Hippocampal expression of murine IL-4 results in exacerbation of amyloid deposition

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Title:
Hippocampal expression of murine IL-4 results in exacerbation of amyloid deposition
Physical Description:
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Language:
English
Creator:
Chakrabarty, Paramita
Tianbai, Li
Herring, Amanda
Ceballos-Diaz, Carolina
Das, Pritam
Golde, Todd E.
Publisher:
BioMed Central

Notes

Abstract:
Background: Pro-inflammatory stimuli, including cytokines like Interleukin-1β, Interleukin-6 and Interferon-γ, in the brain have been proposed to exacerbate existing Alzheimer’s disease (AD) neuropathology by increasing amyloidogenic processing of APP and promoting further Aβ accumulation in AD. On the other hand, antiinflammatory cytokines have been suggested to be neuroprotective by reducing neuroinflammation and clearing Aβ. To test this hypothesis, we used adeno-associated virus serotype 1 (AAV2/1) to express an anti-inflammatory cytokine, murine Interleukin-4 (mIL-4), in the hippocampus of APP transgenic TgCRND8 mice with pre-existing plaques. Results: mIL-4 expression resulted in establishment of an “M2-like” phenotype in the brain and was accompanied by exacerbated Aβ deposition in TgCRND8 mice brains. No change in holo APP or APP C terminal fragment or phosphorylated tau levels were detected in mIL-4 expressing CRND8 cohorts. Biochemical analysis shows increases in both SDS soluble and insoluble Aβ. mIL-4 treatment attenuates soluble Aβ40 uptake by microglia but does not affect aggregated Aβ42 internalization by microglia or soluble Aβ40 internalization by astrocytes. Conclusions: Short term focal mIL-4 expression in the hippocampus leads to exacerbation of amyloid deposition in vivo, possibly mediated by acute suppression of glial clearance mechanisms. Given that recent preclinical data from independent groups indicate engagement of the innate immune system early on during disease pathogenesis may be beneficial, our present study strongly argues for a cautious re-examination of unwarranted side–effects of anti-inflammatory therapies for neurodegenerative diseases, including AD. Keywords: Interleukin 4, Inflammation, Adeno-associated virus, Hippocampus, Amyloid plaque, Amyloid precursor protein
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Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36, pgs.1-12
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/UFir) at the time of funding. The Institutional Repository at the University of Florida community, with research, news, outreach, and educational materials.

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Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36







Hippocampal expression

exacerbation of amyloid (

Paramita Chakrabarty,, Li Tianbail, Amanda Herring', Caro


( AL+-.,+


affect aggregated AI342 internalization by microglia or solI
Conclusions: Short term focal mlL-4 expression in the hip
in vivo, possibly mediated by acute suppression of glial clk
from independent groups indicate engagement of the inr
pathogenesis may be beneficial, our present study strong
side-effects of anti-inflammatory therapies for neurodeger
Keywords: Interleukin 4, Inflammation, Adeno-associated
protein

Background


S 2012 Chakrabarty et al., licensee BioMed
Biole led Central Creative Commons Attribution License (htt
distribution, and reproduction in any media


MOLECULAR
SNEURODEGENERATION






)f murine IL-4 results in

deposition

na Ceballos-Diazl, Pritam Das2 and Todd E Golde*


ji M/- Lrcirisgeriic lyLIU-i\iuo mice WIL pre-exisLiri

02-like" phenotype in the brain and was accompanied
change in holo APP or APP C terminal fragment or
g CRND8 cohorts. Biochemical analysis shows increases
nuates soluble AI340 uptake by microglia but does not
ble AI340 internalization by astrocytes.
ocampus leads to exacerbation of amyloid deposition
irance mechanisms. Given that recent preclinical data
te immune system early on during disease
argues for a cautious re-examination of unwarranted
?rative diseases, including AD.
rus, Hippocampus, Amyloid plaque, Amyloid precursor


immune activation either being the trigger, a homeo-
static response mechanism or a bystander phenomenon
associated with the disease pathology [2].
Anti-inflammatory cytokines, such as Interleukin (IL) -


'creativecommons.org/licenses/by/2.0), which permits unrestricted use,
, provided the original work is properly cited.






Chakrabarty et a. Molecular Neurodegeneration 2012, 7:36
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mechanism to clear AP from the CNS [9]. Data
our lab [10-12] and others [13-17] have demonst:
that glial activation can effectively clear AP plaques.
generally thought that although a sustained inflan
tory response is neurotoxic, activation of the innat(
mune system can indeed have a beneficial function
clearing debris and possibly promoting repair.
In an effort to further understand the role of neu
flammation on AP plaque pathology and specifical
test the role of anti-inflammatory cytokines on
pathology, we utilized recombinant adeno-assoc
virus serotype 1 (rAAV2/1) to overexpress murine
(mIL-4) in the hippocampus of Amyloid precursor
tein (APP) transgenic mice with pre-existing am
plaques. Our results show that mIL-4 expre
resulted in exacerbated AP deposition in APP transl
mice brains after 6 weeks of expression. Biochei
analysis of mIL-4 overexpressing mice brains
phagocytosis assays in primary murine glia suggests
mIL-4 expression leads to increased AP levels pom
as a result of reduced glia phagocytosis.

Results
To explore the role of anti-inflammatory cytokini
regulating AP accumulation in the CNS, we have
recombinant adeno-associated virus serotype 1 (rAA)
to express mIL-4 in the brains of APP trans!
TgCRND8 mice. Recombinant AAV2 plasmids were ]
aged in AAV serotype 1 capsid as described previous)
and rAAV2/1 viruses expressing mlL-4 or EGFP under
control of the cytomegalovirus enhancer/chicken P-
promoter were used for further experiments. i
TgCRND8 mice were stereotaxically injected with AA
constructs (1x10"3 particles/ml) into the CA layer o
hippocampus at 4 months (after AP plaque deposition
started) and were analyzed after 6 weeks (n= 6 for rA
mIL-4; n=6 for rAAV1-EGFP). Immunohistochel
analysis with anti-EGFP antibody shows that the
transgene is predominantly expressed in the hippoca
CA neurons, neuronal projections in the cortex and
amus, and some overlying cortical neurons follc
6 week expression ofAAV1-EGFP (Additional file 1: F
S1, A-I). No detectable expression was noted in the
brain, olfactory bulb or cerebellum. In previous st
AAV2/1-EGFP expression had minimal effects on am
pathology or gliosis when compared to naive uninj,
mice [10,18,19]; so, AAV1-EGFP injected animals
used as the control cohort in this study. For adult inj
mice, the brain was coronally dissected 1 mm anterior
posterior to the point of injection and used for subset
analysis. We did not observe any significant chang
GFAP immunoreactive astrocytes (Figure 1, A-D) or
reactive microglia (Figure 1, E-H) in the hippocamp'
mIL-4 expressing mice compared to control mice. A cE


Page 2 o




*om quantification of the histological staining (GFAP and Ibe
Lted in mouse hippocampus using "Positive Pixel Count" p
[t is eram (ADerio. CA) showed that though mLL-4 express


:hat to previous observations [21].
ibly Analysis of plaque burden showed that there was
33.5% increase in amyloid plaques in the hippocam]
of mIL-4 expressing mice compared to EGFP express
mice (Figure 2, A-E). There was a concomitant incre
in in insoluble AP levels by biochemical analysis 41%
sed crease in AP342 levels and 55% increase in A[40 levels
2/1) the SDS extractable Ap levels (Figure 2, F) respective
enic and 76% increase in AP42 levels and 62% increase
ick- A340 levels in the formic acid extractable A3 levels
10] spectively (Figure 2, G). Interestingly, the number
the Thioflavin S stained "cored" plaques in the hippocam:
ctin of mIL-4 expressing mice (15.2% increase) did not
Jult crease significantly compared to controls (Figure 3, A-
'2/1 We next investigated whether the increase in A3 was d
the to changes in APP expression, APP processing, ApoE lev
has or phagolysosomal dysfunction. Neither APP express
V1- nor CTFa expression or CTFP levels were altered in mll
lical expressing TgCRND8 mice compared to controls (Figure
niral A -R_ Vi r5Alitifinnmhir otptrc F f,-r tho 1vo1i nTf oncenrn







Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36


Control


I-I




) r"
H
;,. .,.,
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DI Control
0.4 I l mlL-4

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cdllb


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37-

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50- __ --P-actin
37-

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6.5- Control *
S 5.5
50
3.53
* 3.0.


51:


,LU %i


Figure 1 AAV2/1 mediated expression of mlL-4 in TgCRND8 mice results in M2 phenotype. A-D. rAAV2/1-mlL-4 or rAAV2/1-EGFP (Control)
was injected into the hippocampus of 4 month old TgCRND8 mice and analyzed after 6 weeks. Representative images of GFAP immunoreactivity
in paraffin embedded whole brain sections (A, C) and higher .. .1i of the hippocampus (B, D) is shown. Scale Bar, 600 pm (A, C) and
25 pm (B, D). (n 5-6/group). E-H. Representative images of Iba-1 immunoreactivity in paraffin embedded sections of 5.5 month old TgCRND8
mice injected with rAAV2/1 mlL-4 or rAAV2/1-EGFP (Control). Whole brain sections (E, G) and the corresponding hippocampus (F, H) are shown.
Scale Bar, 600 pm (E, G) and 25 pm (F, H). (n= 5-6/group). I. Densitometric analysis of GFAP and ba-1 immunostaining is shown. The hippocampa
region was selected and Aperio "positive pixel count" program was used to measure percent positivity by averaging intensity of positive staining
in the annotated region. (n 4/group; p > 0.05, t test). Data represents mean sem. J-K. Representative immunoblot (J) and densitometric analysis
of normalized levels of GFAP and cdl b (K) obtained from 5.5 month old TgCRND8 mice injected with rAAV2/1 mlL-4 or rAAV2/1-EGFP (n= 5/
group; p > 0.05, t test). Data represents mean sem. L. Expression of glial activation markers and cytokines were determined in 5.5 month old
mlL-4 expressing TgCRND8 mice compared to EGFP expressing age-matched controls using real time Q-PCR. Data, expressed as relative levels of
mRNA expression, represents averaged fold change values obtained from mlL-4 expressing mice, relative to averaged values obtained from EGFP
expressing mice. (n 4/group; p < 0.05, t test). Data represents mean sem.


of AP [24]. Since anti-inflammatory cytokines, for example,
IL-4 and IL-13, have been shown to inhibit autophagy [25],
we tested whether changes in autophagic response may ac-
count for increased AP accrual in mlL-4 expressing mice.
No significant changes in the autophagic marker LC3-I


were seen in transgenic TgCRND8 mice injected with
AAV2/1-mIL-4 (Additional file 3: Figure S3, A, D), though
a nonsignificant lowering trend was observed in the non-
transgenic cohort (Additional file 3: Figure S3, B, D). We
were unable to detect LC3-II in the lysates, making it


mlL-4


Page 3 of 12


Control


mlL-4


U-


( -'




( C1


I _







Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36


.
:..*-*


. 4


Page 4 of 12


Control ml L-


C '


* .


