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Oncogene (2001) 20, 2424 ± 2437
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
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AP-1 proteins in the adult brain: facts and ®ction about e€ectors of
neuroprotection and neurodegeneration
Thomas Herdegen*,1 and Vicki Waetzig1
1
Institute of Pharmacology, Hospitalstrasse 4, 24105 Kiel, Germany
Jun and Fos proteins are induced and activated following
most physiological and pathophysiological stimuli in the
brain. Only few data allow conclusions about distinct
functions of AP-1 proteins in neurodegeneration and
neuroregeneration, and these functions mainly refer to cJun and its activation by JNKs. Apoptotic functions of
activated c-Jun a€ect hippocampal, nigral and primary
cultured neurons following excitotoxic stimulation and
destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs
might exert neuroprotection by subsequent omission of cJun activation. Besides endogenous neuronal functions,
the c-Jun/AP-1 proteins can damage the nervous system
by upregulation of harmful programs in non-neuronal
cells (e.g. microglia) with release of neurodegenerative
molecules. In contrast, the di€erentiation with neurite
extension and maturation of neural cells in vitro indicate
physiological and potentially neuroprotective functions of
c-Jun and JNKs including sensoring for alterations in the
cytoskeleton. This review summarizes the multiple
molecular interfunctions which are involved in the shift
from the physiological role to degenerative e€ects of the
Jun/JNK-axis such as cell type-speci®c expression and
intracellular localization of sca€old proteins and upstream activators, antagonistic phosphatases, interaction
with other kinase systems, or the activation of
transcription factors competing for binding to JNK
proteins and AP-1 DNA elements. Oncogene (2001) 20,
2424 ± 2437.
Keywords: Fos; JNK; JunD
Introduction
The pool of data on the true functions of AP-1
proteins in brain physiology, regeneration and degeneration is limited, in spite of innumerable publications
reporting on the expression of AP-1 mRNAs and
proteins in the nervous system and neural cells under
physiological and pathological conditions. This insight
is complicated by the capacity of AP-1 proteins to
exert functionally antagonizing functions. This chapter
critically reviews ®ndings on the involvement of AP-1
proteins in degenerative and regenerative processes in
*Correspondence: T Herdegen
the adult nervous system, and develops scenarios for
the functional transition of AP-1 proteins from
physiological-neuroprotective players to degenerative
e€ectors. One issue deserves particular attention: In
this review, the functions of c-Jun are predominantly
set into the context of the c-Jun N-terminal kinases
(JNK) which are considered as regulators of c-Jun
activity. Recent data, however, are accumulating that
c-Jun and even nuclear JNKs can exert independentdissociating functions. Thus, the interplay within the cJun/JNK-axis might undergo substantial revision in the
future.
General considerations of AP-1 proteins in the brain
Aspects of transcriptional functions
Knock-out of the c-jun gene is lethal for the
developing embryo (Hilberg et al., 1993; Jochum et
al., this issue), whereas inhibition of the N-terminal
phosphorylation by exchange of the serine residues 63
and 73 for alanin (c-JunAA) does not interfere with
the development of an apparently intact and fertile
phenotype (Behrens et al., 1999). Consequently,
functional inactivation of c-Jun with preservation of
its physical presence substantially di€ers from functional inactivation by physical absence: in contrast to
the knock-out of c-jun, c-JunAA can still act as
suppressor and antagonizing inhibitor of other transcription factors (TFs). Thus, for understanding AP-1
functions, we have to consider their elaborate
transcriptional functions (see also Chinenov and
Kerppola, in this issue). As with the majority of
TFs, AP-1 proteins function in several ways:
(a) Induction of transcription. Regulated by their posttranslational modi®cations (mainly by phosphorylation), their partners for the formation of homoand/or heterodimers and sca€old proteins, AP-1
proteins bind to regulatory DNA sequences (AP-1
and ATF/CRE-like consensus motifs) and activate
the basal transcription machinery (Ferrell, 2000;
reviewed in detail by Herdegen and Leah, 1998;
see also M Yaniv, in this issue). In the context of
the transcriptional orchestration, the AP-1 proteins co-determine the time and the amount of
gene transcription.
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
(b) Suppression of transcription. Transcriptional suppression can be achieved by transcription factor
binding to regulatory DNA promoter sequences
without activating of RNA-polymerase II complex. For the distinction of the transcriptional
state (activating versus non-activating), it is
mandatory to visualize the speci®c post-translational modi®cations which underly the activation
of the RNA-polymerase II by individual TFs.
Thus, the mere presence of nuclear TF/AP-1
proteins does not allow conclusions about the
transcriptional state of TFs.
(c) Modulation of transcription beyond the DNApolymerase interface. AP-1 proteins can associate
with other TFs such as steroidhormone-complexes,
and this association prevents the DNA binding of
both partners (see chapters by P Herrlich, A Rao
and H van Dam in this issue).
(d) Non-transcriptional e€ects. The presence of mRNA
and/or proteins of TFs including AP-1 proteins in
neurons outside the nucleus suggest functions of
TFs beyond transcriptional control. For example,
Fra-1 and the 55 kDa c-Fos isoform are present in
synaptosomes and synaptic plasma membrane
fractions from rat cerebral cortex and hippocampus, from where they might be retransported into
the nucleus (Paratcha et al., 2000). FosB immunoreactivities were found in dendrites following
kainic acid-induced seizures (Gass et al., 1993)
and in the nucleolus following axotomy (Robinson, 1995). ATF-2, which might be relevant for cJun mediated transcription (Herdegen et al., 1997),
is transported along the axon-target axis apparently serving as an information shuttle between
perikaryon and axonal-synaptic events.
Temporospatial expression in the brain
The AP-1 proteins substantially di€er in their temporospatial expression patterns. The analysis of their
distribution alleviates the understanding of their
putative functions since the combinatory variety is
limited by the individual expression. In theory, more
than 20 dimers can be formed by heterodimerization of
Fos and Jun proteins or homodimerization of Jun
proteins without considering the splice variants; this
number is furthermore enhanced when we regard the
heterodimers with non-AP-1 proteins (summarized in
Herdegen and Leah, 1998). It may be helpful to
roughly characterize the inducibility of the AP-1
proteins in the brain and their role in brain
pathophysiology (for details see Herdegen and Leah,
1998).
c-Jun Basal expression is detected in various compartments including dentate gyrus, motoneurons, peripheral neurons and nuclei of the autonomic nervous
system. Following transynaptic stimulation, c-Jun
shows a similar induction pattern as c-Fos and JunB.
Stimulation by non-synaptic insults (i.e. transection of
nerve ®bers) selectively induces c-Jun and, to a lesser
extent also JunD, but not other AP-1 proteins
(Herdegen et al., 1997). This selective injury-triggered
induction might be mediated through cis-activation of
the distal AP-1 sequence of the c-jun promoter by cJun (Mielke et al., 1999).
