www.elsevier.com/locate/brainres
Brain Research 1003 (2004) 86–97
Research report
Inhibition of mixed lineage kinase 3 attenuates MPP+-induced
neurotoxicity in SH-SY5Y cells
Joanne R. Mathiasena,*, Beth Ann W. McKennaa, Michael S. Saporitoa,Ghanashyam D. Ghadgeb, Raymond P. Roosb, Beverly P. Holskina, Zhi-Liang Wua,Stephen P. Truskoa, Thomas C. Connorsa, Anna C. Maroneya, Beth Ann Thomasa,
Jeffrey C. Thomasa, Donna Bozyczko-Coynea
aNeurobiology, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USAbDepartment of Neurology, The University of Chicago, Chicago, IL 60637, USA
Accepted 3 November 2003
Abstract
The neuropathology of Parkinson’s Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic
cells in culture treated with the MPTP neurotoxic metabolite, MPP+. MPTP through MPP+ activates the stress-activated c-Jun N-terminal
kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-
induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347
acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK
1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was
examined in MPP+-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative
adenoviral constructs to MLKs. CEP-1347 inhibited MPP+-induced cell death and the morphological features of apoptosis. CEP-1347 also
prevented MPP+-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through
protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP+-
mediated increase in activated JNK levels and inhibited neuronal death following MPP+ addition compared to cultures infected with a control
construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP+-
induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP+-mediated SH-SY5Y cell death in culture
and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.
D 2004 Elsevier B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Degenerative disease: Parkinson’s
Keywords: Adenovirus; CEP-1347; c-Jun N-terminal kinase; Mixed lineage kinase; MPP+; SH-SY5Y
1. Introduction
Degeneration of nigro-striatal dopaminergic (DA) neu-
rons is a neuropathological hallmark of Parkinson’s Disease
(PD). Part of the underlying degenerative process is thought
to be due to a selective vulnerability of DA neurons to
mitochondrial dysfunction. This mitochondrial dysfunction
0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2003.11.073
* Corresponding author. Tel.: +1-610-738-6634; fax: +1-610-344-
0065.
E-mail address: [email protected] (J.R. Mathiasen).
is widespread throughout many cell types of the PD patient
[54,56]. Thus, great interest lies in understanding the key
biochemical events that are triggered following blockade of
mitochondrial respiration and are causal to neuronal death.
Mechanistic studies of DA neuron death akin to PD are
routinely conducted using toxins that interfere with electron
transport at the site of mitochondrial complex I and/or
complex II. Classically, MPTP neurotoxicity has gained
broad acceptance as a model of PD since it is a potent
and selective nigro-striatal DA neurotoxin that produces
PD-like symptoms in humans, non-human primates and
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–97 87
mice [1,4,17,25,26,34]. Moreover, MPP+, the neurotoxic
byproduct of MPTP metabolic oxidation [43,50,62,65], is
selectively taken up into DA neurons and inhibits complex I
of mitochondrial electron transport.
Several intraneuronal signaling pathways have been im-
plicated in MPTP-induced neurotoxicity in vivo. For exam-
ple, mice overexpressing a mitochondrial membrane
signaling protein, Bcl-2, or mice that are deficient in another
signaling peptide, p53, are resistant to MPTP-induced neu-
rotoxicity [44,64,69]. MPTP also activates mitogen-activat-
ed protein kinase kinase 4 (MKK4) and a downstream
substrate, c-Jun N-terminal kinase (JNK) in both the striatum
and substantia nigra of mice [52]. Overexpression of all
members of the mixed lineage kinase (MLK) family leads to
an activation of the JNK pathway [5,9,13,21,27,49,58].
Downstream of the MLKs are the dual-specificity mitogen
activated protein kinase kinases (MKK4 and MKK7) which
phosphorylate JNKs on serine and threonine residues [14].
Gene transfer of JNK-interacting protein-1 (JIP) in SH-
SY5Y cells and mice has implicated the JNK pathway in
MPTP (MPP+)-induced DA cell death [67] and adenoviral
expression of dominant negative c-Jun in an axotomy-
induced rat model of DA cell death has demonstrated
inhibition of c-Jun activation and cell death [8]. Of note, in
a variety of neuronal cell types, signaling through these
pathways manifests in morphological and biochemical fea-
tures of apoptosis, including nuclear chromatin condensa-
tion, membrane blebbing, DNA laddering and activation of
caspase(s) [11,12,23,41,59].
Additionally, an inhibitor of the JNK pathway, the
indolocarbazole CEP-1347/KT7515 [36], attenuates MPTP-
mediated nigrostriatal DA neuron loss in mice [51]. Recent
studies exemplify that CEP-1347 likely inhibits JNK acti-
vation indirectly through the MLK family [37]. To establish
a direct role of MLK in DA neuron death elicited by MPP+,
studies were conducted in neuronally differentiated SH-
SY5Y cells, evaluating CEP-1347 protection against and
interception of a variety of events associated with apoptotic
death. MPP+-treated SH-SY5Y cells were chosen as a
model system to investigate signaling pathways causative
of cell death because these cells exhibit (1) DA neuron
characteristics including dopamine synthesis, (2) expression
of dopamine receptors, (3) specific uptake and sequestration
of dopamine consistent with expression of the dopamine
transporter [3,14,57] and (4) they can be differentiated into a
neuronal phenotype by incubation with retinoic acid [32].
Furthermore, MPP+ induces apoptotic cell death in SH-
SY5Y cells and activates JNK and the early transcription
factor nuclear factor nB [7,30,33,46,55]. The current studies
investigated the ability of CEP-1347 to (1) inhibit the
neurotoxic effects of MPP+ in retinoic acid (RA) differen-
tiated SH-SY5Y cells; and (2) inhibit MPP+-induced JNK
activation. Studies also determined whether SH-SY5Y cells
over-expressing dominant negative forms of MLK family
members would be protected from MPP+-induced JNK
activation and cell death.
