вход по аккаунту


Death-associated protein kinase expression in human temporal lobe epilepsy.

код для вставкиСкачать
Death-Associated Protein Kinase Expression
in Human Temporal Lobe Epilepsy
David C. Henshall, PhD,1 Clara K. Schindler, BS,1 Norman K. So, MB, BChir, MRCP,2
Jing-Quan Lan, MD,1 Robert Meller, DPhil,1 and Roger P. Simon, MD1
Experimental and human data suggest programmed (active) cell death may contribute to the progressive hippocampal
atrophy seen in patients with refractory temporal lobe epilepsy. Death-associated protein (DAP) kinase is a novel
calcium/calmodulin-activated kinase that functions in apoptosis mediated by death receptors. Because seizure-induced
neuronal death involves both death receptor activation and calcium, we examined DAP kinase expression, localization,
and interactions in hippocampal resections from patients with intractable temporal lobe epilepsy (n ⴝ 10) and autopsy
controls (n ⴝ 6). Expression and phosphorylation of DAP kinase was significantly increased in epilepsy brain compared
with control. DAP kinase and DAP kinase–interacting protein 1 (DIP-1) localized to mitochondria in control brain,
whereas levels of both were increased in the cytoplasm and microsomal (endoplasmic reticulum) fraction in epilepsy
samples. Coimmunoprecipitation analysis showed increased DAP kinase binding to calmodulin, DIP-1, and the Fasassociated protein with death domain (FADD) in epilepsy samples. Finally, immunohistochemistry determined DAP
kinase was coexpressed with DIP-1 in neurons. This study provides the first description of DAP kinase and DIP-1 in
human brain and suggests DAP kinase is a novel molecular regulator of neuronal death in epilepsy.
Ann Neurol 2004;55:485– 494
Experimentally evoked brief, repetitive, and/or spontaneous seizures, in addition to prolonged seizures (status
epilepticus), have been shown to cause neuronal loss in
brain.1–3 This may be epileptogenic4 and contribute to
seizure-induced neurological impairments5 and the severity of subsequent epilepsy.6 Neuroimaging has corroborated these experimental data, whereby patients
with suboptimally managed epilepsy experience progressive reductions in hippocampal volume7–10 and
cognitive function.11,12 Accordingly, neuroprotective
interventions may benefit patients after brain injuries
known to precipitate epilepsy and as adjuncts to anticonvulsant therapy after certain seizures.
Neuronal loss after seizures may involve programmed (active) cell death pathways.13,14 Several
classes of cell death regulatory proteins are activated by
seizures, including caspases, death receptors, and Bcl-2
family proteins, and molecular and pharmacological
manipulations of these effectors can reduce postseizure
neuronal death by as much as half.15–20 Accordingly,
defining the relevant molecular pathways and identifying new cell death modulators is a priority for epilepsy
Death-associated protein (DAP) kinase is a novel
160kDa calcium/calmodulin-dependent, death domain–containing serine/threonine kinase that has been
implicated in the control of apoptosis.21 DAP kinase
can be activated downstream of death receptors such as
tumor necrosis factor receptor 1 (TNFR1) and Fas and
has been implicated in premitochondrial events that
promote caspase and Bcl-2–regulated apoptosis,22–24
although antiapoptotic functions have been demonstrated.23 In turn, expression of DAP kinase is negatively regulated by ubiquitination via its E3 ligase,
DAP kinase interacting protein 1 (DIP-1).25 DAP kinase is present throughout the central nervous system
during development, but adult expression is largely restricted to the resistant cornu ammonis 2 region of the
hippocampus and dentate granule cells of the hippocampus.26 –28 Recent studies suggest DAP kinase
may be a target in neurological diseases,27,29,30 and
work by our group in a rodent epilepsy model has implicated DAP kinase in regulating seizure-induced neuronal death, showing DAP kinase overexpression in relation to injury-causing seizures and recruitment to the
TNFR1 complex.28 DAP kinase’s molecular profile
and participation in pathways activated by seizures
makes it an attractive target in the setting of epileptic
From the 1Robert S. Dow Neurobiology Laboratories, Legacy Research; and 2Oregon Comprehensive Epilepsy Program, Neurological Sciences Center, Portland, OR.
Address correspondence to Dr Henshall, Robert S. Dow Neurobiology Laboratories, Legacy Clinical Research and Technology Center, 1225 NE 2nd Avenue, Portland, OR.
Received Aug 20, 2003, and in revised form Nov 11. Accepted for
publication Nov 11, 2003.
Published online Feb 18, 2004, in Wiley InterScience
( DOI: 10.1002/ana.20001
© 2004 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
brain injury. However, there have been no reports of
DAP kinase involvement in human material in the setting of neurological disease. Accordingly, we examined
the expression and activation of DAP kinase in the hippocampus obtained from epilepsy patients who had
undergone surgical resection for intractable seizures.
1mm thickness prepared from the remaining sample for biochemical analysis.
Autopsy control tissue (n ⫽ 6 per group) was obtained
from the University of Maryland Brain and Tissue Bank (see
Table for details). Hippocampal specimens were from individuals without known neurological disease and were also
fresh-frozen, en bloc samples.
