activation and redistribution of c-jun n-terminal kinase/stress activated protein kinase in...

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 435±441 435

Journal of Neurochemistry, 2001, 76, 435±441

Activation and redistribution of c-Jun N-terminal

kinase/stress activated protein kinase in degenerating neurons

in Alzheimer's disease

Xiongwei Zhu,* Arun K. Raina,* Catherine A. Rottkamp,* Gjumrakch Aliev,* George Perry,*Heather Boux² and Mark A. Smith*

*Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

²StressGen Biotechnologies Corporation, Inc., Victoria, British Columbia, Canada

Abstract

Cellular responses to increased oxidative stress appear to be

a mechanism that contributes to the varied cytopathology of

Alzheimer's disease (AD). In this regard, we suspect that

c-Jun N-terminal kinase/Stress activated protein kinase

(JNK/SAPK), a major cellular stress response protein induced

by oxidative stress, plays an important role in Alzheimer

disease in susceptible neurons facing the dilemma of

proliferation or death. We found that JNK2/SAPK-a and

JNK3/SAPK-b were related to neuro®brillary pathology and

JNK1/SAP-Kg related to Hirano bodies in cases of AD but

were only weakly diffuse in the cytoplasm in all neurons in

control cases and in non-involved neurons in diseased brain.

In this regard, in hippocampal and cortical regions of

individuals with severe AD, the activated phospho-JNK/

SAPK was localized exclusively in association with neuro-

®brillar alterations including neuro®brillary tangles, senile

plaque neurites, neuropil threads and granulovacuolar

degeneration structures (GVD), completely overlapping with

t-positive neuro®brillary pathology, but was virtually absent in

these brain regions in younger and age-matched controls

without pathology. However, in control patients with some

pathology, as well as in mild AD cases, there was nuclear

phospho-JNK/SAPK and translocation of phospho-JNK/SAPK

from nuclei to cytoplasm, respectively, indicating that the

activation and re-distribution of JNK/SAPK correlates with the

progress of the disease. By immunoblot analysis, phospho-

JNK/SAPK is signi®cantly increased in AD over control cases.

Together, these ®ndings suggest that JNK/SAPK dysregula-

tion, probably resulting from oxidative stress, plays an

important role in the increased phosphorylation of cytoskeletal

proteins found in AD.

Keywords: Alzheimer's disease, cytoskeleton, oxidative

stress, phosphorylation, signal transduction.

J. Neurochem. (2001) 76, 435±441.

While there are a myriad of lesions in the diseased brain of

Alzheimer's disease (AD), the pathogenesis of these varied

abnormalities is poorly understood. Nonetheless, there are

multiple lines of evidence showing an association between

oxidative stress and neurodegeneration, as well as showing

that oxidative damage is one of the earliest events in the

disease (Nunomura et al. 1999). Mammalian cells respond

to extracellular stressors such as oxidative stress by

activating signaling cascades such as c-Jun N-terminal

kinase/stress-activated protein kinase (JNK/SAPK), ERK

and p38 that are mediated by members of the MAP kinase

family (Robinson and Cobb 1997; Cobb 1999). Members of

the JNK/SAPK subgroup are speci®cally activated in

response to UV irradiation, pro-in¯ammatory cytokines

and certain mitogens, as well as environmental oxidative

stress and such stimuli elicit very different types of cellular

response, ranging from cell proliferation to cell death (Su

and Karin 1996; Minden and Karin 1997), depending on the

cellular and environmental conditions as well as cooperation

with other signaling pathways such as the ERK pathway. In

Received June 12, 2000; accepted August 29, 2000.

Address correspondence and reprint requests to Dr Mark A. Smith,

Institute of Pathology, Case Western Reserve University, 2085 Adelbert

Road, Cleveland, Ohio 44106, USA. E-mail: [email protected]

Abbreviations used: AD, Alzheimer's disease; DAB, 3,3 0-diamino-

benzidine; GVD, granulovacuolar degeneration structures; JNK/SAPK,

c-Jun N-terminal kinase/stress activated protein kinase; NFT, neuro-

®brillary tangles; NGS, normal goat serum; PHF, paired helical

®laments; SDS, sodium dodecyl sulfate; TAK1, TGF-b activating

kinase 1; TBS, Tris-buffered saline.

relation to the pathogenesis of AD, it is notable that

JNK/SAPK parallels MAPK pathways such as ERK and p38

that are activated in AD (Hensley et al. 1999; Perry et al.

