Formation and maintenance of Alzheimer's disease β-amyloid plaques in the absence of microglia

Full text

Page 1

© 2009 Nature America, Inc. All rights reserved.
nature neuroscience  advance online publication
?
Brief communications
CD11b-HSVTK (TK) mice express the thymidine kinase of herpes simplex
virus under the CD11b (also known as ITGAM) promoter. HSVTK is a
suicide gene that converts antiviral nucleotides analog prodrugs such
as ganciclovir (GCV) into a monophosphorylated form, which is then
transformed into a toxic triphosphate by cellular kinases. Thus, GCV
treatment leads to selective ablation of proliferating myeloid cells1. TK
mice were bred with APPPS1 transgenic mice, which develop robust amy-
loid pathology that is accompanied by a strong microglia activation as
early as 2 months of age2. To restrict HSVTK expression in APPPS1; TK
mice to resident microglia and to overcome the reported GCV-mediated
myelotoxicity1, we generated chimeric mice harboring congenic GFP-
labeled wild-type bone marrow (Supplementary Methods). At 5 months,
we treated bone marrow chimeric APPPS1; TK and APPPS1 (negative
littermates without the CD11b-HSVTK gene) mice with oral GCV for
4 weeks. This treatment caused a ~30% decrease in microglia number in the
neocortex (and most other brain regions) in APPPS1; TK mice compared
with APPPS1 control mice (Supplementary Fig. 1). Despite the decrease
in microglia number we found no change in the morphology and number
of congophilic or Aβ-immunoreactive amyloid deposits (Supplementary
Fig. 1). Consequently, no change in transgenic human Aβ40 or Aβ42 was
detected using western blotting and ELISA (Supplementary Fig. 1).
To test whether a more complete microglial ablation induces a
change in cerebral amyloidosis in APPPS1 mice, we used several alter-
native GCV administration protocols in TK mice. The most successful
approach was to deliver GCV directly into the ventricle (intracerebro-
ventricular, icv) via an osmotic pump (50 mg ml−1, 0.25 µl h−1 flow
rate). In contrast with systemic GCV administration, this icv application
benefited from the fact that adoptive bone-marrow transfer appeared
to be dispensable. We found a 40% reduction in the number of Iba1-
positive microglia in the neocortex and in other brain regions after
1 week of icv GCV administration and a 90% reduction after 2 weeks
(Supplementary Fig. 2). Concomitant with the ablation of microglia,
we observed a temporary activation of astrocytes (Supplementary
Fig. 2). We found no apparent change in the number or morphology
of neurons and no alteration in endogenous murine Aβ40 and Aβ42
levels in TK mice lacking microglia (Supplementary Fig. 2).
To investigate the effect of a complete microglial ablation on
cerebral amyloidosis, we icv GCV-treated 3-month-old amyloid-
bearing APPPS1; TK and APPPS1 mice. GCV administration for
2 weeks resulted in a 95% reduction of the number of Iba-1 positive
microglia (Fig. 1a–c). GVC treatment for 4 weeks, which allowed us to
study plaque homeostasis in the absence of microglia, caused a 97%
reduction in the number of microglia in GCV-treated APPPS1; TK
mice compared with APPPS1 mice. Qualitatively similar ablation was
observed using CD11b, F4/80 or CD68 as microglial markers (data not
shown). The efficacy of microglia ablation was confirmed by conven-
tional ultrastructural analysis (detection of microglia was independent
of the expression of activation markers) and by in vivo multiphoton
imaging. Both techniques showed at least a 90% reduction in the
microglial cell number (Supplementary Fig. 3).
This virtually complete ablation of microglia in APPPS1; TK mice
did not change congophilic amyloid load, the distribution and extent
of Aβ-deposition, or plaque morphology after 2 and 4 weeks of GCV
treatment (Fig. 1a–e). Plaque size distribution analysis also revealed
no differences in APPPS1; TK mice harboring or lacking microglia
(Fig. 1f). There was no change in human APP expression and Aβ levels
in the brains of APPPS1; TK mice versus APPPS1 mice (Fig. 1g). We
also found no difference between microglia-deficient APPPS1; TK
mice and APPPS1 mice in terms of number and morphology of dys-
trophic neuritic structures near amyloid plaques (Fig. 1h), consistent
with our ultrastructural observations (Supplementary Fig. 3).
APPPS1 mice start to develop cerebral amyloidosis in the neo-
cortex at 2 months of age. Even at the earliest stages of Aβ deposition,
microglia consistently surround the amyloid plaques2. To determine
the extent to which microglial cells are involved with or are even the
cause of Aβ deposition and plaque formation, we icv GCV-treated
Formation and maintenance of
Alzheimer’s disease β-amyloid
plaques in the absence
of microglia
Stefan A Grathwohl1–3,12, Roland E Kälin4,12, Tristan Bolmont1,2,
Stefan Prokop4, Georg Winkelmann4, Stephan A Kaeser1,2,
Jörg Odenthal1,2, Rebecca Radde1,2, Therese Eldh5, Sam Gandy6,
Adriano Aguzzi7, Matthias Staufenbiel8, Paul M Mathews9,10,
Hartwig Wolburg11, Frank L Heppner4,13 & Mathias Jucker1,2,13
In Alzheimer’s disease, microglia cluster around
-amyloid deposits, suggesting that these cells are
important for amyloid plaque formation, maintenance and/or
clearance. We crossed two distinct APP transgenic mouse
strains with CD11b-HSVTK mice, in which nearly complete
ablation of microglia was achieved for up to 4 weeks after
ganciclovir application. Neither amyloid plaque formation
and maintenance nor amyloid-associated neuritic dystrophy
depended on the presence of microglia.
1Department of Cellular Neurology, Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany. 2German Center for Neurodegenerative Diseases,
Tübingen, Germany. 3Graduate School of Cellular and Molecular Neuroscience, University of Tübingen, Tübingen, Germany. 4Department of Neuropathology,
Charité–Universitätsmedizin Berlin, Berlin, Germany. 5Department of Radiation Oncology, University of Tübingen, Tübingen, Germany. 6Department of Neurology, Mount
Sinai School of Medicine, New York, New York, USA. 7Institute of Neuropathology, Department of Pathology, University Hospital, Zürich, Switzerland. 8Novartis Institutes for
Biomedical Research, Nervous System Research, Basel, Switzerland. 9Nathan Kline Institute, Orangeburg, New York, USA. 10Departments of Psychiatry, New York University
School of Medicine, New York, New York, USA. 11Department of Pathology, University of Tübingen, Tübingen, Germany. 12These authors contributed equally to this work.
13These authors jointly directed the study. Correspondence should be addressed to F.L.H. (frank.heppner@charite.de) or M.J. (mathias.jucker@uni-tuebingen.de).
Received 12 June; accepted 24 September; published online 18 October 2009; doi:10.1038/nn.2432