II
ao

(


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( -

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N~4


SE Ap42
E F G *Ap40
2.0 1200 4000
r

E 800 000

< 400 '2OOO
0.5. W 0" 1000
Control IL-4 Control IL-4 Control IL-4
Figure 2 Increased A3 deposition in AAV1-mlL-4 expressing TgCRND8 mice. A-D. 4 month old TgCRND8 mice were stereotaxically injected
in the hippocampus with either AAV1-mlL-4 (C, D) or AAV1-EGFP (A, B) and sacrificed after 6 weeks (n-5-6/group). Representative brain sections
stained with 33.1.1 antibody (pan AB 1-16) depict increased A3 deposition in mlL-4 expressing mice (C, D) compared to controls (A-B) in the
immediate vicinity of the injection site. Scale Bar, 150 pm. E. A3 plaque burden analysis shows a i I increased amyloid deposition in
mlL-4 injected mice compared to control EGFP injected mice (n5/group). (*p < 0.05, testt. Data represents mean sem. F-G. Biochemica
analyses of A342 and AP40 levels by ELISA show I. increased SDS (F) and formic acid (G) extractable A3 levels in mlL-4 injected mice
compared to controls (n5/group). (*p < 0.05, t test). Data represents mean sem.


difficult for us to infer effects of mlL-4 on autophagic flux
in vivo. p62 (or sequestosome-1), which is a central player
in autophagy was found to be decreased by 47% in mIL-4
expressing mice (Additional file 3: Figure S3, C-D).
A basic tenet of the amyloid cascade hypothesis is that
AP accumulation triggers the onset and severity of neu-
rodegenerative pathology, including tau hyperphosphor-
ylation. Though occasional CP13 immunoreactive glial
cells were visible in both mIL-4 and control mice, mIL-4
mice did not lead to appearance of phosphorylated CP13
immunoreactive tau (phospho Ser202/Thr205) in the
hippocampal neurons in TgCRND8 mice, (Additional file
4: Figure S4, A-B). Immunoblotting also failed to show


any detectable levels of CP13 or MC6 (phospho Ser235)
or PHF1 epitope (phospho Ser396/Ser404) in both
cohorts (data not shown).
To further investigate the mechanism of mIL-4
induced AP accumulation, we treated primary wild type
mouse neuroglial cultures with rAAV2/1-mIL-4 for
72-80 hours. Analysis of the conditioned media of these
cells revealed increased mIL-4 protein (p < 0.01, t test)
but no increases in mIL-6 (data not shown) or mIFN-y
protein compared to control cultures (Figure 5, D). In
addition, primary cultures transduced with mIL-4
showed increased levels of nuclear phosphorylated
STAT6 compared to mIFN-y or untreated cultures


Control


r
~ ,i?._

ILh ~r


mlL-4


^


,i







Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36




Control mlL-4









C
600-








Control mlL-4
Figure 3 No significant change in cored plaques seen in AAV1-
mlL-4 expressing TgCRND8 mice. A-B. Representative fluorescent
images ofThioflavin S stained hippocampi from 5.5 month old
TgCRND8 mice injected with rAAV2/1 mlL-4 (B) or rAAV2/1-EGFP
(Control). Scale Bar, 200 pm. C. A comparison of the total number of
Thioflavin S stained "cored" plaques from the forebrain of 5.5 month
old TgCRND8 mice injected with rAAV2/1 mlL-4 or rAAV2/1-EGFP is
depicted. Data represents mean sem. (n=5/group; p-0.0542, t
test).

(Figure 5, A-C). Quantitative RT-PCR analysis confirm
increased mlL-4 (4.5e3 x) levels as well as increase
levels of cdllb (6 x), scavenger receptor A (3.3 x) an
scavenger receptor B1 (15.6 x) (Figure 5, E). No signifi
cant changes in mouse APP, BACE1, or A3 degradin
enzymes (IDE or Neprilysin) were seen (Figure 5, E
Since increased cdllb and scavenger receptors may re
sult in altered phagocytic potential of mIL-4 expression
glia, we performed phagocytosis assays on mouse pri
mary glia and astrocytes treated with medium alone o
recombinant mIL-4. Since A340 and A342 can have dil
ferent effects on phagocytosis, we used both to test ou
how mIL-4 affects astroglial phagocytois. Primary mous
glia were treated with recombinant cytokines or vehicle
for 10 hours, and fresh media added before incubatio
with fluorescent A3 for different times (Figure 6, Add
itional file 5: Figure S5- Additional file 6: Figure S6
These experiments were performed with cultures con
training >95% CD45 and Cdllb immunopositive gli
(Additional file 5: Figure S5, A-B). Initial microscope
examination showed a decrease in the levels of internal
lized A340 in mIL-4 treated microglia following 15 mir
30 min or 60 min incubation with A340-555 nr
(Additional file 6: Figure S6, A-I). This was confirmed b
flow cytometric analysis which showed a significant de


Page 5 of 12




microglia at these three different timepoints (15 min:
-19%; 30 min: -38%; 60 min: -19%) (Figure 6, A;
Additional file 5: Figure S5, D-L). Since we have previ-
ously shown that mIL-6 treatment augments microglial
A3 phagocytosis [10], we included this as an internal
control for A340 internalization in the flow cytometric
assay (15 min: +29%; 30 min: +13%; 60 min: +41%)
(Figure 6, A; Additional file 5: Figure S5, J- L). Immuno-
blotting of microglial cells following A340 phagocytosis
also showed that mIL-4 treated glia take up A3 less effi-
ciently (Additional file 5: Figure S5, M-N). In order to
test whether fibrillized A342 (fA342) has a different ef-
fect on glial phagocytosis, we performed phagocytosis
assays using A342 fibrils (Figure 6B). Flow analysis
shows that neither mIL-6 (15 min: +25.7%; 30 min:
+28%; 60 min: +8%) nor mIL-4 (15 min: +0.53%; 30 min:
+8.1%; 60 min: +10.4%) significantly affects fA342
internalization by microglia (Figure 6B). Since IL-4 can
potentially activate astrocytic internalization of AP3, we
performed phagocytosis with murine astrocyte cultures
(Figure 6, C-D). Overall, neonatal astrocytes were very
inefficient in internalizing A340 as shown earlier [2,26].
Flow cytometric analysis shows that mIL-4 does not sig-
nificantly alter astrocytic A340 phagocytosis (15 min:
+39.5%; 30 min: +50%; 60 min: +12.2%) (Figure 6, C)
whereas mIL-6 increases astrocytic A340 internalization
significantly at all timepoints tested (15 min: +83.1%;
30 min: +58.3%; 60 min: +85.2%) (Figure 6, C). On the
d other hand, mIL-4 (15 min: -16.8%; 30 min: +3%) as well
d as mIL-6 (15 min: -48.4%; 30 min: -26%) has negligible
d effects on astrocytes phagocytosing fA342 at the two
earlier timepoints tested (Figure 6, D). Only at the last
g timepoint tested, i.e., following 60 min of incubation of
). fA342, both mlL-4 (60 min: +44.4%; p<0.05, 1 way
Anova) and mIL-6 (60 min: +45%) enhances fAP42 up-
g take by primary astrocytes (Figure 6, D).
i-
r Discussion
We have found that overexpression of mlL-4 in the
it hippocampus of plaque-depositing APP CRND8 mice
e increased A3 plaque pathology. We extensively investi-
e gated the likely factors that could be responsible for the
n effects of mIL-4 expression on increased A3 burden
S in vivo. APP, APP CTE BACE, ApoE and A3 degrading
). enzyme levels did not appear to be altered. As IL-4 has
been reported to inhibit autophagy [27], we investigated
a LC3I/II levels, but this was not informative, as we could
c not reliably detect LC3II. Levels of another key regulator
L- of autophagy, p62, were decreased by mIL-4. As p62 has
i, pleiotropic functions in addition to its role in autophagy
n [28] the significance of this finding is unclear. Further-
y more if autophagic pathways were being inhibited, p62
- would be expected to increase, not decrease. Given i)
d that these data indicating that mIL-4 does not appear to






Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36


A Control mlL-4 B


Wipe-wo-


Owl


-APP


-Actin

-CTFp
CTFa

-Actin


(A
04-
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*o 3
N
a 2
E
0 1
z


APP


3 Control
SmlL-4


II o.i


CTFa CTFp


Control mlL-4


---,------ Prion

,Actin


_1F


Control


Control mlL-4
1.2,
0.9

0.8-
-. "--- -CTF cL .8'

IL0.3.
'- "-'----A Actin n 01:3
O.O- -
>


mlL-4


O Control
m mlL-4


Figure 4 mlL-4 does not affect human APP transgene expression or CTF production. A-B. Representative anti CT20 immunoblot showing,
no significant changes in APP, CTFa and CTFP levels in AAV1-mlL-4 expressing TgCRND8 compared to age-matched controls (A). Intensity
analysis (mean sem) of anti CT20 immunoreactive APP and CTFa levels was normalized to B-actin in TgCRND8 mice cohort (B). (n 5/group;
p >0.05, t test). C-D. Representative immunoblot showing no significant changes in prion protein levels in AAV1- mlL-4 TgCRND8 compared tc
age-matched controls (C). Intensity analysis of immunoreactive prion protein levels (mean sem) was normalized to B-actin (D). (n 5/group;
p >0.05, t test). E-F. Representative anti CT20 immunoblot showing no significant changes in APP and CTFa in 5 month old AAVI-mlL-4
expressing wild type B6/C3H mice compared to age-matched controls (E). Intensity analysis of anti CT20 immunoreactive APP and CTFa levels
(mean +sem) was normalized to B-actin in wild type mice cohort (F). (n 5/group; p > 0.05, t test).


be affecting APP processing and is not altering major AP3
chaperones or degrading enzymes and ii) that we and
others have previously linked proinflammatory activation
of microglia to reduced plaque burdens and enhanced
microglial scavenging of A3 [10,11,13,14,17,29,30], we
hypothesize that mlL-4 increases A3 burden via reduc-
tions in glial scavenging of A3. We explored this hypoth-
esis through AP internalization studies in primary


microglia and astrocytes cultures. These studies do show
that mlL-4 abrogates soluble AP40 uptake by microglia
without affecting aggregated fAP42 phagocytosis. Add-
itionally, mlL-4 does not affect the uptake of soluble or
aggregated AP by astrocytes, though on longer incuba-
tion, there was an increasing trend in AP ingestion by
the astrocytes. Given the low number of astrocytes in-
ternalizing AP and the fact that astrocyte cultures may


Page 6 of 12


., .