1999; Zhu et al. 2000) and that JNK/SAPK can phosphoryl-

ate t in vitro (Goedert et al. 1997; Reynolds et al. 1997).

In this study, we investigated the status of the major

isoforms of JNK/SAPK, the ubiquitously expressed JNK1/

SAPK-g and JNK2/SAPK-a and the neuronal isoforms,

JNK3/SAPK-b (Su and Karin 1996), in AD. We found a

pronounced re-distribution of JNK/SAPK isoforms in AD

compared to age-matched controls and, most notably, the

phosphorylation of JNK/SAPK is markedly increased in AD

and is closely associated with degenerating neurons. Taken

together, these data indicate a role of the JNK/SAPK

pathway in disease pathogenesis.

Materials and methods

Brain tissue

Hippocampal, cortical and cerebellar brain tissue obtained

postmortem was either frozen for immunoblot analysis or ®xed in

methacarn (methanol : chloroform : acetic acid, 6 : 3 : 1), embedded

in paraf®n and 6-mm thick consecutive sections were prepared on

silane-coated (Sigma, St Louis, MO, USA) slides for immunocyto-

chemistry. The following cases were used in this study: AD (n � 31;

ages � 64±85; postmortem interval � 1±23 h); young and age-

matched control (n � 24; ages � 7±81; postmortem interval �3±22 h). All cases were categorized based on clinical and

pathological criteria established by CERAD and an NIA consensus

panel (Khachaturian 1985; Mirra et al. 1991). Agonal status and

cause of death were obtained, where available, from postmortem

reports.

Immunocytochemical procedures

Immunocytochemistry was performed by the peroxidase anti-

peroxidase protocol essentially as described previously (Stern-

berger 1986; Nunomura et al. 1999). Brie¯y, following immersion

in xylene, hydration through graded ethanol solutions and

elimination of endogenous peroxidase activity by incubation in

3% hydrogen peroxide for 30 min, sections were incubated for

30 min at room temperature in 10% normal goat serum (NGS) in

Tris-buffered saline (TBS; 50 mm Tris-HCl, 150 mm NaCl,

pH 7.6) to reduce non-speci®c binding. After rinsing brie¯y with

1% NGS/TBS, the sections were sequentially incubated overnight

at 48C with either (i) immunoaf®nity puri®ed rabbit polyclonal

antibody to JNK2/SAPK-a (1 : 200), JNK3/SAPK-b (1 : 200) or

JNK1/SAPK-g (1 : 200) (StressGen Biotechnologies Corporation,

Inc., Victoria, BC, Canada); or (ii) immunoaf®nity puri®ed rabbit

polyclonal antibody to phospho-JNK/SAPK (1 : 300) (New

England Biolabs, Inc., Beverly, MA, USA), which only recognizes

isoforms of JNK/SAPK activated by dual phosphorylation at

Thr180 and Tyr182; or (iii) mouse monoclonal Alz50 (1 : 100) or

AT8 antibody (1 : 2000) to phosphorylated cytoskeletal t protein.

The sections were then incubated in either goat antirabbit (ICN,

Costa Mesa, CA, USA) or goat antimouse (ICN) antisera (1 : 50),

followed by species-speci®c peroxidase antiperoxidase complex

(1 : 250) (Sternberger Monoclonals Inc. and ICN, Cappel).

3,3 0-Diaminobenzidine (DAB) was used as chromagen. For some

experiments, sections were double-labeled with two different

antibodies in which case rabbit antisera were localized using the

peroxidase antiperoxidase method with DAB as the chromogen and

mouse monoclonal antibodies were localized using the alkaline

phosphatase antialkaline phosphatase method using Fast Blue as the

chromogen as previously described (Perry et al. 1999).

