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The Journal of Neuroscience, March 1, 2003, 23(5):1605
Interleukin-1 Mediates Pathological Effects of Microglia on Tau
Phosphorylation and on Synaptophysin Synthesis in Cortical Neurons
through a p38-MAPK Pathway
Yuekui
Li1,
Ling
Liu1,
Steven W.
Barger1, 2, 3, 4, and
W. Sue T.
Griffin1, 2, 3, 4, 5
Departments of 1 Geriatrics and 2 Anatomy,
University of Arkansas for Medical Sciences, and
3 Department of Veterans Affairs Medical Center and
4 Geriatric and 5 Mental Illness Research
Education Clinical Centers, Little Rock, Arkansas 72205
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ABSTRACT |
The presence of tangles of abnormally phosphorylated tau is a
characteristic of Alzheimer's disease (AD), and the loss of synapses
correlates with the degree of dementia. In addition, the overexpression
of interleukin-1 (IL-1) has been implicated in tangle formation in AD.
As a direct test of the requirement for IL-1 in tau phosphorylation and
synaptophysin expression, IL-1 actions in neuron-microglia cocultures
were manipulated. Activation of microglia with secreted  -amyloid
precursor protein or lipopolysaccharide elevated their expression of
IL-1 , IL-1, and tumor necrosis factor (TNF) mRNA. When
such activated microglia were placed in coculture with primary
neocortical neurons, a significant increase in the phosphorylation of
neuronal tau was accompanied by a decline in synaptophysin levels.
Similar effects were evoked by treatment of neurons with recombinant
IL-1. IL-1 receptor antagonist (IL-1ra) as well as anti-IL-1
antibody attenuated the influence of activated microglia on neuronal
tau and synaptophysin, but anti-TNF antibody was ineffective. Some
effects of microglial activation on neurons appear to be mediated by
activation of p38 mitogen-activated protein kinase (p38-MAPK), because
activated microglia stimulated p38-MAPK phosphorylation in neurons, and an inhibitor of p38-MAPK reversed the influence of IL-1 on tau phosphorylation and synaptophysin levels. Our results, together with
previous observations, suggest that activated microglia may contribute
to neurofibrillary pathology in AD through their production of IL-1,
activation of neuronal p38-MAPK, and resultant changes in neuronal
cytoskeletal and synaptic elements.
Key words:
Alzheimer's disease; -amyloid precursor
protein; cortical neuron; interleukin-1; microglia; mitogen-activated
protein kinase; synaptophysin; phosphorylated tau
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Introduction |
Increased levels of proinflammatory
factors such as interleukin (IL)-1 and tumor necrosis factor (TNF), as well as other indications of microglial activation, have
been correlated with several neurodegenerative situations, including
Alzheimer's disease (AD), AIDS-associated dementia, and traumatic
brain injury (Griffin and Mrak, 2002 ). Several lines of evidence
indicate that microglia and their products (cytokines, excitatory amino
acids, and other neurotoxins) can cause neuronal damage (Giulian et
al., 1995; Meda et al., 1995; Barger and Harmon, 1997; Jeohn et al.,
1998; Barger and Basile, 2001; Li et al., 2001). These observations, together with previous findings in AD patients, suggest that a chronic
inflammatory reaction, driven mainly by activated microglia, may
contribute to the process of neuropathological changes in AD brain
(Griffin et al., 1989, 1998).
Neurofibrillary changes, in the form of neuritic plaques, neuropil
threads, and neurofibrillary tangles, are key histological features of
AD. Tau is one of the microtubule-associated proteins that stabilizes
growing axons necessary for the development and growth of neurites.
However, in AD, for unknown reasons, tau becomes excessively
phosphorylated and appears in paired helical filaments, dystrophic
neurites, and neurofibrillary tangles (Braak et al., 1994). This
neurofibrillary pathology suggests a loss of axonal integrity and an
eventual decline in connectivity and synapses, a consistent correlate
of dementia in AD (DeKosky and Scheff, 1990; Terry et al., 1991).
