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The Journal of Neuroscience, June 1, 2002, 22(11):4530-4539
Interferon  Induces Retrograde Dendritic Retraction and
Inhibits Synapse Formation
In-Jung
Kim1,
Hiroko
Nagasawa
Beck2,
Pamela J.
Lein2, and
Dennis
Higgins1
1 Department of Pharmacology and Toxicology, State
University of New York, Buffalo, New York 14214, and
2 Department of Environmental Health Sciences, Bloomberg
School of Public Health, Johns Hopkins University, Baltimore, Maryland
21205
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ABSTRACT |
The expression of interferon  (IFN) increases after neural
injury, and it is sustained in chronic inflammatory conditions such as
multiple sclerosis and infection with human immunodeficiency virus. To
understand how exposure to this proinflammatory cytokine might affect
neural function, we examined its effects on cultures of neurons derived
from the central and peripheral nervous systems. IFN inhibits
initial dendritic outgrowth in cultures of embryonic rat sympathetic
and hippocampal neurons, and this inhibitory effect on process growth
is associated with a decrease in the rate of synapse formation. In
addition, in older cultures of sympathetic neurons, IFN also
selectively induces retraction of existing dendrites, ultimately
leading to an 88% decrease in the size of the arbor. Dendritic
retraction induced by IFN represents a specific cellular response
because it occurs without affecting axonal outgrowth or cell survival,
and it is not observed with tumor necrosis factor  or other
inflammatory cytokines. IFN-induced dendritic retraction is
associated with the phosphorylation and nuclear translocation of signal
transducer and activator of transcription 1 (STAT1), and expression of
a dominant-negative STAT1 construct attenuates the inhibitory effect of
IFN. Moreover, retrograde dendritic retraction is observed when
distal axons are selectively exposed to IFN. These data imply
that IFN-mediated STAT1 activation induces both dendritic atrophy
and synaptic loss and that this occurs both at the sites of IFN
release and at remote loci. Regressive actions of IFN on dendrites
may contribute to the neuropathology of inflammatory diseases.
Key words:
interferon ; bone morphogenetic protein-7; dendrites; sympathetic neurons; hippocampal neurons; STAT1; retrograde
transport
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INTRODUCTION |
Interferon (IFN) is a
proinflammatory cytokine that potently stimulates cell-mediated
immunity (Farrar and Schreiber, 1993 ; Bach et al., 1997; Boehm et al.,
1997). IFN is made primarily by T-lymphocytes and natural killer
cells; because the entry of these cells into the CNS is minimal
under normal circumstances, IFN is usually not detectable in the
brain (Traugott and Lebon, 1988; Fabry et al., 1994). However,
expression of IFN rises rapidly in acute inflammatory reactions
caused by trauma (Lau and Yu, 2001), ischemia (Li et al., 2001), or
viral infection (Binder and Griffin, 2001), and elevated expression
persists in chronic inflammatory diseases such as multiple sclerosis
(MS) (Panitch, 1992; Popko et al., 1997) and in chronic viral infection
such as that produced by human immunodeficiency virus (HIV) (Fuchs et
al., 1991; Fan et al., 1993). Levels of IFN also are increased in
murine trisomy 16, which is considered a model for Down's syndrome (Torre et al., 1995; Hallam et al., 2000). Overexpression of IFN causes abnormal cerebellar and hippocampal development in transgenic mice (Corbin et al., 1996; LaFerla et al., 2000), and administration of
IFN to MS patients aggravates the disease (Panitch et al., 1987).
Thus there is reason to suspect that the elevated levels of IFN that
are associated with inflammatory neural conditions may contribute to
their pathology. However, evaluation of this hypothesis is impeded by a
lack of knowledge of the direct effects of IFN on neurons, and, in
fact, previous studies have noted primarily beneficial effects of
IFN on the survival (Chang et al., 1990), growth (Erkman et al.,
1989), and differentiation of isolated neurons (Barish et al., 1991;
Jonakait et al., 1994).
Dendrites are the primary site of synapse formation in the nervous
system (Purves, 1988), and dendritic atrophy has been observed in
degenerative conditions such as Alzheimer's disease (Flood and
Coleman, 1990) and Parkinson's disease (Patt et al., 1991), in Down's
syndrome (Takashima et al., 1989), in chronic viral infections (Masliah
et al., 1997; Li et al., 1999), and as part of the reaction to acute
traumatic injury (Sumner and Watson, 1971; Yawo, 1987). It is,
therefore, important to characterize molecules that cause regression of
these processes. Previous studies have shown that leukemia inhibitory
factor (LIF), ciliary neurotropic factor, and other members of the
interleukin-6 (IL-6) cytokine family induce dendritic retraction in
cultured sympathetic neurons (Guo et al., 1999) and that overexpression
of LIF causes dendritic atrophy in Purkinje cells (Morikawa et al.,
2000). In this study we consider the possibility that IFN shares
with IL-6-related cytokines the ability to interfere with the growth of
dendritic processes. In addition, we have compared the signal
transduction pathways involved in this action.