Page 2

© 2009 Nature America, Inc. All rights reserved.
? 
advance online publication nature neuroscience
Brief communications
6-week-old APPPS1; TK mice for 3 weeks. Again, the majority of
microglia (95%) were gone. However, ablation of microglia neither
prevented plaque formation nor influenced the number of plaques
developing in the neocortex (Fig. 2a–e). The number and morphology
of the dystrophic neuritic structures associated with congophilic amy-
loid deposits were also independent of microglial presence (Fig. 2f).
The icv GCV treatment of APPPS1; TK mice for 3–4 weeks regularly
resulted in microhemorrhages, which occurred predominately in the
thalamic region. This microbleeding seemed to be the probable cause
of the lethal phenotype of the TK mice after 4 weeks of icv GCV and
may have also facilitated the influx of small conglomerates of Iba1-
positive bone marrow–derived macrophages at that time (data not
shown). To avoid these effects, which may be caused by myelotoxicity
of GCV leaking from the CNS into the periphery1, we treated
APPPS1; TK mice with a lower dose of GCV (1 mg ml−1, 0.25 µl h−1
for 4 weeks). We found no behavioral abnormalities in these GCV-
treated mice. This lower dose only partially ablated microglia in the
neocortex. However, because of its anatomical location close to the
infusion site and the ventricles, we observed an almost complete
depletion of microglia in the dorsal part of the hippocampus, with-
out signs of hemorrhages and in the absence of invading peripheral
cells (Supplementary Fig. 4). Again, no change in the Aβ load and
congophilic amyloid plaques was found (Supplementary Fig. 4).
To exclude the possibility that the rather aggressive amyloidosis in
APPPS1 mice is accountable for the absence of changes on microglia
removal, we crossed a second APP transgenic mouse strain, APP23,
with the TK mice. APP23 mice develop cerebral amyloidosis at
6–8 months of age and have both dense cored and diffuse plaques, as
well as vascular amyloid3. As in APPPS1 mice, congophilic amyloid
lesions in APP23 mice are tightly surrounded by activated microglia4. We
gave 17-month-old APP23; TK and APP23 mice icv GCV (50 mg ml−1,
0.25 µl h−1) for 2 weeks. The efficacy of microglial ablation again
reached >95% in the neocortex (Supplementary Fig. 5). Although
there was no change in congophilic amyloid burden and in vascular
amyloid, there was a small reduction in total Aβ load, mainly a result of
the reduction of diffuse amyloid, which is known to be abundant in this
Alzheimer’s disease mouse model (Supplementary Fig. 5). The number
and morphology of dystrophic neuritic structures associated with the
congophilic amyloid deposits were unaltered in microglia-deficient
APP23; TK compared with APP23 mice (Supplementary Fig. 5). A
second group of APP23; TK mice (24 months of age) received low-
dose icv GCV treatment (1 mg ml−1, 0.25 µl h−1) for 4 weeks. Microglia
reduction was again confined to the dorsal hippocampus, with no
obvious change in congophilic amyloid load (data not shown).
Here, we sought to clarify the controversy over the role of microglial
cells in cerebral amyloidosis and Alzheimer’s disease pathogenesis5.
Our results indicate that a substantial reduction or a virtually complete
ablation of resident microglia (including bone marrow–derived micro-
glia) did not alter amyloid plaque load in two distinct APP transgenic
mouse models over a period of 2–4 weeks. These observations extend
Treatment for 2 weeks
a
c
gh
APPPS1
def
b
Iba1 / Congo red
Congo red
Aβ load
Microglia (×103)
Congo red-covered
area (%)
Aβ-covered area (%)
Plaque distribuition (%)
Treatment for 4 weeks
APPPS1
1,200
APPPS1
APPPS1; TK
APPPS1
APPPS1; TK
APPPS1
APPPS1; TK
900
0.8
HSVTK
APP
2.5
2.0
1.5
1.0
0.5
15
12
9
6
3
0
Densitometry
(arbitary units)
0
GAPDH
Aβ 40
Aβ 42
Aβ 40+42
3.00
60
50
40
30
20
10
0
2.25
1.50
Dystrophic boutons
per plaque
0.75
0
0.6
0.4
0.2
0
600
******
300
2 weeks
+–+–
4 weeks
2 weeks
APP
4 weeks2 weeks
Plaque size (area µm2)
4 weeks
4 weeks
10–500
500–1,000
1,000–1,500 1,500–2,0002,000–2,500 2,500–3,0003,000–6,500
0
APPPS1
APPPS1; TKAPPPS1; TK
APPPS1; TK
Figure 1 Virtually complete microglia ablation
in icv GCV-treated APPPS1; TK mice does not
alter Aβ plaque load. (a,b) Male 3-month-
old amyloid-bearing APPPS1; TK and APPPS1
mice were treated with icv GCV for 2 or 4
weeks. Double staining for Iba1 and congophilic
amyloid (top), congophilic amyloid alone
(middle) and Aβ immunohistochemistry
(bottom) are shown. (c) Quantitative
stereological analysis of the number of
Iba-1 positive microglia (n = 5 per group,
*** P < 0.001). (d,e) No change in congophilic
(d) or total Aβ load (e) was noted in GCV-treated
APPPS1; TK mice compared with GCV-treated
APPPS1 mice. (f) Plaque size distribution did
not change after 4 weeks of GCV. (g) For western
blotting, male APPPS1; TK mice were icv
GCV-treated for 3 weeks. Two mice of each
genotype are shown with GAPDH as a loading
control. Densitometric analysis of all mice
(n = 4–5 per group) revealed no difference in
human APP and Aβ (combined Aβ40 and Aβ42)
between microglia-depleted APPPS1; TK and
control APPPS1 mice. Aβ ELISA of the
soluble fraction revealed a 3–4-fold increase
in Aβ40 (P < 0.05) and Aβ42 (P = 0.05)
in microglia-deficient APPPS1; TK mice (data
not shown); however, this may represent an
artifact resulting from an increased release
of ‘insoluble’ Aβ into the soluble fraction
in the absence of microglia cells.
(h) APP-immunoreactive dystrophic boutons
(black) surrounding congophilic amyloid
plaques (red) appeared to be unchanged
in number and morphology in APPPS1; TK
mice lacking microglia compared with APPPS1
mice after 4 weeks of GCV treatment (n = 5 per
group, P > 0.05). Error bars represent s.e.m.
Scale bars represent 100 µm (a,b), 25 µm
(inserts) and 20 µm (h).