2. Materials and methods
2.1. Cell culture
SH-SY5Y cells (J. Biedler, Memorial Sloan-Kettering
Cancer Center, Rye, NY, USA) were seeded at a density of
4� 104/cm2 in T150 flasks and propagated in Dulbecco’s
Modified Eagle’s Medium (DMEM; Gibco BRL, Gaithers-
burg, MD) containing 10% FBS and 2 mM L-glutamine
(Gibco BRL). Growing SH-SY5Y cells were maintained in
a humidified atmosphere at 37 jC in 10% CO2. Cells were
routinely subcultured once a week and used for assays at
passage 8–37. For assays, SH-SY5Y cells were plated at a
density of 1.2� 105/cm2 onto poly-ornithine/mouse laminin
(Sigma-Aldrich, St. Louis, MO/Becton Dickinson Bioscien-
ces, San Jose, CA) coated plates in Neurobasal medium
(Gibco BRL) with B27 supplement (Gibco BRL) and 2 mM
L-glutamine. Cells maintained in Neurobasal medium were
kept in a humidified atmosphere at 37 jC and 5% CO2. The
cells were allowed to attach for 1 h before the addition of 10
AM (final concentration) all trans-retinoic acid (RA, made in
ethanol as a 10 mM stock solution; Sigma-Aldrich) that was
used to induce neuronal differentiation.
Chinese hamster ovary cells (CHO-K1; ATCC#CCL-61,
American Type Culture Collection, Manassas, VA) were
propagated as previously described [38].
2.2. MPP+ treatment
On 3–5 days in vitro subsequent to plating/differentia-
tion, SH-SY5Y cells were exchanged into fresh Neurobasal
medium with B27 supplement and 2 mM L-glutamine
without RA by serial dilution washing and treated for
indicated times with MPP+ (made as an 11� stock in
Neurobasal medium with supplements mentioned above;
Sigma-Aldrich, previously from RBI). Typically, CEP-1347
(stored as 4 mM in DMSO in amber glass vials), was diluted
to a 4� stock concentration in medium, then added to wells
establishing final 1� concentrations and incubated with
cells for 1 h prior to the addition of MPP+.
2.3. Lactate assay
Lactate measurement in culture medium from differenti-
ated SH-SY5Y cells was determined by a standard lactate
assay (Sigma-Aldrich #826-10) following 4, 24 or 48 h of 3
mM MPP+ treatment. Data are represented as fold increase
in lactate compared to untreated differentiated SH-SY5Y
cells.
2.4. DNA Characterization
For visualization of nuclear DNA, cell cultures were
fixed (10 min) with 4% paraformaldehyde and subsequently
incubated for 15–20 min with 1 Ag/ml bisBenzamide
(Sigma-Aldrich) in PBS. Photomicrographs were taken
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–9788
using a Nikon Eclipse TE300 microscope equipped with
both Hoffman and epifluorescence optics. For DNA ladder-
ing studies, cellular DNA was isolated [29] and resolved on
a 1.2% agarose gel containing 0.01% ethidium bromide. For
RT-PCR analyses, RNA was isolated from differentiated
SH-SY5Y cells and PC12 cells with RNAzol B according to
the manufacturer’s directions (TelTest, Friendswood, TX).
2.5. Real-time RT-PCR
Cells were lysed and RNA was isolated with a Qiagen
RNeasy Mini Kit (#74104, Qiagen, Valencia, CA) according
to the manufacturer’s instructions. Samples were run on a
1.2% agarose gel to check for RNA integrity. RNA was
quantified spectrophotometrically (OD 260). cDNA was
generated using oligo dT in Ambion’s Retroscript kit
(Ambion, Austin, TX). 1.5 Ag of total RNA was used per
20 Al reaction. Gene specific primers were identified by
using the Primer Express software (Applied Biosystems,
Foster City, CA). Real-time PCR was performed in an ABI
PrismR 7000 Sequence Detection System (Applied Biosys-
tems) using SYBRR Green PCR Master Mix (Applied
Biosystems). A 50-Al PCR reaction was run using manu-
facturer’s recommended cycling times. Real-time detection
of gene expression is displayed as a threshold cycle or CT
value. The CT value is the cycle at which the amplification
of the gene enters into the exponential phase. Quantification
of gene expression is possible with the assignment of a CT
value. Hence, relative mRNA expression for each sample
can be calculated by normalization to GAPDH CT values.
The relative quantitation value is expressed as D (delta) CT.
2.6. Cell lysis and immunoblot analysis
For measurement of activated, or phosphorylated JNK
(pJNK), differentiated SH-SY5Y cells (1.5� 106) were
lysed in 125-Al ice cold FRAK buffer (1% Triton X-100,
50 mMNaCl, 30 AM sodium pyrophosphate, 50 mM sodium
fluoride, 1 mM sodium vanadate, 10 mM Tris–HCl, pH 7.6)
containing protease inhibitors (1 mM phenylmethylsulfonyl-
fluoride (PMSF) and 20 Ag/ml aprotinin). Lysates were
passed through a 28-1/2 gauge needle and the Triton insol-
uble fraction was then removed from the lysate by centrifu-
gation at 4 jC (20 min, Effendorf microcentrifuge, 14,000
rpm). Supernatant was collected and an aliquot taken for
protein determination (BCA assay, Pierce, Rockford, IL).
