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Volume 17, Number 2,
Issue of January 15, 1997
pp. 543-552
Copyright ©1997 Society for Neuroscience
An Extracellular Proteolytic Cascade Promotes Neuronal
Degeneration in the Mouse Hippocampus
Stella E. Tsirka1,
Andrew D. Rogove1, 3,
Thomas
H. Bugge4,
Jay L. Degen4, and
Sidney Strickland1, 2
1 Department of Pharmacology, 2 Program in
Genetics, and 3 Medical Scientist Training Program,
University Medical Center at Stony Brook, Stony Brook, New York
11794-8651, and 4 Division of Developmental Biology,
Children's Hospital Research Foundation, Cincinnati, Ohio 45229
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Mice lacking the serine protease tissue plasminogen activator (tPA)
are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the
role of tPA in neuronal cell death. Mice deficient for the zymogen
plasminogen, a known substrate for tPA, are also resistant to
excitotoxins, implicating an extracellular proteolytic cascade in
degeneration. The two known components of this cascade, tPA and
plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA
and protein are present in neurons and microglia, whereas plasminogen
mRNA and protein are found exclusively in neurons. tPA-deficient mice
exhibit attenuated microglial activation as a reaction to neuronal
injury. In contrast, the microglial response of plasminogen-deficient
mice was comparable to that of wild-type mice, suggesting a
tPA-mediated, plasminogen-independent pathway for activation of
microglia. Infusion of inhibitors of the extracellular tPA/plasmin
proteolytic cascade into the hippocampus protects neurons against
excitotoxic injury, suggesting a novel strategy for intervening in
neuronal degeneration.
Key words:
tPA;
kainate;
plasminogen;
hippocampus;
neurons;
microglia;
mouse
INTRODUCTION
Neuronal cell death can lead to devastating human
pathologies that are associated with severe cognitive and motor
deficits. One mechanism by which neuronal death is induced is
overstimulation with glutamate, the primary excitatory neurotransmitter
in the CNS. Recent evidence suggests that various mediators contribute to excitotoxin action, including production of reactive oxygen intermediates, nitric oxide, p53, and cytokines (Coyle and Puttfarcken, 1993 ; Lipton and Rosenberg, 1994; Ankarcrona et al., 1995; Morrison et
al., 1996). An excitotoxic pathway is implicated in ischemic neuronal
death (Oyzurt et al., 1987; Meldrum, 1990) and may also be involved in
other neurodegenerative diseases (Lipton and Rosenberg, 1994).
A region of the mammalian brain that is especially sensitive to
neuronal degeneration is the hippocampus. The serine protease tissue
plasminogen activator (tPA) has been correlated with hippocampal function, because the levels of tPA mRNA are rapidly increased in the
rat and mouse hippocampus on stimulation of neuronal activity (Qian et
al., 1993; Carroll et al., 1994). Excessive neuronal activity can lead
to neuronal death, suggesting a possible connection between tPA and the
degenerative process. Consistent with this idea, mice deficient for tPA
are resistant to neuronal destruction induced by excitotoxins acting
via all three subtypes of glutamate receptors (kainate, AMPA, and NMDA)
(Tsirka et al., 1995). This finding indicates that tPA participates in
the pathway of excitotoxin-mediated neuronal death.
In this report, we show that plasminogen-deficient mice are also
resistant to kainate-induced degeneration, implicating this zymogen
substrate for tPA as an effector of this pathway. Plasminogen mRNA,
which is primarily synthesized in the liver, is also produced in the
hippocampus (Sappino et al., 1993). Plasminogen mRNA is found
exclusively in neurons, whereas tPA mRNA is expressed in both neurons
and microglia. tPA and plasminogen proteins are also found in the
hippocampus, suggesting that their local synthesis is playing a role in
mediating degeneration. Mice lacking tPA show attenuated microglial
activation, but plasminogen-deficient mice exhibit normal microglial
activation. This result indicates that the influence of tPA on
microglial cells is independent of the activation of plasminogen.
Finally, infusion of  2-antiplasmin, an inhibitor of the
protease generated by tPA, into the hippocampus of wild-type mice
confers neuroprotection to excitotoxic damage.
Our findings indicate that one element of excitotoxic damage is
initiation of an extracellular proteolytic cascade whose product is
plasmin. Promotion of plasmin formation or action facilitates neuronal
degeneration, whereas inhibition retards this process. This model has
implications for new therapeutic approaches to excitotoxin-mediated
neuronal injury.