Previously, we reported that secreted -amyloid precursor protein
(sAPP) can cause neuronal cell damage that can be restricted to
functional synapses (Barger and Basile, 2001). This and other
sAPP-evoked neurotoxicity occurs via an indirect mechanism that
involves microglial activation (Barger and Harmon, 1997; Li et al.,
2000a; Barger and Basile, 2001). To further explore these
relationships, we used a microglial-neuronal coculture model to (1)
assess the effects of microglial activation on synthesis of neuronal
synaptophysin and tau, (2) probe the possible link between
phosphorylation and the maintenance of synapses, and (3) determine
potential signal transduction components mediating phosphorylation
events in glial-neuronal interactions.
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Materials and Methods |
Reagents. sAPP was purified from a recombinant
expression system as described previously (Barger and Basile, 2001).
Lipopolysaccharide (LPS) and recombinant rat IL-1 were obtained from
Sigma (St. Louis, MO). Mouse IL-1ra was from R&D Systems
(Minneapolis, MN), monoclonal hamster anti-murine IL-1 antibody was
from Genzyme (Cambridge, MA), and anti-murine TNF
antibody was from PeproTech (Rocky Hill, NJ). SB203580-HCl
was from Calbiochem (Sunnyvale, CA). Monoclonal
anti-phospho-tau antibody AT8 was from RDI (Flanders, NJ); monoclonal
anti-Tau1 antibody was kindly provided by Dr. L. I. Binder
(Northwestern University); monoclonal anti-synaptophysin antibody SY38
was from ICN Biomedicals (Costa Mesa, CA); antibodies against phospho-p38-MAPK or total p38-MAPK were from New England Biolabs (Beverly, MA). Medium, serum, and B27 supplement for
cell cultures were from Invitrogen (Grand Island, NY).
Cell cultures. Primary neuronal cultures were derived from
the cerebral cortex of fetal Sprague Dawley rats
(embryonic day 18), as described previously (Li et al., 1998).
Experiments using primary neuronal cell cultures were performed after
10-14 d in culture. Primary cultures of rat microglia were generated
from the cortical tissue of neonatal (0-3 d) Sprague
Dawley rats, as described previously (Barger and Basile, 2001).
The N9 mouse microglial cell line (Corradin et al., 1993) was
maintained in DMEM (Invitrogen) supplemented with 10%
with fetal bovine serum. Coculture of N9 cells or primary microglia
with primary neuronal cells was performed as described previously (Li
et al., 2000a). Either microglial cell type was grown on semi-permeable
membranes of basket-type cell culture inserts (Falcon; pore size 0.4 µm; Fisher Scientific, Houston, TX), treated with
activation stimuli (LPS or sAPP), and then washed before placement into
culture wells containing neurons. When applied to cocultures, IL1-ra,
anti-IL-1 antibody, anti-TNF antibody, or SB203580-HCl (aqueous
solution) was added to the neuronal culture medium before placement of
the microglial inserts into the neuronal culture wells.
Viability assays. Cell survival was assessed using a
previously described
3-(4-5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay (Li et al., 1998).
RT-PCR amplification. Total RNA was extracted from cultured
cells using TriReagent RNA (Molecular Research Center,
Cincinnati, OH) according to the manufacturer's instructions. RT-PCR
was performed as described previously (Li et al., 2001). Briefly, for
comparisons of mRNA levels among different RNA samples, RT was
performed simultaneously using reagents from a single master mix. PCR
was performed using reagents from Clontech (Palo Alto,
CA). The sequences of primers for both mouse and rat IL-1, IL-1,
and TNF and glyceraldehyde 3-phosphate dehydrogenase (G3PDH)
were used in this study; the mouse sequences were given previously (Li
et al., 2000a, 2001). The following are the sequences of primers used
for rat IL-1: upstream 5'-CT AAG AAC TAC TTC ACA TCC GCA GC-3';
downstream 5'-CTG GAA TAA AAC CCA CTG AGG TAG G-3'; for rat IL-1:
upstream 5'-TGA CTC GTG GGA TGA TGA CG-3'; downstream 5'-CTG GAG ACT
GCC CAT TCT CG-3'; for rat TNF: upstream 5'-GCA CAG AAA GCA TGA TCC
GAG-3'; downstream 5'-CCT GGT ATG AAG TGG CAA ATC G-3'. PCR
amplification was performed through 26 cycles (for IL-1 and IL-1)
or 28 cycles (for TNF) at 94°C for 45 sec, 60°C for 45 sec, and
72°C for 45 sec. For G3PDH, amplification was performed through 26 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 45 sec.