Lysates were prepared for electrophoresis by adding Laem-
meli sample buffer and heating for 10 min at 95 jC. Samples
of equivalent total protein (20 Ag/lane) were run on 4–20%
Tris–Glycine gels (Invitrogen, Carlsbad, CA) and trans-
ferred (60 V/cm2) to 0.2 Am nitrocellulose (BioRad Labora-
tories, Hercules, CA). Membranes were blocked in 3% BSA/
Tris buffered saline (TBS) with 0.1% Triton X-100 followed
by overnight incubation at 4 jC with a polyclonal pJNK
antibody (New England Biolabs, Beverly, MA) diluted
1:1000 in block buffer. Following three 15-min washes in
0.5% Triton X-100/TBS membranes were incubated with a
horseradish peroxidase conjugated secondary antibody
(1:20,000, goat anti-rabbit; Southern Biotechnologies, Bir-
mingham, AL), washed and incubated for 1 min with
enhanced chemiluminescent (ECL) substrate (Amersham,
Buckinghamshire, UK) for imaging with BioMax Film
(Kodak, Rochester, NY). Total JNK levels across samples
were determined following stripping and reprobing mem-
branes with a monoclonal mouse antibody directed against
non-phosphorylated JNK proteins (JNK1/2; 1:1000; BD
PharMingen, Franklin Lakes, NJ). In experiments where
adenoviral infection was included in the protocol, immuno-
blots were stripped a second and/or third time and reprobed
with antibodies directed against MLK3 (Santa Cruz Biotech-
nology, Santa Cruz, CA; rabbit polyclonal sc-536; 1:1000)
and/or h-galactosidase (h-gal; Invitrogen, Carlsbad, CA;
rabbit polyclonal; 1:5000). Immunocytochemistry was per-
formed on AdCMVdnMLK3-infected cells to detect
dnMLK3 expression efficiency (anti-MLK3, Santa Cruz
Biotechnology; 1:300). h-gal histochemistry was performed
on AdCMVLacZ-infected cells to determine the expression
efficiency of LacZ (Invitrogenh-gal staining kit #K1465-01).
2.7. Cell viability assessment
Cell viability was determined based on measurement of
lactate dehydrogenase (LDH) release into the culture medi-
um. LDH assays were conducted in accordance with the
manufacturer’s instructions (Cytotoxicity Detection Kit
(LDH), Roche Diagnostics, Indianapolis, IN). Total LDH
release was obtained by completely lysing cells in desig-
nated wells and data are represented as percent total LDH
release.
2.8. Construction of recombinant replication-deficient
adenovirus
A HindIII to EcoRV fragment from vector pcDNA3EE
containing dnMLK3 cDNA (kinase dead/dominant negative)
[38] was inserted into respective sites of the vector pAdCMV
[18], downstream from the cytomegalovirus (CMV) promot-
er and upstream of the cellular heavy chain enhancer (4F2)
and the bovine growth hormone polyadenylation site.
pAdCMV contains 0–1 and 9–16 map units of the adeno-
virus 5 genome. pAdCMV containing mutant (dn) MLK3
was linearized with NheI and cotransfected, using the calci-
um phosphate precipitation method, with XbaI- and ClaI-
digested adenovirus 5 (sub360) DNA into HEK293 cells, a
trans-complementing cell line for E1 function. Viruses were
purified by CsCl isopycnic centrifugation, dialyzed against
HEPES-buffered saline (10 mM HEPES, 140 mM NaCl, 2
mM MgCl2, pH 7.5) containing 10% glycerol, and stored at
� 70 jC in small aliquots. AdCMVlacZ virus was a gener-
ous gift from Dr. Jerome Schaack (University of Colorado,
Denver, CO [53]). AdCMVdnMLK2 and AdCMVdnDLK
were prepared using the AdEasy Vector System (Quantum
Fig. 1. MPP+ concentration-dependent increases in release of LDH from
RA differentiated SH-SY5Y cell cultures measured 48 h post exposure (A).
CEP-1347-dependent decreases in LDH release are significantly different
from 3 mM MPP+ at concentrations of 10–300 nM, indicating cell survival
promotion (B). Data is representative of three independent experiments
(*= significantly different from basal; **= significantly different from
MPP+ treated; p< 0.05 Student’s t-test).
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–97 89
Biotechnologies, also known as Qbiogene, Carlsbad, CA).
Briefly, MLK2(K125A) or DLK(K162A) cDNAwas cloned
into the pShuttle-CMV transfer vector at the 5VAcc65I and3VXbaI polylinker cloning sites and the positive clones
identified were sequence-verified. These pShuttle-CMV-
MLK2(K125A) or DLK(K162A) constructs were linearized
at the unique PmeI site, dephosphorylated and gel purified.
These constructs were co-transformed with supercoiled ad-
enovirus genome using highly competent BJ5183 E. coli
cells via electroporation. The transformed colonies were
grown on kanamycin plates, the smallest colonies picked
from these plates and amplified in LB/kanamycin medium.
Conventional alkaline lysis miniprep DNA protocols, utiliz-
ing phenol chloroform extractions and ethanol precipitations,
were performed. Following restriction digest and PCR con-
firmation, this DNAwas transformed into competent E. coli
DH5a cells. Positive clones were digested with PacI to
expose their ITRs and transfected into 293A cells using
Qiagen SuperFect reagent (Qiagen, Valencia, CA). Viruses
were purified and stored as described above.
2.9. Adenoviral infection
Differentiated SH-SY5Y cells were infected by removing
the culture medium and incubating cells at 37 jC for 2.5
h with high-titer virus diluted in a small volume of Neuro-
basal medium containing B27 supplement and L-glutamine
providing a multiplicity of infection (MOI) of 1000 based
on optical particle units (OPU; [40]). This MOI was
predetermined to infect f 90–100% of the SH-SY5Y cells.
