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
mate, NMDA and kainate [6,7,12,19,25]. As high con- centrations of glutamate are achieved during ischemia, it
2.1. Cell culture was originally believed that cell death following ischemia
was primarily necrotic. In agreement, z-VAD.fmk was Rat cortical mixed neuronal and glial cultures were
unable to block excitotoxicity induced with NMDA or prepared from gestational day 17 fetal rats. The cortex
kainate [10]. Interestingly, they also showed that if the from each brain hemisphere was dissected under a micro-
excitotoxicity was prevented by the NMDA channel scope and maintained in Hank’s balanced salt solution
blocker MK-801, the surviving neurons gradually under- HBSS Life Technologies, Gaithersburg, MD, on ice
went death which was preventable by the caspase inhibitor. until the dissection was completed. Cortical tissue was
In cultured cortical neurons, glutamate causes cell death then transferred to a tube containing room temperature
which appears to be necrotic and distinct from that induced 0.025 w v trypsin Sigma, St. Louis, MO in HBSS
by STS [19]. However, in cerebellar granule cells, gluta- and mixed by gentle rocking for 10 min. Tissue was
mate at low concentrations has been reported to induce allowed to settle, the trypsin-containing solution was
caspase-3, with resultant apoptosis [8]. Neither NMDA nor aspirated, and the tissue was washed three times with
MK-801 were shown to affect STS-induced cell death in minimum essential media MEM Life Technologies,
hippocampal neurons [23] suggesting that neurons suscep- Gaithersburg, MD. Cells were dissociated by trituration in
tible to apoptosis are of a different phenotype than those MEM containing 25 mM glucose Mallinckrodt Chemical,
which undergo excitotoxicity. Paris, KY and 2 mM
L
-glutamine Sigma, St. Louis, MO The conflicting reports which have emerged concerning
through the mouth of a 10 ml glass pipette. Cells were cell death in in vitro cultures indicate that inducers of
counted and plated in 24-well cell-culture plates Costar,
C .E. Thomas, D.A. Mayle Brain Research 884 2000 163 –173
165
Corning, NY or Lab Tek II 4-well chamber slides Nalge nucleosome ELISA kit Oncogene Research Products,
Nunc International, Naperville, IL, which were previously Cambridge, MA essentially as described by the manufac-
coated with polyethylenimine Sigma, St. Louis, MO and turer. Briefly, the assay measured mono- and oligonucleo-
conditioned with serum-containing media prior to cell somes in cell lysates by capture of free nucleosomes on the
5
culture initiation. Cells were plated at a density of 2.5310 surface of a DNA-binding protein coated microtiter plate
cells well in media composed of MEM, 25 mM glucose, 2 well. Anti-histone 3 biotin-labeled antibody was added,
mM
L
-glutamine, 10 heat-inactivated horse serum and detection was accomplished with streptavidin-linked
Sigma, St. Louis, MO, and 10 heat-inactivated fetal horseradish peroxidase conjugate which catalyzes the
bovine serum Life Technologies, Gaithersburg, MD and conversion of a colorless substrate to yield a product
maintained in a 5 CO , humidified, 378C incubator. Four which, following the addition of an acidified stop solution,
2
days after plating, half of the culture media was removed absorbs at a wavelength of 450 nm.
and replaced with fresh culture media composed of MEM, 25 mM glucose, 2 mM
L
-glutamine, and 10 heat-inacti- 2.4.2. Caspase-3 activity
vated horse serum. Cells were also treated at this time with Caspase-3 activity was measured in cell lysates using
15 mg ml 5-fluoro-29-deoxyuridine and 35 mg ml uridine the fluorogenic substrate N - acetyl - Asp - Glu - Val - Asp -
both obtained from Sigma, St. Louis, MO to minimize 7 - amino - 4 - trifluoromethylcoumarin
Ac-DEVD-AFC non-neuronal cell growth. Additional media changes were
PharMingen International, San Diego, CA. Equal vol- conducted, as described above, at seven and eleven days in
umes of cell lysate and reaction buffer containing 40 mM culture, and cells were treated for experimentation at
HEPES, 200 mM NaCl, 2 mM EDTA, 0.2 w v twelve days in culture.