Page 3

© 2009 Nature America, Inc. All rights reserved.
nature neuroscience  advance online publication
?
Brief communications
recent in vivo multiphoton imaging work6, suggesting that resident
microglia are not important in the de novo formation of amyloid.
However, in contrast with work speculating that microglia (or bone
marrow–derived macrophages7,8) may have a function in restricting
plaque growth6,9 and with compelling in vitro data on Aβ clearance
and phagocytosis capability of microglia5, our results indicate that
de novo formation and progression of cerebral amyloidosis in vivo in
Alzheimer’s disease mouse models are controlled by nonmicroglial
factors or cells other than microglia. Consistent with this, true β-amyloid
phagocytotic activity of microglia and macrophages (engulfment of
amyloid fibrils with the later appearance of fibrils in the lysosomal-
phagosomal compartments) has not been observed in Alzheimer’s
disease brains or in brains of Alzheimer’s disease mouse models10.
Likewise, for peripheral amyloid, there is no undeniable evidence that
macrophages phagocytose amyloid (for example, see ref. 11).
Multiple lines of evidence suggest that exogenous or genetic mani-
pulation of factors involved in innate immune signaling or in neuro-
inflammatory responses, including chemokine and cytokine secretion
presumably mediated by microglia, can affect amyloid deposition in
APP transgenic mice5. Similarly, Aβ vaccination as targeted modula-
tion of the immune system has been shown to prevent or to reduce
cerebral amyloidosis12. As it is of therapeutic importance to under-
stand whether and how a microglia transforms into a potentially
Aβ/amyloid clearing cell, our approach of fully ablating microglia
in vivo will be useful for identifying microglia-dependent or micro-
glia-independent Aβ-clearing mechanisms.
Microglial activation adjacent to amyloid deposits has also
been used to suggest that microglia-mediated neuroinflammatory
responses mediate Alzheimer’s disease–associated neurodegenera-
tion5,13. In particular, it has been argued that cytokines, chemokines
and neurotoxins generated by amyloid-activated microglia cause neu-
ronal damage13. Even if not all aspects of Alzheimer’s disease patho-
logy, such as severe neurodegeneration, are entirely mimicked by APP
transgenic mice, our data argue against substantial microglia-driven
neuritic damage occurring in Alzheimer’s disease, as neural dystrophy
developed and appeared to be unaltered in the absence of microglia
in the Alzheimer’s disease mouse models that we examined.
Finally, our experiments suggest that microglia can be virtually fully
removed from specific brain regions of a living organism for up to
4 weeks. This is rather surprising in light of their highly dynamic
nature under resting condition and their presumed role in surveying
the brain, in repairing clinically silent micro-insults and in monitor-
ing the functional state of synapses14,15. To generalize this observa-
tion, it will be necessary to extend the time span of complete microglia
ablation in the presence or absence of CNS pathology.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
This work was supported by grants to M.J. (BMBF-01GI0705), F.L.H. (SFB-TR43,
Exc 25 and National Institutes of Neurological Disorders and Stroke R01 NS046006)
and P.M.M. (US National Institutes of Health AG017617 and NS045205).
AUTHOR CONTRIBUTIONS
S.A.G., T.B., S.A.K., J.O., R.R., T.E., P.M.M. and H.W. performed the icv
experiments. R.E.K., S.P. and G.W. carried out the oral treatment experiments.
A.A., M.S. and S.G. provided mice and/or advice. Experimental design and
manuscript preparation were mainly carried out by F.L.H. and M.J.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprintsandpermissions/.