MATERIALS AND METHODS
Intrahippocampal injections. Adult male mice,
weighing ~25 gm, were injected intraperitoneally with atropine (0.6 mg/kg of 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 and injected unilaterally with 1.5 nmol of kainic acid in 0.3 µl of PBS into the hippocampus (Andersson et al.,1991; Tsirka et al.,
1995). The coordinates of the injection were bregma  2.5 mm,
medial-lateral 1.7 mm, and dorsoventral 1.6 mm. The excitotoxin was
delivered over 30 sec. After kainic acid was delivered, the injection
needle remained at the above coordinates for another 2 min to prevent
reflux of fluid. After variable lengths of time (indicated in each
experiment), the animals were anesthetized and perfused through the
heart with PBS followed by 4% paraformaldehyde. The brains were
removed and left in 30% sucrose in fixative overnight at 4°C.
Coronal tissue sections (30 µm) were prepared and mounted onto slides
and dehydrated through increasing ethanol gradients. They were then
stained with cresyl violet, which stains rough endoplasmic reticulum in
neuronal cell bodies.
Plasmin (150 ng) was delivered (0.3 µl) unilaterally into wild-type
mice by intrahippocampal injection, as described for the delivery of
kainate.
Intrahippocampal delivery of 2-antiplasmin.
Adult wild-type male mice were anesthetized as above and placed in a
stereotaxic apparatus, and a micro-osmotic pump containing artificial
cerebrospinal fluid (aCSF) (for control animals) or 100 µl of
2-antiplasmin in aCSF (1 mg/ml; Sigma, St. Louis, MO)
was 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 near the midline. The infusion rate was 0.5 µl/hr. The
pump was allowed to infuse the designated solution for 2 d, and
then kainate was injected as described above. Five days after the
kainate injection, the mice were killed, and their brains were examined
for neuronal survival, microglial activation, and tPA activity.
Immunohistochemistry. Brain sections of the mice,
manipulated as described above, were incubated with (1) antiserum
against mouse plasminogen raised in sheep (a gift from E. Reich), at
1:20 dilution, and normal sheep serum (detection of plasminogen
protein), (2) antibody to the mature macrophage/microglia-specific
antigen F4/80 (Harlan Bioproducts, Indianapolis, IN), at the dilutions recommended by the suppliers (1:10), or (3) antibody to rat tPA (Chemicon, Temecula, CA), at the dilutions recommended by the suppliers
(1:100). Biotinylated secondary antibodies were used (Vector
Laboratories, Burlingame, CA), and the avidin-biotin-peroxidase complex (ABC reaction) was visualized with diaminobenzidine and hydrogen peroxide (Vector Laboratories), as described previously (Tsirka et al., 1995).
In situ mRNA hybridization. Plasminogen cDNA was a
generous gift of S. Degen. tPA and plasminogen antisense mRNA probes
were prepared from linearized plasmids and labeled with
digoxygenin-11-UTP. Hybridization and stringency washes were performed
as described (Hébert et al., 1994), except that an
anti-digoxygenin Fab fragment (0.3 mg/ml) was used, followed by
biotinylated secondary antibody and the ABC reaction as above. To
verify probe specificity, antisense mRNAs to bacterial lacZ,
mouse REST (Chong et al., 1995), PLD1 (Hammond et
al., 1995), or PLD2 (W. Colley, T. Sung, S. Hammond, Y. Altshuller, D. Bar-Sagi, A. Morris, M. Frohman, unpublished observations) were used as negative controls.
3H-kainate binding. Determination of
3H-kainate binding was performed as described (Miller et
al., 1990): 12 µm cryostat coronal brain sections (n = 9) were preincubated at 4°C with 50 mM Tris-citrate buffer, pH 7.0, for 1 hr. They were then incubated with 50 nM 3H-kainate (specific radioactivity 60 Ci/mmol) in buffer for 30 min at 4°C. The slides were rinsed four
times with buffer, dried rapidly, and exposed to
3H-sensitive film in x-ray cassettes for a minimum of
20 d. To verify specific binding, adjacent sections were incubated
with a mix of the labeled ligand and 50 µM unlabeled
kainate and were treated in a fashion identical to that of the
experimental sections. The autoradiograms were scanned using a BioRad
imaging densitometer, and the intensity of signal over the areas of
specific hybridization was quantified.
