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Vol. 14, Issue 7, 2665-2676, July 2003
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*Laboratory of Neurobiology and Genetics, The
Rockefeller University, New York, New York 10021; and
Department of Pharmacology,
MSTP Program, University at Stony Brook, Stony
Brook, New York 11794-8651
Submitted December 18, 2002;
Revised February 12, 2003;
Accepted March 3, 2003
Monitoring Editor: Mark Ginsberg
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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5,
1,
1) protects against neuronal death. To investigate
how laminin is involved in neuronal viability, we infused laminin-1
(1,1,1) into the mouse hippocampus. This infusion
specifically disrupted the endogenous laminin layer. This disruption was at
least partially due to the interaction of the laminin-1 1 chain with
endogenous laminin-10, because infusion of anti-laminin 1 antibody had
the same effect. The disruption of the laminin layer by laminin-1 1) did not
require the intact protein because infusion of plasmin-digested laminin-1 gave
similar results; 2) was posttranscriptional, because there was no effect on
laminin mRNA expression; and 3) occurred in both
tPA–/– and
plasminogen–/– mice, indicating
that increased plasmin activity was not responsible. Finally, although
tPA–/– mice are normally
resistant to excitotoxin-induced neurodegeneration, disruption of the
endogenous laminin layer by laminin-1 or anti-laminin 1 antibody
renders the tPA–/– hippocampal
neurons sensitive to kainate. These results demonstrate that neuron
interactions with the deposited matrix are not necessarily recapitulated by
interactions with soluble components and that the laminin matrix is a dynamic
structure amenable to modification by exogenous molecules. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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chains, three
chains, and three chains have been identified, and of the possible 45
potential trimeric molecules that could be generated from these chains, 15
have been observed (Colognato and
Yurchenco, 2000
; Grimpe et
al., 2002). Laminins are known to play an important role in
the nervous system (Venstrom and
Reichardt, 1993), for example, in the neuromuscular junction
(Noakes et al., 1995;
Patton et al., 1998;
Patton et al., 2001;
Sanes and Lichtman, 2001), in
brain development (Luckenbill-Edds,
1997; Miner et al.,
1998; Colognato and Yurchenco,
2000; Liesi et al.,
2001), and in pathology
(Murtomaki et al.,
1992). Laminins have been found in the adult brain, but the role
for these proteins in the adult central nervous system is not well established
(Hagg et al., 1989;
Jucker et al., 1996;
Hagg et al., 1997;
Tian et al.,
1997).
There are some studies that bear on the role of the ECM in adult central
nervous system function. In the mouse hippocampus, injection of glutamate
analogs, known as excitotoxins, can cause massive neuronal death
(Coyle et al., 1978).
It has been shown that mice deficient in the protease tissue plasminogen
activator (tPA) or its zymogen substrate plasminogen are resistant to this
excitotoxin-induced death (Tsirka et
al., 1995; Tsirka et
al., 1997), implicating this extracellular proteolytic system
in neuronal degeneration. Further experiments showed that these proteases
affect neuronal viability by degrading the laminin matrix that is associated
with the neurons (Chen and Strickland,
1997; Nagai et al.,
1999). Thus, there is evidence that in the mouse brain neurons
depend to some extent for survival on their interaction with the ECM, as
reported in other systems (Meredith et
al., 1993; Boudreau et
al., 1995; Coucouvanis and
Martin, 1995; Lukashev and
Werb, 1998; Frisch and
Screaton, 2001).
It is thought that the supramolecular structure of the ECM is maintained by
binding interactions between the various components, such as laminins,
collagens, etc., both to themselves and to other molecules
(Colognato and Yurchenco,
2000). The potential complexity of these interactions makes it
difficult to recapitulate ECM structure and function in cell culture.
Furthermore, there is evidence that cells interact differently with soluble
components of the ECM compared with their interaction with the same molecules
in the context of an insoluble matrix
(Hayman et al.,
1985). Thus, it is important to design ways to evaluate the
maintenance and function of ECM structures in vivo.