Chakrabarty et a. Molecular Neurodegeneration 2012, 7:36
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contain -10-20% microglia, it is difficult to conclude
whether astrocytes by themselves have a critical role in
AB phagocytosis.


20000-

15000-
E
S 10000-

E 5000-

ft


Page 7 of 12


Microglia can scavenge AP via receptor-mediated
phagocytosis as well as macropinocytosis [26]. In vivo
imaging techniques have demonstrated that microglia


0 mlL-4
*mlFN- 1500


,.100


I I r III.


AAV-mlL-4


No virus AAV-mlFN-y


No virus


3


Z

-500 3


-0


98 1II r- Control
on- mIL-4


\VRR 0c<
.> qQ /f
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Figure 5 (See legend on next page)


A
21
0OP




a


,i I







Chakrabarty et al. Molecular Neurodegeneration 2012, 7:36
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(See figure on previous page.)
Figure 5 Characterization of rAAV2/1-mlL-4 treated primary wild tyl
(2x10 viral genomes) was used to transduce primary mouse neuroglial c
STAT6 (568 nm; red fluorescence) could be seen in mlL-4 expressing cult
containing no viruses (A). MAP2 (488 nm; green) and DAPI (350 nm, blue
4 and m FN-y expression in these cultures was verified by ELISA from me
t test). E. Expression of mouse endogenous APP, BACE1, IDE and neprilys
neuroglial cultures compared to untreated control cultures using real tim
levels. Data, expressed as relative levels of mRNA expression, represents a
relative to averaged values obtained from EGFP expressing mice. Data fo
plotted on the right x-axis. Data is representative of two independent ex|


can home onto newly appearing plaques and internalize
A3 [31]. Microglial interaction with A3 occurs through
several cell surface receptors, for example, SR-A, SR-B,
integrins and Toll-like receptors [15,32-34]. Previous
studies have noted that inflammatory cytokines decrease
scavenger receptors levels in microglial cell lines and



A
100-

*80
8.0




0
4 20. I i|


15 min 30 min 60 min

C
100

0 80





20 *
r f.



15 min 30 min 60 min
Figure 6 Differential effect of cytokines on Ap40 and fAp42 uptake
decreases microglial uptake of Ap40 (A) but does not affect fAp42 uptak(
alter fAp42 uptake (B). Primary mouse glia were treated with
Ap42-Hilyte555 for 15 min, 30 min and 60 min. Trypsinized cells were coi
additives were excluded by gating to yield the percentage of fluorescent
representative of three independent experiments. (p < 0.05, One way Ar
treatment does not affect astrocytic uptake of Ap40 (C) but increases fAI
astrocytic Ap40 uptake (C) and only enhances fAp42 uptake after 60 min
recombinant cytokines for 10 hrs and incubated in Ap40-Hilyte555 or Ap-
counted using Accuri6 flow cytometer. Unstained cells and labeled addit
nset depicts GFAP/DAPI labeled astrocyte (C). Results are representative
Tukey's post test). Data represents mean sem.


Page 8 of 12







e mouse neuroglial culture. A-D. rAAV2/1-mlL-4 or rAAV2/1-mlFN-y
Itures for 60 hrs in chamber slides. Upregulation of phosphorylated
res (C) but not in m FN-y expressing cultures (B) or untreated culture
were used to depict neuronal processes and nucleus respectively. mlL-
ia collected from respective cultures (D). ', i 400x. (p < 0.05,
Levels are unchanged in rAAV2/1-mlL-4 expressing primary mixed
Q-PCR. mlL-4 expression augments scavenger receptors and cdl b
eraged fold change values obtained from mlL-4 expressing mice,
mlL-4 RNA is plotted on the left x-axis while the rest of the data are
eriments (t test, *p < 0.05). Data represents mean sem.


inhibit A3 uptake [35]. We find that mlL-4 expression
leads to increased SRs in mixed neuroglial cultures, but
this appears to be linked to modest inhibitory effects on
soluble, but not aggregated, A3 uptake by microglia.
Therefore, it is possible that in the absence of inflamma-
tory mediators, these cells are not competent for AP



B 0 Control
2 100- mlL-4
2 80 mlL-6
0 80.

E .
360.


0








2 20








o -o. .O*nnl nl nnl
15 min 30 min 60 min

D
100e

o 80






20-
0-
15 min 30 in 60 min




nted using Accuri6 flow cytometer. Unstained cells and labeled
ellss in the mix. Inset depicts cd l b/DAPI stained glia (A) Results are
)va with Tukey's post test). Data represents mean sem. C-D. mlL-4
2 uptake after 60 min incubation (D). mlL-6 consistently increases
ncubation (D). Primary mouse astrocytes were treated with
2-Hilyte555 for 15 min, 30 min and 60 min. Trypsinized cells were
es were excluded by gating to yield the percentage of fluorescent cells.
Stwo independent experiments. (p < 0.05, One way Anova with







Chakrabarty et a. Molecular Neurodegeneration 2012, 7:36
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internalization and degradation, even if they express
receptors [36,37]. Indeed, microglia were found ti
ninrli ctmllnt1lnn rilth rt4l,-4nc1 cr -ncincnF in


-,,-
increase in neuritic plaques at autopsy [41]. Our
could provide an explanation for this association.
a* : ,-,,,,,1 ,. .,.,,,t,, tis .... ,Iu^^-^ *-, *_..


in many cases this relationship is assumed and ha!
been formally proven. Increased pro-inflammatory
kines levels (TNFa, Interleukin-6, Interleukin-lp), ,
bined with decreased levels of anti-inflamm:
cytokines (Interleukin-10), have been correlated
cognitive deficits suggesting that early inflanmm
changes may be detrimental [43]. Furthermore p
flammatory stimuli have been linked to overt neul
degeneration [44]. Although proinflammatory fa


risk finding.
:T)
de- Conclusion
on- In summary, we demonstrate that focal overexpress
an of mIL-4 in APP transgenic mouse brains leads to e
lata cerbated AP plaque pathology. As these results are
posite of effects observed with proinflammat
une cytokines, we would suggest that the underlying me
not anism appears to be at least in part a failure of glia
h a successfully clear A3. Our data points to the complex
this lationship between microglial phenotype and the fi
iul", functional outcome, necessitating a more cautious ,
not thorough examination of potential anti-inflammat
yto- therapies for AD.
)m-


in conIrasL Lo our results, ilyo a ei ai, snoweu Mal. 1ii.,- yuanulrative reat-rime rc0L. IUoal La iNWr irum n
expression in the hippocampus led to mitigation of AP hippocampus or primary wild type mouse neuroglial
pathology and improved behavior in APP/PS1 mice [4]. tures was isolated using the RNaqueous kit (Ambi


Page 9 of


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Chakrabarty et al Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/3(



The Q-PCR (initial denaturation cycle of 95 C /i
followed by 40 amplification cycles of 95 C /1!
60 C/ 1 min) was performed with ABI Prism 79,
Time PCR System (Applied Biosystems) using
Green to detect the amplification products. I
' i I.I ''..... of mRNA expression was calculated
ACT method .1. ..i...I by the manufacturer (ABI
7700 Sequence Detection System, User Bulletin #
adjusting the levels to the corresponding intern
control for each sample. Primers and probe
designed ii...i. p; Roche Universal Probe
sequences (Hoffmann-La Roche, Germany).
S. of brain homogenate for .. .:. .
and Af3 ELISA assay. Brain were coronally di
1 mm anterior and posterior to the point of ir
and used for subsequent analysis. Thus, the sam]
immunoblotting were obtained by dissecting the
campus and overlying cortex and thalamus of i
brains. Protein samples (RIPA soluble or 2% Sl
uble) separated on Bis-Tris 12% XT gels (Bio-Rac
were probed with the .--I... .;; CT20 (ar
C-terminal 20 amino acid; T. E. Golde; I :1 Io
(IBL, 1:500), Prion (Abcam; 1:1000); LC3 (MBLI
Cell Signaling, 1:250; Novus, 1:500); p62 (Cell '.i
1 -.') CP1R (iP Dnvipne 1 100' anl anti


Page 10





Smin, averaged by a blinded observer to calculate plaque
s and den. For Thioflavin S quantitation, one section per
) Real ple was used by a blinded observer to manually c
SYBR the plaques using Adobe Photoshop CS5.
dative Primary marine culture and ,.' ..- j-'.' r
by the a '.. .. :.'. assay. Primary microglia, astrocyte
Prism neuronal cultures (mixed with astrocytes) were obt
) after from cerebral cortices of wild type neonate mik
actin described previously [38]. Glial and astrocytic cul
were ,i .:.. i were >90% cdllb (cdllb-APC; 1:200, BD
library sciences) or GFAP -' ..* !! 1:500) immunopositivi
spectively. For phagocytosis assays, microglia
... astrocytes, pre-treated with mIL-4 (R&D Systems,
sected 5 ng/ml for 10 hours) or mIL-6 (R&D Systems,
action 10 ng/ml for 10 hours) were incubated with 0.5
es for Hilyte555-Ap40 (Anaspec, USA) or fibrillar Hilytt
lippo- AP42 (Anaspec, USA). Recombinant fluorescent A[
ejected resuspended in DMSO to 1 mg/ml and dilute
S sol- DMEM I.... i.. to 0.5 tM before addition to
USA) Hilyte555-Ap42 was fibrillized at 370 C for 6 hou
i-APP PBS. Cells were analyzed at three timepoints :.,I'
0m 1 -I 1 -,C ADO -- _- 1. ,--_ -.. Io.