Quantification of neuronal loss over CA1-CA3 hippocampal
subfields. Wild-type (n = 15),
tPA / (n = 15),
plasminogen/ (plg/) (n = 4), uPA/ (n = 2), and wild-type
infused with 2-antiplasmin (n = 2) mice were injected, and the tissue was processed as above. Serial sections of 30 µm were prepared and stained with cresyl violet. Five sections from the dorsal hippocampus of each genotype and treatment of mice were
matched, and the linear distances of intact (completely spared),
partially lost (few intact neurons present), and lost (completely
eliminated) pyramidal cell layers were determined on each section.
Distances were digitized from camera lucida drawings of the
hippocampus. The numbers for each category over each hippocampal region
were averaged across subjects in a group.
In situ labeling of DNA fragmentation. The terminal
transferase-mediated biotinylated-UTP nick end-labeling (TUNEL)
technique was used to assess cell death in the hippocampus of
wild-type, plg+/, and plg/ mice injected
unilaterally with kainate. At 48 hr after injection, the mice were
killed, and their brains were frozen quickly. Cryostat coronal brain
sections (12 µm) through the hippocampus were fixed in 4%
paraformaldehyde and processed using the in situ cell death detection kit, POD, as recommended by the manufacturer (Boehringer Mannheim, Indianapolis, IN).
RESULTS
Plasminogen-deficient mice are resistant to excitotoxin-mediated
neuronal degeneration
In addition to its proteolytic activity, tPA can bind via its
N-terminal noncatalytic region to a number of molecules, including annexin (Hajjar et al., 1994), low density lipoprotein receptor-related protein (Bu et al., 1992; Orth et al., 1992), and heparin
(Andrade-Gordon and Strickland, 1986), as well as a protein component
on the surface of the mouse oocyte (Carroll et al., 1993). It was
possible therefore that the role of tPA in neuronal degeneration was
mediated not by its proteolytic activity but by non-enzymatic
interaction with other hippocampal proteins.
If tPA was functioning as a protease in the hippocampus, it is likely
that its action would be via activation of plasminogen, the known
physiological substrate for tPA (Astrup and Permin, 1947). In this
case, mice deficient for plasminogen, which are viable and develop
normally (Bugge et al., 1995; Ploplis et al., 1995), should display the
same excitotoxin-resistant phenotype as the tPA/ mice.
Therefore, plg/ mice were tested for their response to
excitotoxin by injecting the glutamate analog kainate into the
hippocampus and analyzing neuronal survival 5 d later by cresyl
violet staining. As shown in Figure 1a (low
magnification), control mice heterozygous for the plasminogen mutation
were sensitive to neuronal degeneration (comparable to wild-type mice,
not shown). Their homozygous plg/ litter mates,
however, were resistant to excitotoxic injury (Fig. 1b). The
extent of resistance of the plg/ mice was comparable to
that of tPA/ mice (Table 1).
Fig. 1.
Plasminogen-deficient mice are resistant to
kainate-induced neuronal degeneration. Cresyl violet-stained coronal
sections through the hippocampus reveal the neuronal degeneration
generated by kainate. a, Hippocampus from heterozygous
plg+/ mouse (wt) 5 d after the
injection, showing substantial degeneration on the injected side
(ipsilateral), whereas the uninjected
(contralateral) side remains unaffected (number
of mice injected = 15). CA1, CA2, and
CA3 denote the hippocampal subfields; DG,
dentate gyrus. b, Hippocampus from
plg/ mouse 5 d after the injection,
showing minimal degeneration on the injected side
(n = 4). Arrows show the site of
injection. c, High-magnification photomicrograph of part
of the ipsilateral CA1 subfield of hippocampus from heterozygous
plg+/ mouse (wt) 12 hr after the injection
(n = 3). The pyknotic and refractile neuronal cells
in the wild-type mouse have disappeared completely by 24 hr, showing
that they are not glial cells. d, High-magnification
photomicrograph of part of the ipsilateral CA1 subfield of hippocampus
from heterozygous plg/ mouse
(wt) 12 hr after the injection (n = 3). e, TUNEL labeling, indicating DNA fragmentation in
dying cells (arrows), is observed in the CA1 pyramidal
subfield in wt mice (n = 2) and is absent in
plg/ mice (f) 2 d after
kainate injection (n = 2).
[View Larger Version of this Image (104K GIF file)]
Examination of the injected hippocampus at high magnification showed
that 12 hr after kainate injection, the dying neurons in the control
mice were pyknotic and refractile (Fig. 1c). In contrast,
neurons in the plg/ mice had a normal appearance (Fig.