Materials and Methods
Human Brain Tissue Samples
Western Blotting
This study was approved by the Legacy Health System Institutional Review Board, and informed consent was obtained from all patients. Clinical data are shown in the Table. All patients (n ⫽ 10) were referred for surgical resection
of the temporal lobe by an epileptologist after neurological
assessment, videoelectroencephalogram recording, and magnetic resonance imaging/neuroimaging. Each patient was determined to have medically intractable epilepsy with a history
of recurring seizures. However, no patient had experienced
status epilepticus during the year in which their surgery was
performed. Seizure frequency for each patient was typically
in the range of 1 to 2 per week (range, 2–30 per month). All
patients were taking anticonvulsant medication before surgery. All patients underwent left or right temporal lobe resection, and the hippocampus and in some cases adjacent
temporal cortex were obtained. Specimens were immediately
frozen in liquid nitrogen and stored at ⫺70°C until use. The
hippocampus was separated from adjacent temporal cortex
and was analyzed in its entirety without further microdissection of subfields. Specimens were first sectioned for immunohistochemistry and then coronal slabs of approximately
Western blotting was performed as previously described.31 In
brief, samples were homogenized and lysed in buffer containing a protease inhibitor cocktail and cleared by centrifugation. Protein (50␮g) then was separated using 12 to 15%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE), transferred to polyvinylidene difluoride membranes (BioRad, Hercules, PA) and incubated with monoclonal antibodies against DAP kinase or phospho308DAP kinase
(1:1,000; Sigma, St. Louis, MO) and a custom-made rabbit
polyclonal antibody to DIP-1 (also known as mind bomb32)
that previously has been described.25 As controls, membranes
were also immunoblotted with antibodies against ZIP kinase
(Biomol, Plymouth, PA), and against phospho-p44/42
mitogen-activated protein kinase (p44/42MAPK) and phosphoglycogen synthase kinase-␤ (pGSK␤) (Cell Signaling
Technology, Beverly, MA). Membranes then were incubated
with horseradish peroxidase–conjugated secondary antibodies
(1:2,000 dilution) followed by chemiluminescence detection
(NEN Life Science Products, Boston, MA) and exposure to
X-OMAT film (Kodak, Rochester, NY). Images were collected with a Dage 72 camera and gel bands were analyzed
Table. Data for Controls and Epilepsy Patients
Age (yr)
Cause of Death
PMI (hr)
Cardiac arrest
Pulmonary thromboembolus
Traffic accident
Suicide (hanging)
Gunshot wound (chest)
Traffic accident
Control specimens
Age (yr)
Epilepsy patients
Cause/Risk Factor
Seizure Type
Febrile seizures
Febrile seizures and status
Febrile seizures
Febrile seizures
Febrile seizures
Neocortical lesion
Side of hippocampal resection. (right or left).
PMI ⫽ postmortem interval; CP ⫽ complex partial; GTCS ⫽ generalized tonic-clonic seizures; MRI ⫽ magnetic resonance imaging; MTS ⫽
mesial temporal sclerosis.
Annals of Neurology
Vol 55
No 4
April 2004
using gel-scanning integrated optical density software (Bioquant, Nashville, TN).
Subcellular Fractionation
Subcellular fractionation was performed on a selection of
control (C1, C2, C3) and epilepsy specimens (E2, E3, E4)
according to previous methods with modifications.33,34 In
brief, samples were homogenized in a mannitol/sucrose
buffer containing a protease inhibitor cocktail. Mitochondria
were separated from the homogenate by centrifugation at
10,000g followed by purification through a Percoll gradient
at 41,000g. The crude cytosol fraction was centrifuged at
100,000g to separate microsomes and cytosol. Fractions were
then subjected to immunoblot detection of DAP kinase and
DIP-1 as described above. Fraction quality was verified with
markers for the cytoplasm (␣-tubulin; Santa Cruz Biotechnology, Santa Cruz, CA), mitochondria (cytochrome IV oxidase; Molecular Probes, Eugene, OR; tBid; R & D Systems,
Minneapolis, MN) and the endoplasmic reticulum (caspase12; Cell Signaling Technology).
Immunoprecipitation was performed as previously described.20,28 In brief, samples from the same specimens used
for fractionation were homogenized in lysis buffer containing
1% NP-40 and then protein samples (0.5mg) were incubated
overnight at 4°C with 2 to 5␮g of immunoprecipitation antibodies; anti-actin and anti-Bad (Santa Cruz Biotechnology), anti-calmodulin and anti-FADD (Upstate Biotechnology, Lake Placid, NY), or anti–DIP-1. Samples then were
incubated with protein A/G agarose beads (Santa Cruz Biotechnology) for 1 hour at 4°C. The protein–bead complex
was collected by centrifugation and boiled, and samples were
subjected to Western blotting with anti–DAP kinase. Positive (50␮g whole-cell lysate) and negative (omitting the immunoprecipitation antibody) controls confirmed specificity.
Also, we ensured IgG levels (antibody loading) and levels of
the bait (calmodulin, FADD, DIP-1) were expressed and/or
immunoprecipitated equivalently between control and epilepsy samples.
