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* Sanders-Brown Research Center on Aging,
Department of Neurology, and Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536, USA; and
§ Laboratory of Neurosciences, National Institute on Aging, Baltimore, Maryland 21224, USA
1Correspondence: Laboratory of Neurosciences, National Institute on Aging, GRC 4F01, 5600 Nathan Shock Drive, Baltimore, MD, 21224, USA. E-mail: mattsonm{at}grc.nia.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: NSC-19 • oxidative stress • spinal cord • superoxide dismutase • transgenic mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. As for other
neurodegenerative disorders, a proposed mechanism of neuronal death in
ALS involves reactive oxygen species, membrane lipid peroxidation, and
disruption of ion homeostasis (3
, 4)
. A consequence of
oxidative stress and membrane lipid peroxidation is mitochondrial
dysfunction, initiating a series of events that leads to neuronal
apoptosis (5)
. There are now several lines of evidence
implicating oxidative stress in motor neuron degeneration in ALS. The
evidence includes 1) missense mutations in the antioxidant
enzyme Cu/Zn-superoxide dismutase (Cu/Zn-SOD) occur in a subset of
familial ALS patients (6)
; 2) transgenic mice
expressing mutant human Cu/Zn-SOD develop a clinical phenotype and
neuropathological changes analogous to that seen in humans
(7
8
9
10)
; 3) in vitro studies
revealed increased affinity of mutant Cu/Zn-SOD for hydrogen peroxide
resulting in enhanced hydroxyl radical formation (11
, 12)
;
and 4) increased levels of protein carbonylation
(13)
, free and protein-bound 3-nitrotyrosine
(14)
, and proteins modified by the lipid peroxidation
product 4-hydroxynonenal (HNE) (15)
in the spinal cords of
ALS patients. The cause(s) of oxidative stress in most cases of ALS is
unknown, but it may be related to the absence of the astroglial
glutamate transporter EAAT2 in some patients (16)
.
Elevated glutamate levels would lead to a disruption in calcium ion
homeostasis and an increase in free radical production. Under certain
conditions, glutamate toxicity can manifest as apoptosis in neuronal
cultures (17
, 18)
.
Although there is evidence for apoptosis in ALS (19
20
21)
,
the proapoptotic proteins that mediate neuronal death in this disorder
have not been identified. From the androgen-independent prostatic
cancer cell line, AT-3, a novel gene was isolated by differential
hybridization on a cDNA library prepared from the cells after exposure
to ionomycin to induce apoptosis (22)
. This gene,
designated prostate apoptosis response (par)-4, encodes a 38
kDa protein that belongs to the family of immediate-early gene
products, which include c-Myc, c-Fos, c-Jun, Nur77, and EGR-1
(23)
. Unlike the other immediate-early gene products,
par-4 expression appears to be induced exclusively by
apoptotic stimuli (22
, 24)
. The carboxyl-terminal portion
of the Par-4 protein contains a death domain homologous to that of Fas
and TRADD and may, therefore, initiate a cascade of events analogous to
that of other death domain-containing proteins (23)
.
Within the death domain of Par-4 is a leucine zipper domain that
appears to mediate protein–protein interactions (25)
.
Because of the widespread expression of par-4
(26)
, we hypothesized that the Par-4 protein may play a
role in the pathogenesis of neurodegenerative disorders. Recently, we
observed increased Par-4 protein and mRNA levels in the hippocampus of
a group of Alzheimer’s disease patients relative to the control group
(27)
. Moreover, we demonstrated that over-expression of
full-length Par-4 protein in PC12 cells increases their vulnerability
to apoptosis induced by amyloid ß-peptide and trophic factor
withdrawal (27)
. In the present study, we examined the
levels of Par-4 protein in the spinal cords of ALS patients and
transgenic mice expressing mutant human Cu/Zn-SOD (7)
relative to a control group. We also carried out studies to determine
the response of Par-4 in primary motor neuron cultures and in a motor
neuron cell line, NSC-19 (28)
, exposed to
oxidative/apoptotic insults.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transgenic mice
Heterozygous breeding pairs of transgenic mice expressing the
human Cu/Zn-SOD gene with a G93A mutation were purchased from The
Jackson Laboratory (Bar Harbor, Me.). These mice were generated on a
B6/SJL background. At weaning age, the offspring of the heterozygous
matings were characterized by measuring the levels of Cu/Zn-SOD
activity in tail blood samples [procedure modified from ref
29
]. A subset of mice was paralyzed in one or more limbs
at 4–5 months of age (homozygotes), whereas the remaining mice did not
show the phenotype until 6–8 months of age (heterozygotes; this
corresponded to the age of disease onset in the original
heterozygotes). For Western blot analyses, mice were killed with
inhalation anesthesia and whole spinal cords were removed, placed on
dry ice, and stored at -80°C until use. For immunohistochemical
analyses, mice were perfused transcardially with 4% paraformaldehyde
and whole spinal cords were stored at 4°C in the same solution for
24 h, at which time they were processed for sectioning.