1d), similar to the equivalent cells on the uninjected side,
which are largely unaffected by kainate (Andersson et al., 1991). This
normal appearance persists for at least 30 d (data not shown). In
addition to their normal morphology, the neurons exhibit normal
staining with cresyl violet, which visualizes ribosomal RNA.
The TUNEL labeling procedure, indicating the presence of fragmented DNA
and therefore dying/dead neurons, revealed degenerating neuronal cells
in the CA1 pyramidal subfield of the hippocampus of kainate-injected
wild-type mice (Fig. 1e, arrows). The
contralateral side did not show TUNEL staining, nor did control mice
injected with PBS (data not shown). The staining specific in CA1 is in agreement with the region in which apoptotic death has been detected by
other investigators after subcutaneous injection of kainate using the
same detection method (Morrison et al., 1996). When plg/ mice were subjected to TUNEL labeling, no staining
was observed over the hippocampal neurons, indicating absence of
neuronal death in these mice. These results demonstrate that the
neurons that persist in the tPA/ and
plg/ mice are viable and healthy.
The similarity in phenotype between tPA/ and
plg/ mice, coupled with the documented interaction of
these two proteins, argues that a proteolytic cascade involving tPA and
plasminogen participates in mediating neuronal cell death.
Mice resistant to neuronal degeneration have normal levels of
kainate receptors
One explanation for resistance to kainate in tPA/
and plg/ mice could be attenuated expression of kainate
receptors. In this scenario, decreased numbers of receptors would
result in less binding of excitotoxin, less depolarization of neurons,
and ultimately less death. To evaluate this possibility, brain sections
from tPA/ mice were incubated with
3H-kainate. Strong 3H-kainate binding was
observed on the hippocampal formation, cortex, and caudate-putamen
(Miller et al., 1990) in both wild-type and tPA/ brain
sections. The higher affinity sites of hippocampal binding along the
CA3 region and dentate gyrus were quantitated by scanning these regions
of the autoradiographic slides. As shown in Table 2, the
binding capacity of kainate in the wild-type hippocampus was
indistinguishable from that in tPA/ hippocampus. This
experiment shows that mice resistant to the degenerative actions of
kainate bind the excitotoxin at levels equivalent to those in wild-type
animals, ruling out a gross deficit of the receptors as an explanation
of resistance. Moreover, immunohistochemical experiments using
antibodies against GluR6 (a gift from Dr. J. Prives, State University
of New York, Stony Brook) showed no difference in the levels of this
glutamate receptor subtype between wild-type and tPA/
hippocampi (data not shown).
Mice deficient in uPA or fibrinogen, other components of the
fibrinolytic system, are not resistant to excitotoxin-mediated neuronal
degeneration
Mice express two forms of plasminogen activator, tPA and urokinase
plasminogen activator (uPA). Examination of whole mouse brain (Rickles
and Strickland, 1988) and specifically the hippocampus (Sappino et al.,
1993) has not revealed any uPA mRNA or activity. However, injury in the
brain, such as the injection of excitotoxin, leads to the immigration
of macrophages that can express both tPA and uPA (Vassalli et al.,
1984; Hart et al., 1989). This infiltration of macrophages into the
brain may result in both plasminogen activators being necessary to
mediate neuronal degeneration. If so, mice deficient for uPA should
exhibit resistance to excitotoxicity similar to that of
tPA/ mice.
To examine this possibility, mice deficient for uPA (Carmeliet et al.,
1994) were tested using the unilateral, intrahippocampal delivery of
kainate (data not shown). The hippocampal neurons of these mice
displayed wild-type sensitivity to kainate (Table 1). We also tested
mice deficient for fibrinogen (fibrinogen/ mice) (Suh
et al., 1995). Fibrinogen, a coagulation factor, is the precursor of
fibrin, which binds to tPA and can enhance its activity under certain
conditions. The fibrinogen/ mice were also susceptible
to kainate injection (data not shown) at a level comparable to that of
wild-type mice (no spared neurons were observed over the CA1, CA2, or
CA3 hippocampal subfields). These findings indicate that deficiency in
these members of the fibrinolytic system does not confer excitotoxin
resistance to hippocampal neurons.