Absorption experiments were performed to verify the speci®city

of antibody binding. Brie¯y, the immunostaining protocol was

repeated using absorbed antibody produced by an overnight

incubation of primary antibody with puri®ed JNK2/SAPK-a,

JNK3/SAPK-b or JNK1/SAPK-g peptide (100 mg/mL) at 48C

(StressGen Biotechnologies Corporation, Inc.) or puri®ed phospho-

JNK/SAPK peptide (100 mg/mL) (New England Biolabs). In

parallel, to control against artifactual absorption, we also performed

absorption of JNK/SAPK speci®c antibodies with irrelevant peptide

(TGF-b activating kinase 1 (TAK1) peptide or Ras peptide

(100 mg/mL) (StressGen Biotechnologies Corporation, Inc.) and

absorption of irrelevant antibody [i.e. TAK1 antibody (1 : 200),

phospho-ERK antibody (1 : 200) and phospho-p38 antibody

(1 : 200)] with phospho-JNK/SAPK immunizing peptide

(100 mg/mL) (New England Biolabs).

To determine the speci®city of the phosphorylation-dependent

antisera, some sections were treated with 2 U alkaline phosphatase

(Type III; Sigma) in 100 mL Tris pH � 8.0 and 0.01 m PMSF at

room temperature for variable times from 1 to 72 h prior to

incubation in primary antibody.

Immunoblotting

Tissues from gray matter of temporal cortex of AD (n � 13) and

control cases (n � 16) were homogenized in 10 vol. of TBS

containing 0.02% sodium azide, 0.5% sodium deoxycholate, 0.1%

sodium dodecyl sulfate (SDS), 1% NP-40, 1 mm PMSF, 1 mg/mL

aprotinin and 1 mg/mL antipain. Proteins were separated by

SDS±polyacrylamide gel electrophoresis (10 mg/lane) and electro-

blotted onto Immobilon-p (Millipore, Bedford, MA, USA) by

standard procedures as previously described (Zhu et al. 2000).

Transferred blots were incubated sequentially with blocking agent

(10% non fat milk in TBS-Tween), rabbit anti-JNK1/SAPK-g

(1 : 200), JNK2/SAPK-a (1 : 200), JNK3/SAPK-b (1 : 200) or

phospho-JNK/SAPK antibody (1 : 500) and af®nity-puri®ed goat

antirabbit immunoglobulin peroxidase conjugate preabsorbed to

eliminate human cross-reactivity (1 : 1500) (StressGen Biotechnol-

ogies Corporation, Inc.). Blots were developed by the ECL

technique (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,

USA) according to the manufacturer's instruction. Blots were

striped in stripping buffer (2% SDS, 62.5 mm Tris-HCl, 100 mm

b-mercaptoethanol, pH 6.8) for 30 min at 608C and then probed

with antibody against actin (1 : 1000), which is constitutively

expressed in neuronal cells. Quanti®cation of the results was

performed using a computer-assisted scanning system (PDI,

Huntington Station, NY, USA). The data obtained were expressed

as optical densities and analyzed statistically using one-way

analysis of variance.

Results

The major JNK/SAPK isoforms (JNK1/SAPK-g, JNK2/

SAPK-a and JNK3/SAPK-b) showed speci®c alterations in

436 X. Zhu et al.

q 2001 InteNGS, normal goat serum; rnational Society for Neurochemistry, Journal of Neurochemistry, 76, 435±441

hippocampal diseased tissue in comparison with control.

Both JNK2/SAPK-a (Figs 1a and c) and JNK3/SAPK-b

(results not shown) were related to neuro®brillary pathology

in AD whereas JNK1/SAPK-g was exclusively associated

with Hirano bodies (not shown). In marked contrast, JNK1/

SAPK-g, JNK2/SAPK-a and JNK3/SAPK-b staining were

diffuse and much weaker in the cytoplasm of noninvolved

neurons in AD and all neurons in young and age-matched

controls (Fig. 1b). The speci®city of these antibodies was

demonstrated by signi®cant diminution of signal by

absorption with immunizing peptide (Fig. 1d) with no effect

using irrelevant peptide (result not shown). Immunoblot

analysis further demonstrates the speci®city of the reagents

used and also demonstrates that whereas JNK1/SAPK-g and

JNK2/SAPK-a are increased in total brain homogenate in

AD compared to control cases, JNK3/SAPK-b does not

show increased global levels of protein (Fig. 2).

