text:
MPP+-induced neuronal death in rats involves tyrosine 33 phosphorylation of WW domain-containing oxidoreductase WOX1.
ABSTRACT WW domain-containing oxidoreductase (named WWOX, FOR or WOX1) is a pro-apoptotic protein and tumor suppressor. Animals treated with dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+) develop Parkinson's disease (PD)-like symptoms. Here we investigated whether WOX1 is involved in MPP+-induced neurodegeneration. Upon insult with MPP+ in rat brains, WOX1 protein was upregulated and phosphorylated at Tyr33 (or activated) in the injured neurons in the striatum and cortex ipsilaterally to intoxication, as determined by immunohistochemistry and Western blotting. Also, WOX1 was present in the condensed nuclei and damaged mitochondria of degenerative neurons, as revealed by transmission immunoelectron microscopy. Time-lapse microscopy revealed that MPP+ induced membrane blebbing and shrinkage of neuroblastoma SK-N-SH cells. Dominant-negative WOX1, a potent inhibitor of Tyr33 phosphorylation, abolished this event, indicating a critical role of the phosphorylation in apoptosis. c-Jun N-terminal kinase (JNK1) is known to bind and counteract the apoptotic function of WOX1. Suppression of JNK1 function by a dominant-negative spontaneously induced WOX1 activation. WOX1 physically interacted with JNK1 in SK-N-SH cells and rat brain extracts. MPP+ rapidly increased the binding, followed by dissociation, which is probably needed for WOX1 to exert apoptosis. We synthesized a short Tyr33-phosphorylated WOX1 peptide (11 amino acid residues). Interestingly, this peptide blocked MPP+-induced neuronal death in the rat brains, whereas non-phospho-WOX1 peptide had no effect. Together, activated WOX1 plays an essential role in the MPP+-induced neuronal death. Our synthetic phospho-WOX1 peptide prevents neuronal death, suggestive of its therapeutic potential in mitigating the symptoms of PD.
- Krystyna Szymańska, Krzysztof Szczałuba, Agnieszka Lugowska, Ewa Obersztyn, Marek Radkowski, Beata A Nowakowska, Katarzyna Kuśmierska, Jolanta Tryfon, Urszula Demkow[Show abstract] [Hide abstract]
ABSTRACT: Inherited encephalopathies include a broad spectrum of heterogeneous disorders. To provide a correct diagnosis, an integrated approach including genetic testing is warranted. We report seven patients with difficult to diagnose inborn paediatric encephalopathies. The diagnosis could not be attained only by means of clinical and laboratory investigations and MRI. Additional genetic testing was required. Cytogenetics, PCR based tests, and array-based comparative genome hybridization were performed. In 4 patients with impaired language abilities we found the presence of microduplication in the region 16q23.1 affecting two dose-sensitive genes: WWOX (OMIM 605131) and MAF (OMIM 177075) (1 case), an interstitial deletion of the 17p11.2 region (2 patients further diagnosed as Smith-Magenis syndrome), and deletion encompassing first three exons of Myocyte Enhancer Factor gene 2MEF2C (1 case). The two other cases represented progressing dystonia. Characteristic GAG deletion in DYT1 consistently with the diagnosis of torsion dystonia was confirmed in 1 case. Last enrolled patient presented with clinical picture consistent with Krabbe disease confirmed by finding of two pathogenic variants of GALC gene and the absence of mutations in PSAP. The integrated diagnostic approach including genetic testing in selected examples of complicated hereditary diseases of the brain is largely discussed in this paper.BioMed Research International 01/2014; 2014:424796. · 2.71 Impact Factor - SourceAvailable from: Nan-Shan Chang
Article: Identification of an In Vivo MEK/WOX1 Complex as a Master Switch for Apoptosis in T Cell Leukemia.
Hsin-Ping Lin, Jean-Yun Chang, Sing-Ru Lin, Ming-Hui Lee, Shenq-Shyang Huang, Li-Jin Hsu, Nan-Shan Chang[Show abstract] [Hide abstract]
ABSTRACT: Not all leukemia T cells are susceptible to high levels of phorbol myristate acetate (PMA)-mediated apoptosis. At micromolar levels, PMA induces apoptosis of Jurkat T cells by causing mitochondrial polarization/de-polarization, release of cytosolic granules, and DNA fragmentation. Chemical inhibitors U0126 and PD98059 block mitogen-activated protein kinase kinase 1 (MEK1)-mediated phosphorylation of extracellular signal-regulated kinase (ERK) and prevent apoptosis. Mechanistically, proapoptotic tumor suppressor WOX1 (also named WWOX or FOR) physically interacts with MEK1, in part, in the lysosomes in Jurkat cells. PMA induces the dissociation, which leads to relocation of MEK1 to lipid rafts and WOX1 to the mitochondria for causing apoptosis. U0126 inhibits PMA-induced dissociation of WOX1/MEK1 complex and supports survival of Jurkat cells. In contrast, less differentiated Molt-4 T cells are resistant to PMA-induced dissociation of the WOX1/MEK1 complex and thereby are refractory to apoptosis. U0126 overturns the resistance for enhancing apoptosis in Molt-4 cells. Together, the in vivo MEK1/WOX1 complex is a master on/off switch for apoptosis in leukemia T cells.Genes & cancer 05/2011; 2(5):550-62. - SourceAvailable from: Chun-I SzeM-H Lee, S-R Lin, J-Y Chang, L Schultz, J Heath, L-J Hsu, Y-M Kuo, Q Hong, M-F Chiang, C-X Gong, C-I Sze, N-S Chang[Show abstract] [Hide abstract]
ABSTRACT: The role of a small transforming growth factor beta (TGF-β)-induced TIAF1 (TGF-β1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-β1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-β receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid β (Aβ) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aβ. To test this hypothesis, in vitro analysis showed that TGF-β-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aβ and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.Cell Death & Disease 12/2010; 1:e110. · 5.18 Impact Factor
Page 1
MPP+-induced neuronal death in rats involves tyrosine 33
phosphorylation of WW domain-containing oxidoreductase
Chen-Peng Lo,1,2Li-Jin Hsu,3,4Meng-Yen Li,2Se-Yei Hsu,2Jih-Ing Chuang,5Ming-Shu Tsai,6Sing-Ru Lin,7
Nan-Shan Chang1,4,7,8,* and Shur-Tzu Chen1,2,*
1Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan 70101, ROC
2Department of Cell Biology & Anatomy, National Cheng Kung University, Tainan, Taiwan 70101, ROC
3Department of Microbiology & Immunology, National Cheng Kung University, Tainan, Taiwan 70101, ROC
4Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University Medical College, Tainan, Taiwan
5Department of Physiology, National Cheng Kung University, Tainan, Taiwan 70101, ROC
6Potz General Hospital, Department of Health, Executive Yuan, Chiayi, Taiwan 61347, ROC
7Institute of Molecular Medicine, National Cheng Kung University, Tainan, Taiwan 70101, ROC
8Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA
Keywords: Bcl-2, JNK1, MPP+, neurodegeneration, p53, WOX1, WWOX
WW domain-containing oxidoreductase (named WWOX, FOR or WOX1) is a pro-apoptotic protein and tumor suppressor. Animals
treated with dopaminergic neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+) develop Parkinson's disease (PD)-like symptoms. Here
we investigated whether WOX1 is involved in MPP+-induced neurodegeneration. Upon insult with MPP+in rat brains, WOX1 protein
was upregulated and phosphorylated at Tyr33 (or activated) in the injured neurons in the striatum and cortex ipsilaterally to
intoxication, as determined by immunohistochemistry and Western blotting. Also, WOX1 was present in the condensed nuclei and
damaged mitochondria of degenerative neurons, as revealed by transmission immunoelectron microscopy. Time-lapse microscopy
revealed that MPP+induced membrane blebbing and shrinkage of neuroblastoma SK-N-SH cells. Dominant-negative WOX1, a
potent inhibitor of Tyr33 phosphorylation, abolished this event, indicating a critical role of the phosphorylation in apoptosis. c-Jun
N-terminal kinase (JNK1) is known to bind and counteract the apoptotic function of WOX1. Suppression of JNK1 function by a
dominant-negative spontaneously induced WOX1 activation. WOX1 physically interacted with JNK1 in SK-N-SH cells and rat brain
extracts. MPP+rapidly increased the binding, followed by dissociation, which is probably needed for WOX1 to exert apoptosis. We
synthesized a short Tyr33-phosphorylated WOX1 peptide (11 amino acid residues). Interestingly, this peptide blocked MPP+-induced
neuronal death in the rat brains, whereas non-phospho-WOX1 peptide had no effect. Together, activated WOX1 plays an essential
role in the MPP+-induced neuronal death. Our synthetic phospho-WOX1 peptide prevents neuronal death, suggestive of its
therapeutic potential in mitigating the symptoms of PD.
WW domain-containing oxidoreductase, known as human WWOX (or
FOR) or murine WOX1, is a candidate tumor suppressor (Chang et al.,
2007; Smith et al., 2007). Human WWOX gene, encoding the
WWOX⁄FOR⁄WOX1 protein, is mapped to a fragile site on
chromosome 16q23.2. Homozygous deletion of this gene has been
noted in various types of cancer cells, including ovarian cancer (Paige
et al., 2001), breast cancer (Driouch et al., 2002), non-small cell lung
carcinoma (Yendamuri et al., 2003), gastric carcinoma (Aqeilan et al.,
2004) and many others (Chang et al., 2007; Smith et al., 2007).
WWOX deficiency may be associated with failure of apoptosis in
lymphocytes from patients with thyroid cancer (Sbrana et al., 2006).
Targeted disruption of murine Wwox gene induces spontaneous cancer
formation, indicating that the murine Wwox gene-encoded WWOX⁄
WOX1 protein is a tumor suppressor (Aqeilan et al., 2007).