Our data indicate that IFN adversely affects dendritic maturation
and synapse formation and that it acts by a pathway distinct from that
used by members of the IL-6 family, suggesting possible synergistic
interaction during inflammation. Moreover, we have found that the
regressive signals generated by IFN can be retrogradely transported
from distal axons to neural somata. This retrograde dystrophic
interaction represents a novel method for conveying information about
local injury or inflammation to distant brain loci.
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MATERIALS AND METHODS |
Materials. Recombinant rat IFN, interleukin 1-
(IL-1), and human tumor necrosis factor (TNF) were purchased
from PeproTech (Rocky Hill, NJ). Rat IFN was obtained from PBL
Biomedical Laboratory (New Brunswick, NJ). Recombinant human bone
morphogenetic protein-2 (BMP-2), BMP-6, and BMP-7 were generously
provided by Curis (Cambridge, MA).
Cell culture. Superior cervical ganglia dissected from
perinatal [embryonic day 21 (E21) or postnatal day 1-2 (P1-2)]
Holtzman rats (Harlan Sprague Dawley, Rockford, IL) were dissociated
after treatment with trypsin (2.5 mg/ml) and collagenase (1 mg/ml) for 40 min. Cells were plated onto poly-D-lysine-coated (100 µg/ml) coverslips or compartmented chambers (see below) and
maintained in serum-free medium containing -nerve growth factor
(NGF; 100 ng/ml) (Higgins et al., 1991). Then 1-2 d later,
cytosine--D-arabinofuranoside (1 µm) was added in the
media for 2 d to kill non-neuronal cells. Subsequently, the
cultures were allowed to recover for 1 d and experimental
treatments were begun on the sixth or seventh day in
vitro.
Embryonic hippocampal neurons (E18) were isolated by the method of
Goslin et al. (1998) with the modifications of Bading and Greenberg
(1991). Briefly, hippocampi were dissociated by treatment with trypsin
(2 mg/ml) and deoxyribonuclease (0.6 mg/ml) for 2 min and then exposed
to soybean trypsin inhibitor (1 mg/ml) for 10 min. Cells were plated
onto poly-D-lysine (100 µg/ml) and laminin (7 µg/ml)
and maintained in Neurobasal medium with B27 supplements (Invitrogen,
San Diego, CA) (Brewer et al., 1993).
Compartmented cultures. Compartmented cultures (Campenot
chambers) were set up as described by Senger and Campenot (1997). Briefly, 35 mm dishes were precoated with a layer of ammoniated rat
tail collagen, followed by a layer of air-dried rat tail collagen supplemented with laminin (10 µg/ml). Three-compartmented Teflon dividers (Camp 10, Tyler Research Instruments, Edmonton, Alberta, Canada) were seated on top of parallel tracks scratched in the collagen
substrate with a pin rake (Tyler Research Instruments) and secured in
place with silicone vacuum grease (Dow Corning, Huntington Beach, CA.).
Integrity of the grease seals was assessed by placing culture medium
into the side chambers only and incubating the chambers overnight in a
37°C incubator. Cells were plated only into chambers that did not
leak. Medium was removed from side compartments, and dissociated cells
were plated in the center compartment in serum-free culture medium
containing 100 ng/ml NGF. The next day the integrity of the seals
between the compartments was reconfirmed by checking for leakage from
the center into side compartments. Culture medium was then added to the
side compartments of those cultures exhibiting intact seals between
compartments, and cytosine--D-arabinofuranoside (1 µm)
was added to the medium in the center compartment to eliminate
non-neuronal cells. After axons had extended through the grease seals
into the side compartments (5-7 d after plating), NGF was withdrawn
from the center compartment but was maintained in the side compartment
to encourage growth of neurites into the side compartments and to
eliminate neurons that had failed to extend axons into side
compartments. BMP-7 (10 ng/ml) was added to medium in the center
compartment when extensive axonal growth was evident in side
compartments (within 10-12 d after plating); 4 d later, IFN
(10 or 50 ng/ml) was added to medium in the right side compartment of a
subset of cultures.
Morphological analyses. Cellular morphology was analyzed
using previously described immunocytochemical methods (Lein et al., 1995). Briefly, cells were fixed with 4% paraformaldehyde (15 min at
20°C) and permeabilized with 0.1% Triton X-100 for 3 min. Cells were
reacted with monoclonal antibodies (mAb) that recognize axons or
dendrites and then with rhodamine-conjugated secondary antisera (Roche
Diagnosis, Indianapolis, IN). Monoclonal antibodies to
microtubule-associated protein-2 (MAP-2, SMI-52; Sternberger Immunocytochemicals, Baltimore, MD) or to nonphosphorylated forms of H
and M neurofilament subunits (SMI-32; Sternberger Immunocytochemicals) were used to visualize dendrites. Previous studies have shown that
immunostaining with these antibodies reveals the entire dendritic arbor
(Lein et al., 1995; Bruckenstein and Higgins, 1988b). Total dendritic
length was measured with SPOT software (Diagnostic Instruments, Sterling Heights, MI). A mAb to phosphorylated forms of the H and M
neurofilament subunits (SMI-31; Sternberger Immunocytochemicals) was
used to identify the axons of sympathetic neurons (Osterhout et al.,
1992; Bruckenstein and Higgins, 1988a).