To demonstrate the activation state of JNK/SAPK, an

antibody against phospho-JNK/SAPK was used and, in

clearly de®ned severe AD cases, the immunoreactivity of

phospho-JNK/SAPK was exclusively associated with neuro-

®brillary tangles, senile plaque neurites, neuropil threads

and granulovacuolar degeneration structures, i.e. the classic

neuro®brillary pathology of AD brain (Fig. 3d), paralleling

our ®nding with selective JNK/SAPK reagents. In fact, there

was almost a complete overlap between phospho-JNK/

SAPK and phospho-t recognized by AT8 (Fig. 4) or Alz50

(not shown). However, in mild cases of disease (Fig. 3c),

phospho-JNK/SAPK, in addition to neuro®brillary pathol-

ogy, also localized to neuronal nuclei in areas with little

pathology but not to similar nuclei in regions with intense

neuro®brillary pathology. Similarly, in control cases without

any pathology (Fig. 3a), neuronal or nuclei phospho-JNK/

SAPK was undetected whereas in those control cases with

limited pathology, nuclei were stained (Fig. 3b). In the

cortex, an area that is less affected by AD, even in the most

severe AD cases, nuclei in different neurons, as well as

neuro®brillary tangles (NFT), were stained (result not

shown). Importantly, in the cerebellum, an area that is

exempt from AD pathology, there was no staining for

Fig. 1 While barely detected in control

brain (b), there is prominent JNK2/SAPK±a

in association with neurons containing

neuro®brillary pathology in hippocampal

sections (CA1 area) from an AD patient (a).

Such neuronal staining in the hippocampus

with JNK2/SAPK-a antibody in AD (c) is

almost completely abolished by absorption

with immunizing peptide (d). Asterisks indi-

cate landmark blood vessels in adjacent

sections. Scale bars: a, b, c, d � 100 mm.

Fig. 2 Immunoblots of cortical gray matter from AD (AD) and con-

trol (C) patients probed with antisera against JNK1/SAPK-g (a),

JNK2/SAPK-a (b) and JNK3/SAPK-b (c) show bands at the

expected molecular weights of 46 and 54 kDa. Parallel gel stained

with actin antibody shows that the loading amount is similar.

JNK/SAPK abnormalities in AD 437

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 435±441

phospho-JNK/SAPK in both AD and control cases (result

not shown). To eliminate the possibility that protein could

selectively leach from our sections, we also examined and

immunocytochemically con®rmed the presence of other

soluble proteins such as PDI and cdk7 (Kim et al. 2000; Zhu

et al. 2000b). In all cases, and for all three brain areas

examined, there was no relationship between agonal status,

cause of death or postmortem interval and phospho-JNK/

SAPK staining.

To demonstrate the speci®city of phospho-JNK/SAPK

detection, several control experiments were performed in

parallel. Absorption of the phospho-JNK/SAPK antibody

with the immunizing peptide of phospho-JNK/SAPK essen-

tially abolished immunostaining (Fig. 5) whereas no effect

Fig. 3 Immunocytochemical localization of

phospho-JNK/SAPK in hippocampal neu-

rons in CA1 area in control (a and b) and

AD patients (c and d). Phospho-JNK/SAPK

is not present in most younger controls and

those controls without pathology (a),

whereas in elderly controls with some

pathology (b), nuclear staining is a promi-

nent feature. In areas with low levels of

pathology in mild AD cases (c), phospho-

JNK/SAPK is present in both nuclei (arrow-

head) and cytoplasm (arrow), whereas in

severe AD cases (d), only cytoplasmic neu-

ro®brillary pathology is evident with little or

Fig. 4 Adjacent serial sections of the CA1

area of hippocampus of a case of AD

immunostained with antiphospho-JNK/

SAPK (a and c) and AT8 (b and d) with

landmark vessel (*). (a and b) show an

area of neuro®brillary tangles and (c and d)

show an area of senile plaques. Most of the

same neuro®brillary tangles (arrowhead)

and senile plaques are labeled by both

phospho-JNK/SAPK and AT8. Scale bar:

100 mm.