Nonetheless, WWOX⁄WOX1 levels are upregulated during embry-
onic development and in early stages of cancerous progression, such
as in breast, prostate and skin in humans and mice (Watanabe et al.,
2003; Chen et al., 2004; Chang et al., 2005b; Lai et al., 2005). In
murine gene trap targeting, disruption of murine Wwox gene causes a
higher incidence of B-cell lymphomas in the knockout mice
(Ludes-Meyers et al., 2007). Also, these mice develop testicular
atrophy, suggesting a likely role of murine WWOX⁄WOX1 in organ
In response to stress stimuli, WOX1 undergoes phosphorylation at
Tyr33 (p-WOX1) and probably other conserved serine, threonine or
tyrosine sites. Activated WOX1 is able to induce apoptosis both
in vitro and in vivo (Chang et al., 2001, 2003a,b, 2005a, 2007; Hong
et al., 2007). Stress stimuli increase the binding of WOX1 with tumor
Correspondence: Dr N.-S. Chang,7Institute of Molecular Medicine, as above.
E-mail: firstname.lastname@example.com
*N.-S.C. and S.-T.C. contributed equally to this work.
Received 29 August 2006, revised 13 January 2008, accepted 7 February 2008
European Journal of Neuroscience, Vol. 27, pp. 1634–1646, 2008
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
Page 2
suppressor p53. Both WOX1 and p53 may mediate apoptosis in a
synergistic manner (Chang et al., 2001, 2003a, 2005a). In contrast,
c-Jun N-terminal kinase 1 (JNK1) physically binds and counteracts the
apoptotic function of WOX1 (Chang et al., 2003a).
We have previously demonstrated that WOX1 is expressed at
moderate to high levels in the developing central and peripheral
nervous systems and neural crest-derived structures, suggesting that
WOX1 participates in neuronal differentiation and maturation (Chen
et al., 2004). Upon long-term constant light exposure, WOX1 is
upregulated and activated in damaged photoreceptors in the rodent
retina (Chen et al., 2005). Similarly, WOX1 activation was
observed in chronic inherited retinal degeneration in mice (Chen
et al., 2005). Expression of human WWOX⁄WOX1 and isoform
WOX2, along with Tyr33 phosphorylation, is significantly down-
regulated in the hippocampal neurons of patients with Alzheimer's
disease (AD; Sze et al., 2004). The downregulation is negatively
correlated with upregulation of Tau hyperphosphorylation and
formation of neurofibrillary tangles. These in vivo findings have
been reproduced in neuroblastoma cells. That is, knockdown of
WOX1 expression by small interfering RNA induces Tau phos-
phorylation and tangle formation (Sze et al., 2004). Whether
WOX1 plays a critical role in regulating Tau phosphorylation and
preventing neurodegeneration is unknown and remains to be
Parkinson's disease (PD) is one of the most common forms of
neurodegenerative disorders, second in prevalence only to AD. In
idiopathic PD, mitochondria complex I deficiency has been identified
in the substantia nigra of patients with PD, and the deficiency may be
sufficient to trigger neurodegeneration (Hartley et al., 1994;
Langston, 1996; Swerdlow et al., 1996; Schapira et al., 1998).
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent and
selective nigrostriatal dopaminergic neurotoxin that induces many
neuropathological features of PD in primates and rodents (Heikkila
et al., 1984; Agid, 1991; reviewed by Speciale, 2002). Direct
injection of 1-methyl-4-phenyl-pyridinium (MPP+), a metabolite of
MPTP, into brain striatum produces a similar type of neurotoxicity as
with a systemic treatment with MPTP (Heikkila et al., 1985).
Administration of MPTP⁄MPP+depletes ATP, eliminates mitochon-
drial membrane potential and generates free radicals. Bioenergetic
and oxidative stresses create a context that eventually leads to cell
death (Nicotra & Parvez, 2002; Eberhardt & Schulz, 2003; Vila &
Przedborski, 2003; Schober, 2004). MPTP⁄MPP+also induces
morphological features of apoptosis, including nuclear chromatin
condensation, membrane blebbing and TdT-mediated dUTP-digoxi-
genin nick-end labeling (TUNEL)-positive reaction (Tatton & Kish,
1997; Chen et al., 2002).
It is generally agreed that MPTP-mediated PD models in vivo
invoke several signal pathways that converge to cell death. Among
them, mice, deficient in p53 but overexpressed Bcl-2, are resistant to
MPTP-induced neurotoxicity (Trimmer et al., 1996; Offen et al., 1998;
Yang et al., 1998). Persistent activation of JNK kinases causes
apoptotic cell death. Inhibition of the JNK pathway constitutes a new
strategy for treating PD by blocking the upstream death signal leading
to dopaminergic cell death (Saporito et al., 1999; Xia et al., 2001;
reviewed by Peng & Anderson, 2003). In this study, we utilized
MPP+-mediated injury to the nigrostriatal pathway in rats, and
demonstrated p-WOX1, followed by nuclear translocation. Short
synthetic WOX1 peptides, with or without Tyr33 phosphorylation,
were utilized to modulate MPP+-induced neuronal injury in vivo. In
cell models, MPP+-treated dopaminergic neuroblastoma SK-N-SH and
SH-SY5Y cells were chosen to investigate WOX1 activation and cell
death, and examine its interaction with JNK1.
Materials and methods
Animals and neurotoxin treatment
Handling and utilization of animals for these experiments were
approved by the Intramural Animal Care and Use Committee
(IACUC) of the National Cheng Kung University. Research was
performed according to the guidelines of National Institute of Health
for the Care and Use of Laboratory Animals. Fifty mature male
Sprague–Dawley rats of 300–350 g were used. Animals were
anaesthetized by intraperitoneal injection of sodium pentobarbital
(50 mg⁄kg i.p.). One microliter of MPP+iodide (100 mm in sterile
saline; Sigma-Aldrich) or saline alone was administrated unilaterally
into the corpus striatum through a guided tube with a snug-fitting
trochar and a stainless steel cannula insert (1 lL injection in 5 min).
The coordinates of the striatum were as follows, according to the atlas
of Paxinos and Watson as described previously (Chuang & Chen,
2002): AP, +1 mm from the bregma; ML, )2.8 mm from the midline;
DV, )5.2 mm below the skull surface. Post-injection for indicated
times (i.e. from 1 h to 8 weeks), animals were anaesthetized with
sodium pentobarbital (50 mg⁄kg, i.p.), and killed by intracardial
perfusion with normal saline. Specific regions in the brains were
isolated and then fixed with 4% paraformaldehyde.
Effect of synthetic non-phospho- and phospho-WOX1 peptides
We have made short synthetic peptides of WOX1, without (WWpep)
or with Tyr33 phosphorylation (pY-WWpep; amino acid # 28–38) at
the first WW domain (Chang et al., 2003a). To investigate the effect of
these synthetic WOX1 peptides on neuronal survival and death, rats
were injected with these peptides in the presence or absence of MPP+
as follows: (i) MPP+iodide (50 mm); (ii) WWpep or pY-WWpep
(2 mm); (iii) WWpep (or pY-WWpep) and MPP+or (iv) sterile saline.
Intracranial injection to the brain striatum (1 lL injection in 5 min)
was performed as described above (Chuang & Chen, 2002).
Cell culture and MPP+treatment
SK-N-SH cells were grown in Dulbecco's modified Eagle's medium
(Gibco BRL⁄Invitrogen), supplemented with 10% fetal bovine serum
and 2 mm l-glutamine (Gibco BRL⁄Invitrogen), in a humidified 5%
CO2⁄95% atmosphere incubator at 37 ?C (Sze et al., 2004). For
growing SH-SY5Y cells, they were allowed to adhere onto the surface
of plastic flasks for 1 h prior to the addition of 10 lm all trans-retinoic
acid (10 mm stock solution in ethanol; Sigma-Aldrich) for inducing
neuronal differentiation. Following 3–5 days in culture, cells were fed
with fresh medium containing B27 supplement and 2 mm l-glutamine
without retinoic acid. Where indicated, both types of cells were treated
with 20–2000 lm MPP+for indicated times, and cultured for 1–48 h.
Immunohistochemistry and fluorescence immunostaining
Rats were subjected to MPP+injection to the brains, anaesthetized
(sodium pentobarbital; 50 mg⁄kg), and then killed at indicated times.
The brains were dissected and processed for sectioning in cryostat or
in paraffin blocks. The brain sections were incubated for 2 days at
4 ?C with diluted antisera against a region between 1st and 2nd WW
domains of murine WOX1 (Chang et al., 2001), the first WW domain,
or this first WW domain with p-WOX1 (Chang et al., 2003a). These
antibodies are pan-specific and shown to interact with murine, rat and
human WOX1⁄WWOX. Following incubation, the sections were
incubated with aliquots of biotinylated anti-rabbit IgG (Vector) for
WOX1 in MPP+-induced neuronal death1635
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
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1 h, immersed in an avidin-biotin complex solution for another 1–2 h
at room temperature, and then processed for peroxidase reaction using
the glucose oxidase-nickel-DAB method (Chuang & Chen, 2002;
Chen et al., 2004, 2005). WOX1-expressing cells were counted from
40 to 100 neurons and other cells using Image-Pro-Plus software
(Media Cybernetics). Some of the serial tissue sections obtained from
control and experimental animals were incubated with anti-Bcl-2
(DAKO), caspase-3, -8, -9 (Santa Cruz), and caspase-activated DNase
(CAD, a downstream caspase-3 target) for apoptosis processing, glial
fibrillary acidic protein (GFAP; a marker for astrocytes; Chemicon) or
OX-42⁄CD11b (a marker for activated microglia and macrophages;
Serotec). Control sections were stained with non-immune horse or
goat sera. No controls exhibited positive immunoreactivity. Some of
the sections were stained with Cresyl violet for examining the
cytoarchitectonics of the brain tissues.