1. Heppner, F.L. et al. Nat. Med. 11, 146–152 (2005).
2. Radde, R. et al. EMBO Rep. 7, 940–946 (2006).
3. Sturchler-Pierrat, C. et al. Proc. Natl. Acad. Sci. USA 94, 13287–13292 (1997).
4. Stalder, M. et al. Am. J. Pathol. 154, 1673–1684 (1999).
5. Wyss-Coray, T. Nat. Med. 12, 1005–1015 (2006).
6. Meyer-Luehmann, M. et al. Nature 451, 720–724 (2008).
7. Simard, A.R., Soulet, D., Gowing, G., Julien, J.P. & Rivest, S. Neuron 49, 489–502
(2006).
8. Town, T. et al. Nat. Med. 14, 681–687 (2008).
9. Bolmont, T. et al. J. Neurosci. 28, 4283–4292 (2008).
10. Jucker, M. & Heppner, F.L. Neuron 59, 8–10 (2008).
11. Gruys, E., Timmermans, H.J. & van Ederen, A.M. Lab. Anim. 13, 1–9 (1979).
12. Schenk, D. Nat. Rev. Neurosci. 3, 824–828 (2002).
13. El Khoury, J. & Luster, A.D. Trends Pharmacol. Sci. 29, 626–632 (2008).
14. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Science 308, 1314–1318 (2005).
15. Hanisch, U.K. & Kettenmann, H. Nat. Neurosci. 10, 1387–1394 (2007).
Figure 2 Amyloid plaque formation in microglia-
ablated APPPS1; TK mice. (a) Numbers of
Iba1-positive microglia in 6-week-old male
APPPS1; TK and APPPS1 mice did not differ.
The mice did not exhibit amyloid plaque
deposition. Double staining for microglia (Iba1)
and compact congophilic amyloid (top panels),
congophilic amyloid alone (middle panels) and
Aβ immunohistochemistry (bottom panels) are
shown. (b) Both APPPS1; TK and APPPS1 mice
develop amyloid plaques irrespective of microglia
3 weeks after icv GCV treatment. (c) Quantitative
stereological analysis of total microglia revealed
identical numbers of cells at baseline (0 weeks) in
both genotypes and a 95% reduction in microglia
3 weeks after GCV treatment in APPPS1; TK
mice compared with APPPS1 mice (n = 5 per
group, *** P < 0.001). (d,e) No group difference
in congophilic amyloid and Aβ load was seen
after GCV treatment. (f) APP-immunoreactive
dystrophic boutons (black) surrounding
congophilic amyloid plaques (red) were
indistinguishable in number and morphology in
APPPS1; TK mice lacking microglia and APPPS1
control mice 3 weeks after GCV application (n = 5
per group, P > 0.05). Error bars represent s.e.m.
Scale bars represent 100 µm (a), 25 µm (inserts)
and 20 µm (f).
1,200
Microglia (×103)
Aβ load
Congo red
lba1 / Congo red
Congo red-coverd
area (%)
Aβ-covered area (%)
Dystrophic boutons
per plaque
900
0.20
APPPS1
APPPS1; TK
APPPS1
a
cdef
b
APPPS1; TK
Baseline
APPPS1APPPS1; TK
Treatment for 3 weeks
APPPS1
APPPS1; TK
0.60
15
12
9
6
3
0
0.45
0.30
0.15
0
0.15
0.10
0.05
3 weeks3 weeks3 weeks
0
600
300
0
0 weeks 3 weeks
***

Page 4

1

SUPPLEMENTARY INFORMATION


Formation and maintenance of Alzheimer’s disease β-amyloid plaques
in the absence of microglia

Stefan A Grathwohl1-3,12, Roland E Kälin4,12, Tristan Bolmont1,2, Stefan Prokop4, Georg
Winkelmann4, Jörg Odenthal1,2, Stephan A Kaeser1,2, Rebecca Radde1,2, Therese Eldh5, Sam,
Gandy6, Adriano Aguzzi7, Matthias Staufenbiel8, Paul M Mathews9,10, Hartwig Wolburg11, Frank L
Heppner4,13, Mathias Jucker1,2,13


1Department of Cellular Neurology, Hertie-Institute for Clinical Brain Research, University of
Tübingen, D-72076 Tübingen, Germany.
Tübingen, Germany. 3Graduate School of Cellular and Molecular Neuroscience, University of
Tübingen, Tübingen, Germany. 4Department of Neuropathology, Charité – Universitätsmedizin
Berlin, Germany. 5Department of Radiation Oncology, University of Tübingen, Tübingen, Germany.
6Department of Neurology, Mount Sinai School of Medicine, New York, USA.
Neuropathology, Department of Pathology, University Hospital, Zürich, Switzerland. 8Novartis
Institutes for Biomedical Research, Nervous System Research, Basel, Switzerland. 9Nathan Kline
Institute, Orangeburg, New York. 10Department of Psychiatry, New York University School of
Medicine, New York, NY, USA. 11Department of Pathology, University of Tübingen, Tübingen,
Germany. 12These authors contributed equally to this work. 13These authors jointly directed the
study.