In studies on laminin expression in the mouse hippocampus, we observed that
the prominent, endogenous laminin-10 matrix
(Indyk et al., 2003)
was disrupted by infusion of soluble mouse laminin-1. In this report, we
analyze the mechanism of this observation. Our results indicate that in the
hippocampus, laminin is loosely held in the matrix and can be displaced by
competition with its soluble counter-part. In contrast to the deposited,
insoluble material, the soluble laminin does not protect neurons from
excitotoxic death. The disruption of the laminin matrix sensitizes neurons to
kainate-induced death, further establishing the critical role for the laminin
matrix in neuronal survival.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), or plasminogen-deficient
(plg–/–)
(Bugge et al., 1995;
Ploplis et al., 1995)
male mice (both tPA–/– and
plg–/– were) backcrossed to
C57Bl/6 for eight generations. The mice were injected intraperitoneally with
atropine (0.6 mg/kg body weight) and then were anesthetized deeply with 2.5%
avertin (0.02 ml/gm of body weight). They were placed in a stereotaxic
apparatus (Stoelting, Wood Dale, IL). A microosmotic pump (Alza, Palo Alto,
CA) containing 100 µl of phosphate-buffered saline (PBS) (for control
animals), 100 µl of purified mouse laminin-1 (0.5 mg/ml except 0.1 mg/ml
for one lower dose experiment; Sigma-Aldrich, St. Louis, MO), 100 µl of
anti-laminin 1 polyclonal antibody (0.2 mg/ml; Santa Cruz
Biotechnology, Santa Cruz, CA), 100 µl of plasmin-digested laminin-1 (0.5
mg/ml), or control buffers used for laminin digestion were placed
subcutaneously in the back of the animals. A brain infusion cannula connected
to the pump was positioned at coordinates bregma, –2.5 mm;
medial-lateral, 0.5 mm; and dorsoventral, 1.6 mm to deliver the compound into
the hippocampus. The infusion rate was 0.5 µl/h. The pumps were allowed to
infuse the designated solution for 7 d, and the animals were anesthetized and
perfused through the heart with PBS, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. The brains were removed, postfixed in the same
fixative overnight at 4°C, and then left in 30% sucrose in PBS for 48 h at
4°C. Coronal brain sections (30 µm) were cut on a microtome, collected
in PBS, and then processed for cresyl violet, Fluoro Jade B staining, and
immunohistochemistry. For the kainate injection experiments, the infusion
pumps were allowed to infuse for 5 d and then kainate was injected as
described below. Only mice that showed efficient infusion were used.
Intrahippocampal Injections
Adult mice as indicated in each experiment were anesthetized and infused
with PBS, control buffer, mouse laminin-1, plasmin-digested laminin-1, or
anti-laminin 1 antibody for 5 d as described above. Then a total volume
of 300 nl of 0.83 mM kainate (Tocris Cookson, Ellisville, MO) for a dose of
0.25 nmol was delivered unilaterally using a microinjection apparatus
(Stoelting) via a 2.5 µl-Hamilton syringe equipped with a 33-gauge needle,
over the course of 60 s. In our previous study
(Tsirka et al.,
1995), we injected 1.5 nmol of kainate (Sigma-Aldrich). This
kainate preparation is discontinued, and the new kainate preparation (Tocris
Cookson) is more potent by weight. Injection of 0.25 nmol of kainate induced
similar neurodegeneration in wild-type mice to the 1.5 nmol used previously,
and therefore this lower dose was used in the experiments reported herein.
After retracting the needle 0.1 mm, the needle was kept in place for 2 min to
allow diffusion of the kainate, and then was completely removed. The area was
then cleaned and the wound sutured. To control for mechanical damage,
injections were performed with vehicle alone (PBS) and consistently showed no
neuronal death in the hippocampus (our unpublished data). Two days after the
injection, the animals were sacrificed and their brains were processed as
described above.
Fluoro Jade B Staining
Fluoro Jade B staining was performed as described previously
(Schmued and Hopkins, 2000).