The wells were gently rocked occasionally throughout the
infection period. Following infection warmed Neurobasal
medium containing B27 supplement and 2 mM L-glutamine
was added to the wells bringing the media volume up to
feeder levels (3–4 ml/6-well dish; 200 Al/96-well).For proof of dominant negative (dn) status of the viral
construct (AdCMVdnMLK3), CHO cells were treated in the
following manner. CHO cells were infected twice due to
their continual division. On day 0 cells were plated at 2� 105
cells/well in six-well culture dishes. On day 1 cells were
either uninfected, but subjected to the same conditions, or
infected with either control adenoviral construct, AdCMV-
LacZ (2000 MOI), or AdCMVdnMLK3 (2000 MOI). On
day 2 cells were transfected using Lipofectamine Plus (Gibco
#10964-013), as previously described [38]. All cells were
transfected with a total of 2 Ag of cDNA composed of the
following: 4% MLK3, 20% dnMLK3, and 50% dnMKK4.
The remaining percentage of cDNAwas composed of vector.
The ratio of MLK3 to dnMKK4 was predetermined to be in
the linear range with respect to MLK3 expression and
phosphorylation of the dnMKK4 substrate. A dominant
negative MKK4 substrate was used to prevent downstream
activation that would lead to cell death. The dominant
negative nature of these clones has been previously de-
scribed [38]. Four hours after the transfection, complete
growth medium was added. On day 3, all cells were re-
infected using the same protocol that was performed on day
1. On day 4 cells were lysed and prepared for phospho-
MKK4 ELISA [38] or immunoblot analysis.
2.10. Data computation
Data was computed using Microsoft Excel and statistics
were evaluated using either Student’s or Dunnett’s t-tests.
Significance (*) was based on p < 0.05. The graphics pack-
age used was Prism GraphPad.
3. Results
3.1. CEP-1347 inhibition of MPP+-induced loss in neuro-
nally differentiated SH-SY5Y cell survival, cellular mor-
phology and apoptosis
To establish cytotoxic effects of MPP+ in differentiated
SH-SY5Y cells, increases in the release of cellular lactate
dehydrogenase (LDH) were measured in the medium of
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–9790
cultures treated with increasing concentrations of MPP+ up to
a maximum of 10 mM. At 3 mM MPP+ significant increases
in LDH were observed at 48 h post treatment (Fig. 1A). This
was the lowest concentration of MPP+ that produced a
significant 30–50% increase in LDH release (relative to total
LDH) compared to untreated cultures. Although larger
increases in LDH release were observed at a higher concen-
tration of MPP+ (10 mM), the minimum concentration, 3
mM, which produced a significant increase in cytotoxicity
was used to measure neuroprotective effects of CEP-1347.
CEP-1347 provided significant protection from 3 mM
MPP+-induced LDH release at concentrations of 10, 30, 100
and 300 nM (Fig. 1B). Across multiple experiments 30–100
nM CEP-1347 achieved maximal efficacy against MPP+-
induced cell death ranging from f 40–60% rescue. After
48 h of treatment with 3 mM MPP+ SH-SY5Y cellular
morphology changes, going from adherent process bearing
neuronal-looking cells (Fig. 2A) to rounded, clustered cells
with decreased substrate adhesion and process extension
(Fig. 2C). Moreover, two features indicative of apoptosis
Fig. 2. MPP+-induced changes in cellular morphology and apoptosis in differentia
with Hoffman optics (A) and normal nuclear bis-benzimide (Hoechst) staining (B).
condensed nuclear chromatin. (D) Arrows represent apoptotic cells. Treatment with
(E) and chromatin changes (F). Insert panel D: DNA laddering following 3 mM
bar = 10 Am.
were observed in SH-SY5Y cells following 48 h of treatment
with 3 mM MPP+: (1) Condensed chromatin (Hoechst, Fig.
2B, D, and F) and (2) DNA laddering (Fig. 2D, insert). These
observations confirm reports that SH-SY5Y cells undergo
apoptosis in response to MPP+ treatment [30,33,46,55]. Of
significance, the morphologic changes observed in SH-
SY5Y cells following exposure to 3 mM MPP+ were mostly
prevented by pretreatment of cells with CEP-1347 (Fig. 2E).
Moreover, CEP-1347 (30 nM) prevented nuclear chromatin
condensation in a majority of cells when it was added 1
h prior to 3 mM MPP+ treatment (Fig. 2F), indicative of a
partial inhibition of MPP+-induced apoptosis.
3.2. CEP-1347 acts downstream of MPP+ effects on
mitochondrial demise in differentiated SH-SY5Y cells
MPP+ is a mitochondrial toxin that inhibits respiration at
complex I of the electron transport chain [43]. The resulting
increase in NADH due to this inhibition leads to pyruvate
being metabolized into lactate. To determine whether CEP-
ted SH-SY5Y cells. Untreated control cultures showing normal morphology
Cultures treated with 3 mM MPP+ show degenerating cells at 48 h (C) with
30 nM CEP-1347 1 h prior to 3 mMMPP+ exposure prevents morphologic
MPP+ exposure: lanes (1) MW markers; (2) 0 h; (3) 24 h; (4) 48 h. Scale
Fig. 3. Lack of CEP-1347 effects on cellular lactate production induced by
3 mM MPP+ in differentiated SH-SY5Y cells. Lactate levels in culture
medium were measured at 4, 24 and 48 h post exposure to 3 mM MPP+ in
quadruplicate. Groups as follows: (MPP) 3 mM MPP+; (MPP+/1347) 30
nM CEP-1347 (neuroprotective concentration) was tested with 3 mM
MPP+. Graph represents one of two experiments with the same result.
Fig. 4. JNK activation following 3 mM MPP+ and inhibition by CEP-
1347. Immunoblot analysis of differentiated SH-SY5Y cell lysate for
phosphorylated (activated) JNK and total JNK1/2 protein following 30
min of 3 mM MPP+ and as indicated below a 30-min pretreatment with a
concentration response of CEP-1347 (A). The p46 pJNK band is
quantitated in (B) and represented as a ratio of p46 optical density
relative to total JNK1/2 protein density as a gel loading control.