Immunohistochemistry and DNA Fragmentation
Immunohistochemistry and DNA fragmentation analysis was
performed as previously described.28,31 In brief, brain sections (12␮m) were preblocked in 5% goat serum and then
incubated with antibodies against DAP kinase (1:100),
DIP-1 (1:500), or neuronal nuclear protein (NeuN; Chemicon, Temecula, CA). Sections then were washed and incubated in a 1:500 dilution of goat anti–mouse or anti–rabbit
Cy3 or fluorescein isothiocyante (FITC; Jackson Immunoresearch, Plymouth, PA). Finally, sections were mounted in
4⬘,6 diamidino-2-phenylindole (DAPI; Vector Labs, Burlingame, CA) to assess nuclear morphology, and immunolabeling was studied using a Leica microscope equipped for epifluorescent illumination. Excitation/emission wavelengths
were 340/425nm (blue), 500/550nm (green), and 580/
630nm (red). Images were collected using an Optronics
DEI-750 three-chip camera equipped with a BQ 8000 sVGA
frame grabber and analyzed using an image analysis system
Analysis of cells exhibiting DNA fragmentation was performed using fluorescein-linked terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling (TUNEL;
Roche Molecular Biochemicals, Indianapolis, IN) as previously described.28 TUNEL counts were performed on ten
⫻10 lens fields, and positive cells were further examined under ⫻100 lens magnification to verify and image nuclear features. For phenotype identification, 100 DAP kinase or DIP1–immunopositive cells from sections counterstained with
NeuN were examined from three patient or control sections
under ⫻40 lens magnification.
Data Analysis
Data are presented as mean ⫾ standard error of the mean.
Data were analyzed using a Mann–Whitney U test (StatView
software; SAS Institute, Cary, NC). Significance was accepted at p value less than 0.05.
Increased DAP Kinase Expression in Hippocampi of
Patients with Temporal Lobe Epilepsy
Immunoblotting detected constitutive expression of
DAP kinase at its predicted weight of 160kDa in
whole-cell lysates from all six control brain samples
(Fig 1A). Increased levels of DAP kinase were seen in 8
of 10 epilepsy patient samples, and this was statistically
significant compared with control (see Fig 1A, B).
Because DAP kinase function requires phosphorylation,35 we used a phosphospecific antibody to assess
whether DAP kinase was activated. Phosphorylated
DAP kinase was detected in 8 of 10 epilepsy specimens
examined, and this was significantly different from
controls in which phosphorylated DAP kinase was not
detected (see Fig 1A, C).
Last, we examined expression of DIP-1, the E3 ligase
known to regulate expression of DAP kinase.25 We detected low-level constitutive expression of the approximately 110kDa form of DIP-1 in all control brain
samples. DIP-1 levels were higher than control in 5 of
10 epilepsy samples, but this difference did not reach
statistical significance (see Fig 1A and data not shown).
Expression of ZIP Kinase Is Significantly Lower in
Epilepsy Brain
To verify the specific nature of the changes for DAP
kinase, we examined expression of ZIP kinase,36 another member of the DAP kinase–related family. ZIP
kinase was expressed in all control samples at approximately 55kDa (Fig 2A). Expression of ZIP kinase was
lower in 9 of 10 epilepsy brain samples, and this was
statistically significant compared with controls (see Fig
2A,B). A lower weight band was also apparent in epilepsy specimens, suggestive of proteolysis. Although the
identity of this fragment is uncertain, ZIP kinase immunoblotting using hippocampal lysates from our ex-
Henshall et al: DAP Kinase in TLE
tissue), we examined phosphorylation of two other proteins. Reprobing immunoblots from controls showed
that phosphorylated p44/42 MAPK and GSK␤ could
be detected in these samples (see Fig 2C). Although
levels were quite variable, this was not directly related
to postmortem interval (see Table).
Subcellular Localization of Death-associated Protein
Kinase and DIP-1
For subcellular distribution analysis, quality of fractionation was confirmed by the selective presence of
␣-tubulin in the cytoplasm, cleaved Bid (tBid) in mitochondria (epilepsy samples only), and caspase-12, a
neuronally expressed endoplasmic reticulum–specific
caspase,37 in the microsomal fraction (Fig 3A).
In control brain, DAP kinase (see Fig 3B, C) and
DIP-1 (see Fig 3D, E), were present almost exclusively
within the mitochondrial fraction of the cell. In contrast, levels of both were significantly increased within
the cytoplasm and microsomal fractions from epilepsy
brain (see Fig 3B–E). Mitochondrial levels of DAP kinase and DIP-1 were not different between control and
epilepsy samples (see Fig 3B–E).
Fig 1. Death-associated protein (DAP) kinase expression in
human brain. (A) Representative immunoblots from control
(C) and epilepsy (E) hippocampi showing increased DAP kinase (DAPK) expression (top) and activation/phosphorylation
(center) in epilepsy specimens. Molecular weight markers
(kDa) are depicted to the left. Note, phospho(p)DAPK also
detects a lower weight band (pDAPK*) at approximately
100kDa. DIP-1 levels in epilepsy samples were less noticeably
different from control (bottom). Immunoblot quantification
graphs below show that (B) DAP kinase and (C) phosphoDAP kinase levels are significantly higher in epilepsy (Epil,
n ⫽ 10) specimens versus control (Con, n ⫽ 6). *p ⬍ 0.05,
**p ⬍ 0.01 compared with control.
perimental rat model28 showed the same weight band
appears 24 to 72 hours after seizure cessation (data not
To ensure the lack of DAP kinase phosphorylation
in controls was not caused by a general absence of
phosphorylated proteins in such samples (eg, because
of protracted collection times associated with autopsy
Annals of Neurology
Vol 55
No 4
April 2004
Fig 2. ZIP kinase is downregulated in human epileptic brain.