Cell cultures and experimental treatments
Primary cultures of mixed spinal cord cells were established
from day 12–14 embryos of B6/SJL mice as described (30
, 31)
. Motor neurons were identified in the cultures as described
in our previous studies (31)
. The cultures were maintained
at 37°C in a 5% CO2 atmosphere in Neurobasal
medium and B27 supplements (Life Technologies, Gaithersburg, Md.). The
NSC-19 cell line was generated by somatic cell fusion of mouse
neuroblastoma N18TG2 cells with motor neuron-enriched spinal cord
cultures from embryonic day 12–14 mice (28)
. The NSC-19
cells were maintained at 37°C in a 5% CO2
atmosphere in Dulbecco’s modified Eagle’s medium supplemented with
10% heat-inactivated fetal bovine serum and 50 µg/ml of gentamicin.
Cells were subcultured by removing them from the substratum with
squirts of medium; passages up to 30 were used. A 10 mg/ml stock of HNE
in ethanol was purchased from Cayman (Ann Arbor, Mich.) and was stored
at -80°C. Staurosporine, L-glutamic acid, and
FeSO4 were purchased from Sigma (St. Louis, Mo.)
and stock solutions were prepared in dimethyl sulfoxide (staurosporine)
or sterile water (L-glutamic acid, FeSO4).
Treatments were carried out in Locke’s solution (NaCl, 154 mM; KCl,
5.6 mM; CaCl2, 2.3 mM;
MgCl2, 1.0 mM; NaHCO3, 3.6
mM; glucose, 5 mM; HEPES, 5 mM; pH 7.2). Pretreatment with the Par-4
antisense (5'-ATAGCCGCCGGTCGCCATGTT-3') or nonsense
(5'-CCGTGTCTGATCTTCGTGCGT-3') oligodeoxynucleotides was also carried
out in Locke’s solution. Trophic factor withdrawal was accomplished by
washing the cells six times with Locke’s solution.
Western blot analysis
Human and mouse lumbar spinal cord specimens were homogenized in
a 50 mM potassium phosphate buffer (pH 7.4). The homogenates were then
diluted in a buffer consisting of 62 mM Tris-HCl, 2 mM EDTA, 2.3%
sodium dodecyl sulfate, and 10% glycerol (pH 6.0), and aliquots were
stored at -20°C. Protein content was determined using the Pierce BCA
kit. Proteins were separated by electrophoresis in either a 10%
(human) or 12% (mouse) polyacrylamide gel and transferred to a
nitrocellulose membrane. After an overnight incubation at 4°C in
blocking solution (5% dried milk powder in 1xTTBS), the membrane was
immunoreacted with a rabbit polyclonal antibody against Par-4
(24
, 27)
for 3 h at room temperature. A 1:12,000
dilution of a 6 mg/ml stock of the Par-4 antibody was used. The
nitrocellulose membrane was further processed using a horseradish
peroxidase-conjugated anti-rabbit secondary antibody and a
chemiluminescence detection method (Amersham, Arlington Heights, Ill.).
Densitometric analysis was carried out using Scion Image version 1.59
software.