Plasminogen mRNA is produced in hippocampal neurons, whereas tPA
mRNA is produced in both neurons and microglia
Two aspects of the above results prompted a closer
examination of the site of synthesis of tPA and plasminogen. (1)
Plasminogen is an abundant serum protein synthesized primarily in the
liver (Raum et al., 1980) and is too large to traverse the blood-brain barrier (BBB) without a specialized transport system. Therefore, plasminogen in the hippocampus could be derived from the blood-borne protein via leakage or transport, or from local synthesis. The latter
was suggested by Sappino et al. (1993), who detected low levels of
plasminogen mRNA in the hippocampus by RNase-protection assay. In light
of the resistance of the plg/ mice to excitotoxins,
determining whether plasminogen is synthesized in the brain would help
advance the understanding of the pathway of degeneration. (2) Previous
work has indicated that cells that synthesize both a plasminogen
activator and plasminogen suffer deleterious consequences, possibly
attributable to the generation of intracellular proteolysis (Sandgren
et al., 1991). It was therefore of interest to determine whether the
production of tPA and plasminogen was segregated by cell type.
To determine the sites of synthesis of tPA and plasminogen, antisense
mouse tPA and plasminogen digoxygenin-labeled RNA probes were
hybridized to control or kainate-injected brain sections (Fig.
2). tPA mRNA was detected along the neuronal cell layers in the hippocampus (Fig. 2c), as described previously (Qian
et al., 1993; Sappino et al., 1993). At higher magnification, the pattern of tPA mRNA along the neuronal cell layer had a complex appearance (Fig. 2e), suggesting that synthesis might be
occurring in more than one cell type. In addition, after destruction of neurons by kainate injection, the staining in the larger cells (pyramidal cells) disappeared but remained in the darker and smaller cells (Fig. 2g).
Fig. 2.
tPA mRNA is present in neurons and microglia,
whereas plasminogen mRNA is found only in neurons. Coronal sections
through the hippocampus displaying the site of synthesis of tPA
(c, e, g) and plasminogen (d, f, h). Low
magnification of coronal sections (a-d) and higher
magnification of similar sections (e, f). Other mice were unilaterally injected with kainate to cause neuronal degeneration on one side, and subjected to mRNA ISH 5 d later (g, h). Cresyl violet staining of
kainate-injected consecutive sections confirmed the complete
elimination of the neurons in CA1-CA3 (data not shown). In
a, the probe was control plasmid (pBluescript KS II,
Stratagene, La Jolla, CA), and in b the probe was
digoxygenin-labeled sense tPA cDNA. The arrows in
f indicate dendritic localization of plasminogen
mRNA.
[View Larger Version of this Image (104K GIF file)]
Several lines of evidence indicate that these smaller cells are
microglia. (1) Transgenic mice that express the bacterial lacZ gene under the control of mouse tPA 5
regulatory sequences (tPA/lacZ; Carroll et al., 1994)
express  -gal along the neuronal pyramidal cell layer in cells ~10
µm in diameter, smaller than pyramidal neurons, but of average size
for satellite microglial cells. (2) In the tPA/lacZ
transgenic mice, adjacent coronal brain sections were doubly stained
for -gal and with antibodies to detect markers specific for the
three non-neuronal cell types in the hippocampus (data not shown).
-gal-expressing cells colocalized with perineuronal microglia
(F4/80), but did not colocalize with oligodendrocytes (anti-myelin
basic protein antibody) or astrocytes (anti-glial fibrillary acidic
protein).
These results demonstrate that tPA mRNA is produced both in neurons
(the fraction of the staining that disappears after injection of
kainate) and in the satellite microglia that overlie the neuronal cell
layer (the fraction that persists after kainate injection). The
distribution of tPA mRNA in both cell types is in agreement with the
studies on the localization of tPA in the rat cerebellum and brain stem
(Ware et al., 1995).
Plasminogen mRNA was also detected in the hippocampus along the
neuronal cell layers, indicating local transcription of this gene (Fig.
2d). Plasminogen mRNA, however, was detected only in the
larger pyramidal neurons (Fig. 2f), and the mRNA
staining was completely abolished by kainate destruction of neurons
(Fig. 2h).
An intriguing finding was that plasminogen mRNA was detected in
dendrites emanating from the neuronal cell bodies (arrows in
Fig. 2f and data not shown). This cellular distribution of plasminogen mRNA suggests transport of the message into the dendrites, which could direct local protein production on neuronal activity (Miyashiro et al., 1994; Link et al., 1995; Steward, 1995).
Taken together, these data demonstrate that plasminogen mRNA is
synthesized in the brain exclusively by neurons, whereas tPA mRNA is
produced in both neurons and perineuronal microglia.