(A) Representative immunoblots from control and epilepsy hippocampi show that expression of ZIP kinase (ZIPK) is lower
in epilepsy samples. (B) Graph quantifying ZIP kinase differences. *p ⬍ 0.05 versus control. (C) Detection of phosphorylated proteins in control samples. Control blots were reprobed
with antibodies against phosphorylated p44/42 MAPK and
Death-associated Protein Kinase Interacts with
Calmodulin, FADD, and DIP-1 in Epilepsy Brain
We next examined the interaction of DAP kinase with
proteins related to its activation and function. In control brain, DAP kinase was not immunoprecipitated by
calmodulin (Fig 4A). However, in epilepsy samples, we
detected modest but significant binding of DAP kinase
to calmodulin (see Fig 4A). DAP kinase binding to actin was approximately fourfold higher in epilepsy
brain; however, sample variability precluded this difference reaching statistical significance (data not shown).
Next, we tested whether DAP kinase interacts with
the death receptor adaptor FADD, a phosphoprotein
that links activated death receptors to caspase-8. We
detected very little binding of these two proteins in
control brain; however, in epilepsy brain, DAP kinase
was found to strongly interact with FADD (see Fig
Finally, we found DAP kinase was not present in
control brain DIP-1 immunoprecipitates (see Fig 4C).
However, in epilepsy brain we detected significant
binding of DAP kinase to DIP-1 (see Fig 4C).
Because DAP kinase interactions were consistently
elevated in epilepsy samples, we performed an additional assay to ensure the specificity of these changes.
Binding of 14-3-3 to the cell death promoter Bad (Bcl2–associated death protein) is normally higher in control than seizure hippocampi in the rat.17 Performing
this same binding assay using our human material, we
confirmed that 14-3-3 binding to Bad immunoprecipitates is higher in control human brain than epilepsy
specimens (data not shown).
Fig 3. Subcellular localization of DAP kinase and DIP-1. (A) Verification of fraction quality for cytoplasm (cyto), mitochondria
(mito), and microsomes (micro). (B) Immunoblots showing DAP kinase localization in each fraction from control and epilepsy
brain. (C) Graph quantifying differences in location of DAP kinase between control and epilepsy brain. (D) Immunoblots showing
DIP-1 localization in each fraction from control and epilepsy brain. (E) Graph quantifying differences in location of DIP-1 between control and epilepsy brain. Data are from n ⫽ 3 per group. *p ⬍ 0.05 compared with control.
Henshall et al: DAP Kinase in TLE
Death-associated Protein Kinase and DIP-1
Finally, we used immunohistochemistry to determine
which cells express DAP kinase and to support our coimmunoprecipitation findings. DAP kinase–expressing
cells were detected in all epilepsy samples examined, particularly within the granule cells of the dentate gyrus
(Fig 5C, D, G). Counterstaining using the neuronal
phenotype marker NeuN showed 99% of cells expressing DAP kinase were neurons. Examples of DAP kinase–
expressing neurons are shown in Figure 5E to H.
We next examined DNA fragmentation in these sections. With a single exception, TUNEL-positive cells
were detected on sections from each epilepsy patient
(range, 0 – 4). However, average TUNEL counts were
low (1.7 ⫾ 0.4) and were not significantly different
from numbers in controls (1.0 ⫾ 0.3; range, 0 –3).
Where present, they appeared as intensely stained,
lobed, and/or blebbing, in line with features of a programmed cell death/apoptotic phenotype (see Fig 5B).
No TUNEL-positive cells labeled for DAP kinase.
DIP-1–expressing cells were more abundant in control and epilepsy brain than were DAP kinase–expressing cells (Fig 6). Counterstaining with NeuN showed
that 97% of DIP-1–expressing cells were neurons in
control brain, and 93% were neurons in epilepsy specimens (see Fig 6A–D). Although many DIP-1–expressing cells did not express detectable DAP kinase (data
not shown), DAP kinase–expressing cells invariably colabeled for DIP-1 in both control (98%) and epilepsy
(96%) sections examined (see Fig 6E–L). However,
within individual cells in control and epilepsy brain the
colocalization of these proteins was quite limited, with
DIP-1 largely appearing in a nuclear distribution and
the area immediately surrounding it, whereas DAP kinase appeared in the soma region outside the nucleus
(see Fig 6E–L). Where colocalization was present this
usually appeared as aggregates within the cytoplasm.
Fig 4. Death-associated protein (DAP) kinase binding interactions. Representative immunoblots (WB) showing increased
DAP kinase binding to (A) calmodulin (CaM), (B) FADD,
and (C) DIP-1 immunoprecipitates in epilepsy brain compared with control. Graphs below each panel show quantification of these differences. Data are from n ⫽ 3 per group. *p
⬍ 0.05, **p ⬍ 0.01 compared with control.