To examine synapse formation, hippocampal neurons were plated at
high density (~16,000/cm2) to increase
the frequency of cellular contacts (Fletcher et al., 1994). After
5 d of treatment with IFN (30 ng/ml), the synaptic specializations that formed along dendrites were visualized by double-labeling the cultures with rabbit anti-MAP-2 IgG (a gift from
Dr. Craig Garner, University of Alabama at Birmingham, Birmingham, AL)
and mAb to synaptic vesicle protein-2 (SV-2; Buckley and Kelly, 1985) and then with rhodamine-conjugated antibody to rabbit IgG (Roche Diagnosis) and fluorescein-conjugated antibody to mouse IgG
(Roche Diagnosis).
Cultures were also immunostained with rabbit IgG that specifically
recognizes a phosphorylated form (Tyr 701) of signal transducer and
activator of transcription 1 (STAT1; New England Biolabs, Beverly, MA).
Fixation and permeabilization were performed according to the
manufacturer's protocol.
Each experiment was performed at least three times, and data in the
text and in figures are presented as the mean ± SEM. Statistical significance was assessed with a one-way ANOVA, followed by Tukey's post hoc test.
Western blotting. Cultures were solubilized in lysis buffer
containing 50 mM Tris-HCl, pH 7.4, 1 mM EDTA,
0.1% SDS, and 2.0% -mercaptoethanol. After centrifugation at
15,000 × g, the protein concentration was determined
with the Bio-Rad protein assay (Hercules, CA). Proteins (10 µg/lane)
were separated by SDS-PAGE (7.5%) and then transferred to
nitrocellulose membrane where they were probed with mAb to tau (Tau-2;
Sigma, St. Louis, MO) or a rabbit anti-actin IgG fraction (a gift from
Dr. John Kolega, State University of New York, Buffalo, NY). Next the
membrane was reacted with an appropriate horseradish
peroxidase-conjugated secondary antibody (Amersham, Piscataway, NJ),
and bands were visualized with enhanced chemiluminescent reagent (Amersham).
Transfection. Cells were cotransfected with a plasmid coding
the enhanced green fluorescent protein (pEGFP-N2, Clontech, Palo Alto,
CA) and the expression vector pCAGGS containing HA-STAT1WT (hemagglutinin-tagged wild-type STAT1 gene), HA-STAT3WT, HA-STAT1F (Phe substitution for Tyr701), or HA-STAT3F (Phe substitution for Tyr
705) (Nakajima et al., 1996). Plasmids containing STAT1 and STAT3 were
a generous gift from Dr. Toshio Hirano (Osaka University Medical
School, Osaka, Japan). Transfection was performed using Lipofectamine
2000 (Invitrogen) according to the manufacturer's protocol. Briefly,
cells were treated with 200 µl of DMEM containing 1 µg
of DNA and 4 µg of Lipofectamine. After incubation for 6 hr
cells were washed and allowed to recover for 48 hr before experimental treatments were begun.
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RESULTS |
IFN inhibits initial dendritic outgrowth induced by BMP-7 in
sympathetic neurons
Embryonic rat sympathetic neurons extend only axons (Fig.
1) when grown in serum-free medium in the
absence of non-neuronal cells (Bruckenstein and Higgins, 1988a). In
contrast, exposure to BMP-7, which is produced by glial cells within
sympathetic ganglia (H. Beck and P. Lein, unpublished observation),
causes them to also form dendrites (Fig. 1). With moderate doses (5 ng/ml) of BMP-7, neurons typically extend two to three dendrites within the first week of treatment (Fig. 2).
With maximally effective doses ( 50 ng/ml) there is a greater increase
in both the number of dendrites (Fig. 3)
and total process length, and the neurons eventually form an arbor that
is equivalent in size to that observed in vivo (Lein et al.,
1995).
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Figure 1.
Effect of BMP-7 and IFN on the morphology of
cultured sympathetic neurons. Shown are phase-contrast (A, C, E,
G) and fluorescence (B, D, F, H)
micrographs of neurons immunostained with a monoclonal antibody (mAb)
to MAP-2 after 5 d of treatment. Neurons grown in control medium
lacked dendrites (A, B). Exposure to BMP-7 (5 ng/ml)
caused the neurons to form multiple dendrites (C, D).
IFN (10 ng/ml) alone had no effect on dendritic outgrowth (E,
F), but it inhibited the growth of these processes in
cultures that were exposed to BMP-7 (G, H). Scale
bar, 30 µm.