Plasminogen protein is expressed in a subset of
hippocampal neurons
Because post-transcriptional control can often regulate protein
expression, it was of interest to determine whether tPA and plasminogen
proteins were also present in the hippocampus. The presence of tPA
protein has been examined previously by Sappino et al. (1993), who
determined tPA activity by zymography on tissue sections. Even though
tPA mRNA is observed in the CA1-CA3 regions of the hippocampus and in
the dentate gyrus (Sappino et al., 1993) (Fig. 2c), tPA
enzyme activity is confined to CA2-CA3 and the dentate and is not
detected in the CA1 region. This distribution of tPA activity was
corroborated by subsequent, independent experiments (Tsirka et al.,
1995; Gualandris et al., 1996). Sappino et al. (1993) also showed that
extracts of the CA1 region contained tPA activity; because preparation
of the extracts would dissociate enzyme-inhibitor complexes, they
concluded that inhibitors in the CA1 were masking the tPA activity in
that region. These elegant studies suggest that tPA activity in the
hippocampus is under regulation by inhibitors. Antibodies against tPA
revealed the presence of tPA protein over the CA2-CA3 pyramidal
subfields and dentate gyrus in neuronal as well as microglial cells
(Fig. 3c,e). Injection of kainate resulted in
stronger tPA microglial expression at the ipsilateral side (Fig.
3e), thus confirming that tPA activity is upregulated in
activated microglia (Tsirka et al., 1995).
Fig. 3.
tPA and plasminogen proteins are synthesized
locally in the mouse hippocampus. Immunodetection of tPA and
plasminogen proteins in coronal sections through the hippocampus 12 hr
after kainate injection of a wild-type mouse unilaterally into the
hippocampus. High-magnification photomicrographs of the CA1 field
reacting with (a) IgG, (b) normal sheep
serum, (c) anti-tPA antibody at the contralateral side,
(d) anti-plasminogen antibody at the contralateral side,
(e) anti-tPA antibody at the ipsilateral side, and
(f) anti-plasminogen antibody at the
ipsilateral side. Scale bar, 50 µm.
[View Larger Version of this Image (129K GIF file)]
We have investigated the presence of plasminogen protein by
immunohistochemistry. Plasminogen was detected in neurons in the pyramidal cell layer (Fig. 3). Although plasminogen mRNA is present in
most pyramidal neurons, only a small subset of neurons stained for
plasminogen protein (Fig. 3d). Soon after injection of
kainate, the levels of plasminogen protein expression appear to be
increased (Fig. 3f), suggesting that
post-transcriptional regulation controls its expression. Such
regulation may represent a mechanism for enhanced expression associated
with neuronal activity, in agreement with proposed functions of
tPA/plasmin in plasticity and restructuring in the brain (Krystosek and
Seeds, 1981; Carroll et al., 1994; Frey et al., 1996).
Mice deficient for plasminogen exhibit normal
microglial activation
The injection of kainate into the hippocampus results in local
activation of microglial cells (Andersson et al., 1991), made manifest
by changes in morphology and increased expression of the surface
antigen F4/80 (Lawson et al., 1990) (Fig.
4a-d). Mice deficient for tPA displayed
activation of microglial cells after kainate administration, but the
response was attenuated compared to that of wild-type animals, both in
F4/80 staining intensity and in morphological changes (Tsirka et al.,
1995). These results suggested that tPA participates in the activation
pathway of microglial cells, which in turn could be affecting the
degeneration process.
Fig. 4.
Normal activation of microglia in kainate-injected
plasminogen-deficient mice. Left panels,
Low-magnification F4/80 immunostaining of coronal sections through the
hippocampus 5 d after injection of a wild-type mouse with PBS
(a, b) or with kainate (c, d), and of a
plg/ mouse (e, f) with kainate.
Extensive activation is observed on the ipsilateral side (shown) and
around the injection site. Microglia on the contralateral side are also
activated (not shown), but to a much lower level. Right
panels, High-magnification photomicrographs of representative
activated microglia in the CA1 field of stratum radiatum on the
ipsilateral side of a wild-type (d) and a
plg/ mouse (f).
[View Larger Version of this Image (136K GIF file)]
To examine the effect of plasminogen on microglial activation, F4/80
immunostaining was performed on brain sections from kainate-injected plg/ mice. Both the staining intensity and morphology
of microglial cells from plg/ mice were comparable to
those of wild-type cells (Fig. 4c-f). These results
show that resistance to neuronal degeneration can occur in spite of
normal microglial activation, and that a critical proteolytic event is
downstream of activation. They also suggest that the role of tPA in
microglial activation is independent of its activation of
plasminogen.