Immunochemistry
Immunohistochemical analysis of lumbar spinal cord sections was
performed as described previously (15)
. Fixed spinal cords
from wild-type and transgenic mice were placed in a solution of 30%
sucrose in phosphate-buffered saline (PBS) for 48 h and segments
of the lumbar region were sectioned at 30 µm. A mouse monoclonal
antibody recognizing Par-4 was used for the immunohistochemical
analysis of free-floating spinal cord sections (Santa Cruz
Biotechnology, Santa Cruz, Calif.; 1:1000 dilution of a 200 µg/ml
stock). Cultured cells were fixed in 4% paraformaldehyde, washed three
times with PBS, pretreated for 30 min with a solution of 0.3%
H2O2 in water, and
incubated for 1 h in a blocking solution containing 0.4% Triton
X-100 and 5% normal goat serum in PBS. The cells were then incubated
overnight at 4°C in blocking solution containing the rabbit
anti-Par-4 polyclonal antibody (1:4000 dilution of a 6 mg/ml stock
solution), washed three times in PBS, and incubated for 1 h at
room temperature with a biotinylated secondary antibody in PBS. After
three washes with PBS, cells were incubated for 30 min in ABC reagent
solution (Vector, Burlingame, Calif.), followed by a 5 min incubation
in nickel-enhanced diaminobenzidine tetrahydrocholride solution
(Vector). The immunocytochemical procedure and acquisition of confocal
laser scanning microscope images of glutamate-treated motor neuron
cultures stained with the Par-4 polyclonal antibody were as described
in our previous studies (27)
. Specificity of the
immunoreaction was confirmed by the lack of staining in sections or
cultures where the primary antibodies had been excluded.
Determination of apoptosis and mitochondrial function
Apoptosis was quantified in NSC-19 cell cultures stained with
the fluorescent DNA-binding dye Hoechst 33342 as described previously
(32)
. Cells stained with the Hoechst dye were visualized
and photographed under epifluorescence illumination (340 nm excitation
and 510 nm barrier filter) using a 40x oil immersion objective (200
cells per culture were counted in at least four separate cultures per
treatment condition; analyses were performed without knowledge of
treatment history). The ‘apoptotic’ cells were considered to be those
with condensed and fragmented nuclei. The ability to convert
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide to
formazan crystals was used as a measure of mitochondrial function and
was carried out as described in our previous studies (33
, 34)
. Absorbances were determined with a Bio-tek CERES 900 plate
reader at 592 nm. The absorbance values from treated cultures
(n=8) are expressed as percentages of the average absorbance
value from untreated cultures (n=8).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Par-4 protein was present in all control and in all ALS
spinal cord samples. However, densitometric analysis of the Western
blot revealed a statistically significant increase in the levels of
Par-4 protein in the ALS spinal cord samples relative to the levels in
the control spinal cord samples (P = 0.02 by two-tailed
Student’s t test). It is possible that there is an
up-regulation of Par-4 expression during the postmortem interval and
that the actual difference in Par-4 protein levels between control and
ALS spinal cord is greater than we can detect. Thus, to provide a more
accurate determination of the extent to which Par-4 is increased in ALS
spinal cord, we compared the levels of Par-4 protein in the spinal
cords of transgenic mice expressing a mutant human Cu/Zn-SOD gene
(7)
with the levels of Par-4 protein in the spinal cords
of wild-type mice. Lumbar spinal cord samples from transgenic mice with
hind-limb paralysis had markedly higher Par-4 protein levels than
lumbar spinal cord samples from age-matched wild-type mice (Fig. 1B
). This result indicates that the actual elevation in
Par-4 protein content in the ALS spinal cord is greater than that
observed with the use of human samples. Immunohistochemical analysis
revealed that Par-4 is primarily localized to motor neurons in the
ventral horn region of the lumbar spinal cord (Fig. 2
). Consistent with the Western blot results, little Par-4 staining was
observed in the wild-type mouse spinal cord sections whereas intense
Par-4 immunoreactivity was observed in motor neurons of the transgenic
mouse sections. In contrast, motor neurons were strongly labeled in
both human control and ALS spinal cord sections, and the difference in
staining intensity was not readily detectable (data not shown).