Numbered duplicate wells in A are as follows: (1) and (2) basal untreated
cultures; (3) and (4) 3 mM MPP+; (5) and (6) MPP+ with 0.01 AM CEP-
1347; (7) and (8) MPP+ with 0.1 AM CEP-1347; (9) and (10) MPP+ with
0.3 AM CEP-1347; (11) and (12) MPP+ with 1 AM CEP-1347; (13) and
(14) MPP+ with 10 AM CEP-1347. * indicates significantly different from
basal ( p< 0.05); ** indicates significantly different from MPP+ ( p< 0.05).
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–97 91
1347 altered the toxic effects of MPP+ by interfering with
the initial events of mitochondrial complex I inhibition,
lactate levels were measured in SH-SY5Y cell culture
medium following MPP+ treatment in the presence or
absence of CEP-1347. As early as 4 h after 3 mM MPP+
addition, lactate production increased f 2-fold over basal
and this lactate induction was maintained to 48 h after 3 mM
MPP+ addition (Fig. 3). This increase in lactate production
was not prevented by addition of CEP-1347 to the cultures
(Fig. 3). These data indicate that CEP-1347 does not
interfere with MPP+ toxicity by blocking MPP+ inhibition
of complex I and verifies that CEP-1347 is acting down-
stream of this initial insult. Therefore, the neuroprotective
effects of CEP-1347 were not due to decreases in MPP+
interactions with the mitochondria.
3.3. CEP-1347 inhibits SAPK/JNK pathway activation
following MPP+ treatment in neuronally differentiated SH-
SY5Y cells
Evidence for activation of the SAPK/JNK pathway was
evaluated by immunoblot analysis for pJNK. In SH-SY5Y
cells the total JNK and pJNK antibodies detected two protein
bands of 46 and 54 kDa (Fig. 4). These molecular weight size
bands correspond to the reported sizes of cloned and
expressed JNK [22]. Initial time course experiments showed
JNK activation between 30 min and 4 h following MPP+
exposure to SH-SY5Y cells, with a maximal activation of
2.4-fold above untreated cultures at the 30-min time point.
Typically in MPP+-treated SH-SY5Y cells the p46 kDA band
Fig. 5. Inhibition of plasmid overexpressed wild-type MLK3-stimulated p-
dominant negative (dn) MKK4 by plasmid dominant negative (dn) MLK3
and adenoviral dnMLK3 in CHO cells. Data represent duplicate samples
from one experiment. *= significantly different from MLK3 alone.
**= significantly different from MLK3-stimulated p-dnMKK4 ( p< 0.05,
Student’s t-test).
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–9792
showed a more robust and reliable activation than did the p54
kDA band. CEP-1347 inhibited the pJNK signal seen 30 min
following treatment of cells with 3 mM MPP+ in a concen-
tration-dependent manner from 0.1 to 10 AM with complete
inhibition achieved at 0.1 AMand above (Fig. 4). Notably, the
MPP+-induced pJNK signal fell below basal levels following
treatment with CEP-1347.
3.4. Dominant negative MLK3 inhibits MPP+-induced
toxicity and pJNK in neuronally differentiated SH-SY5Y
cells
To verify the dominant negative action of overexpressed
AdCMVdnMLK3, a CHO cell system was infected and
transfected with the following constructs. CHO cells infected
Fig. 6. AdCMVdnMLK3 inhibition of MPP+-induced cell death in differentiated SH
into culture medium measured 48 h post exposure. AdCMVdnMLK3 (1000 MO
different from 3 mM MPP+ (A). *= significantly different from basal. **= signifi
Student’s t-test. (B) Upper immunoblot shows overexpression of MLK3. Lanes:
(1000 MOI, 48 h), (3) AdCMVdnMLK3-infected SH-SY5Y cells (1000 MOI, 48
immunoblot with same samples in A probed for hgal. Lanes: (1) uninfected SH-
differentiated SH-SY5Y cells immunostained for MLK3 showing very low level
infected differentiated SH-SY5Y cells immunostained with MLK3 antibody show
with AdCMVdnMLK3 were inhibited from wild-type plas-
mid transfected MLK3 phosphorylation of transfected
dnMKK4, MKK4 being a downstream target of MLK3
(Fig. 5). A dominant negative MKK4 substrate was used
to measure overexpressed MLK3 kinase activity so as to
prevent death of the cells through downstream activation
mechanisms. Due to the continual division of CHO cells,
they were subjected to infection on two separate days
bracketing the plasmid transfection day. The MOI was
increased to 2000 per day to provide 90–100% transfection
efficiency demonstrated in parallel sister cultures. Control
viral infection with AdCMVLacZ had no effect on the ability
of MLK3 to phosphorylate dnMKK4. Following confirma-
tion of the dominant negative status of the AdCMVdnMLK3
in CHO cells, overexpression of dnMLK3 in neuronally
-SY5Y cells. MPP+ (3 mM) increased release of LDH from SH-SY5Y cells
I, 48 h expression)-dependent decreases in LDH release are significantly
cantly different from 3 mM MPP+-treated. Significance based on p< 0.05,
(1) uninfected SH-SY5Y cells, (2) AdCMVLacZ-infected SH-SY5Y cells
h). Lower immunoblot, same blot reprobed for phospho-JNK. (C) Separate
SY5Y cells, (2) AdCMVLacZ-infected (1000 MOI, 48 h). (D) Uninfected
s of endogenous MLK3 detected. (E) AdCMVdnMLK3 (1000 MOI, 48 h)
ing 90–100% infection efficiency and expression. Scale bar = 10 Am.