Inhibition of plasmin confers resistance to excitotoxin-mediated
neuronal degeneration in wild-type mice
The above results identify a genetic and cellular pathway,
involving tPA and plasminogen, that can result in neuronal cell death
after excitotoxic injury. To evaluate directly whether plasmin, the
product generated by the action of tPA on plasminogen, is mediating
neuronal death, an inhibitor of the activity of plasmin (2-antiplasmin) was delivered to the brain. Because tPA
is required acutely after excitotoxic insult to mediate neuronal
degeneration (Tsirka et al., 1996), inhibition of tPA/plasmin
proteolytic activity might retard neuronal cell death. Wild-type mice
were infused with 2-antiplasmin, injected 2 d later
with kainate, and then evaluated for their neuronal status in the
hippocampus. Control mice infused with buffer were sensitive to
neuronal degeneration in the hippocampus (Fig. 5,
top). In contrast, 2-antiplasmin retarded
neuronal degeneration induced by kainate (Fig. 5, bottom). The resistance of the protease inhibitor-infused mice was comparable to
that observed with untreated tPA/ mice (Table 1).
Therefore, plasmin is a product of the proteolytic cascade that
promotes hippocampal excitotoxic neuronal death, and inhibition of
plasmin activity in the adult animal can protect against
degeneration.
Fig. 5.
2-antiplasmin can prevent
kainate-induced neuronal degeneration. Cresyl violet-stained coronal
sections through the hippocampus of wild-type mice. The mice were
infused with buffer (aCSF) or 2-antiplasmin for 2 d, kainate was injected, and then the infusion continued for 5 d
more, and the mice were analyzed. Top, Section from a
wild-type mouse infused with aCSF showing extensive kainate-induced degeneration. Bottom, Section from a wild-type mouse
infused with 2-antiplasmin, showing resistance to
kainate-induced degeneration. Arrowheads point to the
site of injection; arrows indicate the site of
infusion.
[View Larger Version of this Image (110K GIF file)]
DISCUSSION
Extracellular proteases and neuronal degeneration
The results presented here demonstrate that extracellular
proteolysis plays a central role in excitotoxin-mediated hippocampal neuronal degeneration. A critical observation in defining this pathway
is the protection against degeneration in mice that lack plasminogen.
The comparable resistant phenotype conferred by both tPA and
plasminogen deficiency is consistent with the function of these enzymes
in other contexts: namely, they operate sequentially within a cascade
whose final proteolytic product is plasmin. This view is strengthened
by the results of local delivery of 2-antiplasmin, because this inhibitor confers resistance to wild-type mice.
Previous work has implicated proteases in neuronal
destruction. (1) The degeneration of ganglion neuronal cells after
transection of the optic nerve can be retarded by the injection of
protease inhibitors into the vitreous body (Thanos, 1991; Thanos et
al., 1993). (2) Protease nexin-1, an inhibitor of plasminogen
activators and plasmin, can protect cultured hippocampal neurons
against hypoglycemic damage, suggesting that proteases can modulate
vulnerability to neurotoxicity (Smith-Swintosky et al., 1995). (3) In
the cerebellum of the mouse mutant weaver, increased
neuronal cell death is evident, coinciding with 10-fold higher than
normal tPA activity. This cell death is also observed in cultured
cerebellar weaver neurons and can be prevented by inclusion
of aprotinin, a serine protease inhibitor, in the culture medium
(Murtomäki et al., 1995). The weaver phenotype results
from a mutation in the potassium channel gene Girk2 (Patil
et al., 1995), which alters the specificity of the channel (Slesinger
et al., 1996) and may result in depolarization of granule neurons
(Goldowitz and Smeyne, 1995). Such a depolarization could lead to
increased tPA levels, because we have found that chemically induced
depolarization of PC12 cells leads to an enhanced rate of tPA secretion
(Gualandris et al., 1996).
Neurons and microglia in neuronal degeneration
Both neurons and microglia synthesize tPA, but it is not known
whether the function of the enzyme from these two sources is the same
or distinct. There are previous reports that microglia participate
directly in neuronal degeneration (Thanos, 1991; Lang and Bishop,
1993). Activated microglia may contribute to neuronal death by
secretion of glutamate (Streit et al., 1992; Patrizio and Levi, 1994),
reactive oxygen intermediates (Piani et al., 1992), or cytokines (Prehn
and Krieglstein, 1994). A requirement for microglia in degeneration
might limit damage to those areas in which both neurons and microglia
have received the appropriate stimulus. It is possible, however, that
microglial tPA does not act directly in the degeneration pathway and
that neuronal tPA is the effector that mediates neuronal death.