2425
JunB Almost absent under basal conditions, this Jun
protein is mainly induced in neurons in a pattern and
half-life very similar to the properties of c-Fos
(Edelstein et al., 2000; Gass et al., 1993; Hata et al.,
2000). Following injury, induction of JunB and c-Fos
might primarily be mediated by transynaptic neuronal
excitation; whether the inducibility of the junB
promoter by IL-6 and CNTF (Co€er et al., 1995) is
relevant for neuronal JunB expression, remains to be
elucidated.
JunD Expressed in neurons and glial cells, JunD
displays a wide basal expression in the majority of cells
in the nervous system. Following transynaptic and
non-synaptic stimulation, JunD expression is enhanced
in a delayed but lasting fashion. The pattern of JunD
expression following non-synaptic degenerative stimulation resembles that of c-Jun. Phosphorylation of
JunD by JNKs, however, is only achieved following
heterodimerization with c-Jun (Kallunki et al., 1996).
c-Fos Due to its fast expression, c-Fos becomes
detectable within 30 ± 35 min following the onset of
an experimental stimulus from rather low or nondetectable basal expression. Its expression is rather
transient, at least following termination of the primary
inducing stimulus (Purkiss et al., 1993). In case of
parallel induction of JunB, the c-Fos proteins might
prefer this Jun isoform to c-Jun. The inducibility of cFos declines with progressing age (Salehi et al., 1999)
and this observation supports the notion that c-Fos
expression widely re¯ects the cellular responsiveness at
the transcriptional level.
FosB The evident characteristic of FosB expression is
its widespread absence in the brain under basal
conditions and its delayed but protracted expression
following intentional stimulation (Gass et al., 1993). In
contrast to other AP-1 proteins, FosB immunoreactivity can display speci®c labeling of the nucleolus
raising the question about its dimerization partner
(Robinson, 1995). Importantly, splicing of the fosB
gene yields truncated DFosB proteins with a comparatively persistent half-life which can confer robust
inhibition of AP-1 dependent transcription (Chen et
al., 1997). Knockout of the fosB gene dramatically
a€ects the maternal behavior towards newborn pups,
and FosB is involved in further physiological functions
such as the circadian rhythm and the imprint of the
addiction memory (Chen et al., 1997; Herdegen and
Leah, 1998).
Fra1, Fra2 The understanding of these AP-1 proteins
is still handicapped by the insucient characterization
of the generation and structure of the proteins, and
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c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2426
there is only recent evidence that Fra isoforms derive
from the fosB gene (Chen et al., 1997). mRNA analysis
demonstrated a lasting expression of these AP-1
members after transient stimuli indicating a particular
role for memory formation. This notion is supported by
presynaptic and dendritic localization of Fra-IR which
changes following behavioral stimuli (Paratcha et al.,
2000).
Counteracting effects of AP-1 proteins in different
populations of CNS cells
Besides neurons, a variety of distinct cell populations
mediate physiological functions and trigger pathological processes in the brain, such as microglia and
astrocytes, oligodendrocytes and Schwann cells, ependymal cells and meningeal cells. It is important to
realize that depending on the cell type similar
functions of TFs might result in functionally opposite
e€ects. Three examples will illustrate this critical issue.
(i) Neurons can be killed by apoptotic programs
which include AP-1 activity (Behrens et al., 1999), but
in parallel, apoptosis is also involved in the
termination of microglial activity resembling the
processes in immune cells (Raivich et al., 1999). In
consequence, inhibition of apoptosis ± one of the
central aims of novel strategies for the treatment of
neurodegenerative diseases ± could also counteract
the termination of microglial activity with prodegenerative e€ects such as persistent phagocytosis
and immune reactions. (ii) AP-1 proteins are essential
for cell cycle progression and are also involved in the
di€erentiation of cells (Hilberg et al., 1993; Leppa and
Bohmann, 1999). In consequence, expression of AP-1
proteins in non-neuronal cells such as astrocytes and
meningeal cells in vivo might trigger fundamental
physiological responses to injury which are inevitably
blocked by AP-1 antagonization. (iii) An important
group of AP-1 target genes comprises the matrix
metalloproteinases which are involved in in¯ammatory
processes as well as in structural guidance depending
on the expressing cell type (Xu et al., 2001).
Inhibition of AP-1 expression (e.g. by antisenseoligonucleotides) negatively a€ects memory formation
(Grimm et al., 1997) and physiological-adaptive
processes such as synchronization of the endogenous
clock (Wollnik et al., 1995). It remains a major
challenge to develop strategies for AP-1 inhibition
which selectively block degenerative functions without
a€ecting physiological functions. Thus, null mutation
of c-fos attenuates phenotypes of pathological activity
such as kindling development as well as physiological
functions such as reorganization of mossy ®bers
(Watanabe et al., 1996).
The functional inhibition of AP-1 proteins (e.g. by
antisense- or decoy-oligonucleotides, inhibition of their
upstream activatory kinases) as a strategy for the
treatment of neurodegenerative disorders demands the
careful analysis of the physiological action of AP-1
proteins for the estimation of harmful side e€ects.
Moreover, this analysis is the premise for answering
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the question to which extent neurodegenerative e€ects
of AP-1 are pathologically enhanced physiological
functions or due to qualitatively di€erent properties
such as binding of novel partners and the subsequent
transcription of novel target genes.
Induction by degenerative stimuli: complex multifactorial
responses
Degenerative processes evoked by one de®ned experimental stimulus, represent a mixture of primary and/
or secondary reactions with speci®c but sometimes
overlapping programs triggered by di€erential cascades of signal-transcription-coupling. Ischemia, for
example, is ®rst of all an abrupt break of
oxygenation and energy delivery; after some time,
energy depletion, breakdown of membrane functions
and rise of intracellular calcium occurs in parallel
with transparency of the blood-brain-barrier, activation of microglia and release of cytokines from
multiple cellular sources (reviewed by Lipton, 1999).
These processes run in parallel and/or subsequently
and can reinforce and/or antagonize each other.
Therefore, it is dicult to precisely attribute transcriptional functions to de®ned pathological processes
or to identify those cellular responses triggered by
individual transcription factors. Finally, as it is true
for the majority of cellular responses, TF-triggered
responses might coincidentally feed into both,
adaptive-protective responses and unbalanced prodegenerative triggers.
Only few experimental paradigms can be considered
as somewhat monofactorial such as axotomy (i.e.
mechanical transection of nerve ®bers) and excitotoxic
stimulation e.g. systemic application of kainic acid. In
these cases, the primary insult ± i.e. axon transection
and transmembranous excitation ± determine the
neuronal response. In contrast, the delayed nonneuronal reaction such as in¯ammation, phagocytosis
or scar formation are the consequence of neuronal
degeneration, i.e. they do not substantially contribute
to the primary neuronal response. Nevertheless, even
the quasi `monofactorial' axotomy can be split into
individual pathophysiological processes such as omission of target-derived trophic factors, structural
damage of the axon and activation of Schwann cells
leading to a release of various cytokines and
chemokines.