438 X. Zhu et al.


was observed by the absorption of (i) the antibody to

phospho-ERK and phospho-p38 with phospho-JNK/SAPK

peptide; or (ii) the antibody to phospho-JNK/SAPK with

JNK3/SAPK-b or Ras peptide (results not shown). To

exclude the possibility that the phospho-JNK/SAPK anti-

body was cross-reacting with t protein, adsorption with t

did not abolish immunoreactivity and immunoblot analysis

using soluble t protein puri®ed from AD and normal brain

did not react with our phospho-JNK/SAPK reagents (not

shown). Finally, to demonstrate the speci®city of this

reagent to the phospho-active form of JNK/SAPK, we

pretreated sections with alkaline phosphatase to remove

endogenous phosphate groups and found that whereas NFT

and senile plaques staining were easily lost after 1 h

treatment with alkaline phosphatase (result not shown),

staining of granulovacuolar degeneration structure was more

stable and can exist, although fainter, even after 3 days

treatment (result not shown). A similar phenomenon was

reported for phospho-ERK and phospho-p38 (Perry et al.

1999; Zhu et al. 2000) and may be related to differential

phosphorylation levels or conformational accessibility of the

phosphate groups.

The speci®city of the phospho-JNK/SAPK antibody is

also demonstrated by immunoblot analysis of cortical brain

homogenates. Immunoblot analysis revealed two major

antiphospho-JNK/SAPK immunoreactive bands with

approximate molecular weights of 54 000 and 46 000 in

the AD and controls. However, the bands were much weaker

in the control brain homogenates (Fig. 6a), consistent with

our tissue-based ®ndings showing an upregulation of

phospho-JNK/SAPK in AD. The statistical analysis of the

quanti®cation result, normalized to actin, shows an approxi-

mate 14-fold increase in total phospho-JNK/SAPK in AD

compared to control cases (p � 0.02) (Fig. 6b).

Fig. 5 Neuronal staining in the hippo-

campus with phospho-JNK/SAPK antibody

in AD (a) is abolished completely by

absorption with immunizing peptide (b).

Asterisk indicates landmark vessel in

adjacent sections. Scale bar: 100 mm.

Fig. 6 (a) Representative results of immunoblots of cortical gray

matter from AD (AD) and control (C) patients probed with antisera

against phospho-JNK/SAPK show bands at the expected molecular

weights of 46 and 54 kDa. The same membrane striped and

reprobed with actin was shown as loading control. (b) Quanti®cation,

which is normalized by actin blot, of phospho-JNK/SAPK immuno-

blots shows a great increase of phospho-JNK/SAPK intensity in AD

(p � 0.02). Quanti®cation is based on total of 13 AD and 16 control

cases and result is shown as Ave ^ SEM.



Discussion

In this study, we demonstrate for the ®rst time the activation

and an altered distribution pattern of JNK/SAPK isoforms in

susceptible neurons in AD compared to younger and age-

matched controls. In AD, pronounced JNK2/SAPK-a and

JNK3/SAPK-b is seen in association with neuro®brillary

pathology. Most notably, the active phospho-JNK/SAPK is

greatly upregulated in AD and shows near complete overlap

with phosphorylated t marking neuro®brillary alterations.

These data not only indicate that almost every pathological

lesion contains increased phospho-JNK/SAPK, but also that

the elevation and phosphorylation of JNK/SAPK is a very

proximal event in AD pathogenesis. Indeed, in vitro, JNK/

SAPK is capable of phosphorylating t protein in a manner

similar to the phosphorylation of PHF-t (Reynolds et al.

2000). While the possibility that differences in phosphatase

activity between AD and control cases accounts for these

alterations in phospho-JNK/SAPK (cannot be ruled out), we

found no signi®cant effect of postmortem interval. Addi-

tionally, it is important to note that phosphorylation of

JNK/SAPK does not necessarily re¯ect catalytic activity.

Nonetheless, the complete overlap of phospho-JNK/SAPK

with phospho-t and the capability of JNK/SAPK to

phosphorylate t in vitro indicates that the greatly upregu-

lated phospho-JNK/SAPK shown in this study is present in

an activated state and therefore may be involved in the

phosphorylation of t in vivo. Moreover, it is interesting to

note that JNK/SAPK can phosphorylate neuro®lament

(Giasson and Mushynski 1996; Julien and Mushynski

1998) and in this context, it is also notable that JNK1/

SAPK-g localizes to Hirano bodies in AD and that

cytoskeletal reorganization is a cardinal feature of AD

(Perry et al. 1985).