The PCR reaction was stopped by final extension for 10 min at 72°C. The PCR cycle for each target gene was determined by sampling 5 µl
aliquots every third cycle from cycle 21 on. The indicated cycles were
verified to be within the linear range of product accumulation for the
specific PCR reaction. Equal volumes of reaction mixture from each
sample were loaded onto 1.5% agarose gels, and fluorescent images were
digitally captured for analysis of intensity with NIH Image software.
Levels of IL-1, IL-1, and TNF mRNA were normalized relative to
G3PDH mRNA in the same sample.
Western immunoblot assay. Proteins were extracted using
lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium
deoxycholate, and 0.1% SDS and quantified using a Micro BCA assay
reagent kit (Pierce, Rockford, IL) as described previously
(Li et al., 1998). Aliquots (40 µg each) were loaded onto a 10%
SDS-polyacrylamide gel, subjected to electrophoresis at 90 V for 1.5 hr, and transferred to Immobilon-P membranes. Membranes were incubated
overnight at 4°C with primary antibody and for 2 hr at room
temperature with secondary antibody and visualized using the
Western-Light Chemiluminescent Detection System (Tropix, Bedford, MA). Films were digitized and analyzed using NIH Image software, version 1.60.
Statistical analysis. Data were analyzed using an unpaired
t test, and values were considered significantly different
when the two-tailed p value was <0.05. Results are
expressed as mean ± SEM.
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Results |
Two strategies were used to assess the capacity of IL-1 to
directly regulate neuronal tau phosphorylation and synaptophysin synthesis and to assess the possibility that p38-MAPK mediates these
events. For the first, we directly applied IL-1 to neuronal cultures,
and for the second, we used a coculture paradigm that allows one cell
type to be treated and analyzed independently of the other, in this
case, activated microglia and neurons. Studies were initiated with the
N9 microglial cell line for the sake of homogeneity (i.e., independence
from astrocytes or other contaminants of primary glial cultures);
additional experiments were performed with rat primary microglia. Both
N9 cells and primary microglia were grown on semi-permeable membranes
of basket-type cell culture inserts and stimulated with either sAPP (10 nM) or LPS (30 ng/ml). Both agonists caused an elevation of
IL-1, IL-1, and TNF mRNA levels in N9 microglia (Fig.
1A). To confirm whether
primary microglial cells have similar responses to sAPP, and whether
there is a chronic production of cytokine expression by primary
microglia after a transient exposure to sAPP, primary microglia were
incubated with sAPP (10 nM) for 2 hr, washed
twice with fresh medium, and then cultured in fresh medium for 6 or 12 hr and harvested for assay of cytokine expression. As shown in Figure
1B, in primary microglia, IL-1 and IL-1 and
TNF remain elevated for 12 hr after a 2 hr treatment with sAPP.
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Figure 1.
Time course of LPS or sAPP induction of IL-1,
IL-1, and TNF mRNA expression in microglia. A, N9
microglial cells were treated with either LPS (30 ng/ml) or sAPP (10 nM) for the indicated times [hours
(h)]. Total RNA was extracted and subjected to
RT-PCR of sequences specific to IL-1, IL-1, or TNF; G3PDH was
amplified to assess equivalency of loading. B, Primary
microglia were treated with sAPP (10 nM) for 2 hr, followed
by two washes with fresh medium. These microglia were then cultured in
fresh medium for up to 12 hr and harvested for cytokine expression
assay. Controls (Con) were untreated sister cultures.
Data are representative of at least two separate experiments.
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There was a significant decrease in the steady-state levels of
synaptophysin and a significant increase in the steady-state levels of
phosphorylated tau in primary neocortical neurons cocultured for 24 hr
with baskets containing LPS- or sAPP-activated microglia (Fig.
2). These changes were accompanied by an
increase in phosphorylation, i.e., activation, of p38-MAPK (Fig. 2), a
signal transduction protein that can phosphorylate tau at five sites
that are phosphorylated in paired helical filaments (Reynolds et al.,
2000). As early as 30 min after application of recombinant IL-1,
there was a marked elevation of tau phosphorylation, increasing to
approximately twofold after 2 hr of incubation, compared with untreated
sister cultures (Fig.