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Figure 2.
IFN inhibits BMP-7-induced dendritic outgrowth.
Sympathetic neurons were treated with BMP-7 (5 ng/ml), IFN (10 ng/ml), or BMP-7 and IFN. On days 8 and 15, cellular morphology
(n 60 cells/condition) was visualized by
immunostaining with mAb to MAP-2. A, Number of dendrites
per cell. B, Total dendritic length. C,
Number of MAP-2-positive cells per culture. *p < 0.05 versus BMP-7.
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Figure 3.
Dose-dependent inhibition of dendritic outgrowth
by IFN. The effects of varying concentrations of IFN were
assessed in cultures treated with either a maximally effective dose of
BMP-7 (50 ng/ml; open circles) or one close to the
ED50 (5 ng/ml; open triangles). After 5 d of cotreatment with BMP-7 and IFN cultures were immunostained with
mAb to MAP-2.
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IFN did not affect the morphological development of sympathetic
neurons grown under control conditions (Fig. 1). However, it profoundly
depressed the initial dendritic outgrowth that occurs in the presence
of BMP-7 (Fig. 1). In cultures that were treated with a maximally
effective dose of BMP-7 (50 ng/ml), there was a 53% decrease in the
number of dendrites per cell in the presence of IFN (Fig. 3). In
cultures treated with lower doses of BMP-7 (5 ng/ml), the
effect was even greater (78% inhibition). Under these conditions total
dendritic length also was reduced (Fig. 2). Inhibition was observed
with concentrations of IFN as low as 0.1 ng/ml; the ID50
(dose causing half-maximal inhibition) was ~1 ng/ml (Fig. 3).
IFN-induced inhibition of initial dendritic growth was long lasting,
with no sign of reversal even after 2 weeks of continuous exposure
(Fig. 2), and IFN also blocked the effects of BMP-2 and BMP-6 (data
not shown), suggesting that it interacts with a common component of the
BMP signaling pathway.
IFN inhibits synapse formation and dendritic growth in
hippocampal neurons
To determine whether IFN also inhibits dendritic growth in CNS
neurons, we examined its effects on cultured embryonic hippocampal neurons. These neurons differ from sympathetic neurons in that they
spontaneously form dendrites in the absence of exogenous BMPs. Exposure
to IFN reduced hippocampal dendritic growth by 36% (Fig.
4B). In addition,
treatment with IFN caused a 37% decrease in the number of
SV-2-positive (Fig. 4C) and synaptophysin-positive (data not
shown) varicosities associated with MAP-2-positive dendrites. Thus in
CNS neurons the IFN-induced inhibition of dendritic growth is
associated with a proportionate decrease in the formation of synapses.
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Figure 4.
IFN inhibits dendritic growth and
synaptogenesis in hippocampal neurons. Hippocampal neurons were grown
in the absence or presence of IFN (30 ng/ml) for 5 d.
A, Fluorescence micrograph of neuron grown in control
medium. Dendritic morphology (n > 25 cells/condition) and presynaptic specializations were analyzed by
double-labeled immunostaining with antibodies to MAP-2
(red) and SV-2 (green).
Cells were examined with a Bio-Rad confocal microscope, using 1 µm
optical sections. SV-2-positive puncta that are associated with
dendrites have been shown previously to represent sites of synaptic
contact (Fletcher et al., 1991). Scale bar, 10 µm. B,
Total dendritic length. C, Synapses per neuron.
*p < 0.05 versus control.
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IFN induces retraction of existing dendritic process
In addition to examining the effects of IFN on initial process
outgrowth, we also wanted to know whether it affected existing processes. Therefore, dendritic growth was initially induced in cultures of sympathetic neurons by a 5 d exposure to BMP-7; this was followed by treatment for 6 d with either BMP-7 alone or BMP-7 with IFN (Fig. 5). In cells treated
with an intermediate dose of BMP-7 (5 ng/ml), IFN caused an ~90%
decrease in both the number of dendrites per cell and in total
dendritic length. Dendritic retraction was also prominent in cultures
exposed to a maximally effective dose of BMP-7, with ~80% of the
processes being eliminated in the presence of IFN. Thus the effect
of IFN on dendritic retraction is at least as great as that on
initial extension. Time course studies revealed that dendritic
retraction was a slow process that began 24-48 hr after initial IFN
exposure and required at least 4 d for completion (data not
shown).
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Figure 5.
IFN induces dendritic retraction. Sympathetic
neurons were treated initially with 5 ng/ml BMP-7 (A, B,
D) or 20 ng/ml (C) for 5 d.
Subsequently, they were divided into two groups. One was treated
continuously with BMP-7, and the other was treated with both IFN (10 ng/ml) and BMP7 for an additional 6 d. Dendritic morphology was
assessed by immunostaining with mAb to MAP-2 (n 60 cells/condition). A, C, Number of dendrites per cell.