ewmicon). 1% Thioflavin S '


I .1 Student's t test was used tor static


kl or ated using Photoshop CS2 (Adobe).
posi-
I con-
Sco- Additional files
>tured
Additional file 1: Figure S1. rAAV2/1- GFP expression n TgCRNDE
rafor- mice hippocampus A-I. Representative image obtained from mice
actions stereotacticaily injected with AAV2/1-EGFP in the hippocampus 4 m
a e od TgCRND8 were injected into the hippocampus and analyzed afi
Saq 6 wee calcu- embedded whole brain section (A), cortex (B), hippocarpal CA neui
)erio). (C- ), midbrain, (F), cerebellum (G), thalamus (H) and oifacto/ bulb (
shown Representative hippocampus from un-njected mice is show
were








Chakrabarty et oa Molecular Neurodegeneration 2012, 7:36
http://www.molecularneurodegeneration.com/content/7/1/36





control (A, nset. iV, cortex layers I to V; CC, corus callosum; Sp, Sep:
[', tTalamus. Scae Bar, 600 Im (A) and 85 Tm (B 1).
Additional file 2: Figure 52. niL-4 expression does not alter ApoE
leve s. A-B. No significant change in ApoE evels was seen in the
hippocampus of ml 4 expressing 5.5 month o d transgenic CRND8 r
or agermatched control cohotts (A). intensity analysis of ApoE eve s
normalized to p actn (B). (n 5 6/group; t test, p > 0.05).


Addil


actn immu noreactviry nas been showr i N). iepresenratve Cara Tro
two irdeperdent experiments have been shown.
Additional file 6: Figure S6. Microscopic ara ysis of Ar(40 phaqoc
by miL-4 treated mouse primary g ia Primary mouse giia were treat


Page 11 o






Authors' contributions
um;
PC conducted the experiments and wrote the manuscript; L- performed
inrmunostaining, primary mouse neutoglia culture, phagocytosis assays a
flow cytrmet'y; AB perforrred mouse brain Q PCR and amyloid burden
analysis; CC-D prepared tecombinat AAV and Q-PCR of neuroglial cuitur
ice PD provided helpful discussion; TEG coordinated the research, supe-vised
'as project and assisted in manuscript preparation. All authors have lead and
approved the final manuscript.


Ig ot Medicine, 4500 San Pablo hd S, Jacksonville, kL 32224, USA.

Received: 15 February 2012 Accepted: 22 July 2012
n Published: 29 July 2012

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Adeno associated virus; ACT: Adult Chan,
Jzheimer's disease; AP: Amyloid P; APP:
: Apolipoprotein c4; BACE: 3 site APP cle,
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ough
doi:10.1186/1750-1326-7-36
-tic V T Cite this article as: Chakrabarty et al Hippocampal expression of mt
IL-4 results in exacerbation of amyloid deposition. Molecular
Neurodegeneration 2012 7'36
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AbstractBackgroundPro-inflammatory stimuli, including cytokines like Interleukin-1β, Interleukin-6 and Interferon-γ, in the brain have been proposed to exacerbate existing Alzheimer’s disease (AD) neuropathology by increasing amyloidogenic processing of APP and promoting further Aβ accumulation in AD. On the other hand, anti-inflammatory cytokines have been suggested to be neuroprotective by reducing neuroinflammation and clearing Aβ. To test this hypothesis, we used adeno-associated virus serotype 1 (AAV2/1) to express an anti-inflammatory cytokine, murine Interleukin-4 (mIL-4), in the hippocampus of APP transgenic TgCRND8 mice with pre-existing plaques.ResultsmIL-4 expression resulted in establishment of an “M2-like” phenotype in the brain and was accompanied by exacerbated Aβ deposition in TgCRND8 mice brains. No change in holo APP or APP C terminal fragment or phosphorylated tau levels were detected in mIL-4 expressing CRND8 cohorts. Biochemical analysis shows increases in both SDS soluble and insoluble Aβ. mIL-4 treatment attenuates soluble Aβ40 uptake by microglia but does not affect aggregated Aβ42 internalization by microglia or soluble Aβ40 internalization by astrocytes.ConclusionsShort term focal mIL-4 expression in the hippocampus leads to exacerbation of amyloid deposition in vivo, possibly mediated by acute suppression of glial clearance mechanisms. Given that recent preclinical data from independent groups indicate engagement of the innate immune system early on during disease pathogenesis may be beneficial, our present study strongly argues for a cautious re-examination of unwarranted side–effects of anti-inflammatory therapies for neurodegenerative diseases, including AD.
http:purl.orgdcelements1.1creator
Chakrabarty, Paramita
Tianbai, Li
Herring, Amanda
Ceballos-Diaz, Carolina
Das, Pritam
Golde, Todd E
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Paramita Chakrabarty et al.; licensee BioMed Central Ltd.
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RESEARCHARTICLEOpenAccessHippocampalexpressionofmurineIL-4resultsin exacerbationofamyloiddepositionParamitaChakrabarty1,LiTianbai1,AmandaHerring1,CarolinaCeballos-Diaz1,PritamDas2andToddEGolde1*AbstractBackground: Pro-inflammatorystimuli,includingcytokineslikeInterleukin-1 ,Interleukin-6andInterferon,inthe brainhavebeenproposedtoexacerbateexistingAlzheimer ’ sdisease(AD)neuropathologybyincreasing amyloidogenicprocessingofAPPandpromotingfurtherA accumulationinAD.Ontheotherhand,antiinflammatorycytokineshavebeensuggestedtobeneuroprotectivebyreducingneuroinflammationandclearing A .Totestthishypothesis,weusedadeno-associatedvirusserotype1(AAV2/1)toexpressananti-inflammatory cytokine,murineInterleukin-4(mIL-4),inthehippocampusofAPPtransgenicTgCRND8micewithpre-existing plaques. Results: mIL-4expressionresultedinestablishmentofan “ M2-like ” phenotypeinthebrainandwasaccompanied byexacerbatedA depositioninTgCRND8micebrains.NochangeinholoAPPorAPPCterminalfragmentor phosphorylatedtaulevelsweredetectedinmIL-4expressingCRND8cohorts.Biochemicalanalysisshowsincreases inbothSDSsolubleandinsolubleA .mIL-4treatmentattenuatessolubleA 40uptakebymicrogliabutdoesnot affectaggregatedA 42internalizationbymicrogliaorsolubleA 40internalizationbyastrocytes. Conclusions: ShorttermfocalmIL-4expressioninthehippocampusleadstoexacerbationofamyloiddeposition invivo,possiblymediatedbyacutesuppressionofglialclearancemechanisms.Giventhatrecentpreclinicaldata fromindependentgroupsindicateengagementoftheinnateimmunesystemearlyonduringdisease pathogenesismaybebeneficial,ourpresentstudystronglyarguesforacautiousre-examinationofunwarranted side – effectsofanti-inflammatorytherapiesforneurodegenerativediseases,includingAD. Keywords: Interleukin4,Inflammation,Adeno-associatedvirus,Hippocampus,Amyloidplaque,Amyloidprecursor proteinBackgroundAmyloid (A )plaquesconstituteahallmarkpathologicalfeatureofAlzheimer ’ sdisease(AD),themost prevalentneurodegenerativedisorder.Neuroinflammationhasbeenhypothesizedtoplayapathogenicrolein thedevelopmentofsporadicAD,particularlybecause pro-inflammatorycytokinesandchemokinescolocalize withneurodegenerativepathologyinbothADpatient brainsaswellasintransgenicmousemodelsofADtype pathology(reviewedin[1]).However,theplacementof theinflammatoryresponseinADneurodegenerative cascadeisstilldebated,withconflictingideasofinnate immuneactivationeitherbeingthetrigger,ahomeostaticresponsemechanismorabystanderphenomenon associatedwiththediseasepathology[2]. Anti-inflammatorycytokines,suchasInterleukin(IL)4,leadtothesuppressionofpro-inflammatoryresponses inmacrophages,microglia,Tcells,andastrocytes[3]. Anti-inflammatorycytokinesarethoughttoenhanceA degradationthroughphagocytosisandreceptormediateduptakeleadingtotheabrogationofA inducedcelldeathinAPPtransgenicmice[4,5]andprimaryglia[6].DownregulationofIL-4receptorscontributestoagingrelatedcognitiveimpairment[7]. Consequently,IL-4orminocyclinetreatmentleadsto restoredsynapticactivityinratsfollowingintracerebroventricularinfusionofA [8].However,emergingevidencehasalsoshownthatactivationoftheinnate immunesystemmayconstituteabeneficialdefense *Correspondence: tgolde@ufl.edu1CenterforTranslationalResearchinNeurodegenerativeDisease,Department ofNeuroscience,UniversityofFlorida,1275CenterDrive,Gainesville,POBox #100159,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Chakrabartyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36 http://www.molecularneurodegeneration.com/content/7/1/36