3A,C).
However, total neuronal tau was unchanged by treatment with IL-1 for
up to 12 hr (Fig. 3A). Treatment of neuronal cells with
IL-1 also resulted in a marked, time-dependent decrease in
steady-state levels of synaptophysin, which was first apparent after a
1 hr incubation (Fig. 3B). After 2 hr of treatment with
IL-1, the synaptophysin level decreased by 55% compared with
untreated sister cultures (Fig.
3B,C).
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Figure 2.
Effects of activated microglia on synaptophysin,
tau, and p38-MAPK in neocortical neurons. N9 microglia were stimulated
with LPS (30 ng/ml) for 2 hr (LPS), washed with fresh
medium, and then placed in coculture with primary neocortical neurons
for 24 hr; untreated sister cultures of microglia were also cocultured
with neurons (Con). Proteins were extracted from the
neurons, and Western blot analysis was performed for the detection of
synaptophysin (Syn), phosphorylated tau
(Tau-phos), or phosphorylated p38-MAPK
(p38-phos). A, Western
immunoblots. B, Relative levels of Syn, Tau-phos, and
p38-phos in treated versus control cultures. Values are expressed as
mean ± SEM (n = 4). **p < 0.01.
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Figure 3.
IL-1 induces tau phosphorylation and decreases
synaptophysin in cortical neurons. A, Illustrations of
phosphorylated tau (Tau-phos) and total tau
(Total-tau) in cortical neurons treated with IL-1 (30 ng/ml) for up to 12 hr. B, Synaptophysin
(Syn) in cultures treated for up to 12 hr.
C, Quantification of protein levels of Syn and Tau-phos
in neuronal cultures treated with IL-1 (30 ng/ml) for 2 hr. Values
are expressed as mean ± SEM of four samples. *p < 0.05; **p < 0.01.
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To test whether IL-1 and TNF play essential roles in the
interactions between activated microglia and neurons, IL-1ra,
anti-IL-1 antibodies, or anti-TNF antibodies were used to block
the actions of these two activated microglia-derived cytokines on
neurons. The presence of IL-1ra in primary neurons cocultured with N9
cells inhibited the IL-1-induced portion of the increase in
phosphorylated tau as well as the decrease in synaptophysin to levels
similar to those in control sister cultures (Fig.
4A,B).
However, addition of anti-TNF antibody to the cocultures did not
show significant protective effects on synaptophysin (Fig.
4A,B). Cocultures of primary
microglia with primary neurons pretreated with IL-1ra yielded similar
results (Fig.
5A,B).
In addition, IL-1ra blocked the IL-1-induced activation of p38-MAPK
(Fig. 5A,B). As confirmation that
sAPP activation of microglia results in similar changes in synaptophysin, tau, and p38-MAPK, coculture of sAPP-activated primary
microglia with primary neurons resulted in a 50% decrease in
synaptophysin and a 50% increase in tau phosphorylation. These were
concomitant with a ~250% increase in activation of p38-MAPK. All of
these changes in neuronal proteins were suppressed by pretreating the
neuronal cultures with either anti-IL- antibody or IL-1ra (Fig.
6 A-C). As in
cocultures with LPS-activated microglia, pretreatment of the neuronal
cultures with anti-TNF antibody did not show these salutary effects,
suggesting that TNF does not play a role in these particular
deleterious effects of microglial activation on neurons (Fig.
6A-C). However, compared with control
sister cultures, neither total p38-MAPK nor total tau levels were
changed in neuronal cultures by 24 hr coculture with sAPP-activated
microglia (Fig. 6A,B).
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Figure 4.
Protective effect of IL-1ra on neuronal
synaptophysin loss induced by activated microglia. A,
Western immunoblots illustrating proteins from cortical neurons
cocultured for 24 hr with naive N9 cells (Con),
LPS-stimulated N9 cells (LPS), LPS-stimulated N9 cells
in the presence of IL-1ra (LPS+ IL-1ra), or
LPS-stimulated N9 cells in the presence of anti-TNF antibody
(-TNF+LPS). B,
Quantification of protein level of synaptophysin. Values are expressed
as mean ± SEM of four to six samples. *p < 0.05; **p < 0.01.