The synthesis of plasminogen by neuronal cells and of tPA by both
neurons and microglia cells suggests that both cell types may be
necessary to effect neuronal degeneration in the hippocampus. Such a
neuronal-microglial cross-talk may limit degeneration to those areas
in which both cell types receive the appropriate stimulus. On neuronal
injury, macrophages cross the BBB and accumulate in the brain. If these
cells were responsible solely for neurotoxicity, they might cause
extensive neuronal degeneration along their migration path. Instead,
the participation of the injured neuronal cells in their own demise (by
presenting the plasminogen component of the cascade) could provide a
means to localize destructive/phagocytic microglia to those areas in
which neurons have been injured. To support this hypothesis, infusion
of tPA alone does not cause neuronal degeneration in the side
contralateral to kainate injection (Tsirka et al., 1996), presumably
because increased plasminogen is also a necessary element in the
pathway.
Another reason for the participation of adjacent cell types is that
production of both tPA and plasminogen in the same cell could lead to
intracellular proteolysis and cell death (Sandgren et al., 1991). We do
not believe, however, that intracellular generation of plasmin plays a
significant role in this system, because administration of
2-antiplasmin retards kainate-induced degeneration, and
this inhibitor can inhibit proteases only in the extracellular space.
Moreover, although tPA and plasminogen mRNAs are expressed by neurons,
it is possible that the two proteins are produced by different neuronal
cell populations. Alternatively, if both tPA and plasminogen are
synthesized in the same neuronal cell, intracellular damage may be
avoided by either translational control of the mRNAs (Huarte et al.,
1987), or by sequestration of the protein products in vesicles (Parmer
et al., 1997).
In most situations in which tPA has been studied, it requires the
presence of plasminogen to be effective, because of the catalytic
amplification generated by zymogen activation. The absence of tPA
attenuates microglial activation, but the absence of plasminogen does
not, indicating that this effect of tPA does not require plasminogen.
Microglial activation thus represents the first example of a
plasminogen-independent role for tPA in vivo.
Synthesis of tPA and plasminogen in the brain
Synthesis of tPA occurs in many tissues, whereas the production of
plasminogen has been thought to occur primarily in the liver (Raum et
al., 1980). If the liver were the sole source of plasminogen, then
delivery to the brain via the circulation would be necessary; however,
access of large molecules from the vasculature to the brain is in
general precluded by the BBB, and the presence of plasminogen in the
brain would be observed only in pathological situations accompanied by
a compromised BBB. Therefore, the hippocampal synthesis of plasminogen
and tPA gives credibility to the presumptive functions of a tPA/plasmin
proteolytic system in physiological settings in plasticity and
remodeling, because all of the components of the cascade are present
locally. This may be only one example of a general phenomenon: that the
brain parenchyma may require local synthesis of necessary proteins,
because its access to the blood-borne material is so restricted.
Various human pathologies involve excitotoxic damage to the brain. The
contribution of extracellular proteases to the degeneration pathway
provides a target for therapeutic intervention. Because delivery of
protease inhibitors protected from excitotoxic neuronal death (Fig. 5)
(Tsirka et al., 1996), it seems reasonable to investigate further the
effectiveness of protease inhibitors for the therapy of
excitotoxic-mediated brain disorders.
FOOTNOTES
Received Aug. 26, 1996; revised Oct. 15, 1996; accepted Oct. 23, 1996.
This work was supported by fellowships from the International Human
Frontier Science Program Organization (S.E.T.), the Danish Medical
Research Council (T.H.B.), and the Medical Scientist Training Program
(A.D.R.), by an Established Investigator Award from the American Heart
Association (J.L.D.), and by grants from National Institutes of Health
(S.S., J.L.D.) and the American Cancer Society (S.S.). We are grateful
to D. Colflesh for valuable photographic expertise and to R. Hart for
generous assistance. We also thank the following for providing
reagents, equipment, advice, and/or helpful discussion: D. G. Amaral,
R. Burwell, S. Degen, J. Engebrecht, M. Frohman, A. Gualandris, B. Hitzemann, P. Rapp, A. Jorgen-Relo, E. Reich, T. Rosenquist, F. Sallés, A. Verrotti, and J. Wells.
Correspondence should be addressed to Sidney Strickland, Department of
Pharmacology, Basic Science Tower, T8, Room 125, State University of
New York at Stony Brook, Stony Brook, NY
11794-8651.
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