Fig. 8. AdCMVdnMLK2 infection of differentiated SH-SY5Y cells.
AdCMVdnMLK2 infection did not inhibit 3 mM MPP+-induced cell death
in SH-SY5Y cells. MPP+ (3 mM) increased release of LDH from SH-SY5Y
cell culture medium measured 48 h post exposure in cells infected with
either AdCMVLacZ (1000 MOI, 48 h) or AdCMVdnMLK2 (250 MOI, 48
h). Immunoblot shows overexpression of MLK2 with an HA antibody
probe. Lanes: (1–5) Adenoviral infection of SH-SY5Y cells for 24 h; (6–
9) adenoviral infection for 48 h. Lane (1) AdCMVLacZ 1000 MOI; (2) and
(6) AdCMVdnMLK2 250 MOI; (3) and (7) AdCMVdnMLK2 1000 MOI;
(4) and (8) AdCMVdnMLK2 2000 MOI; (5) and (9) AdCMVdnMLK2
4000 MOI. *= significantly different from basal. Significance based on
p< 0.05, Student’s t-test.
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–97 93
differentiated SH-SY5Y cells (Fig. 6, compare A lane 1—
endogenous MLK3 with lane 3—overexpressed MLK3 and
C endogenous immunocytochemically detected MLK3 vs.
D overexpressed MLK3) partially suppressed MPP+-in-
duced cell death, comparable to the protective effect of
CEP-1347 on MPP+ toxicity (Fig. 1). MPP+-stimulated
pJNK levels were also partially prevented by expression
of AdCMVdnMLK3 in neuronally differentiated SH-SY5Y
cells (Fig. 7). This dnMLK3 overexpression level did not
decrease basal levels of pJNK (Fig. 6, compare A: lanes 1
and 2 vs. 3). The control construct, AdCMVLacZ, used at
an equivalent MOI (1000) produced similar overexpression
when blots were probed with the h-gal antibody instead of
anti-MLK3 (Fig. 6B) and in histochemical evaluation of the
cells (not shown).
3.5. Real-time RT-PCR expression analysis of SH-SY5Y
cells
The existence of endogenous MLK3 expression in SH-
SY5Y cells was implicit from immunoblot analysis of cell
lysate with anti-MLK3 (Fig. 6). There are, however, no
commercially available antibodies for human MLK1, 2 or
ZPK(DLK). Therefore, real-time RT-PCR was performed on
SH-SY5Y cell lysate with gene specific primers identified
by the Primer Express software provided with the ABI
PrismR 7000 Sequence Detection System. Gene specific
Fig. 7. AdCMVdnMLK3 inhibition of MPP+-induced pJNK response in
differentiated SH-SY5Y cells. Immunoblot analysis of SH-SY5Y cell lysate
for phosphorylated JNK following 30 min of 3 mM MPP+ (A, B).
Numbered wells are as follows: (A1) basal untreated cultures infected with
AdCMVLacZ (1000 MOI, 24 h); (A2) 3 mM MPP+-treated culture infected
with AdCMVLacZ; (B1) basal untreated cultures infected with
AdCMVdnMLK3 (1000 MOI, 24 h); (B2) 3 mM MPP+-treated culture
infected with AdCMVdnMLK3. The p46 band is quantitated from duplicate
distinct samples normalized to total JNK1/2 protein density and graphically
represented as fold over control p46 activated JNK. * indicates significantly
different from AdCMVLacZ ( p< 0.05).
products for MLK1, 2, 3 and ZPK(DLK) were detected in
differentiated SH-SY5Y cells. Two to three separate cell
preparations with three independent measures were gener-
ated and normalized to GAPDH expression to determine the
relative expression of MLK RNA in differentiated SH-
SY5Y cells. MLK1 and MLK3 were equivalently expressed
(5.91F 0.32 and 5.86F 0.21 DCT, respectively) and MLK2
Fig. 9. AdCMVdnDLK infection of differentiated SH-SY5Y cells.
AdCMVdnDLK infection did not inhibit 3 mM MPP+-induced cell death
in SH-SY5Y cells. MPP+ (3 mM) increased release of LDH from SH-SY5Y
cell culture medium measured 48 h post exposure in cells infected with
either AdCMVLacZ (250 MOI, 48 h) or AdCMVdnDLK (500 MOI, 48 h).
Immunoblot shows overexpression of DLK with an HA antibody probe.
Lanes: (1–5) Adenoviral infection of SH-SY5Y cells for 24 h; (6–9)
adenoviral infection for 48 h. Lane (1) AdCMVLacZ 1000 MOI; (2) and (6)
AdCMVdnDLK 250 MOI; (3) and (7) AdCMVdnDLK 500 MOI; (4) and
(8) AdCMVdnDLK 1000 MOI; (5) and (9) AdCMVdnDLK 2000 MOI.
*= significantly different from basal. Significance based on p< 0.05,
Student’s t-test.
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–9794
and ZPK(DLK) were equivalently expressed 1 DCT level
higher than MLK1 and MLK3 (6.83F 1.17 and 6.89F0.31,
respectively). This represents a twofold lower expression of
MLK2 and ZPK(DLK) in differentiated SH-SY5Y cells
than MLK1 and MLK3, as each critical threshold value
represents a doubling of RNA present upon replication in
the PCR reaction. Statistically, differences in MLK2 vs.
MLK3 expression did not reach significance ( p = 0.07),
whereas significant differences were established for MLK1
vs. MLK2; MLK1 vs. ZPK(DLK) and MLK3 vs.
ZPK(DLK).