Annals of Neurology
Vol 55
No 4
April 2004
With evidence accumulating that temporal lobe epilepsy is a progressive disease for brain injury, the impetus to understand the molecular determinants of
seizure-induced neuronal loss intensifies. This study
provides the first description to our knowledge of DAP
kinase, a novel regulator of cell death, and its E3 ligase
DIP-1 in human brain. Our data show that DAP kinase is overexpressed and likely activated in hippocampi obtained from patients with intractable epilepsy. Because DAP kinase is able to regulate apoptosis
in a variety of cell types including neurons, these data
may have significant implications for the treatment of
seizure-induced neuronal death and the design of neuroprotective adjuncts to treat epilepsy or epilepsyprecipitating brain injuries.
Seizures, particularly when prolonged, cause selective
Fig 5. Death-associated protein (DAP) kinase immunohistochemistry. (A) DAPI-stained, low-power (⫻2.5 lens) field photomicrograph from an epilepsy en bloc specimen showing visible blade of the dentate gyrus. (B) Examples of TUNEL-positive nuclei from
epilepsy patient sections. Note lobed/blebbing features suggesting programmed cell death. (C) Single DAP kinase–expressing cell (labeled with Cy3) from the hilar region of the dentate gyrus of an epilepsy patient. (D) Panel showing expression of DAP kinase in a
group of cells within the dentate granule cell layer. (E–H) Phenotype of DAP kinase–expressing cells in epilepsy brain. Counterstaining of sections with NeuN (labeled with FITC) shows DAP kinase is expressed by neurons. Arrows indicate immunostained cells.
Scale bar ⫽ 300␮m (A), 6␮m (B), 10␮m (C, D), 12␮m (E–H).
Fig 6. DIP-1 immunohistochemistry and coexpression in DAPK-positive cells. (A–D) Immunohistochemistry showing phenotype of
DIP-1–expressing cells in epilepsy brain. Counterstaining of sections with NeuN (labeled with Cy3) shows DIP-1 (labeled with
FITC) is expressed by neurons. Arrows indicate neurons staining for DIP-1, whereas an arrowhead identifies a DIP-1–expressing
cell that is not NeuN labeled. (E–L) Colocalization of DIP-1 with DAP kinase in control (middle panels) and epilepsy (bottom
panels) brain. Note DAP kinase largely stains the cytoplasm, whereas DIP-1 appears restricted to a nuclear area. Scale bar ⫽
12␮m (A–D), 8␮m (E–L).
degeneration of neurons within the hippocampus that
is mediated in part by induction of programmed (active) cell death pathways.13,14 Although studies have
challenged whether cell loss is a requirement for the
development of spontaneous seizures (epilepsy),6,38 – 40
new data demonstrate that seizure-induced neuronal
loss can directly cause epilepsy in certain cases.4 Neuronal loss also may underlie seizure-induced cognitive
dysfunction5 and may exacerbate seizure severity in experimental epilepsy.6 Accordingly, targeting apoptosis-
Henshall et al: DAP Kinase in TLE
regulating proteins to prevent seizure-induced neuronal
death as an adjunct to anticonvulsive treatment may
provide a means to reduce the impact of seizures on
brain. Although still controversial,39,41,42 several regulators of programmed cell death, including caspases
and Bcl-2 family proteins, appear to be involved in
seizure-induced neuronal death.16,17,19,33 The initiation site for these pathways may be surface-expressed
death receptors such as TNFR1, which rapidly assemble after seizures, and recruit adaptor proteins, such as
FADD, and initiator caspases.20,28 An ideal death effector target therefore might be one responsive to both
the inevitable calcium overload in the wake of seizure
activity and apoptotic pathways mediated by death receptors. DAP kinase fits this profile because its activity
is stimulated by calcium/calmodulin and it regulates
apoptosis in response to TNF/Fas.22,35
Our analysis of hippocampi from patients with intractable temporal lobe epilepsy provides the first report of DAP kinase upregulation in a human neurological disease. Two additional experiments, showing
increased DAP kinase phosphorylation and binding to
calmodulin, support DAP kinase being functionally active in these specimens. We base this interpretation on
evidence that stimulation of the full-length (wild type)
DAP kinase by calcium/calmodulin increases DAP kinase phosphorylation, phosphorylation of DAP kinase
substrates, and apoptosis.35,43 In most,22,24,29,35 but
not all,23,25 systems, DAP kinase is proapoptotic, and
experimental models show its expression is increased by
cell death–inducing but not benign seizures.28 Accordingly, DAP kinase may be a molecular determinant of
the progressive hippocampal damage seen in temporal
lobe epilepsy patients. In turn, DAP kinase might be a
therapeutic target to treat neuronal injury caused by
brief or prolonged seizures. Promisingly, DAP kinase
inhibition can abrogate neuronal death in response to
stimuli known to be generated by seizures.29,44 Because
DAP kinase may contribute to injury outcome after
stroke,27,30 it also might be a target relevant to other
neurological diseases.