JNK1/SAPK-g, JNK2/SAPK-a and JNK3/SAPK-b show

robust increases and association with neuro®brillary pathol-

ogy and yet only the increase of JNK1/SAPK-g and JNK2/

SAPK-a were detected by immunoblotting. In fact, previous

reports show that JNK3/SAPK-b is highly expressed in almost

all regions of brain in mouse and in the human central nervous

system, while JNK1/SAPK-g is expressed at modest level and

JNK2/SAPK-a is expressed at a low level in mouse but not

detected in human (Kumagae et al. 1999; Lee et al. 1999).

Therefore, it is likely that the relatively high levels of

endogenous neuronal JNK3/SAPK-b `masks' the select

induction in pyramidal diseased neurons when seen in the

context of a brain homogenate. However, perhaps more

importantly, these aspects indicate that JNK3/SAPK-b plays a

role in normal brain physiology whereas JNK2/SAPK-a is

selectively involved in stressed/diseased condition.

Neuronal phospho-JNK/SAPK is not seen in young

controls but is associated with neuro®brillary alterations

found in some elderly controls. Therefore, phospho-JNK/

SAPK may re¯ect a chronic and accumulative stress process

during aging that appears to be an extremely early event in

AD pathology. Normally the inactive JNK/SAPK resides

quiescently in the cytosol and, once activated, translocates

into the nucleus and activates transcription factors such as

c-Jun and ATF-2 (Su and Karin 1996). Therefore, it is

perhaps surprising to ®nd that in severe AD cases, phospho-

JNK/SAPK immunoreactivity is exclusively associated with

cytoplasmic neuro®brillary pathology, but not with nuclei.

Whereas in areas with less pathology in mild cases of AD,

nuclear staining is very prominent. In fact, in these areas the

redistribution of phospho-JNK/SAPK from nucleus to cyto-

plasm can be well appreciated, namely, from nuclear staining

in normal-looking neurons to both nuclear and cytoplasmic

staining neurons to only cytoplasmic staining in pathology-

containing neurons. Such a pattern of re-distribution can also

be appreciated in the AD cortex. This close association of the

re-distribution of phospho-JNK/SAPK and the accumulation

of abnormally phosphorylated t in susceptible neurons in AD

suggests that the activation of JNK/SAPK is an extremely

early event and that the re-distribution of JNK/SAPK after

activation coincides with the formation of neuro®brillary

pathology, i.e. rather than being involved in phosphorylating

physiological targets, JNK/SAPK plays a pathogenic role by

phosphorylating t. Additionally, given that the JNK/SAPK

pathway may play some role in mitogenic signaling (Robinson

and Cobb 1997; Smith et al. 1997; Bost et al. 1999) and its

activation is required in mediating oncogenic ras or SV40

small tumor antigen transformation in some cells (Watanabe

et al. 1996; Xiao and Lang 2000), we suspect along with the

cell cycle-related abnormalities found in susceptible neurons

in AD (Raina et al. 1999), that the activation of JNK/SAPK

may facilitate the re-entry of cell cycle in these susceptible

neurons (Raina et al. 1999, 2000).

The phosphorylation of JNK/SAPK is a response to

cellular stress including oxidative stress. The role of

oxidative stress is well established with damage to proteins

and lipids (Smith et al. 1996; Sayre et al. 1997), as well as

the induction of speci®c antioxidant systems (Pappolla et al.

1992; Smith et al. 1994). One of the major ®ndings of these

studies is that oxidative damage is not limited to the

pathology of AD but rather uniformly involves members of

entire populations of neurons at risk of death in AD

(Nunomura et al. 1999). This abnormal oxidative damage,

as a very early event in AD, may activate the JNK/SAPK in

these neurons, including the apparently normal-looking

neurons, which may account for the observation that nuclear

staining is present in almost all the susceptible neurons in

elderly controls with some pathology and those mild AD

cases. Although the activation of JNK/SAPK indicates an

effort by neurons, in the face of oxidative stress, to

induce protective mechanisms, the ultimate consequence

may vary, depending on the environmental conditions

and the coordination/regulation of other signaling

molecules.

