(Stroke. 1995;26:1901-1907.)
© 1995 American Heart Association, Inc.
Articles |
Three Distinct Phases of Fodrin Proteolysis Induced in Postischemic Hippocampus
Involvement of Calpain and Unidentified Protease
From the Department of Neurosurgery, Hyogo College of Medicine (M.Y., E.T.), the Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science (T.C.S., S.K.), and the Institute of Applied Microbiology, University of Tokyo (K.S.), Japan.
Correspondence to Masayuki Yokota, MD, Department of Neurosurgery, Hyogo College of Medicine, Mukogawa-cho 1-1 Nishinomiya, Hyogo, 663 Japan.
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Abstract |
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Background and Purpose Fodrin, a neuronal cytoskeleton protein, is proteolyzed by calpain after ischemic insult. We examined proteolysis of fodrin induced by global forebrain ischemia in gerbil hippocampus in spatial terms by using the antibody specific to the calpain-proteolyzed form of fodrin.
Methods In gerbils, a 10-minute forebrain ischemia was produced by occlusion of both carotid arteries. After recirculation, the hippocampus was processed for immunohistochemical and immunoblot study with the antibody against the calpain-proteolyzed form of fodrin. Additionally, short-term ischemia was studied to find the threshold of fodrin proteolysis.
Results Three phases of fodrin proteolysis distinct in chronology and distribution arose: (1) an early predegeneration phase in the molecular layer and stratum oriens of the CA1 and CA3 sectors within the first 15 minutes, which lasted up to 4 hours; (2) a late predegeneration phase in the whole CA1 sector, except for the pyramidal cells, between 12 hours and 2 days; and (3) a postdegeneration phase in the cytoplasm of the CA1 neurons, which arose in 3 to 7 days. A 4-minute (not a 3-minute) forebrain ischemia induced the late predegeneration phase of fodrin proteolysis and delayed neuronal death in CA1. Immunoblotting showed that the primary product of calpain action was further proteolyzed by an unidentified protease.
Conclusions Calpain induced proteolysis of fodrin in ischemic hippocampus, and the late predegeneration phase of the proteolysis was closely associated with the delayed neuronal death in the CA1 sector. Calpain and another protease may play a role in the development of neuronal death after transient forebrain ischemia.
Key Words: calpain • cerebral ischemia • fodrin • proteolysis • hippocampus
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transient global forebrain ischemia in rodents induces selective degeneration affecting hippocampal neurons in CA1 with relative sparing of CA3 neurons and dentate granule cells.1 Delayed degeneration affects CA1 neurons, which are normal on light microscopy up to 2 days after the insult. The molecular mechanisms responsible for the selective neuronal vulnerability to ischemic injury are incompletely understood, but pharmacological studies show that glutamate-mediated excitotoxicity is involved.2 3 4 5 6 Glutamate receptors are categorized into two groups, ionotropic and metabotropic. The former group is further subdivided into two distinct types: receptors for NMDA and non-NMDA receptors for AMPA (
-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) and
kainate. Stimulation of NMDA and metabotropic receptors causes
influx of Ca2+ and activates intracellular
Ca2+-dependent cysteine protease, µ-calpain,
which is enriched in the central nervous system.7 8 9 The
activated calpain selectively proteolyzes substrate proteins in
a limited manner.10 Among the calpain-specific
substrates, fodrin, a major cytoskeletal protein underlying the plasma membranes
in brain,11 12 has been most extensively described in
pathophysiological situations. Fodrin undergoes
calpain-catalyzed proteolysis both in long-term potentiation
and postischemic degeneration.13 14
Consequently, its -subunit of 230 kD is converted to a 150-kD
fragment.12 15 On the basis of these observations, we devised a means of capturing the proteolytic process by developing an antibody that specifically distinguishes the proteolyzed fodrin from the intact one.16 Taking advantage of the approach that finally enabled us to analyze the process in spatial terms, we demonstrated that transient ischemia induces at least two distinct phases of fodrin proteolysis in the hippocampus: an early phase in the molecular layer and in the stratum oriens of the CA3 and CA1 sectors arising within the first 15 minutes and a late phase in the entire CA1 sector 4 to 24 hours later. In the present study, we made a more detailed analysis and discovered another distinct phase of fodrin proteolysis that arose along with the neuronal degeneration. Furthermore, we intend to show that the predegeneration phase actually participates in the pathological cascade leading to neuronal death. We also describe our new discovery as to how a secondary proteolytic product derived from the calpain-proteolyzed fodrin accumulates in postischemic brain, raising a new possibility in interpreting the pathological mechanism.