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Figure 5.
Effects of activated primary cortical microglia on
synaptophysin, tau phosphorylation, and p38-MAPK activation in primary
neurons. A, Western blot of phosphorylated tau
(Tau-phos), synaptophysin (Syn), and
p38-MAPK (p38-phos) in cortical neurons
cocultured with naive primary microglia (Con),
LPS-stimulated microglia (LPS), or LPS-stimulated
microglia in the presence of IL-1ra [LPS + IL-1ra
(LPS/IL-1ra)]. Results are representative of two
independent experiments. B, Quantification of protein
levels of Syn, Tau-phos, and p38-phos. Values are expressed as
mean ± SEM (n = 4). *p < 0.05;
**p < 0.01.
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Figure 6.
Effects of sAPP-activated primary microglia on the
levels of neuronal p38-MAPK and tau phosphorylation and of
synaptophysin. A, Western immunoblots of phosphorylated
p38-MAPK (p38-phos), total p38-MAPK
(Total-p38); B, synaptophysin
(Syn), phosphorylated tau (Tau-phos), and
total tau (Total-tau) in cortical neurons cocultured
with naive primary microglia (Con), sAPP-stimulated
microglia (sAPP), sAPP-stimulated microglia in the
presence of IL-1ra (sAPP+IL-1ra), of anti-TNF
(sAPP + -TNF), or of anti-IL-1
antibody (sAPP +
-IL-1). C, Quantification of
protein levels of p38-phos, Syn, and Tau-phos. Values are expressed as
mean ± SEM of three to four samples from two independent
experiments. *p < 0.05; **p < 0.01.
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We next sought to determine whether microglial neuronal toxicity could
account for loss of synaptophysin, increase in tau phosphorylation, and
activation of p38-MAPK. In the present coculture models, no significant
loss in neuronal viability was detected at 24 hr, suggesting that the
loss of synaptophysin, the phosphorylation of tau, and the activation
of p38-MAPK shown here was not caused by cell loss, but in fact
preceded neuron cell death. As further evidence and in agreement with
other studies (Strijbos and Rothwell, 1995), blocking IL-1 activity
with IL-1ra resulted in substantially higher neuronal viability.
Blockade of TNF was somewhat effective in maintaining viability, but
less than blockade of IL-1 (Fig. 7).
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Figure 7.
IL-1ra or anti-TNF antibody attenuates the
neurotoxicity of activated microglia. Survival was assessed by MTT
assay in cortical neurons cocultured with naive N9 cells
(Con), LPS-stimulated N9 cells (LPS),
LPS-stimulated N9 cells in the presence of IL-1ra
(LPS+IL-1ra), or LPS-stimulated N9 cells in the presence
of anti-TNF antibody (LPS+-TNF)
for 24 or 48 hr. Values are expressed as mean ± SEM of four to
eight samples. **p < 0.001.
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Activation of p38-MAPK in neuronal cells has been associated with
responses to stress (Xia et al., 1995; Kawasaki et al., 1997) and, more
specifically, with IL-1 and hyperphosphorylated tau in AD (Sheng et
al., 2001). Because an elevation of phosphorylated p38-MAPK appeared in
coculture of neocortical neurons with microglia (Figs. 2, 5,
6A,C), we tested whether activation
of p38-MAPK participates in IL-1-induced tau phosphorylation and
decreased synaptophysin in neocortical neurons. In case IL-1-induced
activation of astrocytes results in phosphorylation of p38-MAPK, we
carefully prepared our primary neuronal cell cultures and maximally
limited glial contamination using serum-free Neurobasal medium
supplemented with B27 and transiently using
10 5
M cytosine arabinoside as described previously
(Li et al., 1998, 2000b). In the current neuronal cultures, astrocytes
comprised 3% of the total cells, suggesting that the observed
elevation of p38-MAPK phosphorylation was neuronal in origin. At 30 and 60 min after application of IL-1, neurons showed an elevation of
activated p38-MAPK, which returned to basal levels after 2 hr (Fig.
8), whereas the total neuronal p38-MAPK
did not show marked differences after IL-1 treatment (Fig.
8A). Pretreatment of neocortical neurons with
p38-MAPK inhibitor SB203580 significantly suppressed IL-1-induced
changes in tau and synaptophysin (Fig. 8B,C), indicating that activation
of p38-MAPK was involved in the IL-1 signal transduction pathway
regulating these proteins in neurons.
