Supplementary Material
Brain amyloid-β oligomers in aging and Alzheimer’s disease
Sylvain E. Lesné, Mathew A. Sherman, Marianne Grant, Michael Kuskowski, Julie A.
Schneider, David A. Bennett & Karen H. Ashe
This section contains:
1) Supplementary Methods;
2) Supplementary Figure 1;
3) Supplementary Figure 2;
4) Supplementary Figure 3;
5) Supplementary Figure 4;
6) Supplementary Figure 5;
7) Supplementary Figure 6;
8) Supplementary Figure 7;
9) Supplementary Table 1;
10) Supplementary References.
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1) Supplementary Methods
Western blots and immunoprecipitations were performed as described in the main
paper, except that the primary antibodies were identified using infrared dye-conjugated
secondary antibodies (Li-Cor, USA), and detected with the Odyssey system (Li-Cor,
USA).
The following primary antibodies were used here: 6E10 [1:2,500] against the
amino-terminus of Aβ, 4G8 [1:2,500] against the mid-region of Aβ, biotinylated-6E10
[1:2,500] (Covance, USA), A42- and A40-end specific monoclonal antibodies 8G7 and
5C3 [1:1,000] (EMD Biosciences - Calbiochem, USA), APPCter-C17 [1:5,000] against
APP C-terminus (gift of A. Delacourte and N. Sergeant).
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2) Supplementary Figure 1
Comparison of the segregation of membrane-associated proteins from human brain in
the 4-step extraction protocol (Lesne et al., 2006; Sherman & Lesné, 2011) and the 3step extraction protocol (Shankar et al., 2008). Using the 4-step protocol, the NR1
subunit of NMDA receptors, detected using an antibody recognizing the carboxylterminal antibody of the NR1 subunit (NR1ct) and prion protein (PrP), both known
membrane- (MB)-associated proteins, appear selectively in the MB-enriched fraction.
Using the 3-step extraction protocol, these MB-associated proteins segregate into both the
TBS and the TBS-T extracts. In contrast, using the 4-step extraction protocol, these
proteins selectively appear in the MB-associated fraction, but not the extracellular- (EC)associated or the intracellular- (IC)-associated fractions. Although PrP can become
soluble following cleavage of the glycosylphosphatidylinositol (GPI) lipid tethering PrP
to the outer surface of the cell membrane, its absence in the EC-enriched fraction argues
against the possibility that the PrP that is present in the TBS extract is soluble PrP.
Instead, it indicates that some membrane-associated proteins are present in TBS extracts.
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3) Supplementary Figure 2
Levels of NeuN in the inferior temporal gyrus of ROS brain samples. (A) Representative
western blots of the neuron-specific nuclear protein NeuN used for quantification. αtubulin was used as a loading control. (B) Box plots showing the levels of NeuN. Green
indicates N, orange MCI, red AD and blue NAD. Group sizes are shown in parentheses.
Lines within box plots denote median values, upper and lower box boundaries represent
the 75th and 25th percentiles, respectively, and short bars flanking boxes represent the
95th and 5th percentiles. (Kruskal-Wallis followed by Mann-Whitney U test with
Bonferroni corrections). (C) Box plots depicting the levels of NeuN in study participants
segregated by Braak stages. No overall differences were detected across groups.
(Kruskal-Wallis followed by Mann-Whitney U test with Bonferroni corrections).
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4) Supplementary Figure 3
Characterization of Aβ*56 in human brain tissue by direct immunoblotting using various
antibodies. Below each western blot (WB) are the primary antibodies used to probe the
blot. WBs of membrane-enriched brain fractions show the inability of antibodies
recognizing portions of APP that do not contain Aβ, including APPCter and 22C11
antibodies, to detect Aβ*56 in human brain extracts or transgenic (Tg) mouse extracts.
Antibodies that detect the carboxyl-terminus of Aβ, including 5C3 antibodies recognizing
Aβ(x-40) and 8G7 antibodies recognizing Aβ(x-42), also cannot detect Aβ*56, probably
because its folding structure buries the carboxyl-terminus of Aβ. 4G8 antibodies
recognizing the mid-region of Aβ should not be used to detect Aβ*56, because of nonspecific immunoreactivity in the region of the blot where ~56kDa proteins migrate.
However, 6E10 antibodies detect Aβ*56 clearly. These results using human brain tissue
are consistent with previously reported data using Tg2576 mouse brain tissue (Lesné et
al., 2006). WT = non-transgenic mouse. Tg = Tg2576 transgenic mouse. NCI = no
cognitive impairment. Grey dots next to the right border of the blots indicate non-specific
binding.
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5) Supplementary Figure 4
4G8 and 82E1 antibodies recognize Aβ*56. The membrane-enriched fractions of human
brain tissue (profile obtained using inferior temporal gyrus from a subject diagnosed with
MCI) were probed using 4G8 recognizing Aβ(17-24) and 82E1 antibodies recognizing
the amino-terminus end of Aβ (residue +1). Proteins were fractionated by SDS-PAGE
and transferred to a nylon membrane. The detection of Aβ*56 by both antibodies (in
addition to 6E10) provides additional evidence that Aβ*56 is not a cleavage product of
APP, but rather an oligomeric species of Aβ.