The relation of AP-1 proteins and MAP kinases and
their physiological functions
For the analysis of AP-1 functions, it is mandatory
to understand the upstream system of kinases which
is essential for the induction and/or activation of the
transcription factors. The AP-1 dimers are part of
the transcriptional e€ector system of the ERK, JNK
and p38 MAP kinases, i.e. AP-1 proteins ful®ll
(some of) their functions in the context of MAP
kinases. As with other kinase systems, the MAP
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
kinase integrate intra- and extracellular signals and
reinforce or antagonize the (parallel) information
transfer of other signal-transduction pathways (Ichijo,
1999; see also Rincon et al., this issue). Therefore,
the understanding of the e€ector pro®le of MAP
kinases provides crucial insight into the function of
AP-1 proteins. We have to emphasize, however, that
AP-1 proteins can modulate gene transcription
independent of preceding MAP-kinase activation
and, vice versa, MAP kinases have a wide range of
action by post-translational modi®cation of nonnuclear substrates.
2427
The c-Jun/JNK-axis
It is accepted knowledge that the transcriptional action
of c-Jun strictly depends on its phosphorylation of the
serine 63 and 73 residues (Gupta et al., 1996; Kallunki
et al., 1996). Consequently, the study of JNKs in the
brain enlightens the contextual understanding of their
nuclear substrates c-Jun and (in c-Jun heterodimers)
JunD (Kallunki et al., 1996, Figure 1).
However, data are also available on calciumtriggered c-Jun activation independent of both, the
phosphorylation of N-terminal serines 63/73 and the
Figure 1 Modulation of the c-Jun/JNK-axis. This graphic gives modulators of the expression and phosphorylation of c-Jun which
are relevant in the mammalian nervous system. The molecules framed by grey boxes and interconnected by dotted lines form
sca€old complexes. The red molecules o€er a putative experimental and therapeutic approach for JNK/c-Jun inhibition
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c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2428
activation of JNKs (Cruzalegui et al., 1999; Rizzo et
al., 1999), and in PC12 cells, phosphorylation of the cJun serine residues 63 and 73 was found to be
regulated by ERKs (Leppa et al., 1998). Besides these
residues, other amino acids might be relevant for c-Jun
activation since the exchange of threonine residues at
position 91 and 93 to phosphate-mimicking aspartic
acid residues enhances the `gain of function' of the
serines 63 and 73 (Musti et al., 1997). The presence of
JNK-interacting proteins (JIPs), positive as well as
negative regulators of JNK-mediated c-Jun phosphorylation (Dickens et al., 1997) can channel JNK
signaling away from c-Jun as shown in cerebellar
granule cells where an increase in nuclear JNK activity
parallels a decrease in c-Jun phosphorylation and
expression (Co€ey et al., 2000). Finally, primary
hippocampal/cortical neurons from c-JunAA mutants
are protected against kainic acid-induced apoptosis
inspite of unaltered JNK activity (Behrens et al., 1999).
Physiological functions
In the adult mammalian brain, the three JNK genes are
transcribed and are most likely present in their
activated form (Carletti et al., 1995; Martin et al.,
1996; Mielke et al., 2000; Schauwecker, 2000; Yang et
al., 1997) under normal conditions, i.e. during the
absence of any intentional stimulation. One prominent
feature of the c-Jun/JNK-axis is their involvement in
neuronal plasticity and memory formation: (i) JNKs
are largely residing in neurites in vitro (Co€ey et al.,
2000) and are retrogradely transported in neuronal
axons in vivo (Fernyhough et al., 1999), i.e. they act as
signal molecules between the presynaptic terminal and
the perikaryon; (ii) JNKs alter cytoskeletal functions
by transcriptional regulation and phosphorylation of
neuro®laments, tau proteins and microtubulins (Reynolds et al., 2000; Sadot et al., 1998), and inversely (iii)
by inhibition of glucocorticoid receptor transcriptional
activity, c-Jun and JNKs are e€ectors of speci®c cell
programs initiated by depolymerization of the cytoskeleton after con¯uency (Oren et al., 1999), and similarly,
c-Jun and JNKs are activated by microtubular disarray
(Wang et al., 1998; Yujiri et al., 1999); on the other
hand, con¯uency of PC12 cells drastically inhibited cJun phosphorylation by JNKs (Lallemand et al., 1998).
Taken together, the c-Jun/JNK-axis participates in the
sensoring for cytoskeletal alterations; (iv) JNK3
phosphorylates the threonine at position 668 of the
Alzheimer's disease amyloid precursor protein (APP)
(Standen et al., 2001) with physiological functions such
as e€ects on neuronal di€erentiation; (v) JNKs receive
transmembranous signals in microdomains (McDonald
et al., 2000); and ®nally, (vi) JNKs initiate long-lasting
genetic alterations by activation of TFs such as c-Jun,
JunD or ATF-2 (Gupta et al., 1996; Kallunki et al.,
1996). This plethora of properties renders JNKs as
perfect candidate molecules for memory formation. In
fact, visual stimulation, exposure to a novel environment and generation of electro-convulsive seizures (an
experimental paradigm for neuronal plasticity) acti-
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vates JNK with N-terminal phosphorylation of c-Jun
in the rodent brain (Brecht et al., 1999; Xu et al.,
1997). Interestingly, repetition of electro-convulsive
seizures dramatically reduces their e€ectiveness for cJun phosphorylation in the presence of ongoing JNK
activation (Brecht et al., 1999). This habituation of the
transcriptional processes indicates the presence of
regulatory mechanisms for JNK-triggered c-Jun activation which are activated following lasting and/or
repetitive stimulation underlying neuronal plasticity.
It is important in this context that the glutamate
receptor subunit 6 (GluR6, a member of the kainate
receptor family) mediates seizure activity and spontaneous bursts (Telfeian et al., 2000) with reduced
sensitivity to kainic acid in mice with GluR6 knockout
(Mulle et al., 1998). GluR6 transmission is coupled
with JNK activation via PSD-95/SAP90 (a membraneassociated guanylate kinase), MLK2 and JIP (Sheng
and Pak, 2000; Yasuda et al., 1999) Therefore,
inhibition of JNKs or c-Jun might dissociate the
essential axis between synaptic excitation and gene
induction with resistance to seizure activity as seen
following JNK3 knockout and c-JunAA knock in, but
with the potential risk of inhibiting learning and
memory formation.