For dual fluorescence immunostaining, brain tissue sections from
two rats were immersed in blocking solution (1% bovine serum
albumin, 0.1% Triton X-100, 0.1% NaN3in phosphate-buffered saline)
for 1 h, followed by adding specific antibodies for 3 h incubation at
4 ?C, washing with phosphate-buffered saline four times, and probing
with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and
Texas Red-conjugated goat anti-mouse IgG (Chemicon) for 1 h.
Fluorescence microscopy was then performed (Nikon Eclipse TE-
2000S). Specific antibodies were against WOX1, p53 (Ab-3, Onco-
gene), JNK1 and⁄or phospho-JNK1⁄2 (p-JNK, Chemicon). Similar
experiments were carried out using SK-N-SH and SH-SY5Y cells.
Immunoelectron microscopy
Following MPP+injection for the indicated times (e.g. 4 h, 1 and
3 days), the striatal regions from the contralateral and MPP+-injected
(ipsilateral) sides were fixed for 2 h using a freshly prepared 4%
paraformaldehyde solution, and subsequently dehydrated in a graded
series of ethanol and embedded in LR White resin (London Resin
Company, UK) in 50 ?C oven. Ultrathin sections obtained from an
ultramicrotome (Reichert-Jung, Austria) were labeled with WOX1 or
p-WOX1, or indicated antibodies, followed by detecting with protein
A-gold particles, i.e. 10 nm for anti-mouse (or goat) and 20 nm anti-
rabbit gold particles (BBInternational, UK). Immunolabeled sections
were stained with saturated aqueous uranyl acetate and lead citrate at
room temperature. Specimens were then observed under a transmis-
sion electron microscope (JEOL JEM-1200EM, Japan).
Western blotting and co-immunoprecipitation (IP)
Western blotting was used to evaluate the expression of WOX1
protein and the extent of Tyr33 phosphorylation. Twelve rat brain
tissues post-intrastriatal injected with MPP+or saline for the indicated
times (e.g. 1 h, 4 h, 1 day or 3 days) were extracted using a lysis
buffer [5 mm Tris, 2% sodium dodecyl sulfate and 10% glycerol in the
presence of a cocktail of protease inhibitors (Sigma) and phosphatase
inhibitor sodium orthovanadate (500 lm)]. Equal amounts of proteins
from samples underwent electrophoresis and were then transferred
onto nitrocellulose membranes. The membranes were then incubated
with aliquots of WOX1 or p-WOX1 antisera, before staining with
horseradish peroxidase-conjugated goat anti-rabbit IgG. The reaction
was visualized using an ECL detection kit (ECL, Amersham; Chen
et al., 2005).
Where indicated, tissues obtained from the striatum and cortex,
either ipsilateral or contralateral to MPP+injection, were extracted
using a cytoplasmic and nuclear extraction kit (Pierce; Chang et al.,
2001). These protein preparations (1 mg protein) were quantified,
followed by performing co-IP using specific antibodies against
WOX1, JNK1⁄p-JNK or p53.
TUNEL assay
TUNEL assay was performed using sections from each animal
(Chuang et al., 2001). This assay allows detection of internucleosomal
DNA fragmentation caused by neuronal apoptosis by specifically
labeling the 3¢-hydroxyl termini of breakages of the DNA strands
(ApopTag kit, Oncor).
Analysis of DNA fragmentation
Tissues obtained from MPP+-injected striatum (both ipsilateral and
contralateral) were lysed for 20 min at 4 ?C in 10 mm Tris-HCl, 0.5%
Triton X-100 and 5 mm EDTA. After centrifugation at 27 000 g for
15 min at 4 ?C, the supernatants were extracted with an equal volume
of phenol⁄chloroform. DNAwas then precipitated with 0.1 volume of
3 M sodium acetate, pH 5.2, and 2 volumes of ethanol, suspended in a
buffer of 10 mm Tris-HCl and 1 mm EDTA, and treated with
20 lg⁄mL proteinase K for 1 h at 37 ?C. Finally, DNA preparations
were electrophoresed in 2% agarose gels. Gels were stained with
0.5 lg⁄mL ethidium bromide for 15 min, and the fragmented DNA
was visualized under ultraviolet light. Similarly, MPP+-treated neuro-
blastoma cells (e.g. SK-N-SH) were collected, spun down and
processed for DNA fragmentation analysis.
Time-lapse microscopy
We measured MPP+-induced reduction in sizes and membrane
blebbing of SK-N-SH cells by time-lapse microscopy using an
invertedfluorescence microscope
SK-N-SH cells were grown on cover slides, transfected with an
expression vector for enhanced green fluorescence protein (EGFP),
WOX1 (EGFP-tagged) or dominant-negative WOX1 (Dn-WOX1
tagged with EGFP; Chang et al. 2003a), and cultured for 24–48 h.
Dn-WOX1 blocks Tyr33-phosphorylation in WOX1. The cells were
exposed to a relatively high concentration of MPP+at 1 mm. Images
were taken by a CCD camera every 30 s. The overall recording time
was 2 h. Where indicated, cells were pretreated with antioxidant
U74389G (100 lm; Sigma-Aldrich), followed by exposure to MPP+.
(Nikon EclipseTE2000-5).
MPP+-induced neurodegeneration in rats
In agreement with our previous observations (Chen et al., 2002;
Chuang & Chen, 2002), Cresyl violet staining revealed typical
histology of cortical, striatal and nigral neurons in the normal brain
tissues. Four to 24 h post-MPP+injection, increased numbers of
pyknotic neurons were observed in the area surrounding the injection
site. Numerous small degenerative cells were found throughout the
ipsilateral cortex and caudate nucleus 3 days post-injection, indicating
an increased extent of neuronal injury (Fig. 1A and B). These cells in
the brain ipsilateral to injury were small in size, resembling dying
neurons or glia. Slight degeneration of neurons was also observed in
the substantia nigra ipsilateral to the MPP+injury 3 to several days
later, as demonstrated by staining cells with a dopaminergic marker
tyrosine hydroxylase (data not shown). The cells in the injured striatum
had undergone apoptosis as verified by TUNEL assay (Fig. 1C and D).
1636 C.-P. Lo et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 4
Very few or no TUNEL-positive cells were observed in the ipsilateral
substantia nigra, although many tyrosine hydroxylase-immunoreactive
dopaminergic neurons underwent degeneration (data not shown).
Upon injection insult, the presence of numerous inflammatory
microglia⁄macrophages, immunoreactive with OX-42⁄CD11b anti-
body, was found in the injured nigrostriatal regions (data not shown).
GFAP-positive astrocytes were normally present in uninjured striatum
(Fig. 1E). In contrast, there were significantly reduced numbers and
immunostaining intensity of GFAP-positive astrocytes in the injected
area (Fig. 1F).
MPP+upregulates and activates WOX1 in injured neurons
Neurotoxin MPP+suppresses WOX1 expression in nerve bundles, but
upregulates the expression in MPP+-injured neurons. For example, the
the white matter tracts of rat brains (e.g. the nerve fibers of the brain
damage to nerve fibers, resulting in disorientated structures of myelin
reduced (Fig. 1H). Vacuolization occurred in the injured striatum
3–7 days post-injection insult. Post MPP+injection for 8 weeks, the
majority of the white matter tracts disappeared, whereas the residual
WOX1 protein continued to be present.
In the striatum and cortex, WOX1 expression in normal neurons was
relatively low (Fig. 2A and C). These results are in agreement with our
previous observations (Chen et al., 2004, 2005; Sze et al., 2004).
However, WOX1 expression was significantly upregulated in apoptotic
neurons of striatum post-injury for 1–3 days (P < 0.0001, Student's t-
test; quantified by Image-Pro-Plus software from 40 to 100 cells;
Fig. 2B). WOX1 protein was present in the nuclei of these cells
(Fig. 2B), indicative of WOX1 activation (Chang et al., 2003a; Chen
et al., 2005). WOX1 undergoes Tyr33 phosphorylation and nuclear
translocation in response to stress stimuli (Chang et al., 2003a,b, 2007).
Three to 7 days later, there was a continual increase in numbers of
Compared with the healthy neurons in the contralateral side of the
cortex (Fig. 2C), a significantly increased WOX1 expression was
Fig. 1. MPP+intoxication in rat brains. (A) Post-MPP+injection to the rat brains for 3 days, many large and medium size neurons (> 20 lm) in the striatum
contralateral to the MPP+injection site are shown, as determined using Nissl stain. (B) The number of damaged neurons, with pyknotic nuclei, is significantly
increased in the MPP+-injected striatum. (C) Also, essentially no TUNEL-positive cells are present in the contralateral uninjected area. (D) In contrast, numerous
apoptotic cells are shown in the injured striatum as determined by TUNEL assay. (E) GFAP-positive astrocytes are present in the normal uninjured striatum.
(F) However, GFAP-expressing astrocytes are significantly downregulated in the injected or damaged region (asterisk). (G and H) WOX1 immunoreactivity or
expression is primarily distributed in the nerve bundles (arrow) of the intact striatum (G), and MPP+reduced the expression (H). Note that MPP+-induced
vacuolization (arrowheads) is present in the neuropils (H). Scale bar in (A) is also for (B–D); the bar in (E) is for (F–H). Bar: 60 lm.