Briefly, free-floating brain sections were mounted onto slides, dried at
55°C for 2 h, and incubated at room temperature in the following baths: 1%
NaOH in 80% ethanol, 5 min; 70% ethanol, 2 min; H2O, 2 min; 0.06%
potassium permanganate, 10 min; H2O, 2 min; 0.0004% Fluoro Jade B
(Histo-Chem, Jefferson, AR), 20 min; and three H2O washes, 1 min
each. Slides were then dried, immersed in Histoclear, and coverslipped with
DPX mounting medium (Sigma-Aldrich). Fluorescence was visualized using a
fluorescein isothiocyanate filter on an Axioskop2 microscope (Carl Zeiss,
Thornwood, NY).
Immunohistochemistry
Mouse brain sections, manipulated as described above, were incubated with
1) affinity-purified rabbit anti-mouse laminin polyclonal antibody
(Sigma-Aldrich), at 1:1000 dilution; this antibody has previously been shown
to react with both the 1 and 1 chains of laminin-1
(Chen and Strickland, 1997); 2)
affinity-purified rabbit anti-M2 muscarinic acetylcholine receptor polyclonal
antibody (Chemicon International, Temecula, CA), at 1:400 dilution; 3) rabbit
anti-mouse fibronectin polyclonal antibody (Chemicon International), at 1:500
dilution (our unpublished data; control for
Figure 2D). Biotinylated
secondary antibodies were used (Vector Laboratories, Burlingame, CA), and the
avidin-biotin-peroxidase complex (ABC reaction) was visualized using a Nova
Red kit (Vector Laboratories).
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Imaging Analysis
The brain sections through the hippocampus after staining were photographed
using an AxioVision System. The images were processed using Photoshop 5.5 and
figures were prepared using PowerPoint.
Laminin-1 Digestion and SDS-PAGE Analysis
Laminin-1 was purchased from either Sigma-Aldrich or Chemicon
International. For plasmin digestion, laminin-1 was incubated in 100 µg/ml
human plasminogen, 4.8 µg/ml recombinant tPA, 20 mM Tris-HCl, pH 7.2, 150
mM NaCl, 1 mM CaCl2 at 37°C for 4 h. To assess the ability of
tPA to cleave this protein directly, laminin-1 was incubated in the
above-mentioned buffer without human plasminogen. After digestion, the
solutions were denatured at 100°C for 10 min in 100 mM dithiothreitol
(DTT), and then dialyzed against PBS. Residual tPA and plasminogen or plasmin
activity was checked by casein zymography, and revealed that this procedure
completely destroyed tPA and plasminogen or plasmin activity (our unpublished
data). Intact or digested laminin was denatured at 100°C for 10 min in 100
mM DTT and run on a reducing 10–20% gradient SDS-PAGE, stained with
Coomassie Blue, and then destained. The gel was then scanned into
Photoshop.
In Situ Hybridization
mRNA in situ hybridization was performed according to Chen et al.
(1995). DNA clones for mouse
laminin 1 were obtained from Drs. J.H. Miner and J.R. Sanes (Washington
University, St. Louis, MO), and the plasmid DNA was sequenced to verify the
correct insert sequence (Indyk et
al., 2003). Digoxigenin (DIG)-labeled sense or antisense RNA
probes (Roche Applied Science, Indianapolis, IN) were synthesized from laminin
1 chain-specific clones by using T3- or T7-polymerase transcription.
Probes were purified by LiCl/EtOH precipitation, and correct probe size was
verified by formaldehyde gel electrophoresis. In situ hybridizations were
performed on mouse brain sections. Briefly, the animals were anesthetized and
perfused through the heart with diethyl pyrocarbonate (DEPC)-treated PBS,
followed by DEPC-treated 4% paraformaldehyde in 0.1 M phosphate buffer, pH
7.4. The brains were removed, postfixed in the same fixative overnight at
4°C, and then left in DEPC-treated 30% sucrose in PBS for 48 h at 4°C.