2German Center for Neurodegenerative Diseases,
7Institute of








- Supplementary Figures 1-5
- Supplementary Method









Nature Neuroscience: doi:10.1038/nn.2432

Page 5

2


Supplementary Fig 1: Reduction of microglia in systemically GCV-treated bone-marrow chimeric
APPPS1; TK mice does not alter Aβ plaque load. Five month-old β-actin-GFP bone marrow
chimeric male and female APPPS1; TK+ and APPPS1; TK- mice received GCV (60 µg/ml)
systemically (per os) for 4 weeks. (a) Iba1 immunohistochemistry (upper panels) revealed a
decrease in the amount of Iba1-positive microglia in APPPS1; TK+ compared to APPPS1; TK-
mice, while congophilic amyloid burden and Aβ-immunostaining (middle and lower panels) were
not altered. Scale bars: 200 µm. (b) Morphometric analyses revealed a significant reduction of the
area covered by Iba1 staining in APPPS1; TK+ compared to APPPS1; TK- mice (n=9 for
APPPS1; TK+; n=13 for APPPS1; TK-; **p<0.01). (c) No significant difference was found in the
Aβ-load (area covered by Aβ-immunostaining) of APPPS1; TK+ compared to APPPS1; TK- mice
(p>0.05). (d,e) Western blot analysis revealed a reduction of Iba1 levels in APPPS1; TK+
compared to APPPS1; TK- mice (a representative blot with n=2/group is shown; GAPDH was used
as a loading control; for quantitative analysis 8 mice/group were used; ***p < 0.001); (f,g) Western
blot analysis of human APP and human Aβ40/42 showed no significant change in APP and
combined Aβ40/42 (p>0.05). (h,i) ELISA for human Aβ levels comparing APPPS1; TK+ to
APPPS1; TK- mice revealed neither a difference in the soluble nor insoluble fraction (n=8 for
APPPS1; TK+ and n=12 for APPPS1; TK- mice; p>0.05).

Nature Neuroscience: doi:10.1038/nn.2432

Page 6

3

Nature Neuroscience: doi:10.1038/nn.2432

Page 7

4
Supplementary Fig. 2: Microglia ablation in the neocortex of TK transgenic mice after i.c.v.
GCV application (a) I.c.v. GCV application for 1 or 2 weeks (50mg/ml; 0.25μl/h via osmotic
pump) leads to an ablation of microglia (Iba1 immunostaining) exclusively in TK+ mice, while
non-transgenic TK-littermates are not affected. TK+ mice exposed to PBS do not exhibit any
microglia loss (not shown). The same results were observed when microglia were stained using
CD11b or CD68 (not shown). (b) Quantitative stereological assessment of total Iba1-positive
microglia numbers resulted in a 40%, respective 90% reduction of Iba1-positive microglia in the
neocortex after 1 and 2 weeks of i.c.v. GCV treatment (n=4/group; ***p<0.001). Note the
peculiar morphology of residual microglial cells after one week of GCV administration, which is
indicative of an activated microglial state. (c) Increased GFAP-immunoreactivity of adjacent
sections peaking at one week of GCV administration suggests an astrocytic activation, which
decreased slightly after two weeks of GCV application, when the majority of microglia cells was
already ablated. (d) Morphometric analyses showed significant GFAP-increases in TK+ mice
upon 1 and 2 weeks of GCV administration (n=4/group; ***p<0.001). (e) NeuN
immunohistochemical stainings revealed unaltered morphology and number of neurons in TK+
and TK- mice at 1 or 2 weeks of GCV application. (f) ELISA assessing murine Aβ40 and Aβ42
protein levels was identical in GCV-treated and untreated TK+ and TK- mice (n=4/group;
p>0.05). Scale bars: 100 µm.

Nature Neuroscience: doi:10.1038/nn.2432

Page 8

5

Supplementary Fig. 3: Lack of amyloid-associated microglia: ultrastructural assessment and in
vivo imaging. (a) Ultrastructural analysis of amyloid plaques in 4 month-old male APPPS1; TK+
and APPPS1; TK- mice after 4 weeks i.c.v. GCV treatment (50mg/ml; 0.25μl/h). While amyloid
plaques in APPPS1; TK- mice are typically surrounded by microglia cells (identified by their
ultrastructural characteristics; cell bodies are labeled with “M”), almost no microglial cells were
detectable in GCV-treated APPPS1; TK+ mice (panel top left and right). Semiquantitative analysis
revealed a ~90% reduction of microglia cell bodies/amyloid plaque (27 and 37 plaques were
analysed in two APPPS1; TK- and two APPPS1; TK+ mice, respectively; ***p<0.001). The
presence or absence of microglia next to amyloid plaques did not influence plaque morphology;
similarly, the appearance of dystrophic neuritic elements in vicinity of amyloid (arrows) was not
different from plaques lacking or harbouring microglia in APPPS1; TK+ or APPPS1; TK- mice.
Amyloid fibrils seemed also unchanged in APPPS1; TK- and microglia-depleted APPPS1; TK+
mice (panels bottom left and right). Spaces evolving from the disappearance of microglia
processes in GCV-treated APPPS1; TK+ mice appeared to be occupied by other glial and
neuronal elements. Scale bars: 5 µm (top panels); 1 µm (bottom panels). (b) In vivo multiphoton
microscopy of APPPS1; TK+ mice crossed to mice expressing eGFP under the Iba1-promoter
allowed to visualize microglia ablation and amyloid plaque changes in vivo over time. Plaques in
APPPS1; TK+; Iba1-eGFP mice were visualised using i.v. injection of Methoxy-XO4. In addition,
mice received Texas Red dextran i.v., which results in a fluorescent angiogram allowing repeated
imaging of the same sites over time. On day 1 after GCV application of a 4 month-old
APPPS1; TK+; Iba1-eGFP mouse, numerous microglia cells (green) clustering around an amyloid
plaque (blue) were observed. On day 12 of GCV treatment most microglia disappeared while
amyloid plaques remained unchanged. Using x-y-z stacks of 300 x 300 x 100 µm the number of
microglia cells on day 1 compared to day 12 was decreased at >90% (n=5/mouse; 3 mice were
analysed; ***p<0.001). Scale bars: 20 µm.
Nature Neuroscience: doi:10.1038/nn.2432