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Rapid increase in Par-4 levels after exposure of mouse spinal cord
neurons and NSC-19 cells to oxidative/apoptotic insults
If increased Par-4 levels contribute to neuronal degeneration in
ALS, then the levels of Par-4 should increase in living motor neurons
after exposure to insults relevant to ALS. We tested this hypothesis by
exposing spinal cord motor neuron cultures and NSC-19 cells to several
oxidative/apoptotic insults. As shown in Fig. 3
, glutamate caused a concentration-dependent increase in Par-4 levels in
the cultured motor neurons. This effect was observed after only 4 h of treatment and under conditions that preceded apoptosis
(31)
. We then treated the cultures with
FeSO4, which induces hydroxyl radical production
via the Fenton reaction and increases membrane lipid peroxidation in
neuronal cells (32)
. It has been reported that the levels
of membrane lipid peroxidation are increased in lumbar spinal cord
motor neurons of ALS patients (15)
and of Cu/Zn-SOD mutant
mice (35)
. Based on our previous findings that the levels
of HNE-protein conjugates are greater in ALS spinal cord relative to
the levels in control spinal cord (15)
and that HNE can
induce neuronal apoptosis (32)
, we also tested the effects
of HNE on Par-4 levels in both culture systems. A 4 h exposure of
mouse spinal cord cultures to either FeSO4 or HNE
resulted in a pronounced increase in Par-4 protein levels in the motor
neurons (Fig. 3)
. A pronounced increase in Par-4 immunoreactivity was
also observed in NSC-19 cells exposed to either
FeSO4 or HNE (Fig. 4
). Because NSC-19 cells are more resistant to toxic insults than primary
cultures, we treated NSC-19 cells with higher concentrations of both
insults and for a period of 8 h. The levels of Par-4 protein were
also increased greatly in NSC-19 cells exposed to staurosporine for
4 h or subjected to trophic factor withdrawal for 12 h (Fig. 4)
, treatments that induce Par-4 before apoptosis in primary
hippocampal cultures (27
, 36)
.
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Par-4 antisense pretreatment protects motor neurons from
oxidative/apoptotic insults
To further implicate Par-4 in the pathogenesis of neuronal
degeneration in ALS, we determined whether a Par-4 antisense
oligodeoxynucleotide could protect motor neurons against
oxidative/apoptotic insults. The effectiveness of the antisense
oligodeoxynucleotide used here at reducing Par-4 protein levels has
been demonstrated in our previous studies (27
, 37)
. As
shown in Fig. 3
, pretreatment with the Par-4 antisense DNA blocked the
increase in Par-4 levels caused by exposure of the motor neuron
cultures to either FeSO4 or HNE. Consistent with
our previous studies showing specificity of the antisense DNA
(27
, 37)
, pretreatment with a Par-4 nonsense
oligodeoxynucleotide did not affect the FeSO4- or
HNE-induced increase in Par-4 immunoreactivity in these cultures (data
not shown). In cultures treated with FeSO4, there
were many dead or dying motor neurons, whereas in cultures pretreated
with the Par-4 antisense DNA there were considerably more viable cells
(Fig. 3)
. Using NSC-19 cells, we then carried out experiments to
examine the ability of the Par-4 antisense DNA to protect motor neurons
from apoptosis. We assessed two hallmark features of apoptosis, namely
mitochondrial dysfunction and chromatin condensation and fragmentation.
Exposure of NSC-19 cells to staurosporine, FeSO4,
or HNE resulted in a decrease in mitochondrial-reducing potential,
indicated by an impaired ability for MTT reduction, which occurred
within 8 h of treatment (Fig. 5
). Pretreatment of the cells with Par-4 antisense DNA significantly
attenuated the decrease in MTT reduction caused by each insult, whereas
Par-4 nonsense DNA was ineffective (Fig. 5)
. Longer exposures of NSC-19
cells to staurosporine, FeSO4, or HNE or
subjecting the cells to trophic factor withdrawal resulted in chromatin
condensation and fragmentation. This effect was significantly reduced
in cells pretreated with Par-4 antisense DNA but not in cells
pretreated with nonsense DNA (Fig. 5)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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observed increased levels of the
apoptosis-related antigen Le(Y) and fragmented DNA in motor neurons of
the lumbar spinal cord of ALS patients. The abundance of mRNAs encoding
Bax and Bcl-2 are altered in ALS spinal cord motor neurons in a manner
consistent with apoptosis (20)
. In transgenic ALS mice,
over-expression of Bcl-2 delayed motor neuron degeneration and
prolonged survival of the animals (21)
. Expression of a
caspase-1 inhibitor in transgenic ALS mice did not delay disease onset
but did prolong survival (37)
, and a
proteolytic-processing characteristic of activated caspase-1 has been
reported in the spinal cords of transgenic ALS mice (39)
.
