Fibrillar Amyloid-? Peptides Activate Microglia via TLR2:
Implications for Alzheimer’s Disease1
Malabendu Jana, Carlos A. Palencia, and Kalipada Pahan2
Microglial activation is an important pathological component in brains of patients with Alzheimer’s disease (AD), and fibrillar
amyloid-? (A?) peptides play an important role in microglial activation in AD. However, mechanisms by which A? peptides
induce the activation of microglia are poorly understood. The present study underlines the importance of TLR2 in mediating A?
peptide-induced activation of microglia. Fibrillar A?1–42 peptides induced the expression of inducible NO synthase, proinflam-
matory cytokines (TNF-?, IL-1?, and IL-6), and integrin markers (CD11b, CD11c, and CD68) in mouse primary microglia and
BV-2 microglial cells. However, either antisense knockdown of TLR2 or functional blocking Abs against TLR2 suppressed
A?1–42-induced expression of proinflammatory molecules and integrin markers in microglia. A?1–42 peptides were also unable
to induce the expression of proinflammatory molecules and increase the expression of CD11b in microglia isolated from TLR2?/?
mice. Finally, the inability of A?1–42 peptides to induce the expression of inducible NO synthase and to stimulate the expression
of CD11b in vivo in the cortex of TLR2?/?mice highlights the importance of TLR2 in A?-induced microglial activation. In
addition, ligation of TLR2 alone was also sufficient to induce microglial activation. Consistent to the importance of MyD88 in
mediating the function of various TLRs, antisense knockdown of MyD88 also inhibited A?1–42 peptide-induced expression of
proinflammatory molecules. Taken together, these studies delineate a novel role of TLR2 signaling pathway in mediating fibrillar
A? peptide-induced activation of microglia. The Journal of Immunology, 2008, 181: 7254–7262.
by the presence of both neurofibrillary tangles and neuritic plaques
composed of aggregates of amyloid-? (A?) protein, a 40–43 aa
proteolytic fragment derived from the amyloid precursor protein
(1, 2). The importance of A? in AD has been shown by means of
several transgenic animal studies. The overexpression of mutant
amyloid precursor protein in mice results in senile plaques forma-
tion and synapse loss and correlative memory deficits, as well as
behavioral and pathological abnormalities similar to those found in
AD patients (1, 2). Although deposition of A? peptides is one of
the primary causes of neuronal loss in AD (2, 3), the mechanism
by which A? causes neuronal loss has been poorly characterized.
Microglia are considered as CNS-resident professional macro-
phages and sensor cells that respond to many pathological events.
Localized activation of microglia has been implicated in the patho-
genesis of a variety of neurodegenerative diseases, including AD,
Parkinson’s disease, Creutzfeld-Jacob disease, HIV-associated de-
mentia, stroke, and multiple sclerosis. During activation, microglia
lzheimer’s disease (AD)3is a neurodegenerative disor-
der resulting in progressive neuronal death and memory
loss. Neuropathologically, the disease is characterized
are capable of releasing various potentially cytotoxic molecules
such as NO, oxygen radicals, proteases, adhesion molecules, and
proinflammatory cytokines such as TNF-?, IL-1?, LT-?, and IL-6
(4–8). It is believed that excessive production of these neurotoxic
proinflammatory molecules plays an important role in enhancing
the degenerative process in the inflamed CNS of AD patients.
Therefore, understanding mechanisms that regulate microglial ac-
tivation is an important area of investigation that may enhance the
possibility of finding a primary or an adjunct therapeutic approach
against incurable neurodegenerative disorders.
TLR, the mammalian homologs of the Drosophila Toll protein,
serves as an important link between innate and adaptive immunity
(9, 10). TLRs respond mainly to bacteria, bacterial products, virus
and flagellin by transmitting a ligand-induced transmembrane sig-
nal that induces the expression of various cytokines such as
TNF-?, IL-1, IL-6, and IL-12 for host responses. At least 11 dif-
ferent TLRs have been described to date, which display distinct
ligand specificities (11–16). Although all the major CNS cell types
express TLRs, microglia are the only cells in the CNS that express
almost all the TLRs known to date. However, theoretically CNS
microglia should not come in contact to bacterial products barring
couple of pathological conditions such as meningitis and brain
abscess, suggesting that TLRs might be involved in pathogen-in-
dependent brain pathologies as well.