Coronal brain sections (30 µm) were cut on a microtome, collected in
DEPC-treated PBS, and kept in 4°C. For prehybridization treatment, the
brain sections were washed in DEPC-treated PBS for 4 times for 5 min each,
followed by two 5-min incubations in DEPC-treated PBS containing 100 mM
glycine. The brain sections were then treated with DEPC-treated PBS containing
0.3% Triton X-100 for 15 min and washed in DEPC-treated PBS two times for 5
min each. The sections were permeabilized in 100 mM Tris-HCl, 50 mM EDTA, pH
8.0, 15 µg/ml RNase-free proteinase K for 30 min and postfixed for 5 min in
DEPC-treated PBS containing 4% paraformaldehyde. The sections were then washed
in DEPC-treated PBS four times for 5 min each and incubated in 0.1 M
triethanolamine buffer, pH 8.0, containing 0.25% acetic anhydride two times
for 5 min each. The sections were then prehybridized in 4x SSC
containing 50% deionized formamide for 30 min and then incubated overnight at
55°C in hybridization solution containing RNA probe (sense or antisense, 1
µg/ml hybridization solution), 10% dextran sulfate, 20 mM Tris HCl, pH 8.0,
300 mM NaCl, 0.2% sarcosyl, 1x Denhardt's solution, 1 mg/ml salmon sperm
DNA, 1 mg/ml yeast tRNA, 40% formamide, 10 mM DTT. Posthybridization washes at
65°C consisted of 4x SSC and 50% formamide in 2x SSC, followed
by 37°C washes in 10 mM Tris HCl, 1 mM EDTA, 500 mM NaCl. Sections were
then treated with 20 µg/ml RNase A in RNase buffer (10 mM Tris HCl, 1 mM
EDTA, 500 mM NaCl) for 30 min at 37°C, followed by washes in RNase buffer,
50% formamide in 2x SSC at 65°C, and buffer 1 (0.1 M Tris HCl, pH
7.5, 150 mM NaCl). Sections were then blocked with 1.5% blocking reagent
(Roche Diagnostics) in buffer 1 and incubated overnight with anti-DIG antibody
(1:500 anti-DIG Fab fragment-AP conjugated; Roche Applied Science) and 1%
blocking reagent in buffer 1. After several washes in 0.1 M Tris HCl, pH 7.5,
150 mM NaCl, sections were washed in buffer 3 (100 mM Tris-HCl, pH 9.5, 100 mM
NaCl, 50 mM MgCl2) and color reaction performed in the dark by
using 75 mg/ml nitro blue tetrazolium and 50 mg/ml 5-bromo-4-chloro-3-indolyl
phosphate (Roche Applied Science) in buffer 3. Reaction was stopped by washes
in buffer 3 and TE, and sections coverslipped with 10 mM Tris, 1 mM disodium
EDTA buffer/glycerol and photographed. Sense probes were used as negative
controls in all experiments.
Quantification of hippocampal neuronal laminin layers and neuronal loss
C57Bl/6 mice were infused into the hippocampus with intact soluble laminin (n
= 5) or plasmin digested laminin (n = 5) for 7 d, and their brains were
processed for laminin immunostaining. tPA-deficient mice were infused with
control buffer (n = 6), or intact soluble laminin-1 (n = 5), or
plasmin-digested laminin (n = 5) for 5 d, and kainate was injected. Two days
after the kainate injection their brains were processed for cresyl violet
staining. We used camera lucida tracings to quantitate the lengths of neuronal
laminin layers or the numbers of neuronal cell loss
(Tsirka et al., 1995;
Tsirka et al., 1997;
Chen et al., 1999).
Four sections from the hippocampus of each mouse in each group were matched,
and the linear lengths of disrupted neuronal laminin layers or dead pyramidal
cell layers were determined on each section. The values from each category
were averaged across the subjects in a group using the Sigma Plot program
(Jandel Scientific, Corte Madera, CA).
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Nakagami et
al., 2000). This laminin matrix is composed primarily of
laminin-10, containing the 5, 1, and 1 chains
(Indyk et al., 2003).