Besides the adult nervous system, the c-Jun/JNKaxis has important functions for the maturation and
di€erentiation of neurons in vivo and in vitro. In
cerebellar neurons, nuclear JNK activity from MKK7
complexes increases during neuritogenesis (in parallel
with reduced c-Jun activity) (Co€ey et al., 2000); in
PC12 cells, c-Jun and JNK contribute to neurite
outgrowth (Kita et al., 1998; Leppa et al., 1998).
During postnatal development, the three JNK isoforms
decline at various degrees in the brain (Carboni et al.,
1998), and these high perinatal levels indicate a role of
JNKs during developmental neuroplasticity.
Variable activition of c-Jun and JNKs:
questionable reliability
The expression and inducibility of individual JNK
proteins, and in particular of their splice variants,
however, under normal conditions and following
patho-physiological stimuli are not yet resolved. This
is true for in vivo and in vitro systems (Table 1). The
data are also di€ering on the intracellular distribution
of JNKs as well as on the N-terminal phosphorylation
of c-Jun:
(i)
There are studies reporting a weak and limited
distribution of JNK1 in the brain which is also
absent in the hippocampus (Carboni et al., 1998)
while others describe a high JNK1-IR level which
parallels that of JNK3 (Lee et al., 1999) and a
substantial basal JNK1 activity in the hippocampus (Mielke et al., 1999). Inversely, information on
strong and widely distributed JNK2 mRNA
signals (Carboni et al., 1998) is in contrast to
publications of low level JNK2-IR (Lee et al.,
1999).
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2429
Table 1 Expression and intracellular distribution of members of the JNK-MAP-cascade
Isoform
Stimulus/disease
Area/cell type
JIP-1
MKK4
MCA occlusion
MPTP
Cortex (rat)
Striatum (mouse)
:
=
:
MKK4
MPTP
Substantia nigra (mouse)
=
:
MKK4
Anisomycin,
serum deprivation
Postnatal development
Striatal excitotoxic
lesion
MCA occlusion
Cerebellar granule
neurons (rat)
Forebrain, cerebellum (rat)
Substantia nigra (rat)
Total JNK
Anisomycin,
serum deprivation
Postnatal development
Cerebellar granule
neurons (rat)
Forebrain, cerebellum (rat)
Total JNK
Total JNK
Total JNK
MPTP
MPTP
ALS
Striatum (mouse)
Substantia nigra (mouse)
Spinal cord (rat)
=
=
Total JNK
Adriamycin
Trigeminal ganglion
neurons (rat)
Differentiated PC12 cells
PC12 cells
=
MKK7
Total JNK
Total JNK
Total JNK
JNK1
JNK1
Cortex (rat)
NGF deprivation
Neuritogenese (by
protease inhibition
JNK1
Postnatal development Brain (rat)
JNK1
Embryonic development CNS, PNS (mouse)
JNK1
Transient hypoxia
Fetal forebrain,
(JNK3-activity)
cultured cells (rat)
JNK1
JNK1
JNK1
Ionizing radiation
Kainic acid
Kainic acid
Brain homogenates (rat)
Brain homogenates (rat)
Hippocampus (mouse)
JNK1
JNK2
Phosphorylated
JNK1+2
JNK3
JNK3
JNK3
Sodium arsenite
Sodium arsenite
Transient global
ischemia
Hypoxia
Postnatal development
Transient hypoxia
Cortical neurons
Cortical neurons
Hippocampus (gerbil)
JNK3
Sodium arsenite
Hippocampus (human)
Brain (rat)
Fetal forebrain
cultured cells (rat)
Cortical neurons
Expression
Activity+Localisation
:
:
: (JNK1)
;
=
:
Reference
Cytoplasm
Cytoplasm
(only neurons)
Cytoplasm
(only neurons)
Nucleus
Hayashi et al., 2000
Saporito et al., 2000
Nucleus
Nucleus
Coffey et al., 2000
Oo et al., 1999
Saporito et al., 2000
Coffey et al., 2000
:
Cytoplasm,
Hayashi et al., 2000
nucleus
:
Nucleus (associated Coffey et al., 2000
with MKK4)
:
Nucleus (associated Coffey et al., 2000
with MKK7)
:
Saporito et al., 2000
:
Saporito et al., 2000
: (strong in
Migheli et al., 1997
astrocytes)
:
Increase in
Pena et al., 2000
the nucleus
:
Eilers et al., 1998
:
Giasson et al., 1999
;
:
Inhibited
:
during
hypoxia
:
;
: in
Slight : in
susceptible resistent
cells
cells
=
=
=
=
=
:
Nucleus
Carboni et al., 1998
Martin et al., 1996
Chihab et al., 1998
Nucleus
:
Zhang et al., 1998
Carboni et al., 1998
Chihab et al., 1998
:
Namgung and Xia, 2000
;
=
Ferrer et al., 1997
Ferrer et al., 1997
Schauwecker, 2000
Namgung and Xia, 2000
Namgung and Xia, 2000
Sugino et al., 2000
Overview of the expression and the activation of JNKs and their upstream regulators in the brain and in neuronal cells in vitro. : gives an
increase, ; gives a decrease of expression and/or activity; = marks no signi®cant alteration
(ii) The intranuclear presence of JNKs under basal
conditions and the nuclear translocation of JNKs
following stress (with subsequent phosphorylation
of c-Jun and/or JunD) are further issues of
controversial observations: Under basal conditions, JNK3-IR was detected in the cytoplasm
(Martin et al., 1996), whereas JNK1-IR was found
in the cytoplasm and the nucleus (Schauwecker,
2000) or in the cytoplasm and the dendrites (Lee et
al., 1999) or only in the nucleus (Oo et al., 1999).
Visualization of phosphorylated JNK1+JNK2
immunoreactivity (p-JNK-IR) yielded a complete
absence in untreated gerbils, and a nuclear p-JNKIR following ischemia in the hippocampus (Sugino
et al., 2000). Di€ering observations exist on the
absence (Herdegen et al., 1998) or presence
(Schauwecker, 2000) of phosphorylated c-Jun in
the dentate gyrus, an area with high constitutive
expression of c-Jun. Following systemic application of adriamycin, JNK1 is translocated into the
nucleus without alteration of the total JNK1
content in dorsal root ganglia, and in these cells,
JNK1 and phosphorylated c-Jun are concentrated
in nuclear domains enriched in splicing factors and
perichromatin ®brils (Pena et al., 2000). Kainic
acid raised the JNK1-IR in the cytoplasm and the
nucleus of hippocampal neurons based on a
moderate increase in the 46 kDa JNK-fraction
(Schauwecker, 2000), whereas MPTP provoked a
moderate increase in the 54 kDa JNK-fraction in
the substantia nigra (Saporito et al., 2000).