We wondered whether TLRs were playing any role in A?-in-
duced microglial activation. In this study, we report that fibrillar
A?1–42 increased the expression of both proinflammatory mole-
cules and TLR2 in microglia. However, antisense knockdown of
TLR2 suppressed fibrillar A?1–42-induced expression of proin-
flammatory molecules and integrin markers in microglia. Further-
more, microglia derived from TLR2 knockout mice were not ac-
tivated by fibrillar A?1–42. These in vitro data were substantiated
by in vivo findings in which intracortical injection of fibrillar
A?1–42 in TLR2 knockout mice did not exhibit any microglial
Department of Neurological Sciences, Rush University Medical Center, Chicago, IL
Received for publication May 10, 2007. Accepted for publication September
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by Grant IIRG-07-58684 from Alzheimer’s Association
and Grants NS39940 and NS48923 from the National Institutes of Health.
2Address correspondence and reprint requests to Dr. Kalipada Pahan, Department of
Neurological Sciences, Rush University Medical Center, 1735 West Harrison Street,
Suite 320, Chicago, IL 60612. E-mail address: Kalipada_Pahan@rush.edu
3Abbreviations used in this paper: AD, Alzheimer’s disease; iNOS, inducible NO
synthase; A?, amyloid-?; LTA, lipoteichoic acid; poly(I-C), polyinosinic-polycyti-
dylic acid; ASO, antisense oligonucleotide; ScO, scrambled oligonucleotide.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
Materials and Methods
FBS and DMEM/F-12 were obtained from Mediatech. LPS (Escherichia
coli) and polyinosinic-polycytidylic acid (poly(I-C)) were purchased from
Sigma-Aldrich. Human A?1–42 and A?42-1 reverse peptides were ob-
tained from Bachem Bioscience. Lipoteichoic acid (LTA) from Bacillus
subtilis (TLR2 ligand) and FSL1 (follistatin-like 1 ligand for TLR2/6) have
been obtained from Invivogen. Anti-mouse TLR2 Abs were obtained from
eBioscience. Abs against mouse CD11b and inducible NO synthase (iNOS)
were purchased from Calbiochem. TLR2?/?mice and littermate controls
were purchased from Jackson ImmunoResearch Laboratories. Phosphoro-
thioate-labeled antisense and scrambled oligodeoxynucleotides were syn-
thesized in the DNA-synthesizing facility of Invitrogen.
The following antisense oligonucleotide (ASO) and scrambled oligonu-
cleotide (ScO) were used to target MyD88 and different TLRs genes:
MyD88 (ASO) 5?-GGC CGC CAC GGG CGT CCG AG-3?, (ScO) 5?-
GGA CCC CGA GGG CCG CGC TG-3?; TLR1 (ASO) 5?-GGT AGG
TCC TTG GGC ACT CTG-3?, (ScO) 5?-GGC TCC TTT AGG GCC ATG
GTG-3?; TLR2 (ASO) 5?-CTG GAG CGG CCA TCA CAC ACC-3?,
(ScO) 5?-CAT CGC ACG CAG CCGAGC CAT-3?; TLR3 (ASO) 5?-GGC
TGC AGT CAG CTA CGT TG-3?, (ScO) 5?-TGC AGC TAG TGC TAG
GCG TC-3?; TLR4 (ASO) 5?-GCC AGG AGC CAG GGA GGC A-3?,
(ScO) 5?-ACG GCG ACG GCA AGC GGA G-3?; TLR6 (ASO) 5?-GCA
GAG GCT ATC CCA GAG GG-3?, (ScO) 5?-GCG GAA TGC CCG GAA
CGT AG-3?; TLR7 (ASO) 5?-GCA GTC CAC GAT CAC ATG G-3?,
(ScO) 5?-ATC GGA TCC GAC TGA CCA G-3?; and TLR9 (ASO) 5?-
GGA GGG ACA AGG GGT GCA G-3?, (ScO) 5?-AGG GCG GGA TGG
AGC GAG A-3?.
Preparation of fibrillar A?
Fibrillar A?1–42 (Bachem Bioscience) was prepared by incubating
freshly solubilized peptides at 50 ?M in sterile distilled water at 37°C
for 5 days (17).
