Supplementary information to
Immunotherapy alleviates amyloid-associated synaptic pathology in an
Alzheimer’s disease mouse model
MM Dorostkar et al.
Supplementary methods
Surgery
Cranial windows were implanted over the somatosensory cortex as described previously (Bittner et
al., 2009; Burgold et al., 2010). After the surgery animals were allowed to recover for at least three
weeks before the start of experiments. Before surgery mice were group housed under SPF
conditions with ad libitum access to food and water, thereafter they were singly housed. Before
surgery mice were group housed under SPF conditions with ad libitum access to food and water,
thereafter they were singly housed.
In vivo imaging
Amyloid plaques were stained with methoxy-X04 (Klunk et al., 2002). An initial loading dose of 2
mg/kg methoxy-X04 was administered, followed by a weekly maintenance dose of 0.4 mg/kg, applied
24h before each imaging session (Burgold et al., 2010).
Imaging started at 12 months of age, a time when new born plaques are formed and plaques steadily
grow. The imaging was performed using an LSM 7 MP (Zeiss AG, Jena, Germany) equipped with a
ultrafast Ti:Sapphire Laser (MaiTai HP DeepSee, Spectra-Physics, Darmstadt, Germany). In order to
obtain a large field of view in combination with a high optical resolution, a W Plan-Apochromat 20x
1.0 NA objective (Zeiss, Jena, Germany) was used. The pixel scaling in xy direction was adjusted to
0.83 µm and 3 µm for the z direction, respectively. Methoxy-X04 was excited, using two photon
excitation, at 750 nm and fluorescence was detected between 440 and 500 nm. Images were
acquired with 12 bit data depth and corrected for depth-dependent signal strength using the z
correction tool of the Zen Software of the microscope. The images were acquired using the tile scan
mode of the microscope which means that a mosaic of the field of views is imaged in a way that a big
volume is generated. This gives an area of 850x850 µm2 per position and the imaging depth ranged
from 300 to 400 µm. In each mouse three of these big imaging volumes were acquired. During
imaging mice were kept anaesthetized using 1.3% Isoflurane.
Immunohistochemistry
Mice were perfused with ice-cold PBS pH 7.4 and 4% PFA. Brains were removed and post fixed in 4%
paraformaldehyde over night at 4°C. All following steps were performed at room temperature. From
each mouse the hemisphere contralateral to the cranial window was used for
immunohistochemistry. Floating sections, ~50 µm thick, were collected as serial sections,
corresponding to a region from -3.5 to -3 from bregma. Sections were permeabilized in 2% Triton X100 in PBS at pH 7.4 for 3 days and then blocked with 0.3% Triton X-100, 10% normal goat serum in
PBS at pH 7.4.
Primary antibodies were incubated together for 48h (anti-Dlg4 mouse monoclonal antibody, 1:200,
cat. no. 124011; anti-Synapsin 1/2 polyclonal rabbit antibody, 1:500, cat. no. 106002; Synaptic
Systems GmbH, Göttingen, Germany). Secondary antibodies were also incubated for 48 hours (goat
anti-mouse-Alexa647, A21236, goat-anti-rabbit-Alexa488, A11034, 1:500 each). Then a solution of
0.01 mg/ml methoxy-X04 in PBS pH 7.4 was added to stain fibrillar plaques. Sections were mounted
onto glass slides using fluorescent mounting medium (Dako Deutschland GmbH, Hamburg, Germany)
and covered by glass cover slips (no. 1.5, Zeiss, Germany).
Ex vivo immunofluorescence and image analysis
Images were acquired on an inverted confocal microscope (Zeiss LSM 510) equipped with a 40× PlanApochromat, numerical aperture 1.3 (Zeiss, Jena, Germany). Fluorophores were excited at 488 nm
and 633 nm. Emission was collected from 500 to 550 nm and with a 650 nm long pass filter. Plaques
were imaged using two-photon excitation at 750 nm and emission was collected from 435 to 485 nm.
Imaging distant from plaques was done using a 3x zoom which results in a scanned area of 75x75
µm2 with a lateral resolution of 0.098 µm and an axial resolution of 0.3 µm. Plaques were avoided by
manually focusing through the entire thickness of the slice at the lowest zoom setting and areas
devoid of plaques were selected for imaging. Imaging including plaques was performed using 1x
zoom which corresponds to an area of 225x225 µm2. Images were recorded with a lateral resolution
of about 0.110 µm and an axial resolution of 0.3 µm. The differences in lateral resolution to areas
distant from plaques are caused by the different combination of zoom settings and pixel sizes and
are thought to be negligible.
Synapse densities distant from plaques were sampled from four different regions: visual cortex V1
layer 1/2, CA1 regions stratum radiatum and stratum oriens and the molecular layer of the dentate
gyrus. From each of the 5 sections per mouse 3 images per region were acquired. Synapse densities
from the 3 images from one region were averaged giving the mean of this section. The means for
each section were again averaged to obtain the mean for a mouse for each region. These values were
used for statistical testing and are displayed in the figures. For the analysis of the synapse densities
around plaques 9 to 10 images were acquired for each mouse throughout the whole cortex within all
5 sections.
Images were preprocessed by 3D deconvolution in AutoDeblur to improve signal-to-noise ratio as
well as resolution (Media Cybernetics Inc., Rockville MD, USA). Images were further processed in Igor
Pro to detect single puncta, based on published algorithms (Dorostkar et al., 2010). Briefly, the
Laplace operator (which is the sum of the unmixed second partial derivatives in the Cartesian co-
ordinates x and y), was calculated from an image. The result was thresholded at 2×standard
deviation of the pixel values to yield a binary image, from which centers of mass for each puncta
were determined and counted. Synapse densities were normalised to an adjusted image area from
which the areas occupied by blood vessels or cell bodies were subtracted (Supplementary Fig. 2). In
order to relate local synapse density to the distance from plaques, synapses were detected as
described above. Amyloid plaques were detected by simple thresholding at the 99.7th percentile,
followed by manual adjustment of the results. The distance transform of the thresholded image was
calculated and binned into 5µm wide bins. Then, the number of synapses was related to the adjusted
area of each bin.