B, Total dendritic length. D, Number of
MAP-2-positive cells per culture. *p < 0.05 versus
BMP-7 on day 5.
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IFN does not affect axonal growth or cell number
To assess the specificity of action of IFN, we first examined
its effect on the survival of sympathetic neurons. In all experiments neuronal number remained essentially constant (Figs. 2, 5), indicating that IFN was not acting by promoting the survival of a subpopulation of neurons. To assess specificity further, we examined the effects of
IFN on axonal growth. To allow induction of IFN-sensitive genes,
explants of sympathetic ganglion were initially maintained for 2 d
in the presence or absence of IFN. After this pretreatment ganglia
were dissociated, and the cells were plated onto coverslips; treatment with either control medium or IFN was resumed. Axonal length was assessed after 12 hr of growth and found to be unaltered by
exposure to IFN (Fig. 6).
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Figure 6.
Lack of effect of IFN on axonal growth.
A, Explants of superior cervical ganglia were maintained
for 2 d in the presence or absence of IFN (10 ng/ml).
Subsequently, the ganglia were dissociated, and cells were plated onto
coverslips coated with laminin (2 µg/ml). Cells then were exposed
continuously to control medium or IFN for 12 hr. Axons were
immunostained with mAb that recognizes phosphorylated forms of H and M
neurofilament subunits. Cellular morphology was analyzed using Image
software (n 60 cells/condition).
B, Cultures were treated with BMP-7 (5 ng/ml) for 8 d; during the last 3 d of treatment some were also exposed to
IFN (10 ng/ml). Expression of cytoskeletal proteins was detected by
Western blotting with mAb to tau and a polyclonal antibody to actin.
Equal amounts of protein were loaded into each lane.
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To explore further the possibility that IFN selectively affects
dendritic processes, Western blot analysis was used to compare the
expression of cytoskeletal proteins (Fig. 6). The expression of tau,
which is found primarily in axons, was not affected by IFN. The
expression of actin, which is involved in both dendritic and axonal
development, was elevated by treatment with BMP-7 and only slightly
decreased by IFN, as was the expression of the dendritic protein
MAP-2 (data not shown). Moreover, the total protein content of control
and BMP-7-treated cultures was unaffected by IFN exposure (data not
shown), indicating that overall cellular health is not affected by
IFN. These results suggest that IFN selectively affects dendritic outgrowth.
Comparison of the effects of IFN and other cytokines
Like IFN, TNF, IL-1, and IFN have been detected at
sites of inflammatory reactions in the nervous system (Taupin et al., 1993; Rothwell and Hopkins, 1995; Ensoli et al., 1999). We therefore compared their effects on dendritic growth. Neither inhibition of
initial dendritic outgrowth (Table 1) nor
dendritic retraction (Fig. 7) was
observed with TNF, IL-1, or IFN. In addition, we found no
evidence for potentiation of IFN action by TNF or IL-1.
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Figure 7.
TNF does not potentiate the effect of IFN on
dendritic retraction. Sympathetic neurons were exposed to BMP-7 (5 ng/ml) for 9 d. Beginning on day 5, some of them were treated
additionally with IFN (10 ng/ml), TNF (10 ng/ml), or the
combination of both of the agents. On day 9, dendritic morphology was
assessed by immunostaining with mAb to MAP-2. *p < 0.05 versus BMP-7 on day 5.
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Involvement of STAT1 in dendritic retraction
Many cellular responses to IFN are mediated by activation of
the Janus kinase pathway causing the phosphorylation of STAT1, a
transcription factor (Farrar and Schreiber, 1993; Bach et al., 1997;
Schindler, 1999). To determine whether the effects of IFN on
dendrites required the activation of STAT1, we examined the distribution of STAT1 in sympathetic neurons. Immunostaining with an
antibody that recognizes phosphorylated STAT1 revealed that IFN
induced the phosphorylation and nuclear translocation of STAT1 (Fig.
8).
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Figure 8.
IFN induces phosphorylation and nuclear
translocation of STAT1. Sympathetic neurons were treated with BMP-7 (5 ng/ml) for 5 d. Subsequently, IFN (10 ng/ml) was added for 1 hr
to the medium of some cultures (C, D), whereas others
were treated with BMP-7 alone (A, B). The cellular
location of STAT1 was detected by immunostaining with an antibody that
reacts with a phosphorylated form of STAT1 (Tyr 701); the cells were
examined with a Bio-Rad confocal microscope, using 1 µm optical
sections. Shown are phase-contrast (A, C) and
fluorescence (B, D) micrographs.