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mechanismtoclearA fromtheCNS[9].Datafrom ourlab[10-12]andothers[13-17]havedemonstrated thatglialactivationcaneffectivelyclearA plaques.Itis generallythoughtthatalthoughasustainedinflammatoryresponseisneurotoxic,activationoftheinnateimmunesystemcanindeedhaveabeneficialfunctionby clearingdebrisandpossiblypromotingrepair. InanefforttofurtherunderstandtheroleofneuroinflammationonA plaquepathologyandspecificallyto testtheroleofanti-inflammatorycytokinesonA pathology,weutilizedrecombinantadeno-associated virusserotype1(rAAV2/1)tooverexpressmurineIL-4 (mIL-4)inthehippocampusofAmyloidprecursorprotein(APP)transgenicmicewithpre-existingamyloid plaques.OurresultsshowthatmIL-4expression resultedinexacerbatedA depositioninAPPtransgenic micebrainsafter6weeksofexpression.Biochemical analysisofmIL-4overexpressingmicebrainsand phagocytosisassaysinprimarymurinegliasuggeststhat mIL-4expressionleadstoincreasedA levelspossibly asaresultofreducedgliaphagocytosis.ResultsToexploretheroleofanti-inflammatorycytokinesin regulatingA accumulationintheCNS,wehaveused recombinantadeno-associatedvirusserotype1(rAAV2/1) toexpressmIL-4inthebrainsofAPPtransgenic TgCRND8mice.RecombinantAAV2plasmidswerepackagedinAAVserotype1capsidasdescribedpreviously[10] andrAAV2/1virusesexpressingmIL-4orEGFPunderthe controlofthecytomegalovirusenhancer/chicken -actin promoterwereusedforfurtherexperiments.Adult TgCRND8micewerestereotaxicallyinjectedwithAAV2/1 constructs(1x1013particles/ml)intotheCAlayerofthe hippocampusat4months(afterA plaquedepositionhas started)andwereanalyzedafter6weeks( n =6forrAAV1mIL-4; n =6forrAAV1-EGFP).Immunohistochemical analysiswithanti-EGFPantibodyshowsthattheviral transgeneispredominantlyexpressedinthehippocampal CAneurons,neuronalprojectionsinthecortexandthalamus,andsomeoverlyingcorticalneuronsfollowing 6weekexpressionofAAV1-EGFP(Additionalfile1:Figure S1,A-I).Nodetectableexpressionwasnotedinthemidbrain,olfactorybulborcerebellum.Inpreviousstudies AAV2/1-EGFPexpressionhadminimaleffectsonamyloid pathologyorgliosiswhencomparedtonaveuninjected mice[10,18,19];so,AAV1-EGFPinjectedanimalswere usedasthecontrolcohortinthisstudy.Foradultinjected mice,thebrainwascoronallydissected1mmanteriorand posteriortothepointofinjectionandusedforsubsequent analysis.Wedidnotobserveanysignificantchangesin GFAPimmunoreactiveastrocytes(Figure1,A-D)orIba-1 reactivemicroglia(Figure1,E-H)inthehippocampusof mIL-4expressingmicecomparedtocontrolmice.Acareful quantificationofthehistolog icalstaining(GFAPandIba-1) inmousehippocampususing “ PositivePixelCount ” program(Aperio,CA)showedthatthoughmIL-4expressing micehadlessmicroglialactivationinandaroundtheinjectionareatherewerenosignificantchangesineither astrocyteormicrogliallevelsoverall(Figure1,I).Immunoblottinganalysiswithhippoc ampallysatesalsoshowedessentiallyunchangedastrogliosisprofileinmIL-4and controlcohorts(Figure1,J-K).AnalysisofmRNAfrom injectedmicehippocampusshow edsignificantlyincreased levelsofmIL-4(3.13timesovercontrol)andmIL-10 (2.4timesovercontrol)(Figure1,L).Nosignificantchange inlevelsofpro-inflammatorycytokines,TNFandIFN, orcd11b(datanotshown)wasseen.Furthermore,thiswas accompaniedbyanincreaseinArginase(4timesovercontrol)(Figure1,L),suggestinganestablishmentofanalternative “ M2a ” microglialphenotype[1,20].Increasedlevels ofCD200R(4.2timesovercontrol)wasalsonoted,similar topreviousobservations[21]. Analysisofplaqueburdenshowedthattherewasa 33.5%increaseinamyloidplaquesinthehippocampus ofmIL-4expressingmicecomparedtoEGFPexpressing mice(Figure2,A-E).Therewasaconcomitantincrease ininsolubleA levelsbybiochemicalanalysis-41%increaseinA 42levelsand55%increaseinA 40levelsin theSDSextractableA levels(Figure2,F)respectively and76%increaseinA 42levelsand62%increasein A 40levelsintheformicacidextractableA levelsrespectively(Figure2,G).Interestingly,thenumberof ThioflavinSstained “ cored ” plaquesinthehippocampus ofmIL-4expressingmice(15.2%increase)didnotincreasesignificantlycomparedtocontrols(Figure3,A-C). WenextinvestigatedwhethertheincreaseinA wasdue tochangesinAPPexpression,APPprocessing,ApoElevels orphagolysosomaldysfunction.NeitherAPPexpression norCTF expressionorCTF levelswerealteredinmIL-4 expressingTgCRND8micecomparedtocontrols(Figure4, A-B).Weadditionallytestedforthelevelsofendogenous mouseprionproteinasthemutanthumanAPPtransgene isexpressedfrommouseprionpromoter[22].Nosignificantchangeinprionproteinlevelswasapparentbetween themIL-4expressingtransgenicmicebrainsandcontrol cohorts(Figure4,C-D).Additionally,nochangeinmouse endogenousAPPlevelsorCTF levelswereseenin 5montholdmIL-4expressingwildtypeB6/C3HlittermatesofTgCRND8miceinjectedinthecerebralventriclesondayP2(Figure4,E-F),suggestingthatmIL-4doesnot changeeithertheholo-APPlevelsorAPPprocessingor prionpromoterexpressioninvivo.Thoughinflammatory signalingcanmodulateapoElevelsthroughmodulationof Erks[23],nosignificantchangeinApoElevelswereseen inmIL-4injectedcomparedtocontroltransgenicAPP mice(Additionalfile2:FigureS2,A-B).AutophagiclysosomaldysfunctionmayleadtoincreasedaccumulationChakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page2of12 http://www.molecularneurodegeneration.com/content/7/1/36

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ofA [24].Sinceanti-inflammatorycytokines,forexample, IL-4andIL-13,havebeenshowntoinhibitautophagy[25], wetestedwhetherchangesinautophagicresponsemayaccountforincreasedA accrualinmIL-4expressingmice. NosignificantchangesintheautophagicmarkerLC3-I wereseenintransgenicTgCRND8miceinjectedwith AAV2/1-mIL-4(Additionalfile3:FigureS3,A,D),though anonsignificantloweringtrendwasobservedinthenontransgeniccohort(Additionalfile3:FigureS3,B,D).We wereunabletodetectLC3-IIinthelysates,makingit Figure1 AAV2/1mediatedexpressionofmIL-4inTgCRND8miceresultsinM2phenotype.A-D. rAAV2/1-mIL-4orrAAV2/1-EGFP(Control) wasinjectedintothehippocampusof4montholdTgCRND8miceandanalyzedafter6weeks.RepresentativeimagesofGFAPimmunoreactivity inparaffinembeddedwholebrainsections(A,C)andhighermagnificationofthehippocampus(B,D)isshown. ScaleBar, 600 m(A,C)and 25 m(B,D).( n =5-6/group). E-H. RepresentativeimagesofIba-1immunoreactivityinparaffinembeddedsectionsof5.5montholdTgCRND8 miceinjectedwithrAAV2/1mIL-4orrAAV2/1-EGFP(Control).Wholebrainsections(E,G)andthecorrespondinghippocampus(F,H)areshown. ScaleBar, 600 m(E,G)and25 m(F,H).( n =5-6/group). I .DensitometricanalysisofGFAPandIba-1immunostainingisshown.Thehippocampal regionwasselectedandAperio “ positivepixelcount ” programwasusedtomeasurepercentpositivitybyaveragingintensityofpositivestaining intheannotatedregion.( n =4/group; p > 0.05, t test).Datarepresentsmeansem. J-K. Representativeimmunoblot(J)anddensitometricanalysis ofnormalizedlevelsofGFAPandcd11b(K)obtainedfrom5.5montholdTgCRND8miceinjectedwithrAAV2/1mIL-4orrAAV2/1-EGFP(n=5/ group; p > 0.05, t test).Datarepresentsmeansem. L. Expressionofglialactivationmarkersandcytokinesweredeterminedin5.5monthold mIL-4expressingTgCRND8micecomparedtoEGFPexpressingage-matchedcontrolsusingrealtimeQ-PCR.Data,expressedasrelativelevelsof mRNAexpression,representsaveragedfoldchangevaluesobtainedfrommIL-4expressingmice,relativetoaveragedvaluesobtainedfromEGFP expressingmice.( n =4/group;* p < 0.05, t test).Datarepresentsmeansem. Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page3of12 http://www.molecularneurodegeneration.com/content/7/1/36

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difficultforustoinfereffectsofmIL-4onautophagicflux invivo.p62(orsequestosome-1),whichisacentralplayer inautophagywasfoundtobedecreasedby47%inmIL-4 expressingmice(Additionalfile3:FigureS3,C-D). Abasictenetoftheamyloidcascadehypothesisisthat A accumulationtriggerstheonsetandseverityofneurodegenerativepathology,includingtauhyperphosphorylation.ThoughoccasionalCP13immunoreactiveglial cellswerevisibleinbothmIL-4andcontrolmice,mIL-4 micedidnotleadtoappearanceofphosphorylatedCP13 immunoreactivetau(phosphoSer202/Thr205)inthe hippocampalneuronsinTgCRND8mice,(Additionalfile 4:FigureS4,A-B).Immunoblottingalsofailedtoshow anydetectablelevelsofCP13orMC6(phosphoSer235) orPHF1epitope(phosphoSer396/Ser404)inboth cohorts(datanotshown). TofurtherinvestigatethemechanismofmIL-4 inducedA accumulation,wetreatedprimarywildtype mouseneuroglialcultureswithrAAV2/1-mIL-4for 72 – 80hours.Analysisoftheconditionedmediaofthese cellsrevealedincreasedmIL-4protein( p < 0.01, t test) butnoincreasesinmIL-6(datanotshown)ormIFNproteincomparedtocontrolcultures(Figure5,D).In addition,primaryculturestransducedwithmIL-4 showedincreasedlevelsofnuclearphosphorylated STAT6comparedtomIFNoruntreatedcultures Figure2 IncreasedA depositioninAAV1-mIL-4expressingTgCRND8mice.A-D. 4montholdTgCRND8micewerestereotaxicallyinjected inthehippocampuswitheitherAAV1-mIL-4(C,D)orAAV1-EGFP(A,B)andsacrificedafter6weeks( n =5-6/group).Representativebrainsections stainedwith33.1.1antibody(panA 1 – 16)depictincreasedA depositioninmIL-4expressingmice(C,D)comparedtocontrols(A-B)inthe immediatevicinityoftheinjectionsite. ScaleBar, 150 m. E. A plaqueburdenanalysisshowsasignificantlyincreasedamyloiddepositionin mIL-4injectedmicecomparedtocontrolEGFPinjectedmice( n =5/group).(* p < 0.05, t test).Datarepresentsmeansem. F-G. Biochemical analysesofA 42andA 40levelsbyELISAshowsignificantlyincreasedSDS(F)andformicacid(G)extractableA levelsinmIL-4injectedmice comparedtocontrols( n =5/group).(* p < 0.05, t test).Datarepresentsmeansem. Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page4of12 http://www.molecularneurodegeneration.com/content/7/1/36