During excitotoxic challenge in the mouse hippocampus, tPA secretion and
expression is induced (Gualandris et
al., 1996; Parmer et
al., 1997; Baranes et
al., 1998), and due to the presence of plasminogen
(Sappino et al.,
1993; Tsirka et al.,
1997; Matsuoka et
al., 1998), increased plasmin activity is generated
(Chen and Strickland, 1997;
Tsirka et al., 1997).
This increased plasmin activity leads to the degradation of the laminin-10
matrix (Chen and Strickland,
1997; Indyk et al.,
2003) and is a critical component of neuronal death promoted by
excitotoxins.
In investigating the role of laminin in neuronal cell death, we infused
purified soluble mouse laminin-1 into the C57Bl/6 mouse hippocampus.
Surprisingly, infusion of soluble mouse laminin-1 disrupted the normal
structure of the endogenous laminin matrix in the hippocampal CA1 and dentate
gyrus (DG) neuronal cell layers (compare
Figure 1A, infused with buffer,
with B, infused with soluble laminin), whereas the laminin in CA3 was largely
unaffected. The reason for the lack of response in CA3 is unknown, but
previous work has noted that neurons in the CA3 region have stronger laminin
staining and are more resistant to neuronal death than those in CA1
(Chen and Strickland,
1997).
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To determine whether this laminin change was due to neuronal damage or neuronal cell death, we performed cresyl violet staining, which assesses cell viability. The infusion of soluble laminin did not change the neuronal morphology or cause any cell death (Figure 1, C and D). The fact that neurons did not show any dispersal upon disruption of the laminin layer (Figure 1C) indicates that they are fixed in place by intercellular and other matrix interactions and are not dependent on laminin alone for structural integrity.
To further confirm that the change in the laminin matrix was not due to neuronal cell death, we performed Fluoro Jade B staining, which sensitively identifies degenerating neurons. As a positive control for the method, we stained a kainate-injected hippocampus, which showed substantial neuronal death as evidenced by Fluoro-Jade B staining (Figure 1F). In contrast, there was no Fluoro-Jade B staining in the soluble laminin-infused hippocampus (Figure 1E), indicating no cell death. These results indicate that soluble laminin disrupted the endogenous laminin matrix in the hippocampus and that this disruption was not due to and did not lead to neuronal death.
One concern about this observation was the possibility that the large amount of soluble laminin in the infused sample might be exhausting the anti-laminin antibody used for the immunostaining. We considered this possibility unlikely, because laminin in the CA3 layer, which is resistant to disruption, was still stained by the antibody. Nevertheless, to assess the status of our antibody, we first stained laminin-infused sections identical to the method used for Figure 1B, recovered this primary antibody, and used it to stain untreated brain sections. The antibody showed normal staining of the control section (our unpublished data), indicating an abundance of anti-laminin antibodies, and ruling out antibody depletion as contributing to our observations. These results show that infusion of soluble laminin-1 can disrupt the laminin matrix in vivo.
Laminin-1 consists of 1, 1, and 1 chains, whereas the
principal laminin in the hippocampus is laminin-10
(Indyk et al., 2003),
with 5, 1, and 1 chains. It was therefore not clear how
laminin-1 was affecting the laminin-10 matrix. One possibility was that the
effects were mediated by either the 1 or 1 chains, which are
common subunits of both laminin-1 and laminin-10. To test this possibility,
antibody against the 1 chain was infused into the hippocampus. This
antibody was capable of disrupting the endogenous laminin matrix
(Figure 2B, compare with A),
indicating that 1 interactions are important in maintaining this
matrix.
To determine whether soluble laminin infusion also affected other proteins
expressed in the mouse hippocampus, we examined the muscarinic acetylcholine
receptor m2, which is highly expressed in the hippocampus
(Levey et al., 1991).
After laminin-1 infusion, sections stained with anti-muscarinic acetylcholine
receptor m2 antibody showed normal expression of this protein in the CA1
region (Figure 2C).
