Brain Research 884 2000 116–128 www.elsevier.com locate bres
Research report
Seizure-induced neuronal death is associated with induction of c-Jun N-terminal kinase and is dependent on genetic background
Paula Elyse Schauwecker
Department of Cell and Neurobiology , University of Southern California, Keck School of Medicine, BMT 401, 1333 San Pablo Street, Los Angeles,
CA 90033, USA
Accepted 22 August 2000
Abstract
Previous studies have shown that expression of c-Jun protein, as well as the c-Jun amino-terminal kinase JNK group of mitogen-activated protein kinases, may play a critical role in the pathogenesis of glutamate neurotoxicity. In order to define the molecular
cascade that leads to c-Jun activation following excitotoxic injury and delineate whether induction of protein synthesis is related to cell death signaling cascades or those changes associated with increased seizure activity, we examined the expression of JNK-1, as well as its
substrate, c-Jun and N-terminal phosphorylated c-Jun following kainic acid KA administration in two strains of mice. In the present study, we assessed the immunohistochemical expression of these proteins at time points between 2 h and 7 days, in excitotoxic cell
death-resistant C57BL 6 and -susceptible FVB N mouse strains that were systemically injected with saline or kainic acid. No strain-related differences in the immunohistochemical expression of any of the proteins were observed in intact control mice. However,
following KA administration, the magnitude and period of induction of JNK-1 protein was associated with impending cell death, while increased phosphorylation of c-Jun protein was associated with resistance to cell death. In contrast, expression of c-Jun protein does not
appear to be a reliable indicator of impending cell death, as it was expressed in resistant and vulnerable subfields in mice susceptible to kainate injury. These results provide the first evidence that JNK-1 expression may be involved in producing the neuronal cell death
response following excitotoxin-induced injury.
2000 Elsevier Science B.V. All rights reserved.
Theme : Disorders of the nervous system
Topic : Neurotoxicity
Keywords : Hippocampus; Immunocytochemistry; Excitotoxicity; Mouse strain
1. Introduction neuronal death. Although the mode of neuronal cell death
induced by excitotoxins has been widely debated, previous Systemic administration of kainic acid KA, a model of
studies have suggested that this type of cell death exhibits excitotoxicity, induces recurrent seizures and epileptiform
some of the characteristics of both necrosis and apoptosis, activity in rodents. The seizures cause selective excitotoxic
depending on the severity of the stimulation and the levels
21
cell death of pyramidal neurons in the CA1 and CA3 of intracellular free Ca
[14,23,28,42]. subfields and dentate hilar neurons of the hippocampus,
While the molecular mechanisms of cell death are not while sparing neurons in the CA2 subfield and dentate
well understood, excitotoxic cell death may result from the granule neurons [4,31,34,41]. Excitotoxic cell death is
activation of intracellular signaling cascades that may be triggered by a massive release of glutamate, which acti-
genetically determined. One of these signaling cascades, vates glutamate receptors leading to dramatic increases in
the c-Jun amino-terminal kinase JNK SAPK pathway, is
21 21
intracellular Ca [7]. The increased Ca
levels initiate a component of signal transduction cascades that results in
signaling cascades within susceptible neurons resulting in cell stress and death, and can be activated by a variety of
cellular stresses, such as heat shock, osmotic imbalance, oxidative stress, and excitotoxicity [1,15,21,26,35,48]. The
JNK SAPK kinases are composed of three isoforms, JNK-
Tel.: 11-323-442-2116; fax: 11-323-442-3466. E-mail address
: schauwechsc.usc.edu P.E. Schauwecker.