DAP kinase has been reported previously to localize
to the microfilaments of the cytoskeleton and mediate
structural rearrangements of the cell during apoptosis
and autophagy.35,45 Although DAP kinase binds actin
in the hippocampus (our data and Henshall and colleagues28), we found it only within the mitochondrial
compartment in control brain. These data might reflect
mitochondria as a site of (presumably protective) DAP
kinase sequestration. Indeed, other proapoptotic proteins are sequestered to mitochondria,46,47 being released in response to apoptotic stimuli. Alternatively,
DAP kinase may target mitochondrial proteins, identification of which could lend insight into its in vivo
substrates, which remain largely unknown. Surprisingly, mitochondria were not the site of DAP kinase
Annals of Neurology
Vol 55
No 4
April 2004
accumulation in epilepsy brain, and whereas the observed cytoplasmic increases might have been predicted
from previous work on its localization35 its elevation in
the microsomal fraction was unexpected. The endoplasmic reticulum, a major constituent of the microsomal fraction, is a potent initiator of apoptosis when
stressed,48 and data have emerged implicating this organelle in seizure-induced neuronal death.49,50 Indeed,
we have shown TNFR activating factor 2 and apoptosis signal–regulating kinase 1, which both mediate endoplasmic reticulum stress pathways,51,52 are activated
and/or recruited to the TNFR1 pathway after experimental seizures.20 The function of DAP kinase within
the endoplasmic reticulum is unclear, but myosin light
chain kinase, which bears substrate and functional similarity has been shown to coordinate TNFR1 movement within cells,53 and TNFR1 has been shown to
traffic to the microsomes during apoptosis.54 Because
we observed increased binding of DAP kinase to
FADD in the same samples, it is possible that a DAP
kinase–containing death signaling complex (DISC) is
forming in the cytoplasm or microsomal fraction. Indeed, this occurs after experimental seizures,28 and
cleaved caspase-8 can be detected in our epilepsy patient specimens (D.C. Henshall, C.K. Schindler, unpublished observation). We might further speculate
that FADD may be a substrate for DAP kinase. FADD
is a phosphoprotein, and its phosphorylated form interacts with activated Fas receptors,55 suggesting DAP
kinase may act on FADD to influence Fas signaling in
brain. Further studies are now required to elucidate the
significance of these localization changes in terms of
their relation to DISC formation, substrates/targets and
specificity to the environment of the epileptic brain.
DIP-1 was identified recently as the in vivo regulator
of DAP kinase levels through its ability as an E3 ligase
to ubiquitinate and thereby downregulate DAP kinase
expression.25 Here, we provide the first description of
DIP-1 in human brain, showing a close relationship
with DAP kinase in which they were coexpressed in
neurons, similarly distributed in subcellular compartments, and could be coimmunoprecipitated. However,
because DAP kinase levels were raised in epileptic brain
despite their binding, the function of DIP-1 may be
compromised in these patients. Nevertheless, promoting DIP-1 function could yet provide a means to target
the DAP kinase pathway.
Certain limitations should be considered in the interpretation of our findings. Although our autopsy hippocampi were well matched for sex and age, they
would not reflect anticonvulsant medication or surgical/anesthesia effects. However, control animals in our
experimental seizure model undergo a surgical manipulation for placement of cannulas/electrodes and receive anticonvulsant, and this does not activate cell
death pathways.16,17 Additional validation for our find-
ings comes from internal controls (eg, lower ZIP kinase
expression in epilepsy brain compared with control)
and previous work showing expected levels of constitutive and nonconstitutive genes in autopsy brain.31 Second, increased phosphorylation of DAP kinase within
its calmodulin regulatory domain can be inhibitory,43
implying increased DAP kinase phosphorylation may
not be as reliable a marker of activity as calmodulin
binding. Finally, antiapoptotic functions of DAP kinase have been reported.23,25 Our data do not preclude such a role, which could underlie the infrequency of TUNEL in epileptic brain reported here
and previously.31 If this is not the case, single time
point sampling may have underestimated TUNEL, or
the chronically epileptic brain may counter DAP kinase
by adjusting its repertoire of antiapoptotic proteins. Indeed, Bcl-2 levels are increased in epilepsy specimens,31
and Bcl-2 can inhibit DAP kinase–induced neuronal
In conclusion, we provide the first evidence for the
overexpression and activation of DAP kinase in a human neurological disease, epilepsy. DAP kinase’s place
within proapoptotic pathways activated by seizures
mean such changes could underlie the molecular drive
to progressive neuronal loss in patients with refractory
epilepsy. Accordingly, these data lend insight into both
the impact of epilepsy on brain and strategies for the
treatment of injurious seizures.
This work was supported by grants from the NIH (National Institute of Neurological Disorders and Stroke, NS39016, D.C.H.,
R.P.S.; NS41935, D.C.H.).
We thank Drs Rosenban and Abtin for surgical collection of specimens and the University of Maryland Brain and Tissue bank for
autopsy specimens. We also thank Dr P. J. Gallagher for the DIP-1
1. Cavazos JE, Das I, Sutula TP. Neuronal loss induced in limbic
pathways by kindling: evidence for induction of hippocampal
sclerosis by repeated brief seizures. J Neurosci 1994;14:
3106 –3121.
2. Bengzon J, Kokaia Z, Elmer E, et al. Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures. Proc Natl Acad Sci USA 1997;94:10432–10437.
3. Bouilleret V, Loup F, Kiener T, et al. Early loss of interneurons
and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy.
Hippocampus 2000;10:305–324.
4. Sayin U, Osting S, Hagen J, et al. Spontaneous seizures and
loss of axo-axonic and axo-somatic inhibition induced by repeated brief seizures in kindled rats. J Neurosci 2003;23:
2759 –2768.
5. Kotloski R, Lynch M, Lauersdorf S, et al. Repeated brief seizures induce progressive hippocampal neuron loss and memory
deficits. Prog Brain Res 2002;135:95–110.