(iii) Substantial contradiction exists on the alteration of
the expression of JNKs following neuronal stress:
Downregulation of JNK1 expression (Chihab et
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c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2430
al., 1998) and immunoreactivity (Ferrer et al.,
1997) is opposed to enhanced JNK1-IR (Schauwecker, 2000) and an increase in JNK1 activity
(Mielke et al., 1999) in the hippocampus following
kainic acid excitotoxicity and fetal hypoxia. JNK1
expression was upregulated in the substantia nigra
compacta following striatal lesion (Oo et al., 1999),
whereas transcription of all three JNK isoform
mRNAs did not change following severe stress in
neural cell lines (Mielke et al., 2000).
(iv) Data based on the determination of JNK activity
demand critical interpretation: Firstly, immunocomplex assays might display JNK autophosphorylation rather than JNK-triggered phosphorylation of the substrate (mainly GST-c-Jun) (Mielke
et al., 2000). Secondly, analysis of the activity of
individual JNK-isoforms are impeded by lack of
speci®c antibodies. Thirdly, neuronal degeneration
with the induction of one JNK isoform (e.g. JNK3)
in the presence of high basal activity of other
isoforms (e.g. JNK1 and JNK2) does not simply
argue for a speci®c neurodegenrative role of the
induced isoform (Namgung and Xia, 2000).
The reasons for these divergent ®ndings at mRNA
and protein levels as well as in immunocytochemistry
are not clear. At least for immunocytochemistry, most
reports had not been subjected to critical analysis of
immunoreactivities. JNKs are highly conserved essential mediators of cellular information transfer and
responses. Therefore, it is doubtful whether JNKs
operate with such an individual cell-type-dependent
and stimulus-dependent pattern of transcription, translation and intracellular distribution. More likely,
activation of JNKs might be minutely modulated in
`microdomains' depending on the association with
sca€old regulators and upstream kinases. This demands speci®c non-crossreacting tools. Indeed, the
recent analysis of intraneuronal JNK distribution and
activation in cultured cerebellar granule cells has
provided evidence like the existence of di€erent JNKpools de®ned by upstream kinases MKK4 and MKK7
and the JIP sca€old protein, with each pool di€erentially activated following stress and maturation (Co€ey
et al., 2000). Finally, the paradigm of JNKs translocalization into the nucleus in dependence on their
activation has been questioned by the observation that
glucocorticoids inhibit the TNFa-induced phosphorylation of JNKs, and even enhances their nuclear
translocalization (Gonzalez et al., 2000).
c-Fos/ERK-axis
In contrast to the c-Jun/JNK-axis, the control of junB
and c-fos induction and the activation of their proteins
by post-translational modi®cations in the brain is less
clear. Reports on c-Fos activating kinases (Deng and
Karin, 1994) did not ®nd a subsequent echo, and the
role of phosphorylation and dephosphorylation of cFos for DNA binding and activation of the polymerase II-complex in neurons has to be deduced from
Oncogene
non-neuronal cells. The transcriptional regulation of cfos is mediated by ERKs with subsequent activation of
the SRF/TCF complex; members of the TCF complex,
such as Elk-1, are expressed and regulated in the adult
brain including alteration in intraneuronal distribution
(Sgambato et al., 1998). ERKs are supposed to be
protective-physiological counterplayers of the JNK/
p38 kinases (Xia et al., 1995) and are activated
following numerous physiological and pathophysiological stimuli.
Activation of the FosB protein is strictly dependent
on the phosphorylation of C-terminal serine residues
by (a) not yet de®ned kinase(s) distinct from the known
ERKs and JNKs (Skinner et al., 1997).
Role in neurodegeneration
The c-Jun/JNK-axis
Pro-degenerative ®ndings From all AP-1 proteins, the
c-Jun protein is the best studied member concerning
the putative roles in the context of neurodegeneration
and neuroprotection. c-Jun and its activating JNKs
have been determined as functionally synergistic
e€ectors in neuronal cell death: knockout of JNK3
and mutation of the serine residues 63 and 73 into nonphosphorylable alanins (so called c-JunAA mutant)
prevent kainic acid induced neuronal death in the
hippocampal CA3 region (Behrens et al., 1999; Yang et
al., 1997). The elevated resistance to kainic acid
indicates that JNK knockout (Yang et al., 1997) and
c-JunAA knock in (Behrens et al., 1999) have profound
in¯uence on the development of de®ned pre-existing
structures which realize neuronal excitation.
Transfection of sympathetic neurons with wildtype
c-Jun (Ham et al., 1995) or with MEKK1 (and
subsequent activation of JNK and c-Jun) (Eilers et
al., 1998) induced apoptosis even in the presence of
NGF, whereas cell death was prevented by transfection
with dominant negative JNK1 (Eilers et al., 1998).
Numerous publications report on coincident activation
of c-Jun and JNK during (apoptotic) degeneration of
neurons in vivo and in vitro (Ferrer et al., 1997; Harada
and Sugimoto, 1999; Ikeuchi et al., 1998; Levresse et
al., 2000; Martin-Villalba et al., 1999; Mielke and
Herdegen, 2000; Oo et al., 1999; Ozawa et al., 1999;
Saporito et al., 2000; Virdee et al., 1997). As a novel
player of the c-Jun/JNK-axis, the apoptosis signalregulating kinase 1 (ASK1) appears to initiate the
harmful functions of JNKs (Kanamoto et al., 2000;
Nakahara et al., 1999).
Finally, a set of experiments has demonstrated that
inhibition of MLKs by CEP-1347 with subsequent
inhibition of activation of JNK and c-Jun provides
robust neuroprotection (Maroney et al., 1998; Saporito
et al., 2000). At present, molecules of this group of
indirect JNK inhibitors undergo phase II trial, and this
study aims at attenuating the progression of Parkinson's disease.
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
These ®ndings, which underly the reputation of cJun and JNK as neuronal killers, have to be strictly set
into the pathophysiological context and must not be
generally misused as negative characteristics of these
molecules for several reasons. Firstly, the expression
and activation of c-Jun and JNK in the mammalian
adult brain in the absence of any harmful stimulus or
under neuroregenerative conditions as well as in neural
cells cultures during di€erentiation clearly suggest
important physiological-protective roles for these
molecules. Secondly, the dramatic attenuation of
hippocampal CA3 neuronal death by JNK3 knockout
(but not by JNK2 knockout and JNK1 knockout)
(Yang et al., 1997) and c-JunAA mutation (Behrens et
al., 1999) following kainic acid excitotoxicity was
observed in mice with a substantial C57/BL6 genetic
background ± this strain, however, is explicitly
resistant against kainic acid-triggered hippocampal
death (Schauwecker, 2000; Steward et al., 1999;
Herdegen et al., unpublished observations), i.e. similar
as with wild type C57/BL6 mice, the mutants should
not undergo neuronal cell death and the `rescue' of the
mutants might rather reveal natural resistance than
neuroprotection mediated by genetic manipulation.