In cell culture studies, expression of ALS-linked Cu/Zn-SOD mutations
in PC12 cells, hippocampal neurons, and superior cervical ganglion
neurons was found to increase their vulnerability to apoptosis
(40
, 41)
. Collectively, the results of these studies
indicate that oxidative stress triggers a cascade of events leading to
apoptotic neuronal death in ALS. In this article, we have provided
evidence that oxidative stress-induced apoptosis in motor neurons in
ALS is mediated by Par-4. We previously reported higher Par-4 mRNA and
protein content in the hippocampus of Alzheimer’s disease patients
relative to control patients and that there is a rapid increase in the
levels of Par-4 protein in primary hippocampal cultures exposed to the
amyloid ß-peptide (27)
. Thus, Par-4 may be a general
effector of the cell death process in neurodegenerative disorders, but
the extent of this role remains to be determined.
In at least some ALS patients, the pathogenesis of motor neuron
degeneration is likely to involve an excitotoxic component. The initial
evidence in support of this came from a study where elevated
concentrations of glutamate were found in the cerebrospinal fluid of
ALS patients relative to those of normal patients (42)
.
Subsequently, impaired glutamate transport was observed in synaptosomes
prepared from the spinal cords of ALS patients (43)
and
from transgenic ALS mice (44)
. The cause of impaired
glutamate transport in ALS spinal cord was shown to be the result of a
selective loss of the astroglial glutamate transporter protein EAAT2
(45
, 46)
, which appears to result from defects in
EAAT2 mRNA processing (16)
. Pharmacological
inhibition of glutamate transport in rat spinal cord organotypic slice
cultures leads to a slow degeneration of motor neurons over several
weeks (47)
. This toxic effect could be prevented by
non-NMDA receptor antagonists but not by NMDA receptor antagonists,
consistent with the evidence that motor neurons are selectively
vulnerable to AMPA/kainate receptor-mediated injury (30)
.
The use of antisense oligonucletides specific for EAAT2 mRNA
in spinal cord organotypic slice cultures from rat revealed that
astroglial transporters play the major role in protecting motor neurons
from glutamate toxicity (48)
. Although excitotoxicity may
manifest as necrosis in acute neurodegenerative conditions such as
stroke (49)
and severe epileptic seizures
(50)
, mild glutamate toxicity has been shown to cause
apoptosis in primary neuronal cultures (17
, 18)
. Moreover,
glutamate has been shown to contribute to neuronal apoptosis observed
in some animal models of human neurodegenerative conditions (51
, 52)
. Because induction of par-4 gene expression
requires an elevation of intracellular [Ca2+]
(22)
, the initial phase of activation of glutamate
receptors, we propose that Par-4 plays a role in motor neuron
degeneration in ALS cases involving an excitotoxic component. Support
for this hypothesis comes from our observations that glutamate
treatment causes an increase in Par-4 levels in primary cultures of
spinal cord motor neurons (Fig. 3)
at time points preceding apoptosis
(31)
.
