD18
92
male
AD
V/VI
4/4
AD19
74
male
AD
V/VI
4/4
AD20
68
female
AD
VI/VI
4/4
AD21
80
female
AD
VI/VI
4/4
AD22
71
female
AD
VI/VI
4/4
AD23
89
male
AD
V/VI
4/4
Case
Age (y)
Gender Diagnosis
HC6
74
female
HC7
76
HC8
N/A- tissue not available for staging.
Supplemental Methods
Calculating Random versus observed colocalization between synaptic elements and oA
The level of oA colocalization with synaptic elements was higher than expected by chance in
all experiments. To calculate the chance level, we used the density of synaptic markers and the
median size of each synapse at a given distance from plaques to generate a random distribution
of synaptic puncta and overlaid this on a random distribution of the known NAB61 burden for
that plaque distance and predict that ~0.13% of synapsin puncta would be NAB61 positive in
the area within 10 microns from a plaque edge by chance. We found that ~12% of both pre and
postsynaptic elements are positive for NAB61 at the edge of plaques, which is about 90 fold
higher than expected by chance. Parallel calculations with control brain data indicate that
random colocalization between oA and synapsin I puncta would occur at 0.0047%. Thus,
despite the rarity of oApuncta in control brains, our observation that 0.73% of synapsin I
puncta colocalize with NAB61 positive puncta is ~165 fold higher than expected by chance
alone. Taken together, these data strongly suggest that oA, mostly associated with plaques,
targets synapses in the human brain and may be playing a role in synapse loss.
Array tomography Sample preparation
For array tomography, samples were prepared as outlined elsewhere (1-3). Briefly, fresh
tissue samples were collected from the superior temporal gyri of each subject and fixed in 4%
paraformaldehyde and 2.5% sucrose in PBS for 3 h. The samples were dehydrated through
ethanol and into LR White resin (Electron Microscopy Sciences) and polymerized overnight at
53 °C. Embedded blocks were cut into ribbons of 70-nm sections on an ultracut microtome
(Leica) by using a Jumbo Histo Diamond Knife (Diatome, Hatfield, PA).
Array tomography image analysis
Images were analyzed as described elsewhere (1). Briefly, Images were viewed and
analyzed with Image J (National Institutes of Health open software) and MATLAB (Mathworks).
For analysis, each set of images was converted to stacks, and aligned by using the Image J
MultiStackReg and StackReg plug-ins [courtesy of Brad Busse and P. Thevenaz et al. (4)].
Volumes at known distances from a plaque were selected, and an automated, threshold-based
detection program was used to count puncta that appeared in more than one consecutive
section (WaterShed program, provided by Brad Busse, Stephen Smith, and Kristina Micheva,
Stanford University). Synapse densities were calculated by dividing the number of PSD95 or
synapsin I puncta by the volume of tissue sampled. The median synapsin I-positive puncta and
PSD95-positive puncta density were calculated at each volume sampled and the median of
these taken as the volume for each case. Data were further subdivided by plaque distance to
find the median synapse density at each measured distance from a plaque for each case.
Parallel calculations were made for synaptic puncta size and the volume of the sampled
neuropil occupied by NAB61 staining.
Watershed exported a thresholded image stack (separate for each channel) showing
puncta that were present in more than one slice of the array. Using MATLAB, the output stacks
from PSD95, synapsin I, apoE and NAB61 staining for each region of interest were used to
generate matrices, and the number of PSD95 and synapsin puncta with any pixels colocalized
with NAB61, apoE, or both were counted. The minimum distance between each synapsin I
puncta and the nearest PSD95 object was measured and the synapsin I object was classified as
“paired” if there was a PSD95 object within 0.5 micrometers. Conversely, the distance from
each PSD95 object to the nearest synapsin I object was calculated and the PSD95 object
classified as “paired” if a synapsin I object was present within 0.5 micrometers. The sizes of
PSD95 and synapsin I puncta that contacted (or did not contact) NAB61 or apoE deposits were
also determined. The percentage of NAB61-positive PSD95 and synapsin puncta was
calculated by dividing the number of NAB61-positive puncta by the total number of PSD95 or
synapsin puncta in each region of interest. A similar method was use to count the number of
synaptic elements that contacted apoE or NAB61 puncta in images from cultured neurons.
Purification of lipidated apoE particles
Lipidated apoE particles were purified from human apoE2, apoE3 or apoE4 overexpressing
immortalized astrocytes using an affinity column as described elsewhere (5). Briefly, astrocytes
were cultured in Advanced DMEM (Invitrogen) with 10% FBS. After 80-90% confluency, cells
were washed with PBS and further incubated in Advanced DMEM with N-2 Supplement
(Invitrogen) and 3M of 25-hydroxycholesterol (Sigma) for 2-3 days. Collected culture media
were applied onto a column with mouse monoclonal antibody against human apoE (WU E-4,
courtesy of Holtzman, DM). Lipidated apoE particles were eluted from the column with 3 M
sodium thiocyanate, concentrated using Apollo centrifugal quantitative concentrators (QMWL:
150kDa, Orbital Biosciences) and dialyzed against PBS with 0.02% sodium azide.
SUPPLEMENTARY FIGURES
Supplementary Fig. 1: Synaptic localization of A confirmed with two different antibodies
on array tomography.
Array tomograms of human brain samples in which two different antibodies were used to probe
A reveal that oA colocalizes with synaptic elements. R1282, which sees different forms of A
including oA was found to colocalize significantly with NAB61-positive puncta both within
plaques and at synapses (arrow), albeit the signal intensity of R1282 was not as robust as
NAB61 on arrays. Scale bar is 5 m.
Supplementary Fig. 2: Characterization of synaptoneurosome preparations
Synaptoneurosomes are enriched in pre- and post-synaptic marker proteins. For each brain
sample, the total (T) and the synaptoneurosome (S) protein extracts were loaded side-by-side in
SDS-PAGE, followed by Western blotting against synaptic markers synaptophysin and PSD95.
Actin serves as the loading control.
Supplementary Fig. 3: ApoE is enriched at synapses in control and AD brains
Biochemical analysis of cytosolic (cyto) and synaptoneurosome (SNS) fractions of brain
homogenates from AD and control subjects reveal that apoE is enriched at synapses. This
observation is true regardless of APOE genotype and AD diagnosis. Interestingly, fragments of
apoE are enriched at synapses in the brains of AD subjects with APOE 4/4 genotype.
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