Page 9

6

Supplementary Fig. 4: Low dose i.c.v. GCV application results in microglia ablation in the
hippocampus of APPPS1; TK mice and does not affect Aβ plaque load. (a) Three month-old male
APPPS1; TK+ and APPPS1; TK- mice received i.c.v. low dose GCV (1 mg/ml; 0.25 μl/h) for 4
weeks resulting in a site-specific almost complete ablation of microglia in the dorsal hippocampus
of APPPS1; TK+ mice. No difference in congophilic amyloid load or total Aβ deposition was
observed between GCV-treated APPPS1; TK+ and APPPS1; TK- mice. Shown is a double staining
for microglia (Iba1 antibody) and congophilic amyloid (upper panels); single Congo red staining
(middle panels); and Aβ-immunohistochemistry (Aβ load; lower panels). Scale bar: 100 µm. (b)
Quantitative stereological analysis of total microglial cell number in the dorsal hippocampus
revealed a 93% reduction (n=3/group; **p<0.01), while no change in congophilic (c) or total Aβ
load (d) was noted in the dorsal hippocampus between GCV-treated APPPS1; TK+ and
APPPS1; TK- mice (p>0.05).

Nature Neuroscience: doi:10.1038/nn.2432

Page 10

7


























Supplementary Fig. 5: Microglia ablation in APP23 mice. Seventeen month-old female
APP23; TK+ and APP23; TK- mice, which reveal robust cerebral amyloid and amyloid-associated
microgliosis, received GCV i.c.v. (50mg/ml; 0.25μl/h) for 2 weeks. (a) Similar to APPPS1; TK mice,
a dramatic reduction of Iba1-positive microglia in the neocortex of APP23; TK+ mice compared to
APP23; TK- control mice was observed. While congophilic amyloid load was not different between
the groups, Aβ load was slightly reduced in microglia-depleted APP23; TK+ mice. Shown are
double stainings for microglia (Iba1) and congophilic amyloid (upper panels); single Congo red
staining (middle panels); and Aβ-immunohistochemistry (Aβ load; lower panels). Scale bar:
100 µm. (b) Quantitative analysis of total microglia cell numbers revealed a 94% reduction
(n=4/group; ***p<0.001). (c) No significant change in congophilic amyloid, but (d) a small 15%
decrease in Aβ load (mainly in diffuse amyloid) was found (n=4/group; **p<0.01) in GCV-treated
APP23; TK+ compared to APP23; TK- mice. (e) Vascular amyloid (cerebral amyloid angiopathy,
CAA) frequency and severity were not different between the groups (n=4/group; p>0.05). (f) APP-
immunoreactive dystrophic boutons (black) surrounding congophilic amyloid plaques (red)
appeared identical in number and morphology in APP23; TK+ mice lacking microglia and
APP23; TK- control mice. (n=4/group; p>0.05). Scale bar: 20 µm.
Nature Neuroscience: doi:10.1038/nn.2432

Page 11

8

SUPPLEMENTARY METHODS

Mice
Female hemizygous CD11b-HSVTK (TK) mice1 were crossed to male hemizygous APPPS12 or
hemizygous APP23 mice3. While APP23 mice express a transgene consisting of human APP with
the KM670/671NL mutation under a Thy-1 promoter element, APPPS1 mice in addition carry a
mutated human presenilin 1 (PS1) transgene. APPPS1 mice have been generated on a C57BL/6
(B6) background, while APP23 and TK mice were originally derived on a B6D2 background and
were backcrossed to B6 mice for more than 15 generations (APP23) or at least for 12 generations
(TK). For in vivo multiphoton microscopy experiments APPPS1; TK+ mice have been further
crossed to hemizygous B6-Tg(Iba1-eGFP) mice4 (generously provided by Shinichi Kohsaka,
Tokyo, Japan) to visualize microglia cells. Mice were kept under pathogen-free conditions and
experiments were in compliance with protocols approved by the local animal use and care
authorities.

Generation of bone marrow chimeric mice and oral GCV application
Bone marrow chimeric mice were generated as described1. Donor cells were obtained from tibia
and femur of B6-Tg(ACTbEGFP)1Osb mice (Jackson Laboratories, Bar Harbor, MN) was used.
After lethal irradiation with 950 rad mice were treated with antibiotics (Tetracycline 0,1 g/l or Borgal
24%, Intervet/Schering-Plough, Kenilworth, NJ). Successful reconstitution was defined as >94%
engraftment of blood leukocytes by FACS analysis. Per os GCV was given at 60 µg/ml GCV
(Valcyte, generously provided by Roche Pharma, BL, Switzerland) via the drinking water.