Isolation of mouse microglia
Primary microglia were isolated from mixed glial cultures according to the
procedure of Giulian and Baker (18). Briefly, on day 7–9, the mixed glial
cultures were washed three times with DMEM/F-12 and subjected to shak-
ing at 240 rpm for 2 h at 37°C on a rotary shaker. The floating cells were
washed and seeded on to plastic tissue culture flasks and incubated at 37°C
for 2 h. The attached cells were removed by trypsinization and seeded onto
new plates for further studies. The 90–95% of this preparation was found
to be positive for Mac-1 surface Ag (19, 20). Mouse BV-2 microglial cells,
a gift from V. Bocchini (University of Perugia, Perugia, Italy) were also
maintained and induced as indicated.
Semiquantitative RT-PCR analysis
Total RNA was isolated from mouse BV2 microglia and mouse primary
microglial cells using RNA-Easy Qiagen kit following the manufacturer’s
protocol. To remove any contaminating genomic DNA, total RNA was
digested with DNase. Semiquantitative RT-PCR was conducted as de-
scribed earlier (16) using oligo(dT)12–18as primer and MMLV reverse
transcriptase (Clontech Laboratories) in a 20-?l reaction mixture. The re-
sulting cDNA was appropriately diluted, and diluted cDNA was amplified
using Titanium Taq polymerase and the following primers: iNOS (sense)
5?-CAG CTC CTC ACT GGG ACA GCA CAG A-3?, (antisense) 5?-CTT
CCA GCC TGG CCA GAT GTT CCT C-3?; IL-1? (sense) 5?-ATG GCA
ACT GTT CCT GAA CTC AAC T-3?, (antisense) 5?-CAG GAC AGG
TAT AGA TTC TTT CCT TT-3?; TNF-? (sense) 5?-TTC TGT CTA CTG
AAC TTC GGG GTG ATC GGT CC-3?, (antisense) 5?-GTATGA GAT
AGC AAA TCG GCT GAC GGT GTG GG-3?; IL-6 (sense) 5?-CGT CCC
CTG GCA TTC CTA GTG GTG-3?, (antisense) 5?-AAG GGG TGA TCC
AGG CGT GAC ATC-3?; IL-15 (sense) 5?-GGG CTG TGT CAG TGT
AGG TCT CCC T-3?, (antisense) 5?-CCA GCT CCT CAC ATT CCT TGC
AGC C-3?; CD11b (sense) 5?-CAG ATC AAC AAT GTG ACC GTA
TGG G-3?, (antisense) 5?-CAT CAT GTC CTT GTA CTG CCG CTT
G-3?; MyD88 (sense) 5?-GCT GCT GGC CTT GTT AGA CCG TGA
G-3?, (antisense) 5?-GAC GTC ACG GTC GGA CAC ACA CAA C-3?;
TLR1 (sense) 5?-GGA CCT ACC CTTT GCA AAC AA-3?, (antisense)
5?-GGT GGC ACA AGA TCA CCT TT-3?; TLR2 (sense) 5?-TGC TTT
CCT GCT GGA GAT TT-3?, (antisense) 5?-TGT AAC GCA ACA GCT
TCA GG-3?; TLR3 (sense) 5?-TTG TCT TCT GCA CGA ACC TG-3?,
(antisense) 5?-GGC AAC GCA AGG ATT TTA TT-3?; TLR4 (sense) 5?-
ACC TGG CTG GTT TAC ACG TC-3?, (antisense) 5?-CTG CCA GAG
ACA TTG CAG AA-3?; TLR5 (sense) 5?-GAG CTC AAT GGG GGA
CCA GAA CAC-3?, (antisense) 5?-CGG CAG TAC TGA CAC TTG TTG
CGG-3?; TLR6 (sense) 5?-CCA AGA ACA AAA GCCC CTG AG-3?,
(antisense) 5?-TGT TTT GCA ACC GAT TGT GT-3?; TLR7 (sense) 5?-
GGA AAT TGC CCT CGA TGT TA-3?, (antisense) 5?-CAA AAA TTT
GGC CTC CTC AA-3?; TLR8 (sense) 5?-GAA GCA TTT CGA GCA TCT
CC-3?, (antisense) 5?-GAA GAC GAT TTC GCC AAG AG-3?; TLR9
(sense) 5?-ACT GAG CAC CCC TGC TTC TA-3?, (antisense) 5?-AGA
TTA GTC AGC GGC AGG AA-3?; and GAPDH (sense) 5?-GGT GAA
GGT CGG AGT CAA CG-3?, (antisense) 5?-GTG AAG ACG CCA GTG
Amplified products were electrophoresed on a 1.8% agarose gels and
visualized by ethidium bromide staining. Message for the GAPDH gene
was used to ascertain that an equivalent amount of cDNA was synthesized
from different samples.