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(Figure5,A-C).QuantitativeRT-PCRanalysisconfirmed increasedmIL-4(4.5e3x)levelsaswellasincreased levelsofcd11b(6x),scavengerreceptorA(3.3x)and scavengerreceptorB1(15.6x)(Figure5,E).NosignificantchangesinmouseAPP,BACE1,orA degrading enzymes(IDEorNeprilysin)wereseen(Figure5,E). Sinceincreasedcd11bandscavengerreceptorsmayresultinalteredphagocyticpotentialofmIL-4expressing glia,weperformedphagocytosisassaysonmouseprimarygliaandastrocytestreatedwithmediumaloneor recombinantmIL-4.SinceA 40andA 42canhavedifferenteffectsonphagocytosis,weusedbothtotestout howmIL-4affectsastroglialphagocytois.Primarymouse gliaweretreatedwithrecombinantcytokinesorvehicle for10hours,andfreshmediaaddedbeforeincubation withfluorescentA fordifferenttimes(Figure6,Additionalfile5:FigureS5 – Additionalfile6:FigureS6). Theseexperimentswereperformedwithculturescontaining > 95%CD45andCd11bimmunopositiveglia (Additionalfile5:FigureS5,A-B).Initialmicroscopic examinationshowedadecreaseinthelevelsofinternalizedA 40inmIL-4treatedmicrogliafollowing15min, 30minor60minincubationwithA 40-555nm (Additionalfile6:FigureS6,A-I).Thiswasconfirmedby flowcytometricanalysiswhichshowedasignificantdecreaseininternalizedfluorescentA 40inmIL-4treated microgliaatthesethreedifferenttimepoints(15min: -19%;30min:-38%;60min:-19%)(Figure6,A; Additionalfile5:FigureS5,D-L).SincewehavepreviouslyshownthatmIL-6treatmentaugmentsmicroglial A phagocytosis[10],weincludedthisasaninternal controlforA 40internalizationintheflowcytometric assay(15min:+29%;30min:+13%;60min:+41%) (Figure6,A;Additionalfile5:FigureS5,J-L).ImmunoblottingofmicroglialcellsfollowingA 40phagocytosis alsoshowedthatmIL-4treatedgliatakeupA lessefficiently(Additionalfile5:FigureS5,M-N).Inorderto testwhetherfibrillizedA 42(fA 42)hasadifferenteffectonglialphagocytosis,weperformedphagocytosis assaysusingA 42fibrils(Figure6B).Flowanalysis showsthatneithermIL-6(15min:+25.7%;30min: +28%;60min:+8%)normIL-4(15min:+0.53%;30min: +8.1%;60min:+10.4%)significantlyaffectsfA 42 internalizationbymicroglia(Figure6B).SinceIL-4can potentiallyactivateastrocyticinternalizationofA ,we performedphagocytosiswithmurineastrocytecultures (Figure6,C-D).Overall,neonatalastrocyteswerevery inefficientininternalizingA 40asshownearlier[2,26]. FlowcytometricanalysisshowsthatmIL-4doesnotsignificantlyalterastrocyticA 40phagocytosis(15min: +39.5%;30min:+50%;60min:+12.2%)(Figure6,C) whereasmIL-6increasesastrocyticA 40internalization significantlyatalltimepointstested(15min:+83.1%; 30min:+58.3%;60min:+85.2%)(Figure6,C).Onthe otherhand,mIL-4(15min:-16.8%;30min:+3%)aswell asmIL-6(15min:-48.4%;30min:-26%)hasnegligible effectsonastrocytesphagocytosingfA 42atthetwo earliertimepointstested(Figure6,D).Onlyatthelast timepointtested,i.e.,following60minofincubationof fA 42,bothmIL-4(60min:+44.4%; p < 0.05,1way Anova)andmIL-6(60min:+45%)enhancesfA 42uptakebyprimaryastrocytes(Figure6,D).DiscussionWehavefoundthatoverexpressionofmIL-4inthe hippocampusofplaque-depositingAPPCRND8mice increasedA plaquepathology.Weextensivelyinvestigatedthelikelyfactorsthatcouldberesponsibleforthe effectsofmIL-4expressiononincreasedA burden invivo .APP,APPCTF,BACE,ApoEandA degrading enzymelevelsdidnotappeartobealtered.AsIL-4has beenreportedtoinhibitautophagy[27],weinvestigated LC3I/IIlevels,butthiswasnotinformative,aswecould notreliablydetectLC3II.Levelsofanotherkeyregulator ofautophagy,p62,weredecreasedbymIL-4.Asp62has pleiotropicfunctionsinadditiontoitsroleinautophagy [28]thesignificanceofthisfindingisunclear.Furthermoreifautophagicpathwayswerebeinginhibited,p62 wouldbeexpectedtoincrease,notdecrease.Giveni) thatthesedataindicatingthatmIL-4doesnotappearto Figure3 NosignificantchangeincoredplaquesseeninAAV1mIL-4expressingTgCRND8mice.A-B. Representativefluorescent imagesofThioflavinSstainedhippocampifrom5.5monthold TgCRND8miceinjectedwithrAAV2/1mIL-4(B)orrAAV2/1-EGFP (Control). ScaleBar, 200 m. C. Acomparisonofthetotalnumberof ThioflavinSstained “ cored ” plaquesfromtheforebrainof5.5month oldTgCRND8miceinjectedwithrAAV2/1mIL-4orrAAV2/1-EGFPis depicted.Datarepresentsmeansem.( n =5/group;p=0.0542, t test). Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page5of12 http://www.molecularneurodegeneration.com/content/7/1/36

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beaffectingAPPprocessingandisnotalteringmajorA chaperonesordegradingenzymesandii)thatweand othershavepreviouslylinkedproinflammatoryactivation ofmicrogliatoreducedplaqueburdensandenhanced microglialscavengingofA [10,11,13,14,17,29,30],we hypothesizethatmIL-4increasesA burdenviareductionsinglialscavengingofA .WeexploredthishypothesisthroughA internalizationstudiesinprimary microgliaandastrocytescultures.Thesestudiesdoshow thatmIL-4abrogatessolubleA 40uptakebymicroglia withoutaffectingaggregatedfA 42phagocytosis.Additionally,mIL-4doesnotaffecttheuptakeofsolubleor aggregatedA byastrocytes,thoughonlongerincubation,therewasanincreasingtrendinA ingestionby theastrocytes.GiventhelownumberofastrocytesinternalizingA andthefactthatastrocyteculturesmay Figure4 mIL-4doesnotaffecthumanAPPtransgeneexpressionorCTFproduction.A-B. RepresentativeantiCT20immunoblotshowing nosignificantchangesinAPP,CTF andCTF levelsinAAV1-mIL-4expressingTgCRND8comparedtoage-matchedcontrols(A).Intensity analysis(meansem)ofantiCT20immunoreactiveAPPandCTF levelswasnormalizedto -actininTgCRND8micecohort(B).( n =5/group; p > 0.05, t test). C-D. RepresentativeimmunoblotshowingnosignificantchangesinprionproteinlevelsinAAV1-mIL-4TgCRND8comparedto age-matchedcontrols(C).Intensityanalysisofimmunoreactiveprionproteinlevels(meansem)wasnormalizedto -actin(D).( n =5/group; p > 0.05, t test). E-F. RepresentativeantiCT20immunoblotshowingnosignificantchangesinAPPandCTF in5montholdAAV1-mIL-4 expressingwildtypeB6/C3Hmicecomparedtoage-matchedcontrols(E).IntensityanalysisofantiCT20immunoreactiveAPPandCTF levels (meansem)wasnormalizedto -actininwildtypemicecohort(F).( n =5/group; p > 0.05, t test). Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page6of12 http://www.molecularneurodegeneration.com/content/7/1/36

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contain~10-20%microglia,itisdifficulttoconclude whetherastrocytesbythemselveshaveacriticalrolein A phagocytosis. MicrogliacanscavengeA viareceptor-mediated phagocytosisaswellasmacropinocytosis[26].Invivo imagingtechniqueshavedemonstratedthatmicroglia Figure5 (Seelegendonnextpage.) Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page7of12 http://www.molecularneurodegeneration.com/content/7/1/36