WOX1 in MPP+-induced neuronal death1637
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 5
shown in both cytosol and nuclei in the apoptotic neurons (P < 0.001
vs contralateral, Student's t-test; Fig. 2D). WOX1 expression persisted
for 1 or 2 weeks in these apoptotic cells. Degradation of WOX1
appeared to occur in severely degenerated neurons 1–2 months later
(P < 0.001 vs contralateral, Student's t-test; Fig. 2E). Phosphorylation
of WOX1 at Tyr287 allows this protein to undergo polyubiquitination
and proteosomal degradation in vitro (Mahajan et al., 2005). Whether
this event occurs in vivo is unknown. The upregulated WOX1 was
indeed Tyr33-phosphorylated or activated (p-WOX1), as determined
using phospho-Tyr33-specific antibody. The distribution of activated
WOX1 was similar to that of total WOX1 in cells in striatum and
cortex ipsilateral to intoxication (data not shown).
MPP+induces nuclear accumulation of WOX1
during neuronal degeneration
WOX1 was sparely expressed in the cytoplasm of normal striatal
and cortical neurons as determined by immunoelectron microscopy
(data not shown), which agrees with the above observations under
light microscopy (Fig. 2A and C). One day following MPP+
injection, WOX1 was mainly present in the nuclei of neurons in the
injured area (Fig. 3A), suggesting that WOX1 is activated via
translocation from the cytoplasm to the nucleus. Also, WOX1 was
present in the nucleoli (Fig. 3B). These cells exhibited mem-
brane blebbing (Fig. 3B). In the cytoplasm, WOX1 was accumu-
lated in the mitochondria and rough
(Fig. 3C–E; Fig. 3D and E for high magnification), suggesting
that WOX1 also relocates to these organelles. Three days post-
damage, activated WOX1 protein (p-WOX1) was present on the
envelope and matrix of nuclei (Fig. 3F) and in apoptotic bodies
(Fig. 3G) in degenerating neurons. Furthermore, p-WOX1 was
present on the myelin sheaths and in the axon of striatal nerve
bundles (Fig. 3H). At this stage, the axonal myelination appeared to
be relatively intact. Taken together, MPP+-induced neuronal damage
in vivo positively correlates with Tyr33 phosphorylation and
accumulation in the nuclei, mitochondria and rough endoplasmic
endoplasmic reticulum
Fig. 2. Neurotoxin MPP+upregulates WOX1 expression in MPP+-injured neurons. (A and C) WOX1 is expressed at a relatively high level in the nerve bundles
(arrows in A and C) in the saline control or contralateral intact striatum and cortex. (B) One day post-MPP+injection, WOX1 expression was significantly increased
in the apoptotic neurons or glia (***P < 0.0001 vs contralateral, Student's t-test; quantified by Image-Pro-Plus software from 40–100 cells), whereas the WOX1
immunoreactivity in striatal nerve fibers was significantly reduced. (C and D) Similarly, compared with the contralateral uninjected cortex (C), significant
upregulation of WOX1 expression and nuclear accumulation (or activated WOX1) can be observed in the cortex ipsilateral to MPP+injection (***P < 0.001 vs
contralateral, Student's t-test; D). (E) WOX1 expression was significantly reduced 2 months later in the cells of injected side, along with severe cell death. Scale
bar: 50 lm. Cont, contralateral; Ipsi, ipsilateral.
1638 C.-P. Lo et al.
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MPP+induces de-colocalization of WOX1 with JNK1, but not
with p53, in neurons
We have shown the binding and colocalization of WOX1 with JNK1
and p53 both in cultured cells and in brain and other tissue sections
(Chang et al., 2001, 2003a, 2005a; Chen et al., 2005). Also, WOX1
colocalizes with JNK1 in the neurons from patients with AD (Sze
et al., 2004). Colocalization of WOX1 with JNK1 and p53 was shown
in the cytoplasm of striatal neurons, either apparently healthy or
apoptotic, ipsilateral to the MPP+-injected site of rat brain on Day 1
(Fig. 4). On Day 3, de-colocalization of cytosolic WOX1 with JNK1
could occur (Fig. 4; arrows and arrowhead). In contrast, WOX1 and
p53 remained colocalized on Day 3 (Fig. 4). Expression of caspases 3,
8 and 9 was barely increased (less than 10%) in the injured striatum
and cortex. However, no colocalization of caspases and WOX1 was
observed in the degenerative neurons, as determined by immunola-
beling with dual antibodies (data not shown).
of WOX1 and JNK1 by immunoelectron microscopy. Ultrathin brain
and p-JNK1, followed by probing with two different sizes of immuno-
gold particles. Colocalization of WOX1 with p-JNK1 was observed
primarily in the condensed nuclei of striatal and cortical neurons
Fig. 3. MPP+induces WOX1 accumulation in the nuclei in striatal neurons, as determined by transmission immunoelectron microscopy. WOX1 expression is
relatively low in the cytoplasm of normal striatal and cortical neurons (data not shown). (A) One day following MPP+injection, WOX1 was mainly accumulated in
the nuclei (N) of neurons in the injured area (see dark immunogold-labeled WOX1 proteins), suggesting nuclear translocation of WOX1. WOX1 was barely
detectable in the mitochondria (M). (B) At a later stage of less than 3 days, membrane blebbing (arrowhead) occurred in degenerating neurons. (B–E) WOX1 is
present in the nucleoli (see arrow and insert) (B), and cytosolic organelles (C), such as mitochondria (M), (D) and rough endoplasmic reticulum (arrow,
E). (F) WOX1 is indeed Tyr33-phosphorylated (using anti-p-WOX1 antibody) and present in a condensed nucleus (N) and an apoptotic body (A) 3 days post-
injection. (G) Note that WOX1 appeared to be excluded from an apoptotic body (see the gold particles; arrow). (H) MPP+induced membrane blebbing of the
neurites of injured striatum, whereas myelin sheaths remained largely intact and p-WOX1 was present on the sheaths and in the axon (arrow). Scale bars: 500 nm (A,
F, G); 1 lm (B); 200 nm (C, E, H); 100 nm (D).
WOX1 in MPP+-induced neuronal death1639
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European Journal of Neuroscience, 27, 1634–1646
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1–3 days post-injection (Fig. 5A and B). In uninjured control neurons,
very few immunogold particles for WOX1 or p-JNK1 were observed
(data not shown). Colocalization of WOX1 and p-JNK was also shown
in MPP+-treated striatum, including apoptotic bodies (Fig. 5C) and
damaged organelles (Fig. 5D–G) in degenerated neurons. Together, the
agreement with our previous report (Chang et al., 2003a).
MPP+induces WOX1 activation and modulates the binding
of WOX1 with JNK1
Next, we examined the potential roles of JNK1 and WOX1 in the
MPP+-induced neuronal death. These proteins counteract each other's
function in regulating apoptosis of cultured cells (Chang et al., 2003a).
Neuroblastoma SK-N-SH cells were grown overnight and then
synchronized under serum-free conditions for 16–24 h. Stimulation
of these cells with MPP+caused a time-related phosphorylation of
WOX1 and JNK1 (Fig. 6A). Similar results were observed when
stimulating these cells with MPP+in the presence of serum (data not
shown). Also, similar results were observed with SH-SY5Y cells.
SK-N-SH cells were responsive to UV light- and estrogen-induced
activation of WOX1 and JNK1, and the activated proteins interact
with each other (Chang et al. 2003a, b; 2005b; Hsu et al., 2005; Hong
et al., 2007). Suppression of JNK1 function by a dominant-negative
mutant (dn-JNK1; Chang et al. 2003a) spontaneously induced WOX1
phosphorylation and nuclear accumulation in SK-N-SH cells (Fig. 6B)
and HEK-293 fibroblasts (data not shown), again indicating that JNK1
restricts WOX1 activation.
By co-IP using antibody against WOX1, we determined that WOX1
physically interacted with JNK1 in SK-N-SH cells (cultured in the
presence of serum), and that stimulation of cells with MPP+for
30 min reduced the binding (Fig. 6C). These results are in parallel
with the MPP+-induced delocalization of WOX1 with JNK1 in
neurons (Fig. 4). In contrast, there was no reduced binding for WOX1
with p53 (data not shown).
To further confirm the binding of WOX1 with JNK1 in vivo, we
determined that WOX1 physically interacted with JNK1 in the rat
brains, as determined by co-IP (Fig. 7). MPP+rapidly induced the
binding of WOX1 with JNK1 in both the contralateral and ipsilateral
sides in the cortex 1 h post-injection, followed by dissociation in 4 h.
Similar results were observed in the ipsilateral side of striatum. In
contrast, complex formation of WOX1 with JNK1 was increased in
the contralateral side of striatum in 4 h post-injection. In negative
controls, immunobeads (non-immune IgG and protein A agarose
beads) alone did not bind WOX1 and JNK1 (Fig. 7).
Dn-WOX1 blocks MPP+-induced neuronal death
MPP+-induced activation is probably necessary for WOX1 to induce
cell death. To test this hypothesis, SK-N-SH cells were transfected
with our established Dn-WOX1 expression construct (tagged with
EGFP) or EGFP alone (Chang et al., 2003a), cultured for 24 h, and
then stimulated with MPP+for inducing apoptosis. Dn-WOX1 is
known to inhibit phosphorylation of WOX1 at Tyr33 (Chang et al.,
2003a, 2005a). By time-lapse microscopy, we measured MPP+-
induced reduction in cell sizes. At 1 mm, MPP+rapidly induced cell
shrinkage in control cells expressing EGFP in a time-related manner
(Fig. 8). The cells also underwent membrane blebbing (data not
shown). Dn-WOX1 blocked the cell shrinkage (Fig. 8), suggesting a
critical role of Tyr33 phosphorylation of WOX1 in MPP+-induced
apoptosis of SK-N-SH cells. WOX1 alone enhanced MPP+-induced
cell shrinkage and death (data not shown). MPP+induces oxidative
stress that causes organelle damage and eventual death of cells
(Nicotra & Parvez, 2002; Eberhardt & Schulz, 2003; Vila &
Przedborski, 2003). Pretreatment of cells with antioxidant U74389G
(100 lm), followed by exposure to MPP+, prevented the cell
shrinkage and death. MPP+induced typical features of apoptosis as
mentioned above. However, it did not cause internucleosomal DNA
fragmentation effectively (data not shown).
