jkk1, an upstream activator of jnk/sapk, is activated in alzheimer's disease

JKK1, an upstream activator of JNK/SAPK, is activated

in Alzheimer’s disease

Xiongwei Zhu,* Osamu Ogawa,* Yang Wang,� George Perry* and Mark A. Smith*

*Institute of Pathology and �Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA

Abstract

JNK/SAPK has been implicated in the pathogenesis of Alz-

heimer’s disease, but the upstream cascade leading to JNK/

SAPK activation has not been elucidated in the disease. In

this study, we focused on one of the physiological activators of

JNK/SAPK, JNK kinase 1 (JKK1). Although there was no

significant difference in the level and distribution of total JKK1

between Alzheimer’s disease (AD) and age-matched control

cases, increased levels of activated phospho-JKK1 were

specifically localized to neurofibrillary pathology including

neurofibrillary tangles, senile plaque neurites, granulovaualar

degenerations and neuropil threads in severe AD (Braak

stage V–VI), considerably overlapping with its downstream

effector, phospho-JNK/SAPK, suggesting both a functional

and mechanistic link. Nuclear localization of phospho-JKK1

was also found in mild (Braak stage III–IV) but not in severe

AD cases (Braak stage V–VI), suggesting a possible

re-distribution correlating with the progress of the disease.

By immunoblot analyses, phospho-JKK1 was significantly

increased in AD over control cases. Together, these findings

lend further credence to the notion that the JNK/SAPK path-

way is dysregulated in AD and also indicate an active role for

this pathway in disease pathogenesis.

Keywords: Alzheimer’s disease, JKK1, JNK/SAPK, oxidative

stress.

J. Neurochem. (2003) 85, 87–93.

There are now multiple lines of evidence showing an

association between oxidative stress and Alzheimer’s disease

(AD) (Perry et al. 2000a,b). Since oxidative damage appears

as one of the earliest neuronal abnormalities in the disease

process (Nunomura et al. 1999, 2000), we speculate that the

consequence of oxidative stress would result in a patholo-

gical cascade that leads to the varied cytopathology of AD. In

this regard, c-Jun N-terminal kinase/stress-activated protein

kinase (JNK/SAPK) is one of the major signaling cascades

that are specifically activated in response to oxidative stress

(Mielke and Herdegen 2000). The activation of JNK/SAPK

leads to various consequences ranging from cell proliferation

to cell death (Mielke and Herdegen 2000), depending on the

cellular and environmental conditions as well as co-operation

with other signaling pathways. Since susceptible neurons in

AD face the dilemma of proliferation or death (Zhu et al.

1999; Raina et al. 2000), it appears that JNK/SAPK may

play a pivotal role. Importantly, as relates to the pathogenesis

of AD, activated JNK/SAPK can phosphorylate s protein ina manner similar to that found in AD (Goedert et al. 1997;

Reynolds et al. 1997).

Recently, we and others showed the involvement of

downstream players of the JNK/SAPK pathway in the

pathogenesis of AD (Shoji et al. 2000; Zhu et al. 2001a,d).

In this study, to determine involvement of the entire JNK/

SAPK pathway, we investigated the status of the upstream

physiological activator MAPKK, also termed JNK kinase 1

(JKK1). JKK1 is a dual-specificity kinase that phosphory-

lates threonine and tyrosine in the activation loop of JNK/

SAPK and serves to activate JNK/SAPK. JKK1 mRNA is

widely expressed in human tissue, but is especially abundant

in skeletal muscle and brain (Sanchez et al. 1994; Derijard

et al. 1995). Our results show that activated JKK1 is

increased and localized to neurofibrillary pathology in AD.

Importantly, and indicating a mechanistic link, activated

JKK1 shows considerable overlap with phosphorylated

Resubmitted manuscript received December 2, 2002; accepted

December 2, 2002.

Address correspondence and reprint requests to Dr Xiongwei Zhu,

Institute of Pathology, Case Western Reserve University, 2085 Adelbert

Road, Cleveland, Ohio 44106, USA. E-mail: [email protected]; or 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¢-diam-inobenzidine; ERK, extracellular signal regulated kinase; JNK/SAPK,

c-Jun N-terminal kinase/stress-activated protein kinase; NGS, normal

goat serum; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline.