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Figure 8.
IL-1 induction of tau phosphorylation in
cortical neurons is mediated by phosphorylation of p38-MAPK.
A, Time course of IL-1 (30 ng/ml) induction of
p38-MAPK phosphorylation compared with the level of total p38-MAPK
(Total-p38) in the same neuronal cultures;
B, an illustration of inhibition of activation of
p38-MAPK with SB203580 on IL-1-induced suppression of synaptophysin
(Syn) and on IL-1-induced phosphorylation of tau
protein (Tau-phos) after 2 hr treatment.
C, Quantification of protein levels of synaptophysin
(Syn) and phosphorylated tau protein
(Tau-phos). Values are expressed as mean ± SEM
(n = 3-4). *p < 0.05;
**p < 0.01.
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Discussion |
In this study, the effects of glial-neuronal interactions on
phosphorylation of tau protein and maintenance of synaptophysin levels
were dissected in vitro in a glial-neuronal coculture
model. The results demonstrate that (1) activated microglia increase tau phosphorylation and decrease steady-state levels of synaptophysin through release of IL-1; (2) these IL-1-induced changes occur before
significant neuronal cell loss associated with microglial neurotoxicity
is detectable; and (3) the effects of IL-1 on tau and synaptophysin are
mediated, at least in part, through activation of p38-MAPK. These
experimental results, together with previous observations (Griffin et
al., 1989; Sheng et al., 2001; Griffin and Mrak, 2002), suggest that
microglial activation with overexpression of IL-1 contributes to the
neurodegenerative consequences of AD.
The cognitive alterations in patients with AD are closely associated
with synaptic loss and neurofibrillary pathology in the limbic system
and neocortex (Arriagada et al., 1992). Loss of synaptic proteins such
as synaptophysin, syntaxin, and SNAP-25 (soluble
N-ethylmaleimide-sensitive factor attachment protein) in the
frontal neocortex has provided the best neurobiological correlates of
cognitive impairment (DeKosky and Scheff, 1990; Terry et al., 1991;
Mukaetova-Ladinska et al., 2000). However, the molecular and cellular
processes responsible for loss of synapses and the formation of
hyperphosphorylated tau in the early stages of AD, before significant
neuronal cell death, remain undefined. Three possibilities have been
proposed on the basis of the neuropathology: (1) loss of synapses is a
nonspecific consequence of global neurodegenerative changes that
include neuronal loss (Scheff et al., 1996); (2) loss of synapses
results from the direct neurotoxicity of amyloid -peptide (A)
(Mattson, 1997); and (3) loss of synapses results from cytoskeletal
changes caused either actively by tau aggregates or passively by loss
of tau function (Lee et al., 1991; Wischik et al., 1996). Here we
provide evidence that temporal overexpression of cytokines contributes
to a cascade of events resulting in specific patterns of tau pathology
and neurodegeneration.
IL-1 promotes neuronal production of -amyloid precursor
protein and its derivatives (Buxbaum et al., 1992), but our
current findings suggest that IL-1-mediated proinflammatory sequelae
could damage neuronal connectivity via mechanisms beyond neurotoxic effects of A production. Previously, we found that activated microglia can influence properties of neurotransmitters, such as
cholinergic or glutamatergic systems (Li et al., 2000a; Barger and
Basile, 2001). A role for microglia in neuronal cell death is now
widely accepted (Griffin et al., 1989; Giulian et al., 1995; Barger and
Harmon, 1997; Li et al., 2001). Furthermore, activation of microglia by
sAPP can have effects on neuronal function short of outright cell
death, including reduction of the density of functional synapses
(Barger and Basile, 2001). The effects of activated microglia and IL-1
on tau phosphorylation and synaptophysin expression noted here approach
biochemical explanations for this phenomenon.