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Materials and Methods |
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Induction of Ischemia in Gerbil Forebrain
Adult female Mongolian gerbils weighing 50 to 70 g (Japan Clea), housed under diurnal lighting conditions and allowed food and water ad libitum, were anesthetized during the ischemia and reperfusion period by diethyl ether and room air. Body temperature was maintained at 36°C to 37°C with a homeothermic heating blanket (CMA) during the anesthesia. After a midline cervical incision, both common carotid arteries were dissected in 36 gerbils and occluded with small aneurysmal clips. After a 10-minute occlusion, the clips were removed to allow recirculation. In 36 sham-operated animals, common carotid arteries were dissected but not occluded. Four animals from each group (ischemic and sham-operated) were killed at intervals of 15 minutes, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, and 7 days after recirculation. At each time point, the animals were killed with an overdose of sodium pentobarbital (60 mg/kg IP) and then perfused at 4°C through the ascending aorta with 50 mL of 20 mmol/L Tris/HCl buffer (pH 7.6) containing 5 mmol/L EDTA, 5 mmol/L ß-mercaptoethanol, 250 mmol/L sucrose, and 0.1 mmol/L leupeptin followed by 2% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). The perfused brains were removed and fixed with the same fixative for 24 hours. Next, the brains were immersed in 0.01 mol/L PBS containing 10% to 20% sucrose for 24 hours and processed for immunohistochemistry.
In addition, to clarify the threshold of ischemic duration for delayed neuronal death, a short period of forebrain ischemia was induced. Four animals from each group underwent 2, 3, or 4 minutes of transient forebrain ischemia and were killed at same intervals as the animals with 10 minutes of ischemia. The sections of hippocampus were processed for immunohistochemistry.
To assess tissue architecture, we prepared 5-µm sections from each hippocampus after the fixation and stained them with cresyl violet. The number of neurons in the linear length (1 mm) of the CA1 pyramidal layer (neuronal cell density) was counted in each specimen, according to the method of Kirino et al.17
Preparation of the antibody specific for the calpain-proteolyzed
150-kD form of fodrin's subunit was described previously, and its
character has been thoroughly studied.16 The
anti–human fodrin subunit PEST
(proline-glutamate/aspartate-serine-threonine-rich)
sequence antibody,15 which recognizes both the intact
230-kD and proteolyzed 150-kD forms, was also used for the
immunohistochemistry and immunoblot analysis. Antigenic
epitopes of both the antibodies are shown in Fig 1
.
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The animals were carefully handled in a humane fashion and our experimental protocols met the US Public Health Service standards described in the 1985 Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Immunohistochemistry
Immunohistochemistry was performed essentially as previously
described. In brief, the fixed brains were coronally sectioned at 20
µm on a cryostat. Each section was preincubated with 3% normal goat
serum and 0.3% hydrogen peroxide in PBS for 30 minutes at room
temperature. The sections were then incubated with the antibody to the
proteolyzed form of fodrin or to the PEST sequence (1 µg/mL) at room
temperature for 1 night. After being rinsed, sections were incubated
with biotinylated goat anti-rabbit IgG (Vector) for 2 hours and
then with ABC-peroxidase complex (Vector) for 2 hours at room
temperature. After rinsing of the sections, immunolabel was visualized
with 0.015% diaminobenzidine tetrahydrochloride (Sigma) and 0.003%
hydrogen peroxide in 50 mmol/L Tris/HCl buffer (pH 7.6).