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We next determined whether transfection with a dominant-negative mutant
of STAT1 that cannot be phosphorylated on Tyrosine 701 (Y701F)
(Nakajima et al., 1996) reversed the inhibitory effects of IFN on
dendritic outgrowth. Wild-type STAT1 or mutant STAT1 was cotransfected
with EGFP to identify the transfected cells. Forty-eight hours after
transfection cells were treated with BMP-7 (10 ng/ml) in the presence
or absence of IFN (20 ng/ml). Consistent with the previous
observation (Fig. 3), IFN decreased BMP-7-induced dendritic
outgrowth by ~80% in cultures transfected with control vector,
indicating that transfection by itself did not change the response of
sympathetic neurons to either BMP-7 or IFN (Fig. 9A). Overexpression of
wild-type STAT1 potentiated the inhibition by IFN and also slightly
decreased dendritic outgrowth that was induced by BMP-7. In contrast,
transfection of mutant STAT1 itself did not change the response of
BMP-7 but significantly reduced the inhibitory effect of IFN on
dendritic sprouting.
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Figure 9.
Dominant-negative STAT1 overcomes the inhibitory
effect of IFN on dendrites. Sympathetic neurons were cotransfected
with plasmids containing the genes for EGFP and
(A) wild-type (STAT1 WT) or
mutant STAT1 (STAT1 F) or
(B) wild-type or mutant STAT3 or mutant STAT1 by
Lipofectamine reagent. After 48 hr the cells were treated with BMP-7
(10 ng/ml), IFN (20 ng/ml), or BMP-7 and IFN. On day 6, cellular
morphology was visualized by immunostaining with mAb to MAP-2.
Transfected cells were assessed by the expression of EGFP.
*p < 0.05 versus BMP-7 and IFN in the culture
transfected with the control vector.
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Although IFN predominantly activates STAT1, it also affects STAT3 in
some cells (Stephens et al., 1998). Moreover, we observed previously
that the activation of STAT3 by LIF blocks dendritic growth in
sympathetic neurons (Guo et al., 1999). We therefore determined whether
STAT3 plays a role in IFN-induced dendritic inhibition.
Overexpression of mutant STAT3 did not affect the dendritic inhibition
caused by IFN (Fig. 9B). In addition, IFN did not
induce nuclear translocation of STAT3 in sympathetic neurons (data not
shown). These data strongly suggest that the inhibitory effects of
IFN on dendritic growth require the STAT1 activation and thus
transcriptional activity.
Dendrites retract in response to IFN applied selectively to
distal axons
Because many inflammatory reactions occur in white matter,
elevated levels of IFN often are found in the vicinity of axons at a
substantial distance from neuronal somata. Under these conditions IFN signaling must be retrogradely conveyed to the cell body if it
is to affect dendritic morphology. To examine this possibility, we used
compartmented cultures originally developed by Campenot (Campenot,
1977; Senger and Campenot, 1997). This culture system allows physical
separation of the medium that is bathing cell bodies and proximal
processes from the medium that is bathing distal axons and axon
terminals (Fig. 10A).
Thus IFN can be applied selectively to distal axons and terminals.
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Figure 10.
Dendritic retraction in response to retrograde
signaling by IFN. A, Schematic representation of the
three-compartment culture of sympathetic neurons. Dissociated neurons
were plated in the Center compartment, and axons
extended into the Left and Right
compartments. B, Negative control cultures were
maintained in control medium (C2) in
the absence of BMP-7 or IFN; positive control and experimental
cultures were exposed to BMP-7 (10 ng/ml) added to C2 in
the Center compartment for 7 d. Three days after
the addition of BMP-7, IFN (10 or 50 ng/ml) was added to the
C2 in the Right compartment of experimental
cultures (indicated by the arrow in C).
Then 4 d later, dendritic growth was quantified in cultures
immunostained with mAb to MAP-2 (n 60 cells/condition). Addition of IFN to the distal axons and axon
terminals caused a dose-dependent reduction in total dendritic length
(C). *p < 0.05 versus BMP-7 on day
7.
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Neurons plated into the center compartment extend axons into the left
and right compartments within 5-7 d, and extensive axonal growth is
evident in the side chambers within 10-12 d after plating. Neurons
maintained in control medium do not extend dendrites; in contrast,
neurons whose cell bodies are exposed to BMP-7 (10 ng/ml) exhibit
significant dendritic growth (Fig. 10C). Subsequent addition
of IFN (10 or 50 ng/ml) to the medium of the right compartment, which contains distal axons and axon terminals (Fig.
10B), elicits a dose-dependent reduction in the total
dendritic length of those neurons that extend axons into the right
compartment (Fig. 10C). Neurons that extended axons into the
right compartment were identified by the uptake and retrograde
transport of fluorescent beads (Fluorobeads, F8795, 8 µl/ml;
Molecular Probes, Eugene, OR) added to the right chamber 12-16 hr
before the cultures were fixed and immunostained for dendritic growth.
Comparative analyses of dendritic growth in fluorescently labeled
neurons versus nonlabeled neurons (e.g., neurons that extend axons only
into the left compartment) indicated that dendritic growth in the
nonlabeled neurons was not decreased in response to the addition of
IFN to the right compartment (data not shown). This observation
confirms that the effect of IFN on labeled neurons was not mediated
by IFN leaking into the center compartment.