Quantification of total and oligomeric amyloid beta
To generate brain extracts, mice were perfused with ice-cold PBS. Brains were dissected, and
hippocampus samples were flash frozen in liquid nitrogen and stored at -80°C until further use.
Amyloid beta peptides were extracted from Tg2576 hippocampus by homogenizing the brain
material in 3% SDS extraction buffer (0.1g/mL in 50mM Tris, 150mM NaCl, 0.5% Triton X100, 1mM
EGTA, 3% SDS, 1% Na-desoxycholate pH 7.4 with complete protease inhibitor cocktail, Roche
Diagnostics, Germany and 1mM PMSF) with about 20 strokes in a glass potter and afterwards for 30s
with an ultra turrax homogenizer. The 3% SDS samples were incubated for 30min at room
temperature and centrifuged for 10min at 16,000g at 20°C. The supernatants were analyzed for
amyloid beta peptides using quantitative immunoprecipitation: Mouse anticoupled to anti-mouse antibody magnetic beads (Dynabeads, Life Technologies, Darmstadt,
Germany). The samples were incubated with the magnetic beads overnight at 4°C, washed 3x with
PBS, eluted with 50% acetonitrile, 0.5% TFA and subjected to standard quantitative western blot.
Total soluble amyloid beta peptide was determined by the amyloid beta conformer unselective
antibody 6E10 and amyloid beta Globulomer epitopes by A 887755. Note that the background levels
due to unspecific binding were substracted using IgG2a isotype antibody as a negative control.
Amyloid-beta immunization of Tg2576 mice
Antigen preparation: Three mg of Aβ1-42 monomer (H-1368, Bachem, Bubendorf, Switzerland) were
dissolved with 0.75 ml 0.1% NH4OH (vortexed for 1 min), then 2.25 ml phosphate-buffered saline
(PBS) (20 mM NaH2PO4, 140 mM NaCl, pH 7.4) were added and the mixture allowed to incubate at
room temperature for 1 hour. For Aβ20-42 Globulomer immunizations, Aβ20-42 Globulomer
(Barghorn et al., 2005) was diluted in PBS to a final concentration of 0.3mg/ml.
Immunization: Tg2576 mice were immunized at an age of 3 month. To this end, each antigen was
mixed with equal parts (v/v) of complete Freund’s adjuvant (CFA) and 200 µL of this mixture
(equivalent to: 100µg Aβ1-42 monomer or 30µg Aβ20-42 Globulomer) was used for intraperitoneal
immunization-injections followed by three booster injections with the same amount of antigen in
incomplete Freund’s adjuvant (IFA) every third week. No further booster immunizations occurred
until the animals were sacrificed for brain hippocampal Globulomer epitope and total soluble Aβ
peptide content at age of 12 month. Plasma titers at this timepoint for Aβ Globulomer immunized
mice were in the range of 1:200-10.000 and for Aβ monomer immunized mice in the range of 1:2004.000. No adverse effects were observed after vaccination.
Supplementary figure legends
Supplementary Figure 1
Biochemical quantification of total and oligomeric amyloid beta levels from Tg2576 hippocampi (n =
8-12 animals). (A) Time-course of amyloid beta levels in untreated Tg2576 animals,
immunoprecipitated with conformation-unspecific 6E10 antibodies (total Aβ) or oligomer-specific A887755 antibodies (oligomeric Aβ). Error bars show 95% CI. (B, C) Amyloid beta levels in mice
immunized with either full-length amyloid beta (Aβ1-42) or with amyloid globulomers (Aβ20-42),
which generate a monospecific immune response resembling the antibody specificity of A-887755.
(B) Levels of total amyloid beta, detected with 6E10 antibodies. (C) Levels of oligomeric amyloid beta,
detected with A-887755 antibodies. **, P < 0.01; ***, P < 0.001 (ANOVA with Bonferroni’s post-hoc
test; n = 11-14 animals).
Supplementary Figure 2
Background processing of immunohistochemical stains. (A) Synapsin stain of a cortical region. The
contrast setting has been increased for better visualization of “shadow stained” capillaries and
neuronal cell bodies, which appear dark. (B) The background defined as the 10th percentile of
fluorescence values is displayed in blue. (C) The background was then modified so that only
contiguous areas of at least 20 µm2 were considered (displayed in red). This area was subtracted
from the total area in order to determine the synapse density. Scale bars, 10 µm.
Supplementary References
Bittner T, Fuhrmann M, Burgold S, Jung CK, Volbracht C, Steiner H, et al. Gamma-secretase inhibition
reduces spine density in vivo via an amyloid precursor protein-dependent pathway. J Neurosci.
2009;29(33):10405-9.
Burgold S, Bittner T, Dorostkar M, Kieser D, Fuhrmann M, Mitteregger G, et al. In vivo multiphoton
imaging reveals gradual growth of newborn amyloid plaques over weeks. Acta Neuropathologica.
2010;121(3):327-35.
Dorostkar MM, Dreosti E, Odermatt B, Lagnado L. Computational processing of optical
measurements of neuronal and synaptic activity in networks. Journal of Neuroscience Methods.
2010;188(1):141-50.
Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, et al. Imaging Abeta plaques in
living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered
Congo red derivative. J Neuropathol Exp Neurol. 2002;61(9):797-805.