Immunoblotting
For immunoblot analysis, nine animals treated with a
10-minute ischemia period were perfused with Tris/HCl buffer
(pH 7.6) containing 5 mmol/L EDTA, 5 mmol/L ß-mercaptoethanol,
250 mmol/L sucrose, and 0.1 mmol/L leupeptin at intervals of 15
minutes, 1 day, and 7 days (three animals at each interval).
Furthermore, three sham-operated animals were perfused in the same
fashion 1 day after the operation. The hippocampus was dissected and
immersed in liquid nitrogen, and remained frozen at -85°C until
further processing. Western blotting was performed by use of both the
antibodies as described in the previous study.15 18 One
hippocampus in each group was used in every blotting. Each immunolabled
band was densitometrically quantitated by a videodensitometry system
(ACI Japan).
Statistics
Statistical comparisons were made by two-tailed Student's
t test for unpaired variates.
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Results |
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Forebrain ischemia of 10 minutes' duration produced consistent neuronal degeneration in the hippocampus of all animals surviving 3 and 7 days after the ischemic insult (Fig 2
and Table 1). Degeneration of the CA1
neurons started in CA1a on day 3 (Fig 2B) and spread all over the CA1
neurons by day 7 (Fig 2C).
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In the hippocampus of the sham-treated animals, immunoreactivity
corresponding to the 150-kD proteolytic fragment of fodrin (150kD-IR)
was below detectable levels (Fig 3A). Within 15 minutes
after the reperfusion, 150kD-IR appeared in the neuropil of the stratum
lacunosum moleculare, the stratum oriens of the CA3 sector, and the
stratum oriens of the CA1 sector (Fig 3B), and it remained until around
4 hours after the reperfusion, as previously described.16
Between 6 to 12 hours, additional fodrin proteolysis was observed in
the entire CA1 sector (Fig 3C), except for the soma and dendrites of
the pyramidal neurons (Fig 4A), although no
immunoreactivity was observed in the hilus and stratum lucidum where
mossy fibers pass. One day after the ischemic insult, the
proteolysis was observed only in the entire CA1 sector, and it remained
until day 2 (Fig 3D). These two distinct phases are referred to here as
the early and late predegeneration phases because we have now
discovered a third phase that was initiated at the time of the actual
neuronal degeneration.
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Fig 4B
shows that the soma and dendrites of the pyramidal
neurons in the medial part of the CA1 sector (CA1a) became positive for
150kD-IR, while the lateral part (CA1b) showed very little
immunoreactivity on day 3 (Fig 3E). This fodrin proteolysis seems to
have arisen as a result of cell death because the CA1a cells had
started to degenerate with shrunken and hyperchromatic morphology by
this time, as shown by cresyl violet staining (Fig 2B). Seven days
after the ischemia, all the CA1 neurons were degenerated and
some debris of CA1 neurons showed dense 150kD-IR with moderately dense
immunoreactivity in other CA1 regions (Fig 3F). Apparently, this fodrin
proteolysis within pyramidal cells followed the dying
processes and was therefore referred to as the postdegeneration phase.
Each group showed a consistent tendency in the pattern of
immunohistochemical staining, although its intensity varied in some
cases. Immunoblot analysis confirmed the presence of the 150-kD
fragment of fodrin in postischemic brain for up to 7 days
(see below). Throughout the experiment, CA4 neurons, the dentate gyrus,
and the stratum lucidum showed no 150kD-IR, supporting the hypothesis
that calpain plays essential roles in the postischemic
pathological cascade.14 16 Intact fodrin immunoreactivity
stained with anti-PEST antibody showed uniform distribution in the
whole hippocampus (Fig 5). The soma and dendrites of the
pyramidal neurons in all CA sectors and dentate granule
cells showed heavy immunoreactivity (Fig 5A). Little change was
observed after the ischemia until day 3 (Fig 5B, 5C, and 5D).