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DISCUSSION |
Expression of IFN is elevated in murine trisomy 16, a model of
human Down's syndrome (Torre et al., 1995; Hallam et al., 2000), and
overexpression of IFN induces cerebellar and hippocampal malformations in transgenic mice (Corbin et al., 1996; LaFerla et al.,
2000). IFN is known to regulate the maturation of glia (Merrill et
al., 1993; Popko et al., 1997; Benn et al., 2001), and so it is
possible that the developmental defects induced by IFN exposure
arise secondarily from its actions on these cells. Our results
indicate, however, that IFN also has direct inhibitory effects on
neurons and adversely affects their growth in the absence of
non-neuronal cells. These observations suggest a novel mechanism by
which IFN interferes with neural development, and they contrast with
previous studies that have reported primarily beneficial effects of
IFN on the growth and differentiation of isolated neural cell
populations (Erkman et al., 1989; Barish et al., 1991; Jonakait et al.,
1994).
Exposure to IFN produced a profound and long-lasting inhibition of
BMP-7-induced dendritic growth in cultures of sympathetic neurons. This
effect did not reflect compromised cellular viability or health because
it was not associated with changes in cell number, axon elongation, or
the expression of tau, a protein found primarily in axons (Binder et
al., 1985; Brion et al., 1988). Rather, IFN had a selective
morphogenic effect on the development of a restricted cellular
compartment. Although smaller in magnitude, a qualitatively similar
effect of IFN was observed on the growth of dendrites in cultures of
hippocampal neurons, a population of neurons that was not exposed to
BMP-7 and spontaneously forms dendrites in its absence (Banker and
Waxman, 1988). Thus it appears that IFN inhibits dendritic growth in
cells derived from both the central and peripheral nervous systems and
that it affects both BMP-7-induced and spontaneous dendritic growth. It
is, therefore, likely that IFN has widespread effects on dendritic
growth in the nervous system.
Dendrites are the primary site of synapse formation in the mammalian
nervous system (Purves, 1988), and so agents that inhibit dendritic
growth would be expected to reduce synapse formation. Although
sympathetic neurons form functional synapses with each other in tissue
culture, they do not make such connections with each other in
vivo (O'Lague et al., 1974; Higgins et al., 1981). We therefore
did not think it appropriate to examine the effect of cytokines on
synapse formation in the cultured sympathetic neurons. On the other
hand, hippocampal neurons are known to form synaptic contacts with one
another in vivo (Swanson et al., 1987) and they also do so
in vitro (Bartlett and Banker, 1984; Fletcher et al., 1991).
In such cultures, treatment with IFN caused a reduction in the
number of SV-2-positive and of synaptophysin-positive varicosities that
were associated with MAP-2-positive dendrites. Because such
varicosities have been found previously to represent sites of
transmitter uptake and release (Fletcher et al., 1991; Matteoli et al.,
1992; Pyle et al., 1999), these observations indicate that IFN
reduces synapse formation, at least in neurons from the CNS. In
addition, Vikman et al. (2001) have reported recently that, in mixed
neural glial cultures, IFN exposure also causes a decrease in AMPA
receptor clustering, suggesting that IFN-induced decreases in
synapse number may be aggravated by other functional changes.
In addition to having effects on initial process outgrowth, prolonged
exposure to IFN also causes sympathetic neurons to retract most of
their existing dendrites, leading to an ~90% decrease in the size of
the dendritic arbor. These effects were observed with concentrations of
IFN similar to those detected after viral infection in humans and
mice (Lebon et al., 1988; Weller et al., 1991; Rockstroh et al., 1998).
IFN plays a critical role in viral clearance from the CNS (Binder
and Griffin, 2001) and chronic viral infections, such as those
associated with HIV, lead to long-term increases in the expression of
IFN (Fuchs et al., 1991; Fan et al., 1993). Such chronic infections
also induce dendritic atrophy (Masliah et al., 1997; Everall et al.,
1999; Montgomery et al., 1999). It is, therefore, possible that IFN
has a dual-edged effect in the response to viral infection: it promotes
clearance of virions but also contributes to the pathology, and
especially the dementia, by inducing dendritic retraction. In addition,
it is important to note that elevated IFN expression (Kiefer et al.,
1991; Kristensson et al., 1994; Lau and Yu, 2001; Li et al., 2001)
occurs coincident with dendritic retraction (Sumner and Watson, 1971;
Purves, 1975; Yawo, 1987; Kudo et al., 1993; Park et al., 1996) in many
types of acute inflammatory reactions, including those triggered by trauma, stroke, and axotomy.