These ®ndings summarised above raise several questions:
(i)
The substrate speci®city is unclear which underlies
the selective neurodegenerative e€ect of JNK3 in
the hippocampus, an area with coincident expression (Carboni et al., 1998) and activity (Brecht et
al., 1999; Mielke et al., 1999) of JNK1 and JNK2.
One explanation lies in the individual expression
of JIPs, a group of activators and inhibitors of
JNKs with di€erent anities for the individual
JNKs (Kim et al., 1999; Yasuda et al., 1999).
(ii) A similar extent of hippocampal protection by
JNK3 ko (Yang et al., 1997) and by c-JunAA
mutation (Behrens et al., 1999) and the respective
lowering e€ect on seizure-related behavior following kainic acid but not after PTZ suggest that cJun is the mediator of JNK3 neurodegeneration.
In contrast, the analysis of the N-terminal
phosphorylation of c-Jun and JNK activation
following kainic acid in C57/BL6 mice has
revealed the dissociation of this activation since
increased phosphorylation of c-Jun could be
linked with resistance to death, whereas JNK1
activity was associated with impending death
(Schauwecker, 2000). Moreover, the lasting immunoreactivity of phosphorylated JNK1+2 in the
surviving CA3 gerbil hippocampus following
ischemia (Sugino et al., 2000) does not support
the simplifying concept of mere harmful functions
by activated JNKs in the hippocampus.
(iii) Apart from the hippocampus, no data are
available to which extent the knockout of JNKs
alters the c-Jun expression and phosphorylation.
Thus, we do not know whether the activation of cJun downstream from JNK3 is restricted to the
hippocampal CA1 sub®eld or whether the activa-
tion of c-Jun by JNK3 realizes the degenerative
programs in a broader operative range.
(iv) The N-terminal stretches comprising the phosphoacceptor sites are very similar in JunD and cJun (Kallunki et al., 1996). This fact gives rise to
the following scenario: c-Jun serves as a docking
site for JunD (Kallunki et al., 1996) enabling
JNKs to phosphorylate the JunD protein in c-Jun
or c-JunAA containing heterodimers. In this case,
the pattern of phosphorylation is congruent with
the distribution of c-Jun but it derives from JunD.
Still, it cannot be suciently excluded that the
anti-phospho-c-Jun antibodies do not recognize
phosphorylated JunD.
2431
The function of c-Jun and JNKs as degenerativeapoptotic e€ectors is strictly dependent on the context
of pathophysiological processes. As a representative
example, retinal ganglion cells from E12,5 c-jun
nullizygous mice do not exhibit programmed cell death
when transplanted as xenografts in newborn rats
(Herzog et al., 1999).
The e€ector molecules What are the e€ectors of c-Jun
mediated death? The apoptotis-realizing target genes
of c-Jun/AP-1 are not well de®ned. The transcriptional
control of the ®rst line candidates such as Fas ligand
(Herdegen et al., 1998; Kolbus et al., 2000), TNFa
(Falvo et al., 2000) cyclin D1 (Bakiri et al., 2000;
Timsit et al., 1999) p53 (Kirch et al., 1999) by c-Jun is
questioned by reports that these e€ectors are
independently regulated of c-Jun/JNK or are not
involved in neuronal apoptosis (Low et al., 1999;
Napieralski et al., 1999; Small et al., 1999). Beyond
direct transcriptional activation of endogenous neuronal killer proteins as it is likely for the kainic-acid
induced hippocampal death (Behrens et al., 1999;
Yang et al., 1997), pro-degenerative e€ects of c-Jun
can be exerted by transcription of genes which
enhance the pathological stress as demonstrated by
the following examples.
(i)
In immune cells, di€erentiation and the production
of cytokines and/or their corresponding receptors
is co-modulated by JNKs (Sabapathy et al., 1999;
Yang et al., 1998), and this might also be true for
microglia, the resident brain immune cells.
Numerous observations report on neurodegenerative e€ects by (abundant) cytokine production
(Raivich et al., 1999).
(ii) The tissue plasminogen activator (tPA) is a serine
protease which is essential for ®brinolysis, but it is
also involved in neuronal death and excitotoxicity
(reviewed by Gingrich and Traynelis, 2000). The
basal and PMA-stimulated expression of the
plasminogen-activator inhibitor 1 (PAI-1), an
endogenous counterpart of tPA, is under the
control of c-Jun homodimers (Arts et al., 1996).
Thus, it is conceivable that the omission of c-Jun
dependent expression of the tPA-inhibitor in JNK
knockout mice or c-JunAA mutants leads to the
Oncogene
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2432
removal of the endogenous tPA-`brake' with
degeneration of the brain parenchyma following
ischemia. Consistantly, knockout of the PAI-1
gene enlarges the cerebral infarct area after
transient MCA occlusion (Wang et al., 1998).
Such a pathomechanism might explain our ®nding
that JNK knockout does not diminish the size of
infarct following MCA occlusion (unpublished
observation). The concept of a c-Jun/JNK-triggered PAI-1/tPA-interplay, however, does not
hold true for the observation that mice de®cient
in tPA are virtually resistant to kainate-induced
seizure with attenuated activation of microglia
(Tsirka et al., 1995). In this context, we have to
consider the expression of matrix metalloproteinases (e.g. MMP-1, MMP-9) (Xu et al., 2001)
which can trigger in¯ammatroy processes in the
brain following injury.
Finally, direct evidence is lacking whether the
degenerative e€ects of c-Jun/AP-1 in the adult brain
are additionally realized beyond the transcription of
genes e.g. by putative inhibition of other TFs, such as
steroidhormone-ligand-complexes (see also Chinenov
and Kerppola in this issue). It is conceivable that c-Jun
interferes with gonadal steroids which can act as
promoting factors in axonal regeneration (Jones, 1993).
Oncogene
degeneration, but rather to physiological programs and
neuroprotection (Xia et al., 1995). Inhibition of ERK,
however, does not worsen the ischemic progress in the
gerbil (Sugino et al., 2000). On the other hand, push of
calcium/cAMP pathways with activation of the CRE
c-fos promoter site (with coincident activatioin of
ERKs?) can be involved in pathological programs
following calcium overload. (v) Degeneration can be
regarded as a lasting/chronic harmful stimulus ± the
expression of c-Fos protein, however, often habituates
to repetitive stimuli (Hsieh et al., 1998).
Similar as with c-Fos, the data on the expression of
JunB, JunD, FosB and Fra proteins are more or less
correlative, but nevertheless linked to processes involved in regeneration and degeneration: lasting FosB/
JunD-complexes re¯ected the adaptive neuronal responses in the denervated striatum following MPTP
injection (Perez et al., 1998), Fra-2-IR persisted in
neurons surviving an ischemic insult (Pennypacker et
al., 2000). Di€erential e€ects of Jun proteins have been
described for the regulation of hippocampal NGF
expression following limbic seizures with participation
of JunD in activation and of JunB in suppression
(Elliott and Gall, 2000), and JunD might exert a major
role in NGF production with variable AP-1 partners
such as c-Fos or phosphorylated Fra-2 (Boss et al.,
2001).