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6) Supplementary Figure 5
Characterization of soluble oligomers in human and mouse brain using A11 antiserum. A
western blot of MB-associated proteins in human brain from subjects with no cognitive
impairment (N), mild cognitive impairment (M) or AD, and in mouse brain from a
transgene positive and non-transgenic (WT) J20 mice, shows multiple A11immunoreactive bands with molecular masses of 27, 56 and ~110 kDa, corresponding to
putative 6-mers, Aβ*56 and 24-mers of Aβ. A11 detects globular protein assemblies
with molecular masses >20 kDa. In addition to detecting Aβ oligomers, it also detects
oligomers of other proteins; therefore, some bands may represent non-Aβ oligomers.
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7) Supplementary Figure 6
Characterization of soluble Aβ dimers in human brain tissue using 4G8 and carboxyterminus end-specific Aβ antibodies. Immunoblots of human brain proteins (TBS
extracts) from subjects with no cognitive impairment (N), mild cognitive impairment (M)
or AD differentially detect apparent Aβ dimers with a molecular mass ~9 kDa. The use
of the carboxy-terminus end specific Aβ antibodies Mab13.1.1 and Mab2.1.3 further
supports that these soluble species are composed of Aβ. Please note that synthetic Aβ1-42
was not detected by the 40-end specific monoclonal antibody Mab13.1.1.
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8) Supplementary Figure 7
Relationships between tau-Alz50 and Aβ trimers or Aβ dimers in human brain tissue from
75 neurologically intact individuals from 1 – 96 years of age. (A) We found no
relationship between Aβ trimers and soluble tau-Alz50 (rho = 0.24, P = 0.12). (B) We
found no relationship between Aβ dimers and soluble tau-Alz50 (rho = 0.07, P = 0.55).
Black dots represent data from subjects from the NICHD Brain and Tissue Bank for
Developmental Disorders at the University of Maryland, and green dots subjects from the
Religious Order Study Brain Bank.
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8) Supplementary Table 1
Characteristics of subjects in this study from the Religious Orders Study
Group
Age of death (years),
Mean ± SD (Range)
No. of M/F (%)
Last MMSE score,
Mean ± SD (Range)
Years of education,
Mean ± SD (Range)
PMI (hours),
Mean ± SD (Range)
ApoE3/ApoE(2/3/4) (n)
ApoE4/ApoE(2/3/4) (n)
Years since last exam,
Mean ± SD (Range)
NCI
(n = 26)
82.97•± 7.53
MCI
(n = 34)
86.33•± 5.69
AD
(n = 24)
90.27•± 7.20
NAD
(n = 5)
85.19 ± 6.22
P values*
(67-96)
(72-97)
(73-104)
(75-90)
12/14
(46.1%)
28.35•± 1.38
(26-30)
18.3 ± 2.66
(12-23)
5.57•± 2.25
(2-10)
3/17/6
0/6/0
14/20
(41.2%)
26.41•± 2.96
(21-30)
17.6 ± 3.61
(10-24)
4.90•± 2.56
(1-9)
2/20/5
2/5/0
9/15
(37.5%)
12.33•± 8.79
(0-26)
18.0 ± 2.67
(10-22)
4.48•± 1.66
(2-9)
2/14/7
1/7/1
2/3
(40%)
14.20 ± 7.19
(8-26)
18.6•± 2.40
(16-20)
7.14•± 5.54
(5-9)
1/3/2
0/2/0
>0.99
0.51•± 0.25
(0.11-0.93)
0.46•± 0.32
(0.002-1.03)
0.50•± 0.31
(0.008-1.05)
0.24•± 0.14
(0.09-0.77)
>0.99
0.11
<0.01
>0.99
0.19
Abbreviations: NCI, no cognitive impairment; MCI, mild cognitive impairment; AD, Alzheimer’s disease;
NAD, Non‐AD dementia; MMSE, mini‐mental state examination; M/F, male/female ratio; PMI, post‐
mortem interval; ApoE, apolipoprotein E alleles; * Kruskal‐Wallis test followed by Bonferroni‐adjusted test
for multiple comparisons.
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8) Supplementary References
Lesné, S., Koh, M.T., Kotilinek, L., Kayed, R., Glabe, C.G., Yang, A., Gallagher, M.,
and Ashe, K.H. (2006). A specific amyloid- protein assembly in the brain impairs
memory. Nature 440, 352-357.
Shankar, G.M., Li, S., Mehta, T.H., Garcia-Munoz, A., Shepardson, N.E., Smith, I., Brett,
F.M., Farrell, M.A., Rowan, M.J., Lemere, C.A., et al. (2008). Amyloid- protein dimers
isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat
Med 14, 837-842.
Sherman, M.A., and Lesné, S.E. (2011). Detecting A*56 oligomers in brain tissues In
Alzheimer's Disease and Frontotemporal Dementia: Methods and Protocols. E. Roberson,
ed., Methods in Molecular Biology Series, Vol. 670 (Humana Press) pp. 45-56.
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