Intracerebroventricular GCV application
Twenty-four to 48 hours prior to surgery, osmotic pumps (Model 1002 for 2 wks, Model 2004 for 4
wks, 0,25 µl/h; Alzet) were filled with a 1 or 50 mg/ml solution of Valganciclovir (generously
provided by Roche Bioscience, Palo Alto, CA) in PBS and primed in 37°C PBS. Valganciclovir, the
L-valyl ester prodrug of ganciclovir, is rapidly reconverted by esterases into ganciclovir. Mice were
anesthetized using Ketamine/Xylazine (Ketamine 100 mg/kg; Xylazine 10 mg/kg), placed on a
warming pad, and secured on a modified stereotactic apparatus. The skin and the periosteum
were removed and the pump was placed into a pocket, formed on the back of the animal. The
coordinates for the cannula from bregma were AP: +0.1 mm, ML: +1.0 mm and DV: -2.5 mm (1,5
month old mice) or -2.9 mm (>3 month old mice). The cannula (Brain Infusion Kit III 1-3 mm,
Charles River, Germany) was held in place by dental cement (Heraeus, Hanau, Germany).
Nature Neuroscience: doi:10.1038/nn.2432

Page 12

9
Following surgery, mice were treated with Temgesic (i.p. 0,05 mg/kg daily; Essex Pharma GmbH,
Munich, Germany) for three days.

Histology
Animals were perfused with PBS followed by 4% paraformaldehyde (PFA) under deep
Ketamine/Xylazine anesthesia (Ketamine 100 mg/kg; Xylazine 10 mg/kg). Brains were removed
and fixed in 4% PFA for 24 h. Subsequently, brains were either embedded in paraffin and cut
sagittally in 6 µm thick sections (for systemically GCV-treated chimeric mice), or immersed in 30%
sucrose, frozen in 2-methylbutane, and sliced in 25 µm thick coronal sections (for i.c.v. GCV-
treated brains). Immunohistochemical stainings were done using the Vectostain Elite ABC Kits
(Vector Laboratories; Burlingame, CA) except for anti-NeuN that was detected with anti-mouse
Alexa 488 (Molecular Probes, Leiden, Netherlands). The following primary antibodies were used:
Polyclonal anti-human Aß antibody NT12 and DW6 (the latter generously provided by D. Walsh,
Dublin, Ireland); mouse anti-human Aß antibody 4G8 (Covance, Princeton, NJ); polyclonal
antibody to ionized calcium binding adapter molecule 1 (Iba1; Wako, Richmond, VA); rat
monoclonal anti-mouse CR3 (CD11b; Serotec, Oxford, UK); rat monoclonal anti-CD68 (Abcam,
Cambridge, UK); rat monoclonal anti-F4/80 (Serotec, Oxford, UK); polyclonal antibody to glial
fibrillary acidic protein (GFAP; Dako, Hamburg, Germany); monoclonal antibody to NeuN
(Chemicon, Temecula, CA); and polyclonal antibody to APP (A4CT, generous gift of K. Beyreuther,
Heidelberg, Germany). All polyclonal antibodies were used at a dilution of 1:2000 while anti-NeuN
was used 1:1000. All other histological stains (Congo red; cresyl violet, H&E) were done according
to standard laboratory procedures.

Stereological assessment of amyloid load and microglia
For i.c.v. GCV treated mice, Aß plaque load, congophilic amyloid, CAA, and number of microglia
cells were assessed in the neocortex on random sets of every 12th systematically sampled 25-μm-
thick Aß-immunostained, Congo red stained, or Iba1-immunostained section through the neocortex
(yielding typically 12-14 sections/mouse). For the dorsal part of the hippocampus every 6th section
was used starting at the anterior pole of the hippocampus until -2,5 mm from Bregma resulting in
typically 6-7 sections. Analysis was performed with the aid of the Stereologer software and a
motorized x-y-z stage coupled to a video-microscopy system (Systems Planning and Analysis,
Inc., Alexandria, VA). Congophilic amyloid and Aß load were analyzed using the area fraction
technique while the optical fractionator technique was used to count the total number of
microglia5,6. CAA frequency and severity was quantified according to a previously published
grading system7.
Nature Neuroscience: doi:10.1038/nn.2432

Page 13

10
For systemically GCV-treated mice, Aß deposition, congophilic amyloid, and microglia were
assessed by measuring the immunstained area of 3 cortical regions representing the frontal,
parietal and occipital cortex (each 1,4 mm2) of every 50th systematically sampled 6-μm-thick Aß-
immunostained, Congo red stained, or Iba1-immunostained section through the neocortex. The
stereomorphologic analysis of 27 defined regions/per mouse was performed with the aid of the Cell
D software (Olympus, Tokyo, Japan) using the color filter and phase analysis tool.

Plaque size determination
For the determination of plaque size distribution in i.c.v. GCV treated mice every 12th Aβ-
immunostained section through the neocortex was imaged using the AxioFluor Mosaic Software
(Zeiss, Jena, Germany). Individual plaques were separated with the help of ImageJ using the
watershed algorithm (http://bigwww.epfl.ch/sage/soft/watershed) after application of Gaussian
Blurring (sigma=5). Neocortex was delineated and plaque size measured with ImageJ.

Analysis of amyloid-associated neural dystrophy
For the determination of the number of amyloid plaque-associated dystrophic boutons in i.c.v. GCV
treated mice every 12th section throughout the neocortex was stained for APP using the antibody
A4CT, resulting in 12-14 immunostained sections/mouse. Analysis was performed with the aid of
the Stereologer software using the optical fractionator technique (see above). Only APP-positive
dystrophic neural structures (dystrophic boutons) with a diameter of >2,5 µm and in closest vicinity
of an amyloid plaque were counted (on average within 15 µm from the Congo red positive plaque
surface). The mean number of dystrophic boutons/plaque of typically 100 plaques/mouse was
determined.