Journal of Neurochemistry, 2003, 85, 87–93 doi:10.1046/j.1471-4159.2003.01645.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 87–93 87

JNK/SAPK. Taken together, these data indicate the activation

of the entire JNK/SAPK pathway. Now, it will be of great

importance to determine the role(s) that the JNK/SAPK

pathway plays in AD. Because of the bipartite-natured

response of the activation of this pathway, which can lead to

either neurodegeneration or neuroprotection, we suspect that

the activation of the JNK/SAPK pathway initially provokes a

protective response by mobilizing antioxidant defenses, but it

eventually serves as a death cascade when oxidative insults

exceed cellular defense capabilities.

Materials and methods

Brain tissue

Brain tissue from the hippocampal and cortical regions was obtained

postmortem andfixed inmethacarn (methanol/chloroform/acetic acid;

6 : 3 : 1) and embedded into paraffin. Six-micrometer thick consecu-

tive sections were prepared on silane-coated slides (Sigma, St. Louis,

MI, USA) for immunocytochemistry. The tissue used in this studywas

obtained from mild (Braak stage III–IV) and severe AD cases (Braak

stage V–VI) [n ¼ 20; ages ¼ 59–91, mean ¼ 73.3 ± 10.1 (± SEM);

postmortem interval ¼ 3–24 h, mean ¼ 9 ± 6.6 h (± SEM)] and

control [n ¼ 14; ages ¼ 54–91, mean ¼ 69 ± 10.5 (± SEM); post-

mortem interval ¼ 3–25 HR, mean ¼ 7.4 ± 2 h (± SEM)] patients

from the CWRU Brain Bank, as assessed on clinical and pathological

criteria established by CERAD and an NIA consensus panel

(Khachaturian 1985; Braak and Braak 1991; Mirra et al. 1991).

Immunocytochemical procedures

Immunocytochemistry was performed by the peroxidase antiper-

oxidase protocol essentially as described previously (Smith et al.

1994). Briefly, following immersion in xylene and hydrated though

graded ethanol solutions, endogenous peroxidase activity was

eliminated by incubation of the sections in 3% hydrogen peroxidase

for 30 min. To reduce non-specific binding, 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).

After rinsing briefly with 1% NGS, sections were incubated

overnight in one of several primary antibodies including (i) immu-

noaffinity purified rabbit polyclonal antibody to amino acids 347–

363 of human MAP kinase kinase (JKK1) 1 : 100; StressGen

Biotechnologies Corporation, Inc., Victoria, BC, Canada; (ii) immu-

noaffinity purified rabbit polyclonal antibody to JKK1 only when

phosphorylated at Thr261 (1 : 100; Cell signaling Tech., Beverly,

MA, USA); (iii) immunoaffinity purified rabbit polyclonal antibody

to JNK/SPAK dually phosphorylated at Thr183/Tyr185 (1 : 200;

Cell signaling Tech.); or (iv) mouse monoclonal AT8 antibody to

cytoskeletal s protein phosphorylated at serine 202 and threonine

205 (1 : 2000). The sections were then incubated in either goat anti-

rabbit or goat anti-mouse antisera (ICN, Costa Mesa, CA, USA),

followed by species-specific peroxidase antiperoxidase complex

(Sternberger Monoclonals inc. and ICN, Cappel). Antibodies were

localized using 3,3¢-diaminobenzidine (DAB) as a chromogen

(Dako Corporation, Carpinteria, CA, USA).

Absorption experiments were performed to ensure the specificity

of the reactivities observed. In short, the immunostaining techniques

were repeated using absorbed antibody that was produced by

incubation of the primary antibody with its purified immunizing

peptide at 50 lg/mL. In parallel, each antibody was either absorbedwith an irrelevant peptide, either p38 or Ras peptide (StressGen

Biotechnologies Corporation, Inc., Victoria, BC, Canada) and c-Jun

peptide phosphorylated at Ser73 [phospho-c-Jun (73)] (Cell

Signaling Tech., Beverly, MA, USA), or the immunizing peptides

were incubated with an irrelevant antibody (either p38 antibody;

StressGen Biotechnologies Corporation, Inc.; or phospho-c-Jun (73)

antibody; Cell Signaling Tech.) to control against artifactual absorption.