CHAPS, 20 sucrose, 20 mM DTT, and 100 mM Ac- DEVD-AFC were combined in a microtiter plate and
2.2. Treatment of cells with pharmacologic agents incubated at 378C for 1 h. Fluorescence was measured at
an excitation wavelength of 380 nM and an emission 2.2.1. NMDA pre-treatment
wavelength of 508 nm. Background fluorescence values, In order to effect deletion of NMDA-sensitive neurons,
determined by measuring samples of cell lysis buffer cell cultures were exposed to 500 mM NMDA in cell
without cell lysate and reaction buffer, were subtracted culture media for 4 days prior to STS treatment. When the
from fluorescence units obtained from each sample. media change was conducted in pre-treated cultures on
culture day 11, NMDA was added to maintain a 500 mM 2.4.3. Western blot analysis of caspase-3
concentration. Cells were lysed in 0.3 ml of insect cell lysis buffer
PharMingen International, San Diego, CA and samples 2.2.2. Treatment
were stored at 2208C until analyzed. Samples were Cells were exposed to treatment solutions by complete
analyzed for protein content and 2.5 mg total protein were aspiration of cell culture media and replacement with a
mixed with 23Tris–glycine sample buffer NOVEX USA, serum-free balanced salt solution BSS 116 mM NaCl,
San Diego, CA containing 3 b-mercaptoethanol and 5.4 mM KCl, 0.8 mM MgSO , 1.0 mM NaH PO , 0.9
heated to 958C for 5 min. Samples were loaded into a
4 2
4
CaCl , 26 mM NaHCO , 25 mM glucose, pH 7.4. NOVEX
E pre-cast 4–20 Tris–glycine gel and run on an
2 3
Treatment agents Sigma, St. Louis, MO were dissolved electrophoresis apparatus at 125 constant volts for approxi-
in BSS, except for STS which was dissolved in DMSO mately 2 h in NOVEX
E Tris–glycine STS running buffer. final treatment concentrations of DMSO did not exceed
The gel was then removed from its casting and placed in 0.5.
BupH E Pierce, Rockford, IL Tris–glycine transfer buf-
fer. Proteins were transferred to a Hybond-P membrane at 2.3. Cytotoxicity
100 mA for 18 h at 88C. The membrane was blocked with 5 powdered milk, 0.1 Tween 20 in tris buffered saline.
Cellular leakage of LDH was determined by collection Primary antibody for caspase-3, rabbit polyclonal IgG
of an aliquot of the BSS treatment solution at the appro- Santa Cruz Biotechnology, Santa Cruz, CA was diluted
priate timepoint for analysis using the Modified Gay to a concentration of 0.2 mg ml and incubated with the
procedure [3,9] on a Hitachi 911 or Hitachi 914 analyzer membrane for 1 h. After washing, the membrane was
Roche Boehringer Mannhein, Indianapolis, IN. Total incubated with the secondary antibody, donkey anti-rabbit
cellular LDH content was determined by lysis of control IgG-HRP Santa Cruz Biotechnology, Santa Cruz, CA at a
cells with Triton X-100. concentration of 0.2 mg ml for 45 min. Detection was
accomplished with the use of ECL Plus Western blotting 2.4. Apoptosis related assays
detection reagents Amersham, Buckinghamshire, UK. 2.4.1. Oligonucleosome quantitation
2.4.4. TUNEL and DAPI staining DNA fragmentation was quantitated by the use of a
For staining purposes, treatment media was removed
166 C
from cells which had been cultured in 4-well chamber of oligonucleosomes was determined as an indicator of
slides and cells washed once in phosphate buffered saline apoptosis. It can be seen that STS induced a 831 increase
PBS. Cells were then fixed for 30–45 min at room in cleavage of the substrate while NMDA produced a
temperature in 4 paraformaldehyde followed by a single modest increase of 225. The difference in the formation
wash with PBS and storage at 48C until staining procedure of nucleosomes when comparing the two agents was much
was performed. An in situ cell death detection kit from less, with STS treatment increasing nucleosome formation
Boehringer Mannheim Indianapolis, IN was used to to 308 of control as compared to 225 for NMDA.
conduct TUNEL staining. Briefly, terminal deoxynu- The finding that LDH leakage occurred with a pro-
cleotidyl transferase is used to catalyze the polymerization apoptotic agent such as STS led us to examine the
of nucleotides to free 39-OH DNA ends. Fluorescein labels timecourse of LDH leakage and caspase activation, and to
incorporated into the nucleotide polymers are detectable by compare it to other inducers of cell death. In Fig. 1A it is
fluorescence microscopy. Cells were permeabilized with shown that over a 20 h time period there is essentially no
0.1 Triton-X 100 for 2 min, washed twice with PBS, and temporal separation of LDH leakage and caspase-3 activa-
incubated with the TUNEL reaction mixture at 378C in the tion in response to STS. Treatment of the cells with 100
dark for 1 h and rinsed with PBS. The chamber walls were mM hydrogen peroxide resulted in LDH leakage of ap-
removed from each slide and one drop of Vectashield proximately 27 of total cellular LDH Fig. 1B. Caspase-
mounting media with DAPI Vector Laboratories, Inc., 3 activity was also elevated but occurred more slowly than
Burlingame, CA was added to each well. A coverslip was LDH leakage. Exposure of the cultures to the nitric oxide
placed on each slide and cells were examined using an oil donor, spermine NONOate, also lead to a similar degree of
immersion lens on a fluorescent microscope at the follow- cell injury as assessed by LDH leakage Fig. 1C. In
ing wavelengths: TUNEL staining, excitation 450 nm, contrast to STS and peroxide treatment, no caspase-3
emission 565 nm; DAPI staining, excitation 360 nm, activity was detected. We have also determined that no
emission 460 nm. oligonucleosomes are generated via NONOate exposure
which also implies a non-apoptotic death with this agent data not shown. These data suggest that caspase-3
3. Results activity is not simply activated non-specifically in response