ELISA for murine Aβ
Murine Aβ levels were determined by sandwich ELISA using homogenates of snap frozen brain
hemispheres lacking the cerebellum. Aβ from sucrose homogenates of mice was extracted by
diethylamine (DEA)8. Aβ was captured using Aβ carboxy-terminal monoclonal antibodies, which
recognize exclusively either Aβx-40 (JRF/cAb40/10) or Aβx-42 (JRF/cAb42/26). Bound Aβ was
detected by horseradish peroxidase-conjugated antibody M3.2 which specifically recognizes the
amino-terminal 15 residues of murine Aβ. ELISA results are reported as the mean ± SEM in fmol
Aβ per g wet brain, based on standard curves using synthetic murine Aβ1−40 and Aβ1−42 peptide
standards (American Peptide).

Nature Neuroscience: doi:10.1038/nn.2432

Page 14

11
Immunoassays to measure human Aβ
Frozen hemispheres were sequentially extracted in a two step procedure. Frozen hemispheres
were homogenized in TBS pH 8 including protease inhibitors (Roche, Cat. No. 1836153) and
centrifuged at 25,000g for 1 hour. The supernatant was collected as soluble Aβ fraction and the
pellet was resuspended in 70% formic acid. After sonication for 35 s (Labsonic M, Braun Biotech
International, Germany) samples were centrifuged again for 1 hour at 25,000g. The supernatant
was considered the insoluble fraction9. Samples were diluted to fit the standard samples (Abeta
Peptide 3-Plex) and were analysed in triplicates on a MS6000 using MSD 96-well Multi-Spot
Human 6E10 Abeta Triplex Assay (Meso Scale Discovery, Gaithersburg, MD). For the analysis of
systemically GCV-treated mouse brains, Aβ was determined using commercially available ELISA
kits (Genetics Company, Schlieren, Switzerland).

Western Blot
Urea-based electrophoresis and SDS-PAGE was performed to assess Aβ and APP as well as Iba1
levels. Electrophoresis was carried out according to previously published protocols8 using
antibodies 6E10 against Aβ and anti-Iba1 (for details see above). As internal control monoclonal
anti-GAPDH antibody (Hy Test Ltd., Turku, Finland) was used.

Ultrastructural analysis
Mice were deeply anesthetized and perfused with 0.1M phosphate-buffered saline (PBS) followed
by 4% paraformaldehyde in PBS plus 0.1% glutaraldehyde. Brains were removed and fixed
overnight in 4% PFA in PBS. Small cortical specimens were dissected, fixed in 1% OsO4 in
cacodylate buffer for 1h, dehydrated in ascending series of ethanol and propyleneoxide, stained in
uranyl-acetate for 4 h and flat-embedded in Araldite (Serva, Germany). Using an ultramicrotome
(Ultracut, Leica, Bensheim, Germany), semi-(1µm) and ultrathin sections (50 nm) were cut.
Ultrathin sections were stained with lead citrate, mounted on copper grids and finally analysed with
a Zeiss EM 10 (Oberkochen, Germany) electron microscope.

Multiphoton microscopy
In vivo imaging was performed by installing a round cranial window (5 mm diameter)10 adjacent to
the ALZET osmotic pump. Surgery was carried out under isoflurane anesthesia (2–5%). Following
surgery, mice were treated with Temgesic (i.p. 0,05 mg/kg daily; Essex Pharma GmbH, Munich,
Germany) for three days. Prior to imaging mice were anesthetized with isoflurane (induction with
5% and then reduced to 1%) and secured by a custom build head fixation, while the window was
cleaned with ddH2O. Mice were injected i.v. with 10 mg/kg Methoxy-XO4 (75µl) in DMSO/PBS
Nature Neuroscience: doi:10.1038/nn.2432

Page 15

12
(generously provided by B. Schmidt). To facilitate repeated imaging a fluorescent angiogram was
achieved by the injection of Texas Red dextran (70,000 Da molecular weight; 12.5 mg/ml in sterile
PBS; Invitrogen, Carlsbad, CA) intravenously immediately before imaging. Imagery was performed
with a 40x water immersion lens (0.8 numerical aperture, U-V-I 0/D; Leica Microsystems,
Bensheim, Germany). Multiphoton excitation at 910 nm was generated by a Spectra Physics (San
Jose, CA) Mai-Tai laser (tunable 770–990 nm) and for detection two non-descanned detectors
(R6357 P.M.T.; Hamamatsu, Bridgewater, NJ) were used at close proximity to the objective lens.
Visualization of plaques and microglia cells was performed via a FITC/tetramethylrhodamine
isothiocyanate filter (reflection short pass, 560; bandpass, 525/50; bandpass, 610/75). Five
different x-y-z stacks were recorded per mouse. To separate the Methoxy-XO4 signal from eGFP
signal, a spectral unmixing algorithm was used (edited Java plugin for ImageJ allowing for the
deconvolution of bioluminescence images)11.

Statistical analysis
Statistical analysis was performed using JMP 7.1 Software or GraphPad Prism 3.1. The results are
expressed as mean values ± standard errors of the mean (SEM). Statistical significance is
indicated as follows: *p < 0,05, **p < 0,01, and ***p < 0,001.
Nature Neuroscience: doi:10.1038/nn.2432