Immunoblotting

Tissue from the gray matter of temporal cortex of AD (n ¼ 6)

and control cases (n ¼ 5) were homogenized in 10 vol. of lysis

buffer [50 mM Tris-HCl (pH 7.6), 0.02% sodium azide, 0.5%

sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS),

1% NP-40, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride,

1 lg/mL aprotinin 1 lg/mL antipain and 1 mM sodium ortho-

vanadate]. Proteins were separated by sodium dodecyl sulfate

polyacrylamide gel electrophoresis (40 lg/lane) and electroblottedonto Immobilon-P (Millipore, Bedford, MA, USA) by standard

procedures as previously described (Zhu et al. 2001b). Trans-

ferred blots were incubated sequentially with blocking agent (10%

non-fat milk in TBS-Tween), rabbit anti-JKK1 (1 : 200; Stress-

Gen Biotechnologies Corporation, Inc.) or anti-phospho-JKK1

antibody (1 : 200; Cell Signaling Tech.) and affinity-purified goat

anti-rabbit immunoglobulin peroxidase conjugate preabsorbed to

eliminate human cross-reactivity. Blots were developed by the

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

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

stripped in stripping buffer (2% SDS, 62.5 mM Tris-HCl, 100 mM

2-mercaptoethanol, pH 6.8) for 30 min at 60�C and then probed

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

constitutively expressed in neuronal cells. Quantification of the

results was performed using a computer-assisted scanning system

(PDI, Huntington Station, NY, USA). The data obtained were

expressed as optical densities. Results are reported as mean

values ± SEM. The significance of differences between different

groups was assessed by two-tailed Student’s t-test with p < 0.05

considered statistically significant.

Results

The JKK1 phosphorylation state was assessed in brain tissue

from well characterized AD and age-matched control cases

(Fig. 1). Immunocytochemical analysis of phospho-JKK1

was performed using an affinity purified rabbit polyclonal

antibody directed specifically against the phosphorylated

JKK1 epitope. The antiphospho-JKK1 antibody recognized

neurofibrillary tangles, neuritic senile plaques, granulovaua-

lar degeneration and neuropil threads, i.e. the classic features

of the neuropathology of AD brain, in the hippocampus from

19 out of 20 AD cases used in this study (Figs 1a,e and 2b).

The localization of phospho-JKK1 to neurofibrillary pathol-

ogy was confirmed by its considerable colocalization with

phospho-s as stained by AT8 in AD case (Fig. 2). In marked

88 X. Zhu et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 87–93

contrast, immunoreactivity of phospho-JKK1 can barely

be detected in 11 out of 14 age-matched controls (Fig. 1d).

As sometimes found in normal aging, a few pyramidal

neurons contained neurofibrillary tangles in the other three

age-matched controls and they also displayed granular

immunostaining of phospho-JKK1 (results not shown).

Similar to phospho-JNK/SAPK, in addition to neurofibrillary

pathology staining, phospho-JKK1 was also found in nuclei

in pyramidal neurons in mild AD cases, especially in CA4

area (Fig. 1c), but not in severe AD cases (results not

shown). To confirm the specificity of phospho-JKK1

immunocytochemistry, several control experiments were

performed in parallel. Absorption of the phospho-JKK1

antibody with the immunizing peptide of phospho-JKK1

almost completely abolished immunostaining (Figs 1e and f)

whereas no effect was observed by the absorption of the (i)

antibody to c-Jun phosphorylated at Ser73 [phospho-c-

Jun(73)] with phospho-JKK1 peptide; (ii) antibody to

phospho-JKK1 with Ras peptide or phospho-c-Jun(73)

peptide (results not shown). Additionally, in the cerebellum,

an area that is relatively unaffected by AD, there was only

minimal background immunostaining and no difference in

the staining pattern between AD and normal cases. As an

additional control, total JKK1, i.e. phosphorylated and non-

phosphorylated forms, showed a specific neuronal staining,

as previously reported (Lee et al. 1999), however, there was

no significant difference between AD and age-matched

controls (data not shown). In all cases, there was no

(a) (b) (d)

(c)

(e) (f)

Fig. 1 Immunocytochemical localization of phospho-JKK1 in hippo-

campal neurons in AD patients (a, b and c) and control cases (d).