Three days after the insult, the immunoreactivity was faded in the soma
and dendrites of the pyramidal neurons in the CA1a sector
(Fig 5E), and on day 7 reduction of the immunoreactivity spread all
over the CA1 sector (Fig 5F). These findings represent a small
amount of the intact fodrin in the hippocampus, of which distribution
was shown as the late predegeneration phase in immunohistochemical
study with anti–proteolyzed fodrin antibody, and it was
proteolyzed after the ischemia.
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With the aim of determining which of the predegeneration phases is
responsible for the subsequent delayed neuronal death, we examined the
effect of the ischemic duration on fodrin proteolysis and cell
death (Fig 6) with the knowledge that a critical time
point determining life or death of neurons exists around 3 to 4
minutes.19 Short-term ischemia of less than 3
minutes did not induce delayed neuronal death in the CA1 sector (Table 1). On the contrary, the early predegeneration phase of fodrin
proteolysis in the CA3 sector was observed even under these conditions,
suggesting that proteolysis of fodrin at this stage is insufficient to
cause the delayed death of neurons. In clear contrast, the late
predegeneration phase of proteolysis paralleled the cell death;
fodrin proteolysis in the entire CA1 at 24 hours arose only under the
conditions that induced neuronal degeneration: ie, a 4-minute–long
ischemia. The postdegeneration proteolysis obviously correlated
well with the cell death because it appeared only in dying and dead
neurons (data not shown). These results suggest that the late
predegeneration phase may play critical roles in the
postischemic pathological cascade and present important
medical implications (see "Discussion").
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Finally, we analyzed the proteolytic products of fodrin in
the postischemic hippocampus by immunoblotting with the
anti-fodrin antibodies recognizing distinct sites of the molecule
(Fig 1). Each immunoblot study showed a constant tendency, as shown in
Table 2. In accordance with our previous
report,16 a 150-kD fragment recognized by both of the
antibodies was produced in postischemic hippocampus
throughout the entire experiments, although its amount gradually
decreased (Fig 7). Notably, a smaller fragment of
approximately 130 kD recognized by the anti-PEST sequence antibody was
also produced in the postischemic hippocampus (Fig 7B).
Because this fragment was not recognized by the anti–proteolyzed
fodrin antibody, it is devoid of the amino-terminal portion of the
150-kD fragment. Concomitantly, a smaller fragment of 20 kD recognized
by the anti–proteolyzed fodrin antibody, but not by the anti-PEST
sequence antibody, increased, particularly at 24 hours after
ischemia (Fig 7A), presumably representing the
amino-terminal fragment produced by this secondary proteolysis.
Because the distance between the cleavage site attacked by calpain and
the location of the PEST sequence is close to 20 kD,20 21
these data suggest that fodrin was proteolyzed through two steps, as
shown in Fig 8. Importantly, this 20-kD product
presumably contains the entire calmodulin-binding
segment and would no longer bind to actin filaments. It may be simply
complicated with calmodulin in cytoplasm under
postischemic circumstances with elevated
[Ca2+]i. We should not, however,
overestimate the amount of this 20-kD fragment relative to that of the
150-kD fragment in interpreting the immunohistochemical data, because
the efficiency of electroblotting decreases as the molecular weight of
protein increases. The difference in blotting efficiency between 150-kD
and 20-kD proteins could vary by a factor of 10 or more. We should
therefore view the immunohistochemical data as showing the 150-kD and
20-kD fragments together.
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The finding of the secondary proteolysis raises the question of the
identity of the protease involved. Calpain does not usually produce the
130-kD fragment of the fodrin subunit in test
tubes.12 15 Therefore, an unidentified protease other than
calpain is likely to be responsible for this process, although we
cannot totally exclude calpain as a candidate protease because its
action could be varied under the specific in vivo conditions.