Short synthetic p-WOX1 peptide attenuates MPP+-induced
neuronal death
We have shown downregulation of WOX1 in the hippocampal
neurons of patients with AD (Sze et al., 2004). This downregulation
correlates with increased activities of enzymes (e.g. GSK-3b) in
hyperphosphorylating Tau and tangle formation both in cultured cells
and in the AD neurons, raising a likely role of WOX1 in protecting
neurons from degeneration and eventual death in vivo. We examined
the effect of short synthetic peptides, designed at the first WW domain
without (WWpep) or with Tyr33-phosphorylation (pY-WWpep;
Chang et al., 2003a), on MPP+-induced neurotoxicity. These peptides
were injected into rat brains in the presence or absence of MPP+.
Morphologically, as shown above (Figs 1–3), numerous dark-stained
small apoptotic cells appeared in the cortex and striatum ipsilaterally
to the MPP+-injected region (Fig. 9A). Importantly, pY-WWpep alone
did not effectively cause cell death, but significantly blocked MPP+-
induced neuronal apoptosis (Fig. 9A). WWpep could not inhibit the
effect of MPP+(data not shown). To investigate the underlying
mechanisms, we determined that MPP+induced nuclear accumulation
of CAD and p-JNK1 in the rat brain (Fig. 9B), suggesting nuclear
Fig. 4. Colocalization of WOX1 with JNK1 and p53 in neurons. Immuno-
fluorescence staining with dual antibodies was performed. WOX1 colocalized
with JNK1 and p53 in the cytoplasm of striatal neurons ipsilateral to 1-methyl-
4-phenyl-pyridinium ion (MPP+) injection on Day 1 (orange; see merged
pictures). Colocalization was found in both healthy and apoptotic cells (data not
shown for apoptotic cells). De-colocalization of WOX1 with JNK1 could occur
on Day 3 (arrows and arrowhead; red or green), whereas this event did not
appear to occur with WOX1 and p53. Magnification: 1000 ·.
1640C.-P. Lo et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 8
translocation of these proteins and their contribution to neuronal
apoptosis. pY-WWpep suppressed the nuclear accumulation of these
proteins, whereas WWpep had no effect (Fig. 9B). CAD is one of the
key proteins in causing DNA fragmentation during apoptosis (Degen
et al., 2000). JNK family proteins are also involved in neurodegen-
eration (Borsello & Forloni, 2007).
We have previously determined a potential role of p-WOX1 in
constant light-induced neuronal death in the rat retina (Chen et al.,
2005). Here, we further demonstrate that Tyr33 phosphorylation is
upregulated in the neurons upon challenge with dopaminergic
neurotoxin MPP+. MPP+exerts a similar effect as manifested the
neuropathology of PD (Agid, 1991). By transmission immunoelectron
microscopy and co-IP, we determined the binding of WOX1 with
JNK1 and p53 both in neuroblastoma SK-N-SH cells and in the rat
brains. JNK1 blocks WOX1-induced apoptotic cell death (Chang et al.
2003a, b). A dn-JNK1 was used to inhibit the function of wild-type
JNK1 and shown to spontaneously induce WOX1 phosphorylation at
Tyr33 and nuclear accumulation in SK-N-SH cells, again supporting
that JNK1 antagonizes the function of WOX1. MPP+stimulated an
initial increase in the complex formation of WOX1 and JNK1,
followed by dissociation in the cortical and striatal neurons in rats,
suggesting that this dissociation is needed for WOX1 to exert
Fig. 5. Colocalization of WOX1 and p-JNK in MPP+-treated neurons, as determined by immunoelectron microscopy. Ultrathin tissue sections were stained with
specific antibodies against WOX1 ⁄ WWOX and p-JNK1, and then probed with large and small immunogold particles (WWOX IgG for binding with 'small' 10-nm
anti-goat particles and p-JNK IgG for 'large' 20-nm anti-rabbit particles). (A and B) WOX1 and p-JNK1 were found to accumulate in the nuclei of striatal
(A) and cortical (B) neurons 1 day after MPP+injection. Very few or no immunogold particles were observed in the uninjured control neurons at the contralateral
side (data not shown). (C) Three days post-injury, an increased expression of WOX1 and p-JNK is shown in the apoptotic body (arrow, C). The 'insert' shows
colocalization of WOX1 and p-JNK. (D) These proteins are absent in oligodendrocytes (O) and its surrounded axons (a) and dendrites (d). The presence of
WOX1 ⁄ p-JNK1-positive immunogold particles is shown in severely injured organelles (D; see amplification in E). (E) is an amplified image from (D). (F and G)
Similarly, the presence of colocalized WOX1 and p-JNK is shown in the cytoplasm of injured neuron. C; cytoplasm; N, nucleus. Scale bars: 500 nm (A–C and G);
200 nm (D); 100 nm (E and F).
WOX1 in MPP+-induced neuronal death1641
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When Tyr33 phosphorylated and probably overexpressed, full-
length WOX1 protein may cause neuronal death in vivo. In
contrast, WOX1 levels are significantly downregulated in the
hippocampal neurons of patients with AD. This positively correlates
with increased activities of enzymes (e.g. GSK-3b) in hyperphosph-
orylating Tau and tangle formation in these AD neurons, suggesting
that WOX1 protects neurons from degeneration and eventual death
in vivo (Sze et al., 2004). Without doubt, the functional role of
WOX1 requires further delineation using brain conditional knockout
animal models. We postulate that the levels of WOX1 and
isoforms, along with Tyr33 phosphorylation, may be critical. That
is, activated WOX1 protects against chronic neuronal damage under
physiological conditions. Further upregulation of activated WOX1
is needed to enhance the death of damaged neurons in the diseased
We examined the area surrounding Tyr33 phosphorylation in
WOX1, and synthesized a short p-WOX1 peptide of 11 amino acid
residues. Surprisingly, this short peptide significantly prevented
MPP+-induced neuronal death in the rat brains, whereas non-
phospho-WOX1 peptide had no effect. Death of dopaminergic
neurons in the substantia nigra following MPP+injections into the
striatum would provide a better model for PD. However, we could not
observe a significant extent of cell death in the substantia nigra areas.
This is probably due to early death of rats post-MPP+injections in
these particular experiments. Another likely scenario is that the
phospho-WOX1 peptide binds MPP+and blocks the translocation of
MPP+to the substantia nigra. Nonetheless, we further substantiate our
observations by showing that the phospho-WOX1 peptide blocks
nuclear translocation of CAD and p-JNK1 in the rat brains, suggesting
that the phospho-peptide binds CAD and JNK1 and blocks their
activities. Short peptides do not normally possess tertiary structures in
solutions. Thus, they cannot represent the actual functional properties
of the full-length WOX1. Whether the phospho-WOX1 peptide binds
specific enzymes, thereby blocking neuronal death, remains to be
Fig. 6. JNK1 and WOX1 interactions in neuroblastoma cells. Neuroblastoma SK-N-SH cells were stimulated with 1-methyl-4-phenyl-pyridinium ion (MPP+
250 lm) for the indicated times. (A) MPP+rapidly induced phosphorylation of WOX1 and JNK1 approximately 10–30 min post-stimulation (data normalized as:
p-WOX1 vs WOX1; p-JNK1 vs JNK1; averaged from two experiments). The cells were cultured overnight with 10% fetal bovine serum and synchronized under
serum-free conditions for 16–24 h, prior to stimulation with MPP+. Similar results were observed when these cells were cultured in the presence of 10% fetal bovine
serum. (B) SK-N-SH cells were transfected with an empty vector or a dn-JNK1 by electroporation, followed by culturing for 48 h in the presence of 10% fetal
bovine serum. dn-JNK1-expressing SK-N-SH cells showed spontaneous p-WOX1 and its nuclear accumulation (using anti-p-WOX1 IgG; top panel). Expression of
the exogenous dn-JNK1 is shown (using anti-FLAG IgG; bottom panel). (C) By co-immunoprecipitation (IP) using WOX1-specific antibody, we determined that
WOX1 physically interacted with JNK1 ⁄ 2 in SK-N-SH cells, cultured in the presence of 10% fetal bovine serum. Treatment of cells with MPP+(250 lm) for 60 min
reduced the binding of WOX1 with JNK1 ⁄ 2. IP, immunoprecipitation with anti-WOX1 IgG; Pre-IP, pre-immunoprecipitation protein levels of WOX1 and JNK1 ⁄ 2.
1642 C.-P. Lo et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 10
established. We believe that the phospho-WOX1 peptide may have a
therapeutic potential in alleviating the symptoms of PD.
Normally, WOX1 is expressed in the choroid plexus of ventricles,
and the nerve bundles of cortex, striatum, optic tracts, corpus callosum
and subcortical white matter (Chen et al., 2004). MPP+intoxication
rapidly upregulates WOX1 expression and phosphorylation in vivo
(e.g. white matter tracts in the damaged cortical and striatal regions),
followed by downregulation with time. The initial event of WOX1
activation appears to correlate not only with the sensitivity of axons to
neurotoxic injury, but also with the extent of neuronal damage in the
beginning and subsequent time points.