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canhomeontonewlyappearingplaquesandinternalize A [31].MicroglialinteractionwithA occursthrough severalcellsurfacereceptors,forexample,SR-A,SR-B, integrinsandToll-likereceptors[15,32-34].Previous studieshavenotedthatinflammatorycytokinesdecrease scavengerreceptorslevelsinmicroglialcelllinesand inhibitA uptake[35].WefindthatmIL-4expression leadstoincreasedSRsinmixedneuroglialcultures,but thisappearstobelinkedtomodestinhibitoryeffectson soluble,butnotaggregated,A uptakebymicroglia. Therefore,itispossiblethatintheabsenceofinflammatorymediators,thesecellsarenotcompetentforA (Seefigureonpreviouspage.) Figure5 CharacterizationofrAAV2/1-mIL-4treatedprimarywildtypemouseneuroglialculture.A-D. rAAV2/1-mIL-4orrAAV2/1-mIFN(2x108viralgenomes)wasusedtotransduceprimarymouseneuroglialculturesfor60hrsinchamberslides.Upregulationofphosphorylated STAT6(568nm;redfluorescence)couldbeseeninmIL-4expressingcultures(C)butnotinmIFNexpressingcultures(B)oruntreatedculture containingnoviruses(A).MAP2(488nm;green)andDAPI(350nm,blue)wereusedtodepictneuronalprocessesandnucleusrespectively.mIL4andmIFNexpressioninthesecultureswasverifiedbyELISAfrommediacollectedfromrespectivecultures(D).Magnification,400x.(* p < 0.05, t test). E. ExpressionofmouseendogenousAPP,BACE1,IDEandneprilysinlevelsareunchangedinrAAV2/1-mIL-4expressingprimarymixed neuroglialculturescomparedtountreatedcontrolculturesusingrealtimeQ-PCR.mIL-4expressionaugmentsscavengerreceptorsandcd11b levels.Data,expressedasrelativelevelsofmRNAexpression,representsaveragedfoldchangevaluesobtainedfrommIL-4expressingmice, relativetoaveragedvaluesobtainedfromEGFPexpressingmice.DataformIL-4RNAisplottedontheleftx-axiswhiletherestofthedataare plottedontherightx-axis.Dataisrepresentativeoftwoindependentexperiments( t test,* p < 0.05).Datarepresentsmeansem. Figure6 DifferentialeffectofcytokinesonA 40andfA 42uptakebyprimarymousegliaandastrocytes.A-B. mIL-4treatment decreasesmicroglialuptakeofA 40(A)butdoesnotaffectfA 42uptake(B).mIL-6increasedmicroglialA 40uptake(A)butdoesnot significantlyalterfA 42uptake(B).Primarymousegliaweretreatedwithrecombinantcytokinesfor10hrsandincubatedwithA 40-Hilyte555or A 42-Hilyte555for15min,30minand60min.TrypsinizedcellswerecountedusingAccuri6flowcytometer.Unstainedcellsandlabeled additiveswereexcludedbygatingtoyieldthepercentageoffluorescentcellsinthemix.Insetdepictscd11b/DAPIstainedglia(A)Resultsare representativeofthreeindependentexperiments.(* p < 0.05,OnewayAnovawithTukey ’ sposttest).Datarepresentsmeansem. C-D. mIL-4 treatmentdoesnotaffectastrocyticuptakeofA 40(C)butincreasesfA 42uptakeafter60minincubation(D).mIL-6consistentlyincreases astrocyticA 40uptake(C)andonlyenhancesfA 42uptakeafter60minincubation(D).Primarymouseastrocytesweretreatedwith recombinantcytokinesfor10hrsandincubatedinA 40-Hilyte555orA 42-Hilyte555for15min,30minand60min.Trypsinizedcellswere countedusingAccuri6flowcytometer.Unstainedcellsandlabeledadditiveswereexcludedbygatingtoyieldthepercentageoffluorescentcells. InsetdepictsGFAP/DAPIlabeledastrocyte(C).Resultsarerepresentativeoftwoindependentexperiments.(* p < 0.05,OnewayAnovawith Tukey ’ sposttest).Datarepresentsmeansem. Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page8of12 http://www.molecularneurodegeneration.com/content/7/1/36

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internalizationanddegradation,eveniftheyexpressthe receptors[36,37].Indeed,microgliawerefoundtorequirestimulationwithcytokinesoropsoninsforinternalizinganddegradingA efficientlyinamyloid vaccinationparadigms[38].Microglialpopulationsfrom humanADbrainsdisplayanIL-4/IL-13inducedalternateactivationprofile[39]aswellasincreasedIL-1 suggestingthatanimbalancebetweentheinflammatory “ classical ” andanti-inflammatoryalternateactivation profilesmayresultinadysfunctionalglialresponseand “ failed ” phagocytosis.Althoughspeculative,thesestudies mayhaverelevancetohumantherapy.Manystudies showanassociationofnonsteroidalanti-inflammatory drugs(NSAIDS)usewithreductionofdementiarisk [40].However,intheAdultChangesinThought(ACT) study,therewasanincreasedincidenceofall-causedementiaandADdementiainindividualswithheavynonselectiveNSAIDuse,andthiswasassociatedwithan increaseinneuriticplaquesatautopsy[41].Ourdata couldprovideanexplanationforthisassociation. Itisgenerallyacceptedthatanalteredinnateimmune responseisaninherentfeatureofADandmost,ifnot all,degenerative,CNSproteinopathies[42].Thougha largebodyofliteratureonthissubjectreferstothis alteredimmuneresponseas “ pathogenic ” or “ harmful ” inmanycasesthisrelationshipisassumedandhasnot beenformallyproven.Increasedpro-inflammatorycytokineslevels(TNF ,Interleukin-6,Interleukin-1 ),combinedwithdecreasedlevelsofanti-inflammatory cytokines(Interleukin-10),havebeencorrelatedwith cognitivedeficitssuggestingthatearlyinflammatory changesmaybedetrimental[43] Furthermoreproinflammatorystimulihavebeenlinkedtoovertneuronal degeneration[44].Althoughproinflammatoryfactors mayhaveadetrimentalroleonneuronalfunction,primingofCNSmicrogliaandpossiblyastrocytesbyaccumulatingA ,maybeanessentialinnatehostresponsethat triggersglialphagocytosisactivityandclearsA deposits.Indeed,weandothershavepreviouslyshownthat activationoftheinnateimmunesignalingpathwayscan attenuateA accumulationinAPPtransgenicmiceand alterthediseaseprocess[10-14,17,29,30]. mIL-4hasbeenshowntoenhanceA phagocytosisin primaryrodentglialculturesinvitro[45,46].Based largelyonsuchevidence,promotionofaglialM2 phenotypehasbeenproposedtobebeneficialinpreclinicalADmodelsandthisconceptwasdirectlysupported byonerecentstudy,usingasimilarAAVmediatedIL-4 expressionparadigmasusedinthispresentstudy[4].In thisstudyIL-4inducedaTh2likeglialphenotype,but incontrasttoourresults,Kiyotaetal,showedthatIL-4 expressioninthehippocampusledtomitigationofA pathologyandimprovedbehaviorinAPP/PS1mice[4]. Inherentdifferencesintransgenicmiceaswellas experimentalparadigmsmayunderliethedisparate observations.Itispossiblethatpresenilinsmayregulate cytokinesecretion[47],andmutantPS1especiallymay conferalteredsensitivitytoimmunechallengesinthe residentglialcells[48].Additionally,thetwostudies weredistinctwithrespecttoinitiationoftreatment. Kiyotaandcolleaguesperformedaprimaryprevention studyinwhichIL-4expressionwasinitiatedinthepredepositionphase,whereasIL-4expressioninourstudies wasinitiatedinmicewithmodestlevelspre-existingplaques.Inanycase,additionalstudiesofbothIL-4and otheranti-inflammatoryfactorswillberequiredtoaddressthisdiscrepancyandthegeneralizabilityofthis finding.ConclusionInsummary,wedemonstratethatfocaloverexpression ofmIL-4inAPPtransgenicmousebrainsleadstoexacerbatedA plaquepathology.Astheseresultsareoppositeofeffectsobservedwithproinflammatory cytokines,wewouldsuggestthattheunderlyingmechanismappearstobeatleastinpartafailureofgliato successfullyclearA .Ourdatapointstothecomplexrelationshipbetweenmicroglialphenotypeandthefinal functionaloutcome,necessitatingamorecautiousand thoroughexaminationofpotentialanti-inflammatory therapiesforAD.MethodsMice. Allanimalhusbandryproceduresperformedwere approvedbytheInstitutionalAnimalCareandUse Committee. AAV1preparationandinjection .AAV1virusesexpressingmIL-4orEGFP,underthecontrolofthecytomegalovirusenhancer/chicken actinpromoterwere generatedasdescribedpreviously[19].Forstereotaxic injections,TgCRND8mice(n=6/group)wereanesthetizedwith1.5%isofluranein1%oxygenandsecuredinto aKopfapparatus(Model900SmallAnimalStereotaxic Instrument DavidKopfInstruments).Thecoordinates forinjectionwere 1.7caudal,-1.6lateraland 1.2ventralfromthebregma.AUMP2MicrosyringeInjector andMicro4Controller(WorldPrecisionInstruments, USA)wasusedtoinject2 l(1010viralgenomes)of virusataconstantrateovera10minuteperiod.After allowinganadditional10minutes,theneedlewasraised andthescalpincisionwasclosedaseptically.NontransgenicmiceusedinthestudywereinjectedwithrAAV2/ 1-mIL-4ondayP2intothecerebralventriclesandaged till5monthsofage. Quantitativereal-timePCR. TotalRNAfrommice hippocampusorprimarywildtypemouseneuroglialcultureswasisolatedusingtheRNaqueouskit(Ambion) andreversetranscribedusingSuperscriptIII(Invitrogen).Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page9of12 http://www.molecularneurodegeneration.com/content/7/1/36