Phospho-JKK1 is not present in most age-matched controls (d), but is

seen localized to neurofibrillary tangles and senile plaque neurites

(a) and neuropil threads and granulovacular degeneration (b) in AD

cases. Nuclear staining (c) is readily seen in mild but not in severe AD

cases. These pictures are representatives of 20 AD and 14 control

cases. Neuronal staining in the hippocampus with phospho-JKK1

antibody in AD (e) is abolished completely by absorption with

immunizing peptide (f); *indicates landmark vessel in adjacent

sections. Scale bar for (a)–(f) ¼ 100 lm.

JKK1 in Alzheimer’s disease 89


relationship between agonal status or postmortem interval

and phospho-JKK1 immunoreactivity. Since JKK1 is the

upstream activator of JNK/SAPK kinase and JNK/SAPK

kinase is activated in AD (Shoji et al. 2000; Zhu et al.

2001a,d), we compared the distribution of phospho-JKK1

and phospho-JNK/SAPK and found that they completely

overlap with each other in AD cases (Fig. 3).

Immunoblot analyses of phospho-JKK1 using homogenates

from temporal cortical gray matter of AD and control cases

(Fig. 4) revealed a band at the approximate molecular weight

of 46 kDa in AD brain that was much weaker in control brain.

These data not only verified the specificity of the antibody but

also demonstrated the increased phosphorylation of JKK1 in

AD. Statistical analysis of the data, normalized with the level

of the constantly expressed protein actin, demonstrated a

significant (p ¼ 0.007) increase in the level of the phospho-

JKK1 in AD in comparison to control brain (Fig. 4b). JKK1

antibody also recognized one band at approximately 46 kDa in

both AD and control (Fig. 4a) but no significant difference

(p ¼ 0.80) was found between the two groups (Fig. 4c),

consistent with the immunocytochemical results.

Discussion

This study is focused on the JNK/SAPK pathway, a major

stress activated signal transduction pathway present ubiqui-

tously in mammalian tissue and involved in stimulating cell

defense as well as cell death induced by mitogens or cellular

and environmental stress. Previously, we and others had

Fig. 2 Adjacent serial sections of the CA1 area hippocampus of an

AD case immunostained with AT8 (a) and phospho-JKK1 (b) with

*landmark blood vessel. Most of the same neurofibrillary pathologies

are labeled by both AT8 and phospho-JKK1. The experiments were

repeated three times with different AD cases and similar results were

obtained. Scale bars for (a) and (b) ¼ 50 lm.

Fig. 3 Adjacent serial sections of the CA1 area hippocampus of an

AD case immunostained with antiphospho-JKK1 (a) and its down-

stream kinase, antiphospho-JNK/SAPK (b) *with landmark blood

vessel. Most of the same neurofibrillary tangles are labeled by

both phospho-JKK1 and phospho-JNK/SAPK. The experiments were

repeated three times with different AD cases and similar results were

obtained. Scale bars for (a) and (b) ¼ 100 lm.

90 X. Zhu et al.


shown that JNK/SAPK is activated in AD (Shoji et al. 2000;

Zhu et al. 2001a,d). The far upstream activator, Cdc42/Rac is

also altered (Zhu et al. 2000a) and the downstream effector,

c-Jun is also associated with neurofibrillary pathology and

colocalize with DNA damage in AD (Anderson et al. 1994;

Anderson et al. 1996). In this study, we show, for the first

time, the activation of JKK1, the upstream activator of JNK/

SAPK, in susceptible neurons in AD compared to age-

matched controls. The phospho-JKK1 is seen localized to

neurofibrillary tangles, senile plaque neurites, granulovaualar

degeneration and neuropil threads. It is reported that a portion

of JKK1 is present in nuclei (Lee et al. 1999) and our results

confirmed the nuclear localization of phospho-JKK1, especi-

ally in pyramidal neurons in CA4 area, in mild AD cases.