JNK, a component of the SAPK system, induces both anti-apoptotic
and pro-apoptotic signal pathways. Transient JNK1 activation is pro-
survival (Bode & Dong, 2007), whereas sustained JNK1 activation
induces apoptosis (Glotin et al., 2006). In the apoptotic pathway,
JNK1 phosphorylates and inactivates the anti-apoptotic Bcl-xL
(Tournier et al., 2000). Also, JNK1 may phosphorylate and activate
the pro-apoptotic p53 (Gallo & Johnson, 2002). MPTP⁄MPP+induces
sequential phosphorylation and activation of JNK kinase (MKK4),
JNK1, c-Jun and caspases, and that inhibitors of JNK1 activation
diminish the effects of neurotoxic chemicals on dopaminergic
terminals (Saporito et al., 1999; Nicotra & Parvez, 2002; Eberhardt
& Schulz, 2003; De Girolamo & Billett, 2006). A portion of JNK1
physically interacts with WOX1 in cultured cells (Chang et al.,
2003a). Here we determined the binding in neuroblastoma cells and in
the rat brains. Presumably, the physical association could provide a
dynamic balance in deciding cell fate in promoting either growth or
Caspases are crucial proteases that are involved in the apoptotic
process. In the MPTP-treated mice and in post mortem PD brain
tissues, caspase-3, -8 and -9 are activated following the release of
cytochrome c from the mitochondria (Hartmann et al., 2001;
Viswanath et al., 2001). Overexpression of WWOX⁄WOX1 may
cause caspase activation in apoptotic cells at a terminal death stage
(Chang et al., 2007). However, WOX1 alone does not induce
caspase activation (Chang et al., 2001). We did not observe the
colocalization of WOX1 with caspases (e.g. caspase-3, -8 and -9) in
the neurotoxin-injected area. Nonetheless, upregulation of CAD and
its colocalization with WOX1 could be found after MPP+injury
(data not shown).
We showed the MPP+-induced TUNEL-positive cells in the brain
tissue sections, whereas no DNA fragmentation was observed using
brain extracts and SK-N-SH cells, as determined by agarose gel
electrophoresis (?30 times of experiments; data not shown). The
absence of DNA fragmentation pattern is probably due to insufficient
internucleosomal DNA breakage points. In time-lapse microscopy, we
determined that MPP+induced shrinkage and membrane blebbing of
SK-N-SH cells. We do not exclude the possibility that MPP+
stimulates an untypical pattern of apoptosis.
WOX1 interacts with the proline-rich region of p53 via its WW
domain, wherein WOX1 and p53 synergistically mediate apoptosis
(Chang et al., 2001, 2005a). Suppression of WOX1 function by
antisense mRNA or a Dn-WOX1 abrogates the apoptotic function of
p53, revealing a partnership between p53 and WOX1 in mediating cell
death. Here, the presence of p53 and WOX1 in apoptotic neurons
indicates that both proteins participate in neuronal death in vivo. We
have recently demonstrated that the expression profile of WOX1
protein was not altered by comparing p53 wild-type and knockout
mice, suggesting that p53 does not control the transcription of WOX1
expression (Chen et al., 2004).
Fig. 7. MPP+modulates the binding of WOX1 with JNK1 in vivo. One
microliter of 100 mm MPP+was injected into rats' striatum (see Materials and
methods). Tissues from cortex and striatum were isolated after 1 or 4 h,
followed by preparing tissue extracts for IP using WOX1 IgG antibody. (A)
MPP+increased the binding of WOX1 with JNK1 in both the contralateral and
ipsilateral sides in the cortex 1 h post-injection, followed by dissociation in 4 h.
(B) Similar results were observed in the ipsilateral side of the striatum. In
contrast, the complex formation of WOX1 with JNK1 was increased in the
contralateral side of striatum in 4 h post-injection. In negative controls, non-
immune IgG-coated protein A agarose beads were used for IP.
Fig. 8. Dn-WOX1 blocks MPP+-induced SK-N-SH cell death. SK-N-SH cells
were transfected with Dn-WOX1 (tagged with EGFP) or EGFP alone by
electroporation. These cells were cultured for 24 h, and then stimulated with a
high dose of MPP+at 1 mm to induce cell death. By time-lapse microscopy,
MPP+rapidly induced shrinkage of control EGFP-expressing cells in a time-
related manner, and Dn-WOX1 blocked the shrinkage.
WOX1 in MPP+-induced neuronal death1643
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European Journal of Neuroscience, 27, 1634–1646
Page 11
Fig. 9. Small Tyr33-phosphorylated WW domain peptide blocked 1-methyl-4-phenyl-pyridinium ion (MPP+)-induced neuronal degeneration and death. We made
short synthetic peptides at the first WW domain without (WWpep) or with Tyr33-phosphorylation (pY-WWpep; Chang et al., 2003a), and examined their effects on
MPP+-induced neurodegeneration. These peptides (4 mm) were injected into rat brains in the presence or absence of MPP+(1 m; injection rate, 1 lL in 5 min). Nissl
staining shows dramatic neuronal death occurred in the MPP+-injected striatum (b) and cortex (insert) in rats, as compared with saline control (a) and pY-WWpep-
treated (c) groups (A). Notably, pY-WWpep suppressed MPP+-induced neurodegeneration or cell death in the striatum (d) and cortex (insert). Shown in the bar
graph is the quantitative analysis of cell death (n ¼ 5; Student's t-test; experiments vs controls). By Western blotting using specific antibodies, MPP+induced nuclear
accumulation of caspase-activated deoxyribonuclease (CAD) and phosphorylated c-Jun N-terminal kinase (p-JNK) (B). pY-WWpep blocked the nuclear
accumulation of these proteins (C), whereas WWpep had no effect (B).
1644C.-P. Lo et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 12
The mechanism by which MPTP selectively induces degeneration
of dopaminergic neurons in the substantia nigra is largely unknown.
The plasma membrane dopamine transporter is involved in accumu-
lation of the active metabolite MPP+in neurons (Liu & Edwards,
1997). In contrast, the vesicular amine transporter sequesters the toxin
from its primary site of action in mitochondria, thereby sustaining cell
survival. In addition, mitochondrial anti-apoptotic Bcl-2 blocks MPTP
neurotoxicity in transgenic mice (Offen et al., 1998; Yang et al.,
1998). We have also established that Bcl-2 is upregulated in the
neurons and activated astrocytes⁄microglia in the neurotoxin-affected
areas including striatum, cortex and substantia nigra (Chuang et al.,
2001; Chuang & Chen, 2002). MPP+is known to be selectively
delivered to substantia nigra by dopamine transporters through the
nigrostriatal tract.
MPTP intoxication in animal models for PD involves oxidative
processes. This leads to activation of NADPH-oxidase in microglial
cells and increases in the intracellular levels of superoxide radicals and
nitric oxide for damaging mitochondria in neurons (reviewed by Tieu
et al., 2003). Activated microglia release pro-inflammatory molecules,
such as TNF-a, IL-1b and nitric oxide. Microglia activation for the
inflammatory response occurs in the MPP+-injured nigrostriatal
pathway (Chuang et al., 2001). Here, we determined that WOX1
activation occurs in striatal and cortical neurons, but not OX-
42⁄CD11b-positive microglia or GFAP-labeled astrocytes, suggesting
that WOX1 is not directly involved in brain inflammation.
When overexpressed, oxidoreductases, including WOX1, apopto-
sis-inducing factor and bacterial redox protein azurin (Yamada
et al., 2002), are known to induce apoptosis. Nonetheless, these
proteins may possess pro-survival functions. For example, apopto-
sis-inducing factor scavenges free radicals, thereby protecting
against peroxide-mediated cell death both in vivo and in vitro
(Klein et al., 2002; Lipton & Bossy-Wetzel, 2002). WOX1 is
upregulated in the brain and other organs during embryonic
development (Chen et al., 2004), suggesting that under physiolog-
ical conditions WOX1 functions in an anti-apoptotic manner (Chang
et al., 2007).
Taken together, we have demonstrated a likely role of WOX1 in the
pathogenesis of neuronal damages in PD. p-WOX1 is crucial in this
process, and JNK1 appears to play a counteractive role in binding and
blocking the pro-apoptotic function of WOX1.
This work was supported by the National Science Council of Republic of China
(NSC91-2320-B006-058 and NSC92-2320-B006-079) to S.-T.C. L.-J.H. was
supported by grants from the Ministry of Education (91-B-FA09-1-4) and
National Science Council, Taiwan (NSC95-2320-B-006–072-MY2). N.-S.C.
was supported in part by the American Heart Association, the Department of
Defense (DAMD17-03-1-0736), the Guthrie Foundation for Education and
Research, and the National Science Council, Taiwan (NSC96-2320-B-006-014,
96-2628-B-006-045-MY3 & 96n ˜2628-B-006-041-MY3). We appreciate the
outstanding technical assistance of Ms Sakaya Hsu of Taiwan Instrument
dn-JNK1, dominant-negative JNK1; Dn-WOX1, dominant-negative WOX1;
EGFP, enhanced green fluorescence protein; GFAP, glial fibrillary acidic
protein; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; MPP+,
1-methyl-4-phenyl-pyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine; PD, Parkinson's disease; p-JNK, phospho-JNK1; p-WOX1,
Tyr33-phosphorylated WOX1; TUNEL, dUTP-digoxigenin nick-end labeling;
WOX1 (WWOX ⁄ FOR), WW domain-containing oxidoreductase.
Alzheimer's disease;CAD, caspase-activated deoxyribonuclease;
Aqeilan, R.I., Kuroki, T., Pekarsky, Y., Albagha, O., Trapasso, F., Baffa, R.,
Huebner, K., Edmonds, P. & Croce, C.M. (2004) Loss of WWOX expression
in gastric carcinoma. Clin. Cancer Res., 10, 3053–3058.
Aqeilan, R.I., Trapasso, F., Hussain, S., Costinean, S., Marshall, D., Pekarsky,
Y., Hagan, J.P., Zanesi, N., Kaou, M., Stein, G.S., Lian, J.B. & Croce, C.M.