Real-time PCR analysis
It was performed using the ABI Prism 7700 sequence detection system
(Applied Biosystems) as described earlier (21). Briefly, reactions were per-
formed in 96-well optical reaction plates on cDNA equivalent to 50 ng of
DNase-digested RNA in a volume of 25 ?l, containing 12.5 ?l of TaqMan
Universal Master mixture and optimized concentrations of FAM-labeled
probe, forward and reverse primers, following the manufacturer’s protocol.
All primers and FAM-labeled probes for mouse TNF-?, iNOS, IL-6, IL-
1?, and GAPDH were obtained from Applied Biosystems. The mRNA
expression of different genes was normalized to the label of GAPDH
mRNA. Data were processed by the ABI Sequence Detection System 1.6
software and analyzed by ANOVA.
Immunofluorescence analysis was performed as previously described (21).
Briefly, wells containing 4–5 ? 105cells/well were fixed with 4% para-
formaldehyde for 20 min followed by treatment with cold ethanol (?20°C)
for 5 min and two rinses in PBS. Samples were blocked with 3% BSA in
PBS-T (PBS-Tween 20) for 30 min and incubated in PBS-T containing 1%
BSA and goat anti-CD11b (1/100), and mouse anti-iNOS (1/75). After
three washes in PBS-T (15 min each), wells were further incubated with
Cy5 and Cy2 (Jackson ImmunoResearch Laboratories). For negative con-
trols, a set of culture wells was incubated under similar conditions without
primary Abs. The samples were mounted and observed under an Olympus
IX81 fluorescence microscope.
Microinjection of A? into the cortex of wild-type and TLR2?/?
Wild-type and TLR2?/?B6.129 mice (8- to 10-wk-old) were anesthetized
with i.p. injection of ketamine and xylazine and underwent cortical oper-
ations in a Kopf small animal stereotaxic instrument (Kopf Instruments).
Briefly, the animal was mounted in a stereotaxic frame on a heating blan-
ket. Body temperature was maintained at 37 ? 0.5°C during the time of
surgery. A midsagittal incision was made to expose the cranium, and a hole
?0.5 mm in diameter was drilled with a dental drill over the frontal cortex
according to the following coordinates: 1.5 mm anterior to bregma, 1.5 mm
lateral to bregma, and 1 mm ventral (see Fig. 8A). Either A?1–42 (1 ?g)
or A?42–1 (1 ?g) in 2 ?l of saline solution was injected using a 5-?l
syringe (Hamilton) over a period of 3 min, and the needle was held in place
for another minute before withdrawing it from the skull to prevent reflux up
the needle tract. Similarly, control mice received 2 ?l of saline solution.
The incision was closed with surgical staples and covered with a mixture
of Bacitracin and Hurricane (20% benzocaine).
After 24 h of microinjection, six mice from each of the following groups
(saline and A?) were anesthetized with a mixture of ketamine (66.6 mg/kg)
and xylazine (6.66 mg/kg) by i.p. injection. The cortex was dissected from
each mice model after perfusion with PBS (pH 7.4) and then with 4% (w/v)
paraformaldehyde solution in PBS (22). The tissues were further fixed for
at least 2 h in the same fixative at room temperature followed by three PBS
washes (15 min each). Routine histology was performed to obtain mor-
phological details of cortex tissues. Paraformaldehyde-fixed tissues were
embedded in paraffin, and serial sections (5 ?m) were cut. Sections were
stained with Abs against CD11b and iNOS.
Data are expressed as mean ? SD. Statistical comparisons were made
using Student’s t test (SAS system). Differences between mean were con-
sidered significant at p ? 0.05.
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7262A? ACTIVATES MICROGLIA VIA TLR2