Brain Research 884 2000 163–173 www.elsevier.com locate bres
Research report
NMDA-sensitive neurons profoundly influence delayed staurosporine- induced apoptosis in rat mixed cortical neuronal cultures
Craig E. Thomas , Douglas A. Mayle
Investigative Toxicology , Lilly Research Laboratories, A Division of Eli Lilly and Company, 2001 W. Main St., Greenfield, IN 46140, USA
Accepted 8 August 2000
Abstract
We have investigated cell killing in cultured rat embryonic cortical neurons exposed to the protein kinase inhibitor staurosporine, the excitatory amino acid N-methyl-
D
-aspartate NMDA, or a combination thereof. Our data indicate that there are several populations of neurons that differ in their response to these agents. Cultures exposed to NMDA undergo cell death typified by lactate dehydrogenase
LDH leakage which is likely primarily necrotic in that little caspase-3 activation or oligonucleosome formation is observed even when followed for 48 h. Cells exposed to staurosporine STS exhibit rapid, extensive activation of caspase-3 with coincident LDH leakage,
oligonucleosome formation and TUNEL staining. Both LDH leakage and oligonucleosome content were significantly more elevated at 48 h than at 20 h with STS treatment while caspase-3 activity peaked early 8–20 h and declined markedly by 48 h. Deletion of
NMDA-responsive neurons by pre-treatment of the cultures with NMDA for 4 days prevented the late phase 20–48 h increases in LDH leakage and oligonucleosomes in the remaining neuronal population. Caspase-3 activity was also completely abolished by NMDA
pre-treatment. These results indicate that cells susceptible to acute NMDA-induced toxicity can be killed by non-apoptotic means when exposed to NMDA; however, they undergo a delayed, apoptotic death when exposed to STS. Interestingly, removal of NMDA-responsive
cells prevents the processing of procaspase-3; thus, STS-induced apoptosis in cells resistant to NMDA-mediated killing proceeds independent of caspase-3 activation. The data indicate that nearly all neurons in these mixed cultures can undergo apoptosis in response to
appropriate stimuli such as STS but that the temporal nature, and the pathways activated in response to STS, vary amongst the subpopulations of neurons. These findings may help to explain the simultaneous appearance of features of both apoptosis and necrosis
observed in vivo following cerebral ischemia.
2000 Elsevier Science B.V. All rights reserved.
Theme : Disorders of the nervous system
Topic : Ischemia
Keywords : Neuron; Apoptosis; Necrosis; Staurosporine; N-Methyl-
D
-aspartate; Caspase
1. Introduction exhibiting features of apoptosis. Interestingly, caspase-8
activation occurred beginning at 6 h and was primarily Neuronal cell death following insult such as ischemia or
localized to the large pyramidal neurons while caspase-3 trauma is a complex series of integrated events which can
activation was not detected until 24 h and was localized to lead to neuronal loss via apoptosis and or necrosis. Velier
lamina II III. In a model of controlled cortical impact et al. [28] have recently examined cell death and caspase
injury in the rat, DNA damage indicative of both apoptotic activation in cortical regions following focal cerebral
and necrotic cell death were observed at the site of impact ischemia. It was observed that there appeared to be an
[20]. These findings indicate that delayed cell death early non-apoptotic, likely necrotic, loss of cortical neu-
following ischemia is highly dependent upon the cell type rons followed by a progressive elimination of neurons
and that conclusions concerning cell death can be strongly influenced by the time and region in which cell death is
examined.
Corresponding author. Lilly Development Centre, Parc Scientifique de
Ample in vitro evidence also suggests a multiplicity of
Louvain-la-Neuve, Rue Granbonpre, 11, 1348 Mont-Saint-Guibert, Bel-
pathways leading to cell death in cultured neurons. Treat-
gium. Tel.: 132-10-47-6496; fax: 132-10-47-6925. E-mail address
: cthomaslilly.com C.E. Thomas.
ment of neurons with a variety of insults ranging from
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 3 4 - 1
164 C
glutamate [2,24] to serum withdrawal [29] has provided apoptosis, such as STS, and necrosis, such as NMDA, may
evidence for aspects of both apoptosis and necrosis. The affect cells differently depending upon cell type and
protein kinase inhibitor staurosporine STS has been concentration of the activating agent. In an effort to more
utilized by a number of investigators as an inducer of fully characterize the response of a mixed neuronal popula-
neuronal apoptosis [13,15,23]. In cortical neurons, STS has tion to presumably apoptotic and necrotic insults we have
been shown to activate apoptosis in both neurons [19,21] carefully studied STS and or NMDA-induced cell death in
and, at high concentration, in astrocytes [21]. DNA primary rat cortical neurons by monitoring LDH leakage,
alterations and morphologic appearance were consistent caspase-3 activation and oligonucleosome formation. A
with apoptosis. In PC12 cells, STS induced caspase-3 time-course experiment indicated that there appeared to be
activity and, accordingly, the peptide inhibitor z-VAD.fmk, two phases to the STS-induced apoptotic process; the first
a pan-selective inhibitor of caspases, prevented cell death occurred during the initial 24 h period of STS treatment
[17]. In contrast, cell death elicited by the lipid peroxida- and was followed by increasing levels of oligonucleosome
tion product 4-hydroxynonenal did not activate caspase-3 formation over an additional 24 h of treatment. NMDA
nor did the caspase inhibitor prevent cell death. McManus treatment elicited cell death which showed little evidence
et al. [19] compared STS-induced cell death to glutamate of apoptosis. Pre-treatment of cultures with NMDA prior
toxicity in cortical cells and concluded that glutamate to STS demonstrated that the latter phase of cell death
caused cell killing by non-apoptotic means as no evidence following STS was most likely due to the death of NMDA-
for DNA laddering was observed and the appearance of the sensitive neurons which were exhibiting a delayed, apop-
nuclei was distinct from that following STS treatment. totic death in response to STS. Furthermore, NMDA-
While STS treated neurons are typified by features responsive neurons were obligatory for activation of
associated with apoptosis, the exact mechanism of cell caspase-3 by STS. These findings indicate that: 1 most
death remains unknown. In rat hippocampal neurons, the neurons undergo apoptosis in response to STS, 2 neurons
caspase inhibitor DEVD-CHO prevented the STS-induced which die in a non-apoptotic fashion in response to NMDA
increase in caspase-3 activity but did not ameliorate cell have the capacity to undergo delayed 20–48 h apoptosis,
death [16]. Choi et al. [10] have reported that z-VAD.fmk, 3 caspase-3 is activated specifically in NMDA-sensitive
but not z-DEVD.fmk, a caspase-3-like protease inhibitor, neurons and 4 non-NMDA-sensitive neurons which
can prevent STS-induced cell death in mouse neocortical undergo apoptosis with STS do so in a caspase-3 in-
cultures. In SH-SY5Y cells exposed to STS, the protein dependent manner. Thus, it is clear that neuronal apoptosis
synthesis inhibitor cycloheximide did not prevent cell in the in vivo setting is likely to involve both apoptosis and
death [26] whereas it effectively does so in other cell types necrosis, and that the pattern observed is a reflection of the
[10]. These disparate findings further suggest that induc- cell phenotypes and the nature of the activators of cell
tion of apoptosis can be inducer and cell-type specific; death which are particular to the system under inves-
even in neurons. tigation.
An alternative mode of cell death occurring in neurons is necrosis which is generally considered to occur in
neurons following exposure to excitotoxins such as gluta-
2. Materials and methods