1, JNK-2, and JNK-3, all of which have been proposed to
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 8 8 - 2
P .E. Schauwecker Brain Research 884 2000 116 –128
117
be important mediators of stress-activated signal transduc- served as subjects. All mice were housed individually on a
tion pathways [20]. 12-h light dark schedule. Water and food were available ad
JNKs are also important post-translational modifiers of libitum.
c-Jun activity [3,11], in that they can increase the tran- scriptional activity of c-Jun by phosphorylation on serine
2.2. Drug administration 63 and serine 73, located within the amino terminal
activation domain [8,22,37,43]. Increased c-Jun expression All experimental procedures were performed in accord-
has been identified both in neuronal populations with the ance with approved institutional animal research protocols.
capacity to survive an excitotoxic insult [44], as well as Kainic acid was dissolved in isotonic saline pH 7.3 and
those populations where selective neuronal degeneration administered subcutaneously. Previous studies have de-
occurs [9]. However, the association between expression of fined seizure thresholds and revealed consistent seizures in
immediate early genes, such as c-Jun, and their putative both strains with a mortality rate of less than 25 at a dose
role as cell death-effectors or -repressors following ex- of 30 mg kg, s.c. [39]. Following KA administration, mice
citotoxic cell death remains unclear. were monitored continuously for 3–4 h for the onset and
While the environmental and genetic cues leading to in extent of seizure activity. Seizures were rated according to
vivo cell death of neurons are not well understood, our a previously defined scale [38]: Stage 1, immobility; Stage
model of strain-related differences in susceptibility to 2, forelimb and or tail extension, rigid posture; Stage 3,
excitotoxic cell death provides a means for differentiating repetitive movements, head bobbing; Stage 4, rearing and
between those molecular events that govern cell death and falling; Stage 5, continuous rearing and falling; and Stage
protection versus those that occur in response to cell stress. 6, severe tonic-clonic seizures. All mice included in the
Previously, we had found that susceptibility to KA-induced study exhibited at least 1 h of continuous Stage 5 seizures.
cell death is strain-dependent, yet seizure activity is comparable between strains [39,40]. Thus, we were inter-
2.3. Histological staining and immunohistochemistry ested in determining how the genetic program in C57BL 6
mice a representative resistant strain is altered to allow Following survival times of 2, 6, 24, 48, 72, or 168 h
protection against excitotoxic cell death. The present n55 or 6 per time point, mice received an overdose of
experiments were performed to evaluate the relationship sodium pentobarbital and were perfused transcardially with
between seizure induction, expression of the immediate 4 paraformaldehyde in 0.1 M phosphate buffer pH 7.4
early gene c-Jun, phosphorylation of c-Jun, and its following saline or KA administration. Brains were imme-
upstream regulator JNK, and cell loss within the hip- diately removed and post-fixed overnight in the same
pocampus in mouse strains resistant and susceptible to fixative before being cryoprotected in 30 sucrose in 0.1
kainic acid-induced excitotoxicity. M phosphate buffer pH 7.3. Horizontal sections 35 mm
In this article, we show that increased expression of were cut on a sliding microtome Leica, Deerfield, IL and
JNK-1 occurred only in mice susceptible to excitotoxic cell incubated in 0.1 M phosphate buffer pH 7.4 prior to
death and was never solely associated with a cell stress immunohistochemical staining. For histological assess-
response. In contrast, enhancement of c-Jun phosphoryla- ment, every sixth section was stained with cresyl violet to
tion was observed only in those cells resistant to seizure- determine neuronal cell loss and the general histological
induced cell death or that survived the excitotoxic insult. features of the tissue. For immunohistochemistry, every
Expression of c-Jun protein does not appear to be indica- sixth section was rinsed in 0.1 M phosphate buffer pH
tive of susceptibility or resistance to excitotoxic cell death 7.4 three times for 15 min each, incubated for 2 h on a
as protracted expression of c-Jun protein was observed in rotary shaker in a blocking buffer containing 0.5 Triton-
both damaged and resistant cell populations in mice X and 5 normal goat serum in 0.1 M phosphate buffer
susceptible to excitotoxic cell death. These findings dem- pH 7.4. Sections were then incubated overnight at 48C
onstrate that increased expression of JNK-1 is associated with one of the following polyclonal antibodies: c-Jun
with KA-induced cell death and provides further support 1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
for the hypothesis that the JNK SAPK cascade is involved JNK-1 1:500; Santa Cruz Biotechnology, Inc., Santa
73
in excitotoxic cell death. Cruz, CA, or phospho-c-Jun Ser ; 1:1000; Upstate
Biotechnology, NY in the presence of 5 normal goat serum in 0.1 M phosphate buffer pH 7.4. Sections were
2. Materials and methods rinsed in 0.1 M phosphate buffer pH 7.4, incubated for 2