3.6. Dominant negative MLK2 and dnDLK do not inhibit
MPP+-induced toxicity in differentiated SH-SY5Y cells
AdCMVdnMLK2 and AdCMVdnDLK represent domi-
nant negative adenoviral constructs of MLK family mem-
bers that are related to MLK3. The kinase domains of
MLK3 and MLK2 have 75% sequence homology and
MLK3 and DLK share 42% sequence homology. The
leucine zipper regions share similar homology with MLK3
and MLK2 having 68% homology while MLK3 and DLK
share only 32% homology. It is theoretically possible that
dnMLK2 or dnDLK would cross react by heterodimerizing
with endogenous MLK3 in SH-SY5Y cells and provide
some protection against MPP+. This was not the case over a
wide range of overexpressed protein (125–1000 MOI). In
the higher MOI conditions (500–1000 MOI), dnMLK2 and
dnDLK were toxic, especially when combined with MPP+
(data not shown). At MOI levels that were not toxic to
basal untreated cells, AdCMVdnMLK2 (Fig. 8) and
AdCMVdnDLK (Fig. 9) did not protect differentiated SH-
SY5Y cells against MPP+-induced death. An adenoviral
construct containing dnMLK1 was not tested in these
studies.
4. Discussion
CEP-1347 has been identified as a direct inhibitor of the
mixed lineage kinase (MLK) family and has previously
been found to attenuate MPTP-mediated DA neuronal death
and activation of the JNK signaling pathway in vivo
[38,51,52]. The present studies expand on those findings
by demonstrating that CEP-1347 and a dominant-negative
MLK3 adenovirus construct inhibit MPP+-induced death
and JNK signaling in SH-SY5Y cells. These studies further
implicate the MLK/JNK signaling pathway in MPP+-in-
duced neuronal death in vitro and suggest that this pathway
may be active in degenerating DA neurons in PD. Further,
these studies demonstrate the potential value of CEP-1347
as a neuroprotective compound in PD.
MPP+-treated SH-SY5Y cells are a useful in vitro model
for studying neurodegenerative events that may occur in PD
[7,30,33,46,55]. In the present study, CEP-1347 partially
prevented MPP+-induced cell death in retinoic acid differ-
entiated SH-SY5Y cells at low nanomolar concentrations.
CEP-1347 demonstrates neuroprotective properties in a
variety of primary neurons in culture including dorsal root
ganglion, sympathetic and motor neurons, and PC12 cells
after trophic factor withdrawal, DNA damage, or oxidative
stress [6,36,37]. CEP-1347 also maintains survival of motor
neurons in several in vivo models of programmed cell death,
such as developmental cell death of postnatal rat motor
neurons or of chick lumbar motor neurons in ovo and adult
rat hypoglossal neurons subjected to axotomy [19].
Morphologically, differentiated SH-SY5Y cells treated
with 3 mM MPP+ lose their processes, cluster, and eventu-
ally lift off the plate. This morphological change was
inhibited by pretreatment with CEP-1347. Additionally,
apoptotic indicators were demonstrated to be present fol-
lowing MPP+ addition to RA differentiated SH-SY5Y
cultures. CEP-1347 visually decreased the number of cells
displaying condensed chromatin, indicating that CEP-1347
may be preventing programmed cell death initiated by
MPP+ treatment. Inhibition of apoptotic cell death may be
important because markers of apoptosis have been observed
in many neurodegenerative diseases including PD [28].
However, others have shown that evidence of apoptosis
was missing in samples of patients with PD [15]. While a
snapshot of postmortem tissue is difficult to reconcile with
an ongoing neurodegenerative disease process, coupled with
the unknown clearance of apoptotic cells, it is difficult to
ascribe PD to a strictly apoptotic process. In other studies
CEP-1347 suppressed CHO cell-transfected MLK3-driven
apoptotic death at concentrations that inhibited MLK3
kinase activity [38]. Transient transfection of naive and
neuronally differentiated PC12 cells with MLK family
members showed apoptotic responses including membrane
blebbing, pyknotic nuclei and positive Hoechst staining in
addition to cell death [68], thereby relating MLK family
activation with potential apoptotic cell death.
To begin to address the mechanism of CEP-1347 inhibi-
tion of the MPP+ insult it was important to evaluate whether
the actual mitochondrial insult still occurred in the presence
of CEP-1347. When MPP+ interferes with mitochondrial
function at the level of complex I, lactate levels increase as a
result of an increase in glycolysis and increases in NADH
[45]. To determine whether CEP-1347 acted downstream of
mitochondrial demise, lactate levels were measured follow-
ing MPP+ treatment in the presence or absence of CEP-
1347. Lactate levels were increased twofold over basal
levels at 4, 24 and 48 h post MPP+. This increase was
unaffected by pretreatment with a neuroprotective concen-
tration of CEP-1347. These data indicated that CEP-1347
effects on cellular death pathways were downstream of this
initial toxic event.
CEP-1347 is a known inhibitor of the SAPK/JNK
signaling pathway and in vitro studies have demonstrated
inhibition at the level of the MLK family [38]. Increased
activity of JNK has been demonstrated following MPP+
treatment in undifferentiated SH-SY5Y cells [7,67]. In these
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–97 95
studies, undifferentiated cells were used in addition to
slightly higher concentrations of MPP+ (5 mM). In addition,
adenoviral gene transfer of the JNK binding domain of
JNK-interacting protein-1 (a scaffold protein and inhibitor
of JNK) inhibited MPP+-induced activation of JNK, c-Jun
and caspase and inhibited cell death in undifferentiated SH-
SY5Y cells [67]. The present study confirms these findings
by demonstrating that (1) MPP+ elevated pJNK in differen-
tiated SH-SY5Y cells, (2) that this could be partially
inhibited by adenoviral gene transfer of dominant negative
MLK3 and (3) completely blocked by an inhibitor of the
MLK family, CEP-1347/KT7515. It has been demonstrated
that all three JNK isoforms are represented in SH-SY5Y
cells [39]. What has not been determined is whether the
expression of these isoforms changes upon differentiation or
after MPP+ treatment. It is interesting that JNK activation
was completely inhibited by pretreatment with CEP-1347,
often with inhibition below basal conditions. CEP-1347
inhibition of JNK activation in differentiated SH-SY5Y
cells following MPP+ treatment was concentration depen-
dent with significant inhibition beginning at 10 nM and
maximal inhibition at 300–1000 nM. CEP-1347 does not
directly inhibit JNK in CHO cells as measured by a c-Jun
luciferase reporter construct [38] driven by c-Jun phosphor-
ylation, which is one of the immediate downstream targets
of JNK. This report [38] showed that only MLK3-induced
JNK activation could be inhibited by CEP-1347 and not
activation produced by MEKK1. More recently, there has
been described a lack of JNK activation in SH-SY5Y cells
following MPP+ [20]. These studies, however, were accom-
plished again in undifferentiated SH-SY5Y cells with much
lower concentrations of MPP+ (5 AM), which the authors
claim was insufficient to decrease mitochondrial function or
activate oxidative stress.