The promiscous expression of the c-Fos protein, and the
remaining AP-1 proteins
Role of the c-Jun/JNK-axis in neuroprotection
The expression pattern of c-Fos, the main representative of the Fos family, is well characterized following
numerous neurodegenerative events. Its putative pathogenic role, however, is still enigmatic. Null mutation of
the c-fos gene attenuates the kindling development and
kindling-induced structural plasticity (Watanabe et al.,
1996), and this phenotype resembles the resistance to
kainic acid seizures following JNK3 knock-out and cJunAA mutation (Behrens et al., 1999; Yang et al.,
1997). Thus, similar to the c-Jun/JNK-axis, the
expression of c-Fos is involved in excitotoxicity
(depending on dimerization with c-Jun?). In further
comparison with c-Jun, the insight into functions of cFos is much less clari®ed. (i) There is still no marker
available which indicates the transactive form of c-Fos
corresponding to the N-terminal phosphorylation of cJun. (ii) Very often, pathological stimuli can be
regarded as enhanced physiological stimuli; in consequence, pathological input should include the activation of both, physiological responses and pathologicaldegenerative programs. Thus, expression of c-Fos or
JunB can represent the physiological and/or the
degenerative part of the stimulus. (iii) Following some
degenerative processes with selective induction of c-Jun
(e.g. axotomy), the absence of c-Fos indicates an active
suppression rather than the mere lack of the inducing
input (reviewed by Herdegen et al., 1997) and the
possibility of c-Fos degradation by c-Jun (Tsurumi et
al., 1995). (iv) In contrast to JNK pathways, the
upregulation of ERKs cannot be linked to neuronal
Besides physiological functions in the `normal' brain, cJun and its upstream activators JNKs are also involved
in processes underlying neuroregeneration or neuroprotection. Conditioning ischemia with the rescue of
hippocampal neuronal death is linked to the selective
induction of c-Jun in the surviving CA1 neurons which
otherwise die in the absence of c-Jun (Sommer et al.,
1995). The lasting expression of c-Jun in successfully
regenerating neurons including intrinsic central neurons such as retinal ganglion cells or thalamic neurons
(Vaudano et al., 1998), the enhanced re-induction of cJun following spinal cord transection even months
after the previous injury (Houle and Ye, 1999) or the
protection of ischemic retinal ganglion cells by
intravitreal BDNF in the presence of c-Jun (Kurokawa
et al., 1999) suggest functions of c-Jun in survival and
regeneration rather than for axotomy-triggered degeneration (reviewed in detail by Herdegen et al., 1997). In
PC12 cells, neurite formation has been shown to be
dependent on the ASK1-JNK pathway, Rac-JNK
pathway (Kita et al., 1998) or MEK1-c-Jun pathway
(Leppa et al., 1998). This model, however, cannot be
simply transferred to axonal re-growth in the nervous
system, since numerous molecules including inhibitors
of mitosis are e€ective unspeci®c inducers of neuritogenesis.
Recent ®ndings have set the expression and activation of c-Jun/JNK into a novel protective-regenerative
context. The c-Jun expression following axon-disruption can be mimicked by colchicine, a microtubule-
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
interfering agent, even when the neuron-target-axis is
morphologically intact (Leah et al., 1991). Full-length
MEKK1 (i.e. not cleaved by caspase-3) with downstream activation of JNK protects embryonic stem cells
against apoptosis caused by microtubule toxins (Yujiri
et al., 1999). Thus, axotomy-triggered expression of cJun and retrograde transport of JNK (Herdegen et al.,
1998; Kenney and Kocsis, 1998) most likely function as
a sensor for axonal integrity. The action of c-Jun is
supported by the re-appearance of ATF-2 in regenerating neurons (Kreutz et al., 1999), whereas the early and
rapid disappearance of ATF-2 is a feature of neuronal
injury (Herdegen et al., 1997). Finally, the antiapoptotic molecule Bcl-3 has been identi®ed as a
neuroprotective e€ector of c-Jun; interestingly, apoptosis caused by IL-4 deprivation induces downregulation of c-Jun and Bcl-3, but up-regulation of cFos (Rebollo et al., 2000). Besides c-Jun, the family
member JunD can confer protection by antagonisation
of the p53 expression; in consequence, c-Jun might be
involved in neuroprotection as a docking molecule for
JNKs allowing these kinases to activate JunD
(Kallunki et al., 1996).
Pathological responses and AP-1 expression following
intentional stimulation: the problem of linkage
Strictly speaking, the visualization of AP-1 expression
allows only the conclusion that an intentional stimulus
(which usually starts a complex cascade of various
pathophysiological refunctions) causes the de novo
synthesis of AP-1 proteins. The critical issue of the
meaning of AP-1 expression for the cellular/neuronal
reaction can be exempli®ed by the persistance of c-Jun
in axotomized neurons ± its lasting expression for
weeks in surviving neurons or until the fragmentation
of dying neurons allows a bipartite conclusion: (i) cJun is involved in the regenerative-protective e€orts of
injured neurons which last until realization of death by
unbalanced degenerative programs or until the recovery with/without structural regeneration; (ii) c-Jun is
involved in degenerative genetic programs which last
until the `successful' (apoptotic) termination or which
come to an end with the onset of the neuronal recovery
by protective-regenerative programs. And this observation is also true for both, the N-terminal phosphorylation of c-Jun and the activation of JNK (Herdegen et
al., 1997, 1998).