440 X. Zhu et al.


In conclusion, the colocalization of phospho-JNK/SAPK

and phospho-t and the close association of phospho-JNK/

SAPK redistribution with the progression of AD demon-

strated here indicate a key role for JNK/SAPK in the

pathogenesis of AD. The consequences of such activation

and redistribution of JNK/SAPK certainly merits future

study.

Acknowledgements

This work was supported through funding from the National

Institutes of Health (NS3868) and the Alzheimer's Association

(IIRG-98±136 and ZEN-99±1789).

References

Bost F., McKay R., Bost M., Potapova O., Dean N. M. and Mercola D.

(1999) The Jun kinase 2 isoform is preferentially required for

epidermal growth factor-induced transformation of human A549

lung carcinoma cells. Mol. Cell Biol. 19, 1938±1949.

Cobb M. H. (1999) MAP kinase pathways. Prog. Biophys. Mol. Biol.

71, 479±500.

Giasson B. I. and Mushynski W. E. (1996) Aberrant stress-induced

phosphorylation of perikaryal neuro®laments. J. Biol. Chem. 271,

30404±30409.

Goedert M., Hasegawa M., Jakes R., Lawler S., Cuenda A. and Cohen

P. (1997) Phosphorylation of microtubule-associated protein tau

by stress-activated protein kinases. FEBS Lett. 409, 57±62.

Hensley K., Floyd R. A., Zheng N.-Y., Nael R., Robinson K. A.,

Nguyen X., Pye Q. N., Stewart C. A., Geddes J., Markesbery W.

R., Patel E., Johnson G. V. W. and Bing G. (1999) p38 kinase is

activated in the Alzheimer's disease brain. J. Neurochem. 72,

2053±2058.

Julien J. P. and Mushynski W. E. (1998) Neuro®laments in health and

disease. Prog. Nucl. Acid Res. Mol. Biol. 61, 1±23.

Khachaturian Z. S. (1985) Diagnosis of Alzheimer's disease. Arch.

Neurol. 42, 1097±1105.

Kim H. T., Russell R. L., Raina A. K., Petersen R. B., Smith M. A. and

Perry G. (2000) Protein disul®de isomerase in Alzheimer's

disease. Antiox. Redox Signal. 2, 485±489.

Kumagae Y., Zhang Y., Kim O. J. and Miller C. A. (1999) Human c-Jun

N-terminal kinase expression and activation in the nervous

system. Brain Res. Mol. Brain Res. 67, 10±17.

Lee J. K., Park J., Lee Y. D., Lee S. H. and Han P. L. (1999) Distinct

localization of SAPK isoforms in neurons of adult mouse brain

implies signaling modes of SAPK pathways. Brain Res. Mol.

Brain Res. 70, 116±124.

Minden A. and Karin M. (1997) Regulation and function of the JNK

subgroup of MAP kinases. Biochim. Biophys. Acta 1333, F85±F104.

Mirra S. S., Heyman A., McKeel D., Sumi S. M., Crain B. J., Brownlee

L. M., Vogel F. S., Hughes J. P., van Belle G. and Berg L. (1991)

The Consortium to Establish a Registry for Alzheimer's Disease

(CERAD). Part II. Standardization of the neuropathologic

assessment of Alzheimer's disease. Neurology 41, 479±486.

Nunomura A., Perry G., Pappolla M. A., Wade R., Hirai K., Chiba S.

and Smith M. A. (1999) RNA oxidation is a prominent feature

of vulnerable neurons in Alzheimer's disease. J. Neurosci. 19,

1959±1964.

Pappolla M. A., Omar R. A., Kim K. S. and Robakis N. K. (1992)

Immunohistochemical evidence of oxidative stress in Alzheimer's

disease. Am. J. Pathol. 140, 621±628.

Perry G., Rizzuto N., Autilio-Gambetti L. and Gambetti P. (1985)

Paired helical ®laments from Alzheimer disease patients contain

cytoskeletal components. Proc. Natl Acad. Sci. USA 82, 3916±3920.