(2007) Targeted deletion of Wwox reveals a tumor suppressor function.
Proc. Natl Acad. Sci. USA, 104, 3949–3954.
Bode, A.M. & Dong, Z. (2007) The functional contrariety of JNK. Mol.
Carcinog., 46, 591–598.
Borsello, T. & Forloni, G. (2007) JNK signalling: a possible target to prevent
neurodegeneration. Curr. Pharm. Des., 13, 1875–1886.
Chang, N.S., Doherty, J. & Ensign, A. (2003a) JNK1 physically interacts with
WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-
mediated apoptosis. J. Biol. Chem., 278, 9195–9202.
Chang, N.S., Doherty, J., Ensign, A., Lewis, J., Heath, J., Schultz, L., Chen.
S.T. & Oppermann, U. (2003b) Molecular mechanisms underlying WOX1
activation during apoptotic and stress responses. Biochem. Pharmacol., 66,
Chang, N.S., Doherty, J., Ensign, A., Schultz, L., Hsu, L.J. & Hong, Q. (2005a)
WOX1 is essential for tumor necrosis factor-, UV light-, staurosporine-, and
p53-mediated cell death, and its tyrosine 33-phosphorylated form binds and
stabilizes serine 46-phosphorylated p53. J. Biol. Chem., 280, 43100–43108.
Chang, N.-S., Hsu, L.J., Lin, Y.S., Lai, F.J. & Sheu, H.M. (2007) WW domain-
containing oxidoreductase: a candidate tumor suppressor. Trends Mol. Med.,
13, 12–22.
Chang, N.S., Pratt, N., Heath, J., Schultz, L., Sleve, D., Carey, G.B. & Zevotek,
N. (2001) Hyaluronidase induction of a WW domain-containing oxidore-
ductase that enhances tumor necrosis factor cytotoxicity. J. Biol. Chem., 276,
Chang, N.S., Schultz, L., Hsu, L.J., Lewis, J., Su, M. & Sze, C.I. (2005b)
17beta-Estradiol upregulatesand
WOX2 ⁄ WWOXv2 in vitro: potential role in cancerous progression of
breast and prostate to a premetastatic state in vivo. Oncogene, 24, 714–723.
Chen, S.T., Chuang, J.I., Cheng, C.L., Hsu, L.J. & Chang, N.S. (2005) Light-
induced retinal damage involves tyrosine 33 phosphorylation, mitochondrial
and nuclear translocation of WW domain-containing oxidoreductase in vivo.
Neuroscience, 130, 397–407.
Chen, S.T., Chuang, J.I., Hong, M.H. & Li, E.I. (2002) Melatonin attenuates
MPP+-induced neurodegeneration glutathione impairment in the nigrostriatal
dopaminergic pathway. J. Pineal Res., 32, 262–269.
Chen, S.T., Chuang, J.I., Wang, J.P., Tsai, M.S., Li, H. & Chang, N.S. (2004)
Expression of WW domain-containing oxidoreductase WOX1 in the
developing murine nervous system. Neuroscience, 124, 831–839.
Chuang, J.I. & Chen, S.T. (2002) Differential expression of Bcl-2 and APP
immunoreactivity after intrastriatal injection of MPP+in the rat. Neurochem.
Int., 40, 169–179.
Chuang, J.I., Chen, S.T., Chang, Y.H. & Jen, L.S. (2001) Alteration of Bcl-2
expression in the nigrostriatal system after kainate injection with or without
melatonin co-treatment. J. Chem. Neuroanat., 21, 215–223.
De Girolamo, L.A. & Billett, E.E. (2006) Role of extracellular-regulated kinase
and c-Jun NH2-terminal kinase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-
dine-induced neurofilament phosphorylation. J. Neurosci. Res., 83, 680–693.
Degen, W.G., Pruijn, G.J., Raats, J.M. & van Venrooij, W.J. (2000) Caspase-
dependent cleavage of nucleic acids. Cell Death Differ., 7, 616–627. Review.
Driouch, K., Prydz, H., Monese, R., Johansen, H., Lidereau, R. & Frengen, E.
(2002) Alternative transcripts of the candidate tumor suppressor gene,
WWOX, are expressed at high levels in human breast tumors. Oncogene, 21,
Eberhardt, O. & Schulz, J.B. (2003) Apoptotic mechanisms and antiapoptotic
therapy in the MPTP model of Parkinson's disease. Toxicol. Lett., 139,
Gallo, K.A. & Johnson, G.L. (2002) Mixed-lineage kinase control of JNK and
p38 MAPK pathways. Nat. Rev. Mol. Cell Biol., 3, 663–672.
Glotin, A.L., Calipel, A., Brossas, J.Y., Faussat, A.M., Treton, J. & Mascarelli,
F. (2006) Sustained versus transient ERK1 ⁄ 2 signaling underlies the anti-
and proapoptotic effects of oxidative stress in human RPE cells. Invest.
Ophthalmol. Vis. Sci., 47, 4614–4623.
Hartley, A., Stone, J.M., Heron, C., Cooper, J.M. & Schapira, A.H.V. (1994)
Complex I inhibitors induce dose-dependent apoptosis in PC12 cells:
relevance to Parkinson's disease. J. Neurochem., 63, 1987–1990.
Hartmann, A., Troadec, J.D., Hunot, S., Kikly, K., Faucheux, B.A., Mouatt-
Prigent, A., Ruberg, M., Agid, Y. & Hirsch, E.C. (2001) Caspase-8 is an
Y. (1991) Parkinson's disease:pathophysiology.
activatesWOX1 ⁄ WWOXv1and
WOX1 in MPP+-induced neuronal death1645
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646
Page 13
pathway inhibition results in neuronal death. J. Neurosci., 21, 2247–2255.
Heikkila, R.E., Hess, A. & Duvoisin, R.C. (1984) Dopaminergic neurotoxicity
of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine in mice. Science, 224,
Heikkila, R.E., Nicklas, W.J., Vyas, I. & Duvoisin, R.C. (1985) Dopaminergic
toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their
stereotaxic administration to rats: implication for the mechanism of 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine toxicity. Neurosci. Lett., 62, 389–394.
Hong, Q., Hsu, L.J., Schultz, L., Pratt, N., Mattison, J. & Chang, N.S. (2007)
Zfra affects TNF-mediated cell death by interacting with death domain
protein TRADD and negatively regulates the activation of NF-kappaB,
JNK1, p53 and WOX1 during stress response. BMC Mol. Biol., 8, 50.
Hsu, L.J., Schultz, L., Mattison, J., Lin, Y.S. & Chang, N.S. (2005) Cloning and
characterization of a small size peptide Zfra that regulates the cytotoxic
function of tumor necrosis factor by interacting with JNK1. Biochem.
Biophys. Res. Commun., 327, 415–423.
Klein, J.A., Longo-Guess, C.M., Rossmann, M.P., Seburn, K.L., Hurd, R.E.,
Frankel, W.N., Bronson, R.T. & Ackerman, S.L. (2002) The harlequin mouse
mutation downregulates apoptosis-inducing factor. Nature, 419, 367–374.
Lai, F.J., Cheng, C.L., Chen, S.T., Wu, C.H., Hsu, L.J., Lee, J.Y., Chao, S.C.,
Sheen, M.C., Shen, C.L., Chang, N.-S. & Sheu, H.M. (2005) WOX1 is
essential for UVB irradiation-induced apoptosis and down-regulated via
translational blockade in UVB-induced cutaneous squamous cell carcinoma
in vivo. Clin. Cancer Res., 11, 5769–5777.
Langston, J.W. (1996) The etiology of Parkinson's disease with emphasis on
the MPTP story. Neurology, 47, S153–S160.
Lipton, S.A. & Bossy-Wetzel, E. (2002) Dueling activities of AIF in cell death
versus survival. DNA binding and redox activity. Cell, 111, 147–150.
Liu, Y. & Edwards, R.H. (1997) The role of vesicular transport proteins in
synaptic transmission and neural degeneration. Annu. Rev. Neurosci., 20,
Ludes-Meyers, J.H., Kil, H., Nunez, M.I., Conti, C.J., Parker-Thornburg, J.,
Bedford, M.T. & Aldaz, C.M. (2007) WWOX hypomorphic mice display a
higher incidence of B-cell lymphomas and develop testicular atrophy. Genes
Chromosomes Cancer, 46, 1129–1136.
kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquiti-
nation of tumor suppressor Wwox. Cancer Res., 65, 10514–10523.
Nicotra, A. & Parvez, S.H. (2002) Apoptotic molecules and MPTP-induced cell
death. Neurotoxicol. Teratol., 24, 599–605.
Offen, D., Beart, P.M., Cheung, N.S., Pascoe, C.J., Hochman, A., Gorodin, S.,
Melamed, E., Bernard, R. & Bernard, O. (1998) Transgenic mice expressing
human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and
1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity. Proc. Natl
Acad. Sci. USA, 95, 5789–5794.
Paige, A.J.W., Taylor, K.J., Taylor, C., Hillier, S.G., Farrington, S., Scott, D.,
Porteous, D.J., Smyth, J.F., Gabra, H. & Watson, J.E. (2001) WWOX: a
candidate tumor suppressor gene involved in multiple tumor types. Proc.
Natl Acad. Sci. USA, 98, 11417–11422.
Peng, J. & Anderson, J.K. (2003) The role of c-Jun N-terminal kinase (JNK) in
Parkinson's disease. IUBMB Life, 55, 267–271.
Saporito, M.S., Brown, E.M., Miller, M.S. & Carswell, S. (1999) CEP-
1347 ⁄ KT-7515, an inhibitor of c-jun N-terminal kinase activation, attenuates
the 1-methyl-4-phenyl tetrahydropyridine-mediated loss of nigrostriatal
dopaminergic neurons in vivo. J. Pharmacol. Exp. Ther., 288, 421–427.