To determine whether infusion of another ECM protein would affect
endogenous laminin, we infused purified fibronectin into the mouse
hippocampus. The infusion was efficient as revealed by anti-fibronectin
antibody staining (our unpublished data), but had no effect on the laminin
matrix (Figure 2D). These
results together indicate that the effects observed are specific with respect
to the infused laminin and the targeted laminin matrix, and are due at least
in part to interactions with the 1 chain.
Plasmin-digested Laminin Disrupts the Laminin Matrix
To investigate the structural requirements for the effect of infused
laminin, we examined whether digestion of laminin-1 with plasmin would affect
its ability to disrupt endogenous laminin when infused into the mouse
hippocampus. We first digested purified laminin-1 with plasmin. Our previous
studies have shown that after plasmin digestion, there was a lack of
intermediate bands except for two bands at around 80- and 20-kDa when
visualized by anti-laminin antibody staining
(Chen and Strickland, 1997). It
was possible that the antibody could not recognize the digested fragments, and
therefore other intermediate bands could not be visualized. To address this
question, we electrophoresed both intact and plasmin-digested laminin on
SDS-PAGE, and stained the gel with Coomassie Blue. Laminin-1 occurred as an
1 band at 
400 kDa, and a combined 11 band at 220
kDa (Figure 3A, lane 2). After
digestion with tPA alone, laminin-1 remained intact, demonstrating that tPA
was not able to cleave laminin-1 (Figure
3A, lane 3). However, in the presence of both tPA and plasminogen,
laminin-1 was digested to multiple intermediates. The 1 chain was
completely cleaved (compare lane 4 with lane 2). For the 11 band,
there was some undigested material, which could be due to incomplete digestion
or resistance of one of the chains to plasmin cleavage. However, when
laminin-1 was digested for longer times, the 11 bands were
completely degraded, indicating that both chains are susceptible to plasmin.
These results clearly show that plasmin can cleave laminin-1 at multiple
sites.
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To determine whether the effect of soluble laminin-1 was dependent on its heteromeric structure, plasmin-digested laminin-1 was infused into the hippocampus. The plasmin-digested material was equally capable of disrupting the laminin matrix as the intact soluble molecule (Figure 3, compare B and C). Quantification of these results showed the following percentages of neuronal disruption: intact laminin, 59.4 ± 5.5; plasmin-digested laminin, 61.3 ± 4.8. Thus, the effect of laminin-1 does not require the intact molecule.
Soluble Laminin Does Not Affect Laminin mRNA
The above-mentioned results showed that infusion of soluble laminin-1
specifically disrupts endogenous laminin in the mouse hippocampus. The
maintenance of the laminin matrix is likely to be a steady-state balance
between deposition and degradation. The infused laminin could act by reducing
laminin mRNA expression, thereby reducing laminin protein synthesis, which
might lead to the eventual disappearance of the protein in the matrix.
Therefore, we examined whether soluble laminin could affect endogenous laminin
mRNA synthesis. To this end, we performed laminin 1 chain in situ
hybridization to determine whether the expression of this mRNA is affected.
After laminin infusion, even though the endogenous laminin was clearly
disrupted in the CA1 region of the hippocampus
(Figure 4A), the adjacent
sections hybridized to an antisense laminin 1 probe showed no
significant changes compared with the CA1 region of the noninfused side
(Figure 4B). Consistent with
our previous results, the laminin 1 mRNA showed a neuronal pattern.
Hybridization to a sense probe showed no significant staining (our unpublished
data). The above-mentioned results showed that infusion of soluble laminin-1
did not affect the transcription of the endogenous laminin.
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Soluble Laminin-1 Disrupts the Laminin Matrix in
Plasminogen–/– Mice
Previous results showed that during excitotoxic neurodegeneration, the
tPA/plasmin proteolytic cascade is up-regulated and degrades laminin.
Therefore, another explanation for the effects of soluble laminin could be
that it induced tPA or plasmin generation, thereby leading to disappearance of
the laminin matrix. To test this possibility, soluble laminin-1 was infused
into plasminogen–/– mice. In
these mice, the endogenous laminin matrix was disrupted without cell death by
infusion of soluble laminin equivalent to that observed in wild-type mice
(Figure 5). These results
exclude the possibility that the soluble laminin is acting via an induction of
plasmin activity, since there can be no plasmin generated in the
plasminogen–/– mice.