6. Zhang X, Cui SS, Wallace AE, et al. Relations between brain
pathology and temporal lobe epilepsy. J Neurosci 2002;22:
6052– 6061.
7. Kalviainen R, Salmenpera T, Partanen K, et al. Recurrent seizures may cause hippocampal damage in temporal lobe epilepsy.
Neurology 1998;50:1377–1382.
8. Tasch E, Cendes F, Li LM, et al. Neuroimaging evidence of
progressive neuronal loss and dysfunction in temporal lobe epilepsy. Ann Neurol 1999;45:568 –576.
9. Bernasconi A, Tasch E, Cendes F, et al. Proton magnetic resonance spectroscopic imaging suggests progressive neuronal
damage in human temporal lobe epilepsy. Prog Brain Res 2002;
10. Briellmann RS, Berkovic SF, Syngeniotis A, et al. Seizureassociated hippocampal volume loss: a longitudinal magnetic
resonance study of temporal lobe epilepsy. Ann Neurol 2002;
51:641– 644.
11. Jokeit H, Ebner A. Long term effects of refractory temporal
lobe epilepsy on cognitive abilities: a cross sectional study.
J Neurol Neurosurg Psychiatry 1999;67:44 –50.
12. Hermann BP, Seidenberg M, Bell B. The neurodevelopmental
impact of childhood onset temporal lobe epilepsy on brain
structure and function and the risk of progressive cognitive effects. Prog Brain Res 2002;135:429 – 438.
13. Bengzon J, Mohapel P, Ekdahl CT, et al. Neuronal apoptosis
after brief and prolonged seizures. Prog Brain Res 2002;135:
14. Liou AKF, Clark RS, Henshall DC, et al. To die or not to die
for neurons in ischemia, traumatic brain injury and epilepsy: a
review on the stress-activated signaling pathways and apoptotic
pathways. Prog Neurobiol 2003;69:103–142.
15. Morrison RS, Wenzel HJ, Kinoshita Y, et al. Loss of the p53
tumor suppressor gene protects neurons from kainate-induced
cell death. J Neurosci 1996;16:1337–1345.
16. Henshall DC, Bonislawski DP, Skradski SL, et al. Cleavage of
Bid may amplify caspase-8-induced neuronal death following
focally evoked limbic seizures. Neurobiol Dis 2001;8:568 –580.
17. Henshall DC, Araki T, Schindler CK, et al. Activation of Bcl2-associated death protein and counter-response of Akt within
cell populations during seizure-induced neuronal death. J Neurosci 2002;22:8458 – 8465.
18. Roy M, Hom JJ, Sapolsky RM. HSV-mediated delivery of virally derived anti-apoptotic genes protects the rat hippocampus
from damage following excitotoxicity, but not metabolic disruption. Gene Ther 2002;9:214 –219.
19. Narkilahti S, Nissinen J, Pitkanen A. Administration of caspase
3 inhibitor during and after status epilepticus in rat: effect on
neuronal damage and epileptogenesis. Neuropharmacology
2003;44:1068 –1088.
20. Shinoda S, Skradski SL, Araki T, et al. Formation of a tumour
necrosis factor receptor 1 molecular scaffolding complex and
activation of apoptosis signal-regulating kinase 1 during seizureinduced neuronal death. Eur J Neurosci 2003;17:2065–2076.
21. Cohen O, Kimchi A. DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer. Cell
Death Differ 2001;8:6 –15.
22. Cohen O, Inbal B, Kissil JL, et al. DAP-kinase participates in
TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol 1999;146:141–148.
23. Jin Y, Blue EK, Dixon S, et al. Identification of a new form of
death-associated protein kinase that promotes cell survival.
J Biol Chem 2001;276:39667–39678.
24. Raveh T, Droguett G, Horwitz MS, et al. DAP kinase activates
a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 2001;3:1–7.
25. Jin Y, Blue EK, Dixon S, et al. A DAPK interacting protein,
DIP-1, is an E3 ubiquitin ligase that promotes TNF-induced
apoptosis and regulates the cellular levels of DAPK. J Biol
Chem 2002;277:46980 – 46986.
Henshall et al: DAP Kinase in TLE
26. Sakagami H, Kondo H. Molecular cloning and developmental
expression of a rat homologue of death-associated protein kinase in the nervous system. Brain Res Mol Brain Res 1997;52:
249 –256.
27. Yamamoto M, Takahashi H, Nakamura T, et al. Developmental changes in distribution of death-associated protein kinase
mRNAs. J Neurosci Res 1999;58:674 – 683.
28. Henshall DC, Araki T, Schindler CK, et al. Expression of
death-associated protein kinase and recruitment to the tumor
necrosis factor signaling pathway following brief seizures.
J. Neurochem. 2003;86:1260 –1270.
29. Pelled D, Raveh T, Riebeling C, et al. Death-associated protein
(DAP) kinase plays a central role in ceramide-induced apoptosis
in cultured hippocampal neurons. J Biol Chem 2002;277:
30. Schumacher AM, Velentza AV, Watterson DM, et al. DAPK
catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury.
Biochim Biophys Acta 2002;1600:128 –137.
31. Henshall DC, Clark RS, Adelson PD, et al. Alterations in bcl-2
and caspase gene family protein expression in human temporal
lobe epilepsy. Neurology 2000;55:250 –257.