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Discussion |
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The nature of calpain-catalyzed proteolysis is not digestive, but it usually proceeds in a limited manner, resulting in alteration of the structure and function of the substrate proteins rather than destruction.10 Fodrin provides a number of functional proteins involved in signal transduction with multiple sites for interactions; it acts as part of an anchor for membrane receptors,22 23 regulates actin filaments,11 and undergoes phosphorylation by protein kinase A,24 protein kinase C, and tyrosine kinase.25 26 Limited proteolysis by calpain takes place as a unique mechanism to regulate this interface protein, leading to alterations in receptor geometry and membrane morphology.27 28 We therefore believe that the role of fodrin proteolysis in ischemia-induced neurotoxicity is not simply a destruction of cellular structures but rather a mediation of signals resulting in neuronal degeneration.
Hippocampal CA1 neurons are located at the end of a trisynaptic
chain of excitatory synapses that consists of dentate granule cells,
CA3 neurons, and CA1 neurons. Many reports demonstrate that the
trisynaptic chain is closely involved in development of delayed
neuronal death.29 30 We also demonstrated that two kinds
of protein kinase are activated in the gerbil trisynaptic chain
after forebrain ischemia.31 32 However, in this
study the proteolysis of fodrin was not observed in the dentate gyrus
and mossy fiber system throughout the time course (Fig 3), whereas
intact fodrin exists in the whole hippocampus (Fig 5). This may be due
to lack of Ca2+ accumulation in the dentate gyrus
after the ischemia.33 Amaral and
Witter34 showed that CA3 pyramidal neurons
receive input directly from the perforant path, not via the dentate
gyrus and mossy fibers. Therefore, our results may indicate that a
pathway other than a trisynaptic chain of excitatory synapses is
involved in the development of the delayed neuronal death.
We have demonstrated that short-term (less than 3 minutes) forebrain ischemia, which does not induce delayed neuronal death, produces an early but not a late predegeneration phase of proteolysis. In contrast, the long-term (more than 4 minutes) forebrain ischemia, which consistently induces delayed neuronal death, produced both early and late predegeneration phases of proteolysis. These observations suggest that the late predegeneration phase of fodrin-proteolysis is closely associated with the development of the delayed degeneration of CA1 pyramidal cells. This observation is in agreement with the report that Ca2+ accumulation is observed in the CA1 sector between 2 and 7 days after the 10-minute forebrain ischemia,33 which may continuously activate calpain and cause proteolysis of the fodrin. It is difficult to clarify whether calpain-degraded fodrin has any pathological role in the ischemic neuronal damage or is just a marker for calpain activation. Calpain-degraded fodrin causes morphological changes in erythrocytes,35 and it may play a role in development of long-term potentiation.36 However, its pathological role has not been reported. Microinjection of it into the CA1 sector may be useful to examine its toxicity. Lee et al37 demonstrated that an inhibitor of calpain protected ischemic neuronal damage and proteolysis of fodrin. Therefore, activation of calpain is closely involved in development of ischemic neuronal death, at least as a mediator of the pathological signals stated above. At any rate, if this late predegeneration phase proves to play an essential rate-limiting role, inhibition of calpain action before this stage in postischemic brain could be a possible therapeutic strategy in clinical medicine, giving hope for a successful postinjury treatment. Certainly further studies to evaluate, for instance, the effect of calpain inhibitors on specific proteolysis and cell death will be necessary to establish this assumption.
Finally, we have discovered a secondary processing of fodrin in postischemic brain by which the 150-kD fragment produced by calpain action is further converted to 130-kD and 20-kD fragments. Because the 20-kD fragment seems to carry an entire calmodulin-binding segment and because its localization would no longer be restricted to cytoskeletal structures, it may play a specialized role in the pathological cascade by associating with calmodulin. It is also possible that conversion of the 150-kD fragment to a 130-kD fragment may destabilize the protein structure and thus facilitate further degradation. Identification of the protease responsible for this step would be a subject of future studies.
Received March 13, 1995; revision received May 23, 1995; accepted June 16, 1995.
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[Abstract/Free Full Text]
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