However, in severe AD cases, nuclear localization was barely

detected, indicating a possible re-distribution correlating with

the progress of the disease which is similar to that is seen for

phospho-JNK/SAPK (Zhu et al. 2001a). Importantly, from a

functional and mechanistic view, we found considerable

overlap between phospho-JKK1 and phospho-JNK/SAPK in

susceptible neurons in severe AD. These data provide a key

missing element for the upstream event leading to JNK/SAPK

activation in AD and give further credence that the entire

JNK/SAPK pathway is abnormally activated in AD.

Due to the limitation always inherent in postmortem

studies, the current data do not allow us to unambiguously

define the nature of the JKK1 dysregulation. The observed

abnormalities could be due to an abnormally activated JKK1,

a disturbance of phosphatase activity in vivo, a disturbance of

JKK1 dephosphorylation postmortem, or a combination of

the above. In spite of these caveats, it is interesting to note

that phospho-activation of JKK1 can be initiated by diverse

stress stimuli including oxidative stress. Since oxidative

stress is one of the most prominent features in AD as

evidenced by the extensive damage to proteins, lipids and

nucleic acids (Perry et al. 2000a,b), and is one of the earliest

events in disease pathogenesis (Nunomura et al. 1999,

2000), we suspect that the activation of JKK1/JNK/c-Jun

pathway in AD is a response to oxidative stress. This notion

is supported by our recent finding that JNK is strongly

activated in AbPP transgenic mice with extensive iron

accumulation and oxidative damage but not in AbPPtransgenic mice with little iron accumulation and oxidative

damage (Smith et al. 1998; Zhu et al. 2001c). While the

prominent increase in neuronal oxidative damage of AD

would seem to mark an unstable state leading to cell death,

instead, the actual cell death at any given time is rare (Perry

et al. 1998, 2001). This aspect may be due to the protection

provided by extensive compensatory mechanisms partly

mediated by JNK/SAPK activation such as induction of

specific antioxidant enzymes for example heme oxygenase 1

(HO-1) and specific heat shock proteins (Pappolla et al.

1992; Smith et al. 1994; Premkumar et al. 1995). Indeed,

while the activation of the JKK1/JNK/c-Jun pathway was

initially implicated in neuronal death following stress, it is

now apparent that JNK/SAPK can also affect protective

responses depending on the cellular and environmental

conditions as well as cooperation with other cell signaling

pathways. For example, dietary chemopreventive compounds

at low concentrations activate the JNK/SAPK pathway and

lead to the induction of phase II and other defensive genes

such as HO-1 and enhanced cell survival while those

Fig. 4 (a) Representative results of immunoblots of temporal cortical

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

against phospho-JKK1 and JKK1 show bands at the expected

molecular weights of 46 kDa. The same membrane stripped and

reprobed with actin was shown as loading control. (b) Quantification,

which is normalized by actin blot, of phospho-JKK1 immunoblots

shows an increase of phosphorylation of JKK1 in AD (*p ¼ 0.007). (c)

Quantification, which is normalized by actin blot, of JKK1 immunoblots

shows no change of JKK1 level in AD compared to control (p ¼ 0.80).

Quantification is based on total of six AD and five control cases and

result is shown as average ± SEM.



compounds at higher levels actually kill cells (Owuor and

Kong 2002). Oxidative stress may exert a similar effect on

the JNK/SAPK pathway in AD, i.e. at levels that are

tolerable to neuronal cells, oxidative stress may induce

antioxidant defense such as HO-1 and heat shock proteins via

the activation of the JNK/SAPK pathway (Oguro et al. 1998;

Tekin et al. 2001; Zhang et al. 2002). That vulnerable

neurons show activation of the JNK/SAPK pathway, as well

as extracellular signal regulated kinase (ERK) and p38

pathways (Hensley et al. 1999; Perry et al. 1999; Zhu et al.

2000b, 2001b; Ferrer et al. 2001), denotes a survival response

associated with the phosphorylation of tau that is consistent

with the protracted nature of AD where neuronal death is an

inconsistent feature (Perry et al. 1998, 2001). In this regard,

one might consider the pleotrophic nature of pathological

changes in AD as a display of neuronal oxidant defenses.

Acknowledgements

Sources of Support: NIH (NS38648) and Alzheimer’s Association

(ZEN-99–1789, IIRG-00–2163, NIRG-02–3923).

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jkk1, an upstream activator of jnk/sapk, is activated in alzheimer's disease

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