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Soluble Laminin-1 and Anti-1 Chain Antibody Promotes
Neurodegeneration
Previous results have shown that degradation of the laminin matrix via
plasmin can sensitize neurons to cell death. Because soluble laminin can also
disrupt the laminin matrix, we examined whether this material could also
facilitate neuronal degeneration after kainate challenge. For these
experiments, we used tPA–/– mice,
in which hippocampal neurons are normally resistant to kainite-induced
degeneration. We speculated that
tPA–/– mice, which cannot degrade
laminin during excitotoxic injury and are resistant to neurodegeneration,
would become sensitive to death if their neuronal laminin were disrupted by
soluble laminin infusion. To test this hypothesis, we infused soluble
laminin-1 into the hippocampus of
tPA–/– mice for 5 d and then
injected kainate. In parallel controls, the mice were infused with buffer for
5 d and then kainate was injected. As expected,
tPA–/– mice infused with buffer
were resistant to neurodegeneration (Figure
6, A–C). However, mice infused with soluble laminin-1 mice
were now sensitive to neurodegeneration, mostly in the CA1 region where the
endogenous laminin disruption is most dramatic
(Figure 6, D–F). As shown
above, plasmin-digested laminin-1 can also disrupt endogenous laminin, and we
therefore also tested whether plasmin-digested laminin-1 could promote
neurodegeneration. Plasmin-digested laminin-1 also promoted neurodegeneration
in the tPA–/– mice
(Figure 6, G–I), and the
effect of intact laminin and plasmin-digested-laminin were similar (percentage
of hippocampal neuronal death: buffer, 4.2 ± 1.3; intact laminin, 65.6
± 5.2; and plasmin-digested laminin, 67.4 ± 6.1). These results
demonstrate that when endogenous laminin is disrupted either by intact soluble
laminin or plasmin-digested-laminin, the neurons are sensitized to neuronal
injury.
|
When tPA–/– mice were infused with a reduced dose of laminin-1, the laminin matrix was disrupted in a very limited region of CA1, and as expected did not cause any cell death. Kainate injection into these mice caused neurodegeneration in precisely the region of CA1 where the matrix was disrupted, whereas neurons survived in adjacent areas in which the matrix was not perturbed (our unpublished data). This result strengthens the conclusion that disruption of the laminin matrix was responsible for sensitizing neurons to cell death.
To further investigate the mechanism of laminin-1 on neuronal death, we
infused antibody against the laminin 1 chain into
tPA–/– mice and then injected
kainate into some of these mice. The infused antibody was not toxic alone
(Figure 7, A and C), but
promoted neuronal death when the
tPA–/– mice were injected with
kainate (Figure 7, B and D).
This result, coupled with the observation that this antibody disrupts the
laminin matrix (Figure 2),
indicated that the 1 chain is a critical component for maintaining the
laminin layer and protecting neurons against excitotoxic death.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1 chain antibody into the mouse hippocampus can disrupt
the laminin matrix that normally lies along the neuronal cell layers. This
disruption is specific Because other ECM proteins such as fibronectin had no
effect on the laminin matrix, and the soluble laminin did not affect the
hippocampal expression of other proteins. The effect did not require the
intact heterotrimeric molecule, because laminin-1 extensively digested with
the protease plasmin was still capable of disrupting the in vivo matrix. There
is substantial precedence for small regions of matrix proteins being able to
function as effective cellular ligands
(Ruoslahti, 1996).