Perry G., Roder H., Nunomura A., Takeda A., Friedlich A. L., Zhu X.,

Raina A. K., Holbrook N., Siedlak S. L., Harris P. L. R. and Smith

M. A. (1999) Activation of neuronal extracellular receptor kinase

(ERK) in Alzheimer disease links oxidative stress to abnormal

phosphorylation. Neuroreport 10, 2411±2415.

Raina A. K., Monteiro M. J., McShea A. and Smith M. A. (1999) The

role of cell cycle-mediated events in Alzheimer's disease. Int. J.

Exp. Pathol. 80, 71±76.

Raina A. K., Zhu X., Rottkamp C. A., Monteiro M., Takeda A. and

Smith M. A. (2000) Cyclin 0 toward dementia: cell cycle abnor-

malities and abortive oncogenesis in Alzheimer disease. J. Neurosci.

Res. 61, 128±133.

Reynolds C. H., Utton M. A., Gibb G. M., Yates A. and Anderton B. H.

(1997) Stress-activated protein kinase/c-Jun N-terminal kinase

phosphorylates t protein. J. Neurochem. 68, 1736±1744.

Reynolds C. H., Betts J. C., Blackstock W. P., Nebreda A. R. and

Anderton B. H. (2000) Phosphorylation sites on tau by nano-

electrospray mass spectrometry: differences in vitro between the

mitogen-activated protein kinase ERK2, c-Jun N-terminal kinase

and p38, and glycogen synthase kinase-3b. J. Neurochem. 74,

1587±1595.

Robinson M. J. and Cobb M. H. (1997) Mitogen-activated protein

kinase pathways. Curr. Opin. Cell Biol. 9, 180±186.

Sayre L. M., Zelasko D. A., Harris P. L. R., Perry G., Salomon R. G.

and Smith M. A. (1997) 4-Hydroxynonenal-derived advanced

lipid peroxidation end products are increased in Alzheimer's

disease. J. Neurochem. 68, 2092±2097.

Smith M. A., Taneda S., Richey P. L., Miyata S., Yan S.-D., Stern D.,

Sayre L. M., Monnier V. M. and Perry G. (1994) Advanced

Maillard reaction end products are associated with Alzheimer

disease pathology. Proc. Natl Acad. Sci. USA 91, 5710±5714.

Smith M. A., Perry G., Richey P. L., Sayre L. M., Anderson V. E., Beal

M. F. and Kowall N. (1996) Oxidative damage in Alzheimer's.

Nature 382, 120±121.

Smith A., Ramos-Morales F., Ashworth A. and Collins M. (1997) A

role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-

dependent cells. Curr. Biol. 7, 893±896.

Sternberger L. A. (1986). Immunocytochemistry, 3rd edn. Wiley, New

York.

Su B. and Karin M. (1996) Mitogen-activated protein kinase cascades

and regulation of gene expression. Curr. Opin. Immunol. 8,

402±411.

Watanabe G., Howe A., Lee R. J., Albanese C., Shu I. W., Karnezis A.

N., Zon L., Kyriakis J., Rundell K. and Pestell R. G. (1996)

Induction of cyclin D1 by simian virus 40 small tumor antigen.

Proc. Natl Acad. Sci. USA 93, 12861±12866.

Xiao L. and Lang W. (2000) A dominant role for the c-Jun

NH2-terminal kinase in oncogenic ras-induced morphologic

transformation of human lung carcinoma cells. Cancer Res. 60,

400±408.

Zhu X., Raina A. K., Boux H., Takeda A., Perry G. and Smith M. A.

(2000a) Activation of p38 pathway links tau phosphorylation,

oxidative stress and cell cycle related events in Alzheimer disease.

J. Neuropathol. Exp. Neurol. 59, 880±888.

Zhu X., Rottkamp C. A., Raina A. K., Brewer G. J., Boux H. and Smith

M. A. (2000b) Cdk7 expression in hippocampal neurons is related

to aging and Alzheimer's disease. Neurobiol. Aging 21, in press.



activation and redistribution of c-jun n-terminal kinase/stress activated protein kinase in...

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