Many of the effects of IFN are mediated by activation of the STAT1
signaling pathway (Farrar and Schreiber, 1993; Bach et al., 1997;
Schindler, 1999). However, there is also evidence for the involvement
of STAT3 in some IFN actions (Stephens et al., 1998) as well as for
STAT-independent effects (Ramana et al., 2000; Gil et al., 2001). Our
examination of the pathways involved in IFN-induced dendritic
retraction revealed that IFN stimulated the phosphorylation and
nuclear translocation of STAT1, but not STAT3, in cultures of
sympathetic neurons. In addition, dendritic retraction was blocked by
expression of a dominant-negative STAT1 construct, but not by a similar
STAT3 construct, strongly suggesting that the former is the primary
signaling pathway involved in process elimination. We reported
previously that members of the IL-6 family of cytokines induce
dendritic retraction via activation of STAT3 (Guo et al., 1999). Thus
there appear to be independent routes by which these two classes of
cytokines can cause dendritic atrophy, and this raises the possibility
of synergistic interactions. In this respect it is important to note
that there is frequent coexpression of IFN with IL-6-related
cytokines in inflammatory reactions. For example, astrocytes and
Schwann cells release IFN and IL-6 after traumatic injury or
ischemia (Banner and Patterson, 1994; Curtis et al., 1994; Sun et al.,
1996; Lau and Yu, 2001). The pathways leading from STAT activation to
dendritic retraction are unclear, but there is reason to suspect the
involvement of the paralogous transcriptional regulators p300 and CREB
binding protein (CBP). BMP-induced dendritic growth requires the
phosphorylation and nuclear translocation of SMAD1 (Guo et al., 2001),
and activated SMADs are known to bind to p300 (Janknecht et al., 1998;
Kawabata et al., 1999). Activated STATs also bind p300, and the
expression of type I collagen that is induced by SMAD3 is negatively
regulated by STAT1 activation (Ghosh et al., 2001). Thus competition
between activated STATs and SMADs for interaction with limiting amounts of cellular p300/CBP may account for some of the effects of IFN on
dendritic growth in sympathetic neurons.
Target-derived growth factors regulate the development of many, if not
most, classes of neurons (Oppenheim, 1991; Korsching, 1993). Such
agents typically signal in a retrograde manner, with information being
conveyed from distal axons to neural somata. Previous studies have
focused on the beneficial nature of retrograde signals and have
emphasized their role in promoting neuronal survival (Hendry and
Campbell, 1976; Chun and Patterson, 1977) and differentiation (Hendry,
1977) and in stimulating initial process extension (Campenot, 1977,
1987) and regeneration (Smith and Skene, 1997; Cafferty et al., 2001).
In contrast, observations of sympathetic neurons grown in compartmented
Campenot chambers indicate that, when IFN was applied to
compartments containing distal axons, it led to dendritic retraction in
compartments containing cell bodies and proximal processes. This is a
novel variant of retrograde interaction in which cytokine-induced
signals that are generated in axon terminals cause atrophy of other
parts of the cell. It implies that retrograde interactions with
proinflammatory cytokines can be "dystrophic" and can lead to
elimination of distant processes and their associated synaptic contacts.
Retrograde dystrophic interactions could play an important role in
neural disease because they convey information about injury or
inflammation in one part of the brain to another, thereby eliciting reactive changes in distal regions. For example, axotomy induces the
expression of IFN (Kiefer et al., 1991; Kristensson et al., 1994)
and also causes dendritic retraction (Purves, 1975; Yawo, 1987).
Because retrograde effects of IFN induce dendritic retraction in vitro, it seems likely to contribute to this process
in vivo. Similarly, in MS, IFN is highly expressed in
demyelinating lesions in white matter (Traugott and Lebon, 1988; Popko
et al., 1997), whereas dendritic atrophy has been observed in gray
matter (Peterson et al., 2001). Retrograde effects of IFN acting on
distal axons provide a potential mechanism for linking these phenomena
and for explaining some of the cognitive deficits that are manifest in
this disease (Rao et al., 1991; Ron et al., 1991). The mechanisms underlying such retrograde interactions are currently unclear, and it
will be important to determine whether they involve retrograde transport of IFN, its receptor, STAT1, or some combinations of these
signaling elements (Ahn et al., 2000).
| |
FOOTNOTES |
Received Dec. 3, 2001; revised March 14, 2002; accepted March 18, 2002.
This study was supported by National Science Foundation Grant IBN
01-21210 and a Johns Hopkins University Bloomberg School of Public
Health Faculty Innovation Award (P.L.). We thank Dr. Toshio Hirano and
Dr. Katsuhiko Ishihara for kindly providing plasmids carrying wild-type
and mutant STAT1 and STAT3 genes. The antibody to SV-2 was obtained
from the Developmental Studies Hybridoma Bank at University of Iowa
(Department of Biological Sciences, Iowa City, IA).
Correspondence should be addressed to Dr. In-Jung Kim, Department of
Pharmacology and Toxicology, State University of New York at Buffalo,
102 Farber Hall, 3435 Main Street, Buffalo, NY 14214. E-mail:
ikim{at}acsu.buffalo.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