The MLK family has alternate downstream targets other
than JNK such as the transcription factor NF-nB and p38
[10,24,42,61]. Others have shown dominant negative ver-
sions of MLK3 prevent JNK activation induced by Rac and
Cdc42 but not JNK activation induced by MEKK1 [60].
Taken together, it seems that acute mitochondrial damage
initiates multiple intracellular signaling pathways leading to
cell death. Complete inhibition by CEP-1347 of the pJNK
induced by MPP+ mitochondrial damage, while only pre-
venting up to 70% of the cell death induced by MPP+,
suggests that another cell death pathway (other than through
JNK activation) is induced by MPP+ in these cells. CEP-
1347 can completely inhibit basal and MPP+-stimulated
JNK activation while at the same time only partially
prevents MPP+-induced cell death and condensed chroma-
tin. This study demonstrates that the JNK pathway disrup-
tion is critical but may not be sufficient to completely
protect from MPP+ toxicity. Others have shown that when
JNK is activated following UV light the resulting apoptosis
is JNK dependent [63]. They showed that the absence of
JNK1/2, through JNK1/2 null murine embryonic fibroblasts
prevented UV-induced apoptosis and DNA fragmentation
indicating that JNK is required for the normal apoptotic
response of fibroblasts to UV light. JNK null fibroblasts
were also resistant to the apoptotic effects of a genotoxin
(methyl methanesulfonate) and anisomycin, while the Fas-
death signaling pathway remained intact.
Utilization of an adenoviral dominant negative MLK3,
which infects >90% of differentiated SH-SY5Y cells and
allows considerable overexpression of this protein has shed
some light on the role of MLK3 in MPP+-induced cell
death. The dimerization of adenoviral overexpressed dom-
inant negative MLK3 with endogenous MLK3 [35] is
expected to prevent downstream phosphorylation of targets
such as MKK4 and/or MKK7. Leung and Lassam [35] have
shown that MLK3 homodimerization is crucial for down-
stream activation of the SAPK/JNK pathway. It is clear that
MLK3 partially contributes to the toxic effects of MPP+
through activation of JNK as demonstrated by our dominant
negative MLK3 data. Similar to CEP-1347, overexpression
of dnMLK3 prevented some, but not all, of the MPP+-
induced cell death. In contrast to CEP-1347, dnMLK3 did
not completely inhibit elevated levels of pJNK. CEP-1347 is
an inhibitor of the MLK family and would therefore be
expected to have effects beyond a single member such as
MLK3. Through a real-time RT PCR analysis of differen-
tiated SH-SY5Y cell lysate, expression of MLK1, MLK2,
MLK3 and ZPK(DLK) was measured. Expression of MLK1
and MLK3 had the higher relative expression seen among
these MLKs. It is theoretically possible for dnMLK2 or
dnDLK to heterodimerize with endogenous MLK3 to pre-
vent downstream activation of MKK4 or MKK7. Previous
studies using the same kinase dead (dominant negative)
mutations of MLK2 or DLK in plasmid transfection studies
with MLK3-activated JNK in PC12 cells [68] showed cross
reactivity. Others have shown cross reactivity with dominant
negative DLK prevention of MLK3-induced JNK activity in
293T cells [58]. Even though these dominant negative
adenoviral constructs overexpressed a considerable amount
of the dominant negative proteins in differentiated SH-
SY5Y cells, they were not protective following MPP+
treatment; in fact, they became toxic themselves at high
MOI concentrations. This toxicity was not seen with the
control construct, AdCMVLacZ or the positive test con-
struct, AdCMVdnMLK3, demonstrating that it was not
toxicity of a viral origin. CEP-1347 may be acting upon
other as yet undiscovered MLK family members or alternate
kinases.
This study demonstrates that the indolocarbazole CEP-
1347, likely through its inhibition of the MLK family,
completely inhibited the SAPK/JNK pathway and partially
prevented the neurotoxic effects of MPP+ in differentiated
SH-SY5Y cells. While CEP-1347 shows tendencies towards
an inverted U-shaped concentration response curve when
measuring cell survival, this is not demonstrated when
measuring JNK inhibition. At very high concentrations of
CEP-1347, other pharmacological rather than physiological
events could be occurring that are not addressed in this work.
J.R. Mathiasen et al. / Brain Research 1003 (2004) 86–9796
Adenoviral overexpression of dominant negative MLK3 also
partially protected differentiated SH-SY5Y cells from cell
death and pJNK activation indicating that MLK3 has a role
in MPP+-induced cell death. These results taken with previ-
ous studies indicate that CEP-1347 may be protective in
neurodegenerative diseases such as Parkinson’s Disease that
may involve the SAPK/JNK pathway leading to apoptosis.
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