32. Itoh M, Kim C-H, Palardy G, et al. Mind bomb is a ubiquitin
ligase that is essential for efficient activation of notch signaling
by delta. Dev Cell 2003;4:67– 82.
33. Henshall DC, Bonislawski DP, Skradski SL, et al. Formation of
the Apaf-1/cytochrome c complex precedes activation of
caspase-9 during seizure-induced neuronal death. Cell Death
Differ 2001;8:1169 –1181.
34. Chen D, Minami M, Henshall DC, et al. Upregulation of mitochondrial base-excision repair capability within rat brain after
brief ischemia. J Cereb Blood Flow Metab 2003;23:88 –98.
35. Cohen O, Feinstein E, Kimchi A. DAP-kinase is a Ca2⫹/
calmodulin-dependent, cytoskeletal-associated protein kinase,
with cell death-inducing functions that depend on its catalytic
activity. EMBO J 1997;16:998 –1008.
36. Kawai T, Matsumoto M, Takeda K, et al. ZIP kinase, a novel
serine/threonine kinase which mediates apoptosis. Mol Cell
Biol 1998;18:1642–1651.
37. Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates
endoplasmic-reticulum-specific apoptosis and cytotoxicity by
amyloid-beta. Nature 2000;403:98 –103.
38. Andre V, Ferrandon A, Marescaux C, et al. Vigabatrin protects
against hippocampal damage but is not antiepileptogenic in the
lithium-pilocarpine model of temporal lobe epilepsy. Epilepsy
Res 2001;47:99 –117.
39. Ebert U, Brandt C, Loscher W. Delayed sclerosis, neuroprotection, and limbic epileptogenesis after status epilepticus in the
rat. Epilepsia 2002;43:86 –95.
40. Brandt C, Potschka H, Loscher W, et al. N-methyl-D-aspartate
receptor blockade after status epilepticus protects against limbic
brain damage but not against epilepsy in kainate model of temporal lobe epilepsy. Neuroscience 2003;118:727–740.
41. Fujikawa DG, Ke X, Trinidad RB, et al. Caspase-3 is not activated in seizure-induced neuronal necrosis with internucleosomal DNA cleavage. J Neurochem 2002;83:229 –240.
Annals of Neurology
Vol 55
No 4
April 2004
42. Puig B, Ferrer I. Caspase-3-associated apoptotic cell death in
excitotoxic necrosis of the entorhinal cortex following intraperitoneal injection of kainic acid in the rat. Neurosci Lett 2002;
43. Shohat G, Spivak-Kroizman T, Cohen O, et al. The proapoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. J Biol Chem 2001;276:47460 – 47467.
44. Stoica BA, Movsesyan VA, Lea PMt, et al. Ceramide-induced
neuronal apoptosis is associated with dephosphorylation of Akt,
BAD, FKHR, GSK-3beta, and induction of the mitochondrialdependent intrinsic caspase pathway. Mol Cell Neurosci 2003;
45. Inbal B, Bialik S, Sabanay I, et al. DAP kinase and DRP-1
mediate membrane blebbing and the formation of autophagic
vesicles during programmed cell death. J Cell Biol 2002;157:
455– 468.
46. Noshita N, Sugawara T, Fujimura M, et al. Manganese superoxide dismutase affects cytochrome c release and caspase-9 activation after transient focal cerebral ischemia in mice. J Cereb
Blood Flow Metab 2002;21:557–567.
47. Cao G, Luo Y, Nagayama T, et al. Cloning and characterization of rat caspase-9: implications for a role in mediating
caspase-3 activation and hippocampal cell death after transient
cerebral ischemia. J Cereb Blood Flow Metab 2002;22:
534 –546.
48. Ferri KF, Kroemer G. Organelle-specific initiation of cell death
pathways. Nat Cell Biol 2001;3:E255–E263.
49. Pelletier MR, Wadia JS, Mills LR, et al. Seizure-induced cell
death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores. J Neurophysiol 1999;81:3054 –3064.
50. Kitao Y, Ozawa K, Miyazaki M, et al. Expression of the endoplasmic reticulum molecular chaperone (ORP150) rescues hippocampal neurons from glutamate toxicity. J Clin Invest 2001;
108:1439 –1450.
51. Yoneda T, Imaizumi K, Oono K, et al. Activation of caspase12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent
mechanism in response to the ER stress. J Biol Chem 2001;
52. Nishitoh H, Matsuzawa A, Tobiume K, et al. ASK1 is essential
for endoplasmic reticulum stress-induced neuronal cell death
triggered by expanded polyglutamine repeats. Genes Dev 2002;
53. Jin Y, Atkinson SJ, Marrs JA, et al. Myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor-1. J Biol Chem 2001;
54. Storey H, Stewart A, Vandenabeele P, et al. The p55 tumour
necrosis factor receptor TNFR1 contains a trans-Golgi network
localization signal in the C-terminal region of its cytoplasmic
tail. Biochem J 2002;366:15–22.
55. Kischkel FC, Hellbardt S, Behrmann I, et al. Cytotoxicitydependent APO-1 (Fas/CD95)-associated proteins form a
death-inducing signaling complex (DISC) with the receptor.
EMBO J 1995;14:5579 –5588.
Без категории
Размер файла
1 367 Кб
expressions, death, associates, protein, temporal, human, epilepsy, lobel, kinases
Пожаловаться на содержимое документа