One function of mouse hippocampal laminin is to provide survival support
for neurons. When excitotoxins are injected into the hippocampus, tPA activity
is dramatically increased leading to plasmin generation, and to degradation of
the laminin matrix (Chen and Strickland,
1997; Nagai et al.,
1999). This loss of matrix renders the neurons sensitive to
neuronal death. If laminin degradation is prevented by lack or inhibition of
the proteases responsible, neurons are protected. These results indicate that
the neuron-matrix interaction is an important aspect of cell viability, as has
been observed in many other systems
(Ernsberger et al.,
1989; Meredith et
al., 1993; Boudreau et
al., 1995; Coucouvanis and
Martin, 1995; Frisch and
Screaton, 2001). In support of this concept, disruption of the
laminin matrix with soluble laminin also facilitates neuronal death after
kainate injection, even in animals that lack tPA. Thus, two different
experimental paradigms, one involving proteolytic degradation and one
competitive disruption, demonstrate that the integrity of the laminin matrix
in the hippocampus is critical for protecting neurons against
degeneration.
There are numerous mechanisms by which a soluble matrix protein could affect the amount of that protein in the matrix in vivo. It is possible that transcription of the gene encoding the protein could be affected, leading to less mRNA and eventually less protein in the matrix. However, it is clear from in situ hybridization that the soluble laminin did not affect laminin mRNA levels in the hippocampus. Another possibility is that the soluble protein could induce the tPA/plasminogen system, leading to degradation of the matrix. However, laminin-1 infusion led to disappearance of the matrix laminin in plasminogen–/– mice, indicating that at least this proteolytic system is not responsible.
The most likely mechanism for the observed effect is that the high concentration of soluble laminin competes with the matrix laminin for binding sites with other components of the ECM. Once dissociated from the matrix, these previously tethered laminin molecules might be free to diffuse out of the hippocampus or be degraded by proteases. The infused molecule presumably would not have the same affinity for interaction with other matrix components, so it would not be effectively bound to the ECM, and would not remain with the neuronal cell layers.
With respect to how laminin might protect neurons against excitotoxic
death, it is likely that neuronal laminin receptors mediate this effect. There
have been multiple studies indicating that integrins 31,
61, and 64 are the major receptors for laminin-10
(Ferletta and Ekblom, 1999;
Gu et al., 1999;
Tani et al., 1999;
Kikkawa et al., 2000;
Pouliot et al., 2000;
Nielsen and Yamada, 2001;
Doi et al., 2002).
However, it is not known which, if any, integrin might be playing a primary
role in the hippocampus. The infusion of antibodies against potential receptor
molecules, as with the anti-laminin 1 experiments shown in Figures
2 and
7, might help to identify which
receptors are most important for interaction of laminin with neurons.
These results suggest a dynamic and plastic nature for the hippocampal ECM.
If the matrix proteins were extremely fixed, one would not expect them to be
dislodged by the presence of a competing soluble molecule. On the other hand,
if a dynamic equilibrium exists in which association of the components is
favored, but dissociation is also significant, then a high concentration of a
soluble but imperfect molecule could foster the dissociation without being
held firmly by the association forces. In this sense, the soluble molecule
would function as an antagonist of ECM maintenance. It has been shown in other
contexts that a soluble ECM protein can have different effects that the same
protein embedded in a matrix (Hayman
et al., 1985).
Such a dynamic view of the ECM has obvious implications for tissue
architecture, especially in the brain. It has been shown that tPA can affect
new synapse formation (Baranes et
al., 1998; Müller
and Griesinger, 1998), long-term potentiation
(Huang et al., 1996),
and learning and memory (Madani et
al., 1999). The plasticity demonstrated by these studies
could be partially dependent on proteolytic remodeling of the matrix that
surrounds the neurons, and laminin degradation can also affect LTP
(Nakagami et al.,
2000). Thus, to achieve a structure that can be modified with
experience, it would be important to have a supporting matrix that is stable
but not too stable. Our results provide clear evidence from in vivo
experiments that this situation exists in the mouse hippocampus.
In addition, our experimental paradigm provides a strategy for unraveling some of the complex intermolecular interactions in the matrix in vivo. Other ECM proteins can be tested for their effect on the matrix, and for those that are active, small regions or even peptides that recapitulate the effect could be determined. Putting these data together should improve our understanding of how the ECM is maintained and provide reagents for disassembling the matrix to further study its structure and function.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
|---|
Corresponding author. E-mail address:
strickland{at}rockefeller.edu.
|
REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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