In the present study, decreased synaptophysin synthesis was accompanied
by elevation of phosphorylated tau and preceded significant neuronal
cell death, suggesting that phosphorylation of tau and loss of synaptic
proteins are early events in the process of neuronal cell death. This
is consistent with the hypothesis that altered expression of synaptic
proteins occurs early in the neurodegenerative sequelae of AD (Masliah
et al., 2001). The induction of tau phosphorylation by IL-1 shown here
in vitro and previously in vivo (Sheng et al., 2001) indicates that IL-1 might potentially contribute to the reorganization of the cytoskeleton, interrupt normal microtubule assembly and axon stabilization, and eventually result in loss of
synaptic proteins and synapses. This is supported by the observations in AD that a loss of synaptophysin is observed in tangle-bearing neurons (Callahan and Coleman, 1995) and that activated microglia correlate with neurofibrillary pathology (DiPatre and Gelman, 1997),
including intracellular tau pathology (Sheng et al., 1997). Furthermore, in a study comparing neuropathology in AD patients with
that in cognitively normal individuals with abundant A deposits, the
latter group was found to have normal synapse densities and scant
evidence of inflammation (Lue et al., 1996), suggesting that
inflammatory events contribute to neuronal abnormalities that lead to
clinical symptoms. This suggestion is consistent with our finding that
preincubation of neuronal cells with IL-1ra suppressed the effect of
activated microglia on synthesis of neuronal synaptophysin. Although
blocking IL-1 or TNF enhanced neuronal viability, TNF seemed to
have no significant influence on synaptophysin synthesis or tau
phosphorylation, suggesting that not all cytokines released by
activated microglia use the same pathways to achieve their deleterious
effects on neurons.
Activation of p38-MAPK is involved in neuronal responses to various
stresses (Xia et al., 1995; Kawasaki et al., 1997), and this kinase is
closely related to hyperphosphorylated tau protein in AD (Sheng et al.,
2001). Like several other members of the MAPK family, p38-MAPK is
activated by dual phosphorylation; cytokines, including IL-1, can
effect this activation. Here phosphorylation of p38-MAPK was
significantly increased in neuronal cells either treated with
recombinant IL-1 or cocultured with activated microglia, indicating
that activation of p38-MAPK is involved in IL-1 signal transduction in
neurons. The finding that inhibition of p38-MAPK significantly
suppressed IL-1-induced tau phosphorylation and IL-1-decreased
synaptophysin adds to the evidence for a role for this kinase in neural
physiology and connectivity. Interestingly, treatment of neuronal cells
with recombinant IL-1 resulted in transient increases in activated
p38-MAPK, whereas neuronal cells cocultured with activated microglia
showed a sustained increase that could be blocked by IL-1ra. This
suggests that persistent microglial activation such as that proposed
for AD may result in continued release of IL-1 with neurodegenerative
consequences. This observation further confirms our previous finding
that chronic activation of microglia causes significant and prolonged
microglial production of cytokines, which leads to neuronal cell damage
and further microglia activation (Li et al., 2000a; Barger and Basile, 2001). All of this points to the existence of neurodegenerative cycles
that are dependent on neuronal-microglial interactions such as those
proposed in AD and other neurodegenerative conditions (for review, see
Griffin et al., 2000).
The IL-1-mediated interactions shown here provide experimental support
for the idea that microglial-neuronal relationships and the
concomitant overexpression of IL-1 observed in AD brain contribute to
the neuronal dysfunction and loss characteristic of AD, particularly
those involved in formation of neurofibrillary tangles and loss of
synapses. Our findings regarding the involvement of IL-1 and p38-MAPK
in microglial activation-induced neuronal tau phosphorylation and
synaptic function suggest that pharmacological approaches more directly
targeting excessive expression of cytokines, e.g., cytokine-suppressant
anti-inflammatory drugs, should be developed against brain inflammatory
conditions, which are neuropathogenic not only in AD but also in early
Down's syndrome, in AIDS, in head injury, and in demyelinating
diseases such as multiple sclerosis.
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FOOTNOTES |
Received Aug. 29, 2002; revised Dec. 17, 2002; accepted Dec. 19, 2002.
This research was supported in part by National Institutes of Health
Grant AG12411 and the Donald W. Reynolds Foundation. We thank Pam Free
for secretarial support.
Correspondence should be addressed to Prof. W. Sue T. Griffin, Donald
W. Reynolds Center on Aging, Room 3103, 629 South Elm Street, Little
Rock, AR 72205. E-mail: griffinsuet{at}uams.edu.
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TOP
ABSTRACT
Introduction