Oxidative damage to proteins and nucleic acids appears to be a common
feature of ALS. The fact that transgenic mice expressing human
Cu/Zn-SOD with familial ALS mutations develop a clinical phenotype and
neuropathological changes seen in humans (7
8
9
10)
supports
a causative role for oxidative stress in motor neuron degeneration in
this disorder. Familial ALS mutations in Cu/Zn-SOD result in a higher
affinity for H2O2 relative
to wild-type Cu/Zn-SOD (12)
, conferring on the mutant
enzyme increased ability to catalyze the oxidation of substrates by
H2O2 and enhanced free
radical production (11
, 12)
. Indeed, fibroblasts from ALS
patients, particularly those harboring Cu/Zn-SOD mutations, are more
sensitive to oxidative stress caused by
H2O2 (53)
. In
the spinal cords of transgenic ALS mice, greater oxyradical production
and lipid peroxidation were found, which preceded onset of motor neuron
degeneration (54
, 55)
. Enhanced free radical production by
mutant Cu/Zn-SOD is consistent with the observations of increased
levels of free and protein-bound 3-nitrotyrosine (56
, 57)
and protein carbonyl groups (58)
in the spinal cords of
transgenic ALS mice relative to control mice. The cause(s) of
oxidative stress in most cases of ALS is unknown, but increased levels
of protein carbonylation, nuclear DNA 8-hydroxy-2'-deoxyguanosine,
3-nitro-4-hydroxyphenylacetic acid, free and protein-bound
3-nitrotyrosine, and malondialdehyde-modified proteins have been
reported in the spinal cords of both familial and sporadic ALS patients
(13
, 59)
. We previously reported a higher concentration of
free HNE in the cerebrospinal fluid of ALS patients relative to
controls (59)
and increased protein modification by HNE in
ALS spinal cord relative to control spinal cord (15)
. The
latter findings are of particular interest because one of the proteins
modified by HNE in ALS spinal cord appears to be EAAT2, and HNE has
been shown to impair glutamate transport in NSC-19 cells
(60)
. We have also previously reported that HNE impairs
the function of ion-motive ATPases, glucose and glutamate transporters
in primary neuronal systems (61
62
63
64)
, and glutamate
transporters in astrocytes (33)
, indicating that HNE
promotes excitotoxic damage.
Our data suggest that oxidative stress and membrane lipid peroxidation
are potent inducers of par-4 expression and/or translation
in motor neurons, because both Fe2+ and HNE
caused rapid and large increases in the levels of Par-4 protein in
primary motor neuron cultures and NSC-19 cells. Given the elevations in
free and protein-bound HNE in ALS samples relative to control samples
(15
, 59)
and the ability of HNE to induce apoptosis in
neuronal cells (32
, 60)
, Par-4 may be a mediator of
HNE-induced motor neuron apoptosis initiated by oxyradical attack on
membrane lipids. Administration of vitamin E (65)
or
carboxyfullerenes (66)
to the transgenic ALS mice delays
the onset of disease, further suggesting a role for oxidative stress in
the pathogenesis of ALS. If oxidative stress is a key factor in causing
an increase in Par-4 levels in ALS motor neurons, then antioxidants
would be expected to block the effect concomitant with a delay in
disease onset. Recent studies from our laboratory indicate that Par-4
can be rapidly and greatly induced by oxidative insults at the
translational level (37)
. Further elucidating the
mechanisms by which Par-4 protein levels are regulated may provide
effective targets for the treatment of ALS.
The mechanism by which Par-4 causes neuronal death is unclear, but
evidence suggests that it may have adverse effects on mitochondrial
function. In PC12 cells, expression of full-length Par-4 exacerbates
accumulation of reactive oxygen species and mitochondrial membrane
depolarization (27)
, suggesting an action on cellular
oxyradical metabolism and/or mitochondrial function. Here, we show that
a Par-4 antisense oligodeoxynucleotide can prevent mitochondrial
dysfunction in NSC-19 cells that precedes apoptosis. Disruption in
mitochondrial energy metabolism is proposed to occur early in the
neurodegenerative process (67)
, thus triggering a series
of events culminating in cell death. However, evidence suggests that
metabolic dysfunction is not a general feature of ALS
(68)
, and Par-4 may have more direct effects on the
neuronal apoptotic biochemical cascade. Evidence from non-neuronal
systems suggests that Par-4 induces apoptosis by down-regulating MAP
kinase activity and up-regulating p38 kinase activity in a
p53-independent pathway (23)
. In NIH-3T3 cells, Par-4
expression causes morphological changes indicative of apoptosis upon
its interaction with the atypical isoforms of protein kinase C
(25)
. We previously reported that over-expression of the
leucine zipper domain of Par-4 protected PC12 cells from apoptosis
induced by the amyloid ß-peptide, suggesting a necessary interaction
between Par-4 and another protein in its proapoptotic action in neurons
(27)
. Recently, expression of Par-4 in NIH-3T3 cells was
reported to prevent activation of the neuroprotective transcription
factor NF-
B (69)
. In conclusion, despite limited
knowledge of its mechanism of action in inducing neuronal apoptosis,
our results suggest that Par-4 is a critical link in the chain of
events leading to motor neuron degeneration in ALS.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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