Sbrana, I., Veroni, F., Nieri, M., Puliti, A. & Barale, R. (2006) Chromosomal
fragile sites FRA3B and FRA16D show correlated expression and associ-
ation with failure of apoptosis in lymphocytes from patients with thyroid
cancer. Gene Chromosome Cancer, 45, 429–436.
Schapira, A.H., Gu, M., Taanman, J.W., Tabrizi, S.J., Seaton, T., Cleeter, M. &
Cooper, J.M. (1998) Mitochondria in the etiology and pathogenesis of
Parkinson's disease. Ann. Neurol., 44, S89–S98.
Schober, A. (2004) Classic toxin-induced animal models of Parkinson's
disease: 6-OHDA and MPTP. Cell Tissue Res., 318, 215–224.
Smith, D.I., McAvoy, S., Zhu, Y. & Perez, D.S. (2007) Large common fragile
site genes and cancer. Semin. Cancer Biol., 17, 31–41.
Speciale, S.G. (2002) MPTP: insights into parkinsonian neurodegeneration.
Neurotoxicol. Teratol., 24, 607–620.
Swerdlow, R.H., Parks, J.K., Miller, S.W., Tuttle, J.B., Trimmer, P.A., Sheehan,
J.P., Bennett, J.P. Jr, Davis, R.E. & Parker, W.D. Jr (1996) Origin and
functional consequences of the complex I defect in Parkinson's disease. Ann.
Neurol., 40, 663–671.
Sze, C.I., Su, M., Pugazhenthi, S., Jambal, P., Hsu, L.J., Heath, J., Schultz, L. &
Chang, N.S. (2004) Down-regulation of WW domain-containing oxidore-
ductase induces Tau phosphorylation in vitro. A potential role in Alzheimer's
disease. J. Biol. Chem., 279, 30498–30506.
Tatton, N.A. & Kish, S.J. (1997) In situ detection of apoptotic nuclei in the
substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-
treated mice using terminal deoxynucleotidyl transferase labeling and
acridine orange staining. Neuroscience, 77, 1037–1048.
Tieu, K., Ischiropoulos, H. & Przedborski, S. (2003) Nitric oxide and reactive
oxygen species in Parkinson's disease. IUBMB Life, 55, 329–335.
Tournier, C., Hess, P., Yang, D.D., Xu, J., Turner, T.K., Nimnual, A., Bar-Sagi,
D., Jones, S.N., Flavell, R.A. & Davis, R.J. (2000) Requirement of JNK for
stress-induced activation of the cytochrome c-mediated death pathway.
Science, 288, 870–874.
Trimmer, P.A., Smith, T.S., Jung, A.B. & Bennett, J.P. Jr (1996) Dopamine
neurons from transgenic mice with knockout of the p53 gene resist MPTP
neurotoxicity. Neurodegeneration, 5, 233–239.
Vila, M. & Przedborski, S. (2003) Targeting programmed cell death in
neurodegenerative diseases. Nat. Rev. Neurosci., 4, 365–375.
Viswanath, V., Wu, Y., Boonplueang, R., Chen, S., Stevenson, F.F., Yantiri, F.,
Yang, L., Beal, M.F. & Andersen, J.K. (2001) Caspase-9 activation results in
downstream caspase-8 activation and bid cleavage in 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine-induced Parkinson's disease. J. Neurosci., 21,
Watanabe, A., Hippo, Y., Taniguchi, H., Iwanari, H., Yashiro, M., Hirakawa,
K., Kodama, T. & Aburatani, H. (2003) An opposing view on WWOX
protein function as a tumor suppressor. Cancer Res., 63, 8629–8633.
Xia, X.G., Harding, T., Weller, M., Bieneman, A., Uney, J.B. & Schulz, J.B.
(2001) Gene transfer of the JNK interacting protein-1 protects dopaminergic
neurons in the MPTP model of Parkinson's disease. Proc. Natl Acad. Sci.
Yamada, T., Goto, M., Punj, V., Zaborina, O., Chen, M.L., Kimbara, K.,
Majumdar, D., Cunningham, E., Das Gupta, T.K. & Chakrabarty, A.M.
(2002) Bacterial redox protein azurin, tumor suppressor protein p53, and
regression of cancer. Proc. Natl Acad. Sci. USA, 99, 14098–14103.
Yang, L., Matthew, R.T., Schulz, J.B., Klockgether, T., Liao, A.W., Martinou,
J.C., Penney, J.B. Jr, Hyman, B.T. & Beal, M.F. (1998) 1-Methyl-4-phenyl-
1,2,3,6-tetrahydropyride neurotoxicity is attenuated in mice overexpressing
Bcl-2. J. Neurosci., 18, 8145–8152.
Yendamuri, S., Kuroki, T., Trapasso, F., Henry, A.C., Dumon, K.R., Huebner,
K., Williams, N.N., Kaiser, L.R. & Croce, C.M. (2003) WW domain
containing oxidoreductase gene expression is altered in non-small cell lung
cancer. Cancer Res., 63, 878–881.
1646C.-P. Lo et al.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 1634–1646


text:
The Keurig Brewed® Logo Is The Mark Of Genuine Keurig® Quality. (Graphic: Business Wire)
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subset: commoncrawl


text:
How many apps are installed on your iPhone? [Poll]
Some of us just can't get enough of free and paid apps. If you happen to launch Settings on your iPhone, go to General, and then tap About, you can scroll down a bit and see just how many applications you have installed. I currently have 243 -- how about you?
Source: http://tuaw.com/
Software Updates
more updates- Apple Remote Desktop updated with Yosemite support
- OS X Yosemite 10.10.2, iOS 8.1.3 updates now available
- Sports Illustrated 120 SPORTS channel comes to Apple TV
- Logic Pro X update brings AirDrop support, new effects, tools, and more
- Parallels Access 2.5 released, adds file manager, computer-to-computer remote access
- The Google Translate iOS app is about to get a lot smarter


text:
Lightweight, jersey knit fabric has a slub texture for a stylish twist, forming a bloused liner with an elasticized hem. The prettiest flounce is draped on top, forming a swing silhouette with a gorgeous, crocheted lace applique atop a flirty front cut-out!
Size small measures 14" from shoulder to hem.
100% rayon / hand wash


text:
Title: | Dodge Charger R/T 440 (XS29) 1970 (Download) |
Category: | /Cars/Dodge |
Author: | Mad_Driver10 Website | Email |
Date: | 02.01.2014 | 40527 Downloads |
Filesize: | 5.062 MB |
Rating: | 9.59 based on 164 votes |
= Quality convert from GRID 2
= Qualitatively retextured interior = 2-colors support = 1 bonus paintjob + template = Quality interior lighting = Perfectly fits in SA Atmosphere = SA License Plates support = SA Colors support = Mip-mapping support = 2 extras = Fully working steering wheel, speedometer, windshield wipers, brakepads = Fully working brakelights, taillights, headlights, foglights, reverselights, turn indicators = Perfectly tailored ped_frontseat = Custom Shadow, Collision, chassis_vlo = Realistic damage and handling = Perfectly adapted for ENB Series and without him = Adapted for Active Dashboard 3.2b = Adapted for Improved Vehicle Features 2.0.2 = Adapted for High Quality Light Mod 2.0 = Adapted for Vehicles Special Abilities Editor v1.1 = Replaces: BLADE |
Oh @Mr. Raspberry Jam you should put cars(blade) id lolz..................
niceeee mod yours 10/10
Super Great Car! I always like the old classic muscles they're the best, but why the lights won't turn up I already use that VSA thing
Best Charger mod! 1970 chargers r very less here and this one is off the charts and MAD DRIVER'S mod has always made me feel great about the respect shown to these classic cars keep it up great job.
real cool 10/10
good job, good conversation, keep good works :)
very nice mod...
i can melt aspalt with this car. very nice,good job.
video http://www.youtube.com/watch?v=OzQj_UzeCt0 :)
Always love this generation of B-Body Chargers.


text:
Sponsored Links | |||
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early pinto racks
The early ones are lighter, smaller tie rods and used the flex cable connection instead of a u joint, It's impossible to tell the safety of an old rusty flex connection. Ford went to the larger size when the started using engines larger than 1.6 and 2.0 4 Cyl. There is a reason no one uses the early ones on street rods.
http://cachassisworks.com/p-1768-bil...ect-width.aspx How much you lookin to spend?Reason being Im lookin to sell this never used one.Few yrs old but hey
I dont recall the size but could find out.Im going power.
The problem with installing the Pinto/Mustang 2 rack is that the inner tie rod pivot point does not match the width of the Camaro inner tie rod pivot points. The result will be bump steer. Flaming River has what they call a ''Travel bar'' system that locates your inner tie rod ends in the original position.
Here This set up is expensive, but could be duplicated at home relatively cheap. |
drag strip only |
bump steer
On My buick powered willys you could crank the steering wheel with body lift on acceleration and the keep the wheels straight ahead. the problem is WHEN the front end comes down fast for any reason you don't have enough time to correct and Keep it straight. There is a WIKI here on using a cavilier center pivot rack and making a center traveler bar that you can drill and taper ream to get your geometry right.
The a arms and the tie rods will travel in different arcs, changing the toe as the suspension moves. Get it right and you'll have a safe and fast car. |
stock bump steer.
I don't know how good the stock suspension is on your car, You can clamp the brake rotor so it doesn't turn, fasten a laser pointer to the rotor then jack the car up to see how it points . usually you can take out the coil spring to get full suspension travel to check it out and determine where the best pivot location is
Recent Suspension - Brakes - Steering posts with photos |
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