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TheQ-PCR(initialdenaturationcycleof95C/10min, followedby40amplificationcyclesof95C/15sand 60C/1min)wasperformedwithABIPrism7900Real TimePCRSystem(AppliedBiosystems)usingSYBR Greentodetecttheamplificationproducts.Relative quantificationofmRNAexpressionwascalculatedbythe CTmethoddescribedbythemanufacturer(ABIPrism 7700SequenceDetectionSystem,UserBulletin#2)after adjustingthelevelstothecorrespondinginternalactin controlforeachsample.Primersandprobeswere designedfollowingRocheUniversalProbeLibrary sequences(Hoffmann-LaRoche,Germany). Preparationofbrainhomogenateforimmunoblotting andA ELISAassay. Brainwerecoronallydissected 1mmanteriorandposteriortothepointofinjection andusedforsubsequentanalysis.Thus,thesamplesfor immunoblottingwereobtainedbydissectingthehippocampusandoverlyingcortexandthalamusofinjected brains.Proteinsamples(RIPAsolubleor2%SDSsoluble)separatedonBis-Tris12%XTgels(Bio-Rad,USA) wereprobedwiththeantibodyCT20(anti-APP C-terminal20aminoacid;T.E.Golde;1:1000);82E1 (IBL,1:500),Prion(Abcam;1:1000);LC3(MBL;1:500; CellSignaling,1:250;Novus,1:500);p62(CellSignaling, 1:250),CP13(P.Davies;1:500)andanti -actin (Sigma,1:1000).Relativebandintensitywasquantified usingImageJsoftware(NIH). A levelsweredeterminedbiochemicallyusinghuman A end-specificsandwichELISAaspreviouslydescribed [19]onsamplessequentiallyextractedwithRIPAbuffer, 2%SDSand70%FormicAcid.AllELISAresultswere analyzedusingSoftMaxProsoftware(Molecular Device). Immunohistochemicalimagingandimageprocessing. Brainswerecoronallydissectedatthepointofinjection foranalysis.Immunohistochemicalstainingwasdone usingpanA antibody33.1.1(1:1500,T.Golde),82E1 (1:500,IBL),Iba-1(1:1000;Wako),GFAP(1:500;Chemicon),pSTAT6(1:500,CellSignaling)andMAP2(1:1000; Chemicon).1%ThioflavinS(Sigma)stainingwasdone onparaffinembeddedbrainsectionsusingestablished protocols. ImmunohistochemicallyandfluorescentstainedsectionswerecapturedusingtheAperioScanscopeXTor FLimagescannerandanalyzedusingeitherAperiopositivepixelcountorImageJprogram.Brightnessandcontrastalterationswereappliedidenticallyoncaptured imagesusingAdobePhotoshopCS3. QuantificationofA depositionandgliosis. Paraformaldehydefixedparaffinembeddedbraintissuesections wereimmunostainedwith33.1.1antibody.A plaque burdenandintensityofastrogliosisstainingwascalculatedusingthePositivePixelCountprogram(Aperio). Atleastthreesectionspersample,30 mapart,were averagedbyablindedobservertocalculateplaqueburden.ForThioflavinSquantitation,onesectionpersamplewasusedbyablindedobservertomanuallycount theplaquesusingAdobePhotoshopCS5. Primarymurinecultureandmicroglia/astrocyte phagocytosisassay. Primarymicroglia,astrocytesor neuronalcultures(mixedwithastrocytes)wereobtained fromcerebralcorticesofwildtypeneonatemiceas describedpreviously[38].Glialandastrocyticcultures typicallywere > 90%cd11b(cd11b-APC;1:200,BDBiosciences)orGFAP(Sigma,1:500)immunopositiverespectively.Forphagocytosisassays,microgliaor astrocytes,pre-treatedwithmIL-4(R&DSystems,USA; 5ng/mlfor10hours)ormIL-6(R&DSystems,USA; 10ng/mlfor10hours)wereincubatedwith0.5 M Hilyte555-A 40(Anaspec,USA)orfibrillarHilyte555A 42(Anaspec,USA).RecombinantfluorescentA was resuspendedinDMSOto1mg/mlanddilutedin DMEMmediumto0.5 Mbeforeadditiontocells. Hilyte555-A 42wasfibrillizedat370Cfor6hoursin PBS.Cellswereanalyzedatthreetimepointsfollowing additionofA tothecultureat370C:15min,30min and1hr.Cellswerewashed,gentlytrypsinisedtoremovecellsurfaceassociatedfluorescententities,fixedin paraformaldehydeandmountedinDAPIcontaining mediumforvisualization.Fluorescenceintensityofindividualcellsfromatleast5ormorefieldsofviewsper samplewerecalculatedfromImageJandaveraged.For FACSanalysis,followingwashes,cellswerecollectedby trypsinizationandresuspendedinFACSbuffercontainingBSAandscanned.Scanswerecollectedusing Accuri6andanalyzedwithFCSExpress4FlowResearch (BDBiosciences).Neuronalculturesweretransduced withAAV(mIL-4ormIFN;108viralgenomes)onday 7for60hoursandfixedwithparaformaldehydeforsubsequentimmunocytometry.CytokineELISAwasperformedusingBDBioscienceOptiEIAreagents.StatisticalanalysisOne-wayAnova(withTukey ’ spost-hoctest)ortwotailedStudent'sttestwasusedforstatisticalcomparison (SigmaStat3.0version).Graphicalanalysesweredone usingPrism4(GraphPadSoftware)andfinalimagescreatedusingPhotoshopCS2(Adobe).AdditionalfilesAdditionalfile1: FigureS1. rAAV2/1-EGFPexpressioninTgCRND8 micehippocampus. A-I. Representativeimageobtainedfrommice stereotacticallyinjectedwithAAV2/1-EGFPinthehippocampus.4month oldTgCRND8wereinjectedintothehippocampusandanalyzedafter 6weeks.RepresentativeimagesofEGFPimmunoreactivityonparaffin embeddedwholebrainsection(A),cortex(B),hippocampalCAneurons (C-E),midbrain,(F),cerebellum(G),thalamus(H)andolfactorybulb(I)are shown.RepresentativehippocampusfromuninjectedmiceisshownasChakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page10of12 http://www.molecularneurodegeneration.com/content/7/1/36

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control(A,inset).I-V,cortexlayersItoV;CC,corpuscallosum;Sp,Septum; Th,thalamus. ScaleBar, 600 m(A)and85 m(B-I). Additionalfile2: FigureS2. mIL-4expressiondoesnotalterApoE levels. A-B. NosignificantchangeinApoElevelswasseeninthe hippocampusofmIL-4expressing5.5montholdtransgenicCRND8mice orage-matchedcontrolcohorts(A).IntensityanalysisofApoElevelswas normalizedto -actin(B).( n =5-6/group; t test, p > 0.05). Additionalfile3: FigureS3. mIL-4expressiondoesnotalterlevelsof theautophagicmarkerLC3butdecreasesp62proteinlevels. A-B. No significantchangeintheautophagicmarker(microtubule-associated proteinlightchain3,LC3)wasseeninmIL-4expressing5.5monthold transgenicCRND8mice(TG,A)ornontransgenic5monthold nontransgeniccohorts(NTG,B). C. p62proteinlevelsdecreaseinmIL-4 expressing5.5montholdtransgenicCRND8mice(TG). D. Intensity analysisofantiLC3andp62immunoreactivebandafternormalizationto -actinimmunoreactivity.( n =4-6/group).(* p < 0.05; t test). Additionalfile4: FigureS4. mIL-4expressiondoesnotalterlevelsof phosphorylatedtau.Nosignificantchangeinphosphorylatedtau(using CP13antibody)wasseeninthehippocampusofmIL-4expressing 5.5montholdtransgenicCRND8mice( B )orage-matchedcontrol cohorts( A ).OccasionalCP13immunoreactivitywasseeninglialcellsin thepyramidallayerofthehippocampus(inset). ScaleBar, 150 m(A,B) and25 m(inset).( n =6/group). Additionalfile5: FigureS5. Analysisofprimarymousemicroglial culturefollowingA 40phagocytosis. A-B. Representativeflowcytometric analysisofunstained(A)andCD45-FITCstainedmousemicroglia(B).Glial populationsusedforsubsequentphagocytosisexperimentswere > 95% positiveforbothCD45andcd11b.InsetinAdepictsarepresentative unstainedprimarymouseglialculture.Magnification,200x. C-L. Primary mousegliawerestimulatedwithmediumalone(Control;D-F)or recombinantmIL-4(5ng/ml;G-I)ormIL-6(10ng/ml;J-L).After10hrsof stimulation,cellswereincubatedwithfluorescentA 40for15min(D,G, J),30min(E,H,K)or60min(F,I,L).Cellsweretrypsinizedforthe analysisofinternalizedfluorescentA 40(D-L)byFACS.Unstainedcells andadditiveshavebeengatedforexclusion(C).Representativedata fromthreeindependentexperimentshavebeenshown.Quantifieddata hasbeenplottedinFigure6A. M-N. Immunoblotanalysistodetect presenceofA inprimaryglialcellsatdifferenttimepointsfollowing A 40phagocytosisinthepresenceorabsenceofmIL-4(M).Intensity analysisof82E1immunoreactiveA monomerafternormalizationto actinimmunoreactivityhasbeenshown(N).Representativedatafrom twoindependentexperimentshavebeenshown. Additionalfile6: FigureS6. MicroscopicanalysisofA 40phagocytosis bymIL-4treatedmouseprimaryglia.Primarymousegliaweretreated with5ng/mlmIL-4for10hrsandincubatedwithfluorescentA 40for 0min(A-B),15min(C-D),30min(E-F)or60min(G-H).Followinggentle trypsinization,cellswerefixed,stainedwiththenuclearstainDAPIand visualized.Inset(C)depictsmIL-6treatedgliaafter15minincubation withA 40.Quantitationofaveragefluorescentcountisdepicted(I). Magnification,400x.(* p < 0.05; t test). Abbreviations AAV:Adeno-associatedvirus;ACT:AdultChangesinThought; AD:Alzheimer ’ sdisease;A :Amyloid ;APP:Amyloid precursorprotein; ApoE:Apolipoprotein E 4;BACE: -siteAPPcleavingenzyme;CTF:C-terminal fragment;EGFP:Enhancedgreenfluorescentprotein;Erk:Extracellularsignalregulatedkinases;FACS:Fluorescenceactivatedcellsorting;GFAP:Glial fibrillaryacidicprotein;IL-4:Interleukin-4;IFN:Interferon; IACUC:Institutionalanimalcareandusecommittee;Iba-1:Ionizedcalcium bindingadaptorprotein1;LC3:Microtubule-associatedproteinlightchain3; NSAIDS:Nonsteroidalanti-inflammatorydrugs;SR:Scavengerreceptor; STAT:SignalTransducerandActivatorofTranscription;TNF :Tumornecrosis factor Competinginterests Theauthorsdeclarenocompetinginterests. Authors ’ contributions PCconductedtheexperimentsandwrotethemanuscript;LTperformed immunostaining,primarymouseneuroglialculture,phagocytosisassaysand flowcytometry;ABperformedmousebrainQ-PCRandamyloidburden analysis;CC-DpreparedrecombinantAAVandQ-PCRofneuroglialculture; PDprovidedhelpfuldiscussion;TEGcoordinatedtheresearch,supervisedthe projectandassistedinmanuscriptpreparation.Allauthorshavereadand approvedthefinalmanuscript. Acknowledgements ThisworkwassupportedbyMayoClinic(TEG),NationalInstitutesofHealth/ NationalInstituteonAgingGrants(RO1AG18454,RO1AG29886,P01AG25531; TEG)andAmericanHealthAssistanceFoundationGrantA2009061(PD). Authordetails1CenterforTranslationalResearchinNeurodegenerativeDisease,Department ofNeuroscience,UniversityofFlorida,1275CenterDrive,Gainesville,POBox #100159,FL32610,USA.2DepartmentofNeuroscience,MayoClinicCollege ofMedicine,4500SanPabloRdS,Jacksonville,FL32224,USA. 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Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Chakrabarty etal.MolecularNeurodegeneration 2012, 7 :36Page12of12 http://www.molecularneurodegeneration.com/content/7/1/36