2433
Regulation of pathological-degenerative functions of the
c-Jun/JNK-axis in the brain
The data on the role of c-Jun under physiological and
pathological conditions provoke the essential question:
which molecular mechanisms determine the tripartite
functional transition of c-Jun from a physiological
player to a neurodegenerative e€ector or to a
modulator of neuroregeneration? JNKs form a central,
but not exclusive, role for the c-Jun activation, which is
also true for the nervous system. Therefore, we review
the regulation of c-Jun with particular focus on its
upstream N-terminal activators, JNKs. Several components can be de®ned for the regulation of the c-Jun/
JNK-axis (Figure 1; Table 2):
(i)
Induction. The classi®cation of AP-1 proteins as
immediate-early genes (IEGs) re¯ected their rapid
de novo synthesis following serum deprivation or
TPA stimulation (Almendral et al., 1988). The
rather uniform expression is based on similar
DNA response elements with the primarily
functional designation of the TPA response
element (TRE). This temporally staggered, but
nevertheless congruent, AP-1 expression pattern is
visible at mRNA and protein level following
transynaptic stimulation e.g. following kainic acid
or noxious stimulation (Beer et al., 1998; Herdegen and Leah, 1998). One essential particularity of
c-Jun is its selective induction in the absence of
other AP-1 proteins. Recently, we have de®ned the
distal jun2 (and to a lesser extent the proximal
jun1) response element as regulatory elements for
ATF-2 and c-Jun mediated c-jun induction in the
brain. Besides ATF-2 and c-Jun, the p38 pathway
with the transcription factor MEF2C has emerged
as a novel regulator of the c-jun induction (Han et
Table 2 Physiological-protective and apoptotic-degenerative modulation of c-Jun functions
Protective-regenerative
or physiological actions
Induction of c-jun
N-terminal phosphorylation of c-Jun
Target genes of c-Jun
via MEF2C
ERKs
GAP-43, bcl-3
Formation of dimers
Cell specific expression
Modulation of the c-Jun/JNK-axis
JunD, ATF-2
Thioredoxin, GSTpa
Bipotential actions b
via ATF-2
JNKs
MMPs, cyclin D1
metallothioneins, tau
Neurons
Activation of p38
Activation of ASK
Apoptotic-degenerative actions
TNFa, TNFa-R, Fas-L, p53
Inhibition of steroidhormone-receptors
Activated microglia
Suppression of ATF-2, inhibition of
ERK and of Akt/PKB
Hypothetical functions of c-Jun and its expression related to physiological and regenerative-protective functions at the one hand, and apoptoticdegenerative e€ects at the other hand. aBy inhibition of expression and/or activation of c-Jun and/or JNK. bPotentially physiological/protective
or harmful actions
Oncogene
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
2434
Oncogene
al., 1997; Marinissen et al., 1999); interestingly,
MEF2C can mediate neuronal survival and neurite
elongation during development (Mao et al., 1999).
The down-regulation of ATF-2, a nuclear substrate of p38, is strictly coincident with c-Jun
induction following neuronal injury and this might
substantially change the competitive situation of
p38 substrates e.g. with shift from ATF-2 to
MEF2C phosphorylation with initiation of c-jun
induction in the absence of ATF-2 and c-Jun.
(ii) Activation by N-terminal phosphorylation.
. Sca€old proteins. The N-terminal phosphorylation is the crucial step in the transcriptional
activation of c-Jun, but the operative range of
JNKs and ERKs and the c-Jun amino residues
involved has still to be de®ned. Nevertheless, the
activation of JNKs might be the most relevant
step in the instrumentalization of c-Jun as a prodegenerative e€ector. Therefore, the regulation of
JNKs deserves particular attention. By complexation with distinct sca€old proteins, JNKs are
coupled to individual signal-transduction units
with di€erent upstream activators. The group of
JNK-interacting proteins (JIPs) selectively mediates signaling by the mixed-lineage kinase
(MLK)4MAP kinase kinase 7 (MKK7)4JNK
pathway, and their oligomeric complexes accumulate in peripheral cytoplasmic projections (Yasuda
et al., 1999); interestingly, MLK2/3 can associate
with the GluR6-PSD95 sca€olds (Liu YF, oral
communication), and the activation of JNKs by
MLK2 partially mediates apoptosis by polyglutamine-expanded huntingtin (Liu et al., 2000). Apart
from renal jip1 mRNA, the JIP1-3 proteins are
highly expressed in the brain (Kim et al., 1999)
with cytoplasmic and axonal-presynaptic distribution (Pellet et al., 2000), and complexes with
MLK2 and activated JNK have been found along
microtubules in ®broblasts (Nagata et al., 1998).
Expression of JIPs can be enhanced at a
transcriptional level following ischemia (Hayashi
et al., 2000), neuronal denervation (Anguelova et
al., 2000) or inhibition of JNK (Levresse et al.,
2000) with an immediate impact on JNK activation, since the overexpression of JIPs relative to
JNKs blocks JNK functions (Ferrell, 2000). The
JNK-associated protein 1 (JSAP1), which is
synonymous to JIP-3 and displays a preference
for JNK3 (Ito et al., 1999), antagonizes the
activation of ERKs by inhibition of the Raf-1
mediated MEK1 phosphorylation (Kuboki et al.,
2000). Finally, the û-arrestin 2 protein has recently
been identi®ed as a sca€old for JNK3 in the
murine brain which mediated the cytosolic retention of JNKs and the activation by ASK1
(McDonald et al., 2000).
In principle, sca€olds have the potential to
facilitate signaling ± but ¯uctuations of the
pathway components will inevitably lead to a less
ecient signal transfer. The facilitation of signal
response by sca€olds ultimately leads to an
ultrasensitivation with potential Hill coecients
of more than 10 (Ferrell, 2000). Thus, an increase
in expression and activation of individual cascade
members might be without relevance in the
absence of concomitant stocheometric alterations
in sca€old proteins or decrease ecacy of signal
transduction by disturbance of the balance of
sca€old complexes.
. Antagonistic phosphatases. Besides inhibition of
phosphorylation by sca€olds, activation of JNK
can be terminated by the speci®c MAP kinase
phosphatases 3/6 (MKP3/6) (Muda et al., 1996)
and unspeci®cally by MKP1 or MKP2 (Keyse,
1998). Recently, we have shown an increase of
nuclear MKP1 following transection of central
nerve bundles in surviving mamillary neurons, but
not in dying neurons of the substantia nigra
compacta (Winter et al., 1998). On the other
hand, downregulation of MKPs constitutes an
e€ective step for the activation of JNKs as it has
been reported following protein-damaging conditions (Meriin et al., 1999).
(iii) Parallel activation of agonistic or antagonistic
signal cascades. Numerous signal-transduction
cascades have the potential to activate JNKs,
and ± in the case of nuclear translocation ±
with subsequent activation of c-Jun. In the
context of c-Jun/JNK functions activated during
neurodegenerative processes, the in¯uence of the
three activating cascades p38-ATF2/MEF2C,
ASK1-JNK and Akt-JNK deserves particular
interest.
First of all, activation of p38 can enhance the
induction of c-jun by phosphorylation of ATF-2
or MEF2C; furthermore, the phosphorylation of
ATF-2 could interfere with the competition of cJun for binding to or being phosphorylated by
JNKs (Gupta et al., 1996). Importantly, inhibition of p38-catalyzed substrate phosphorylation
substantially protects against ischemic injury in
the gerbil (Sugino et al., 2000).
Secondly, ASK1 was colocalized with activated
JNK or activated p38 in apoptotic cells following spinal cord injury (Nakahara et al., 1999).
Microtubular disarray, a putative feature of
neuronal degeneration, induced c-Jun dependent
transcription via JNK and synergistic but parallel
activation of ASK1 (Wang et al., 1998). ASK1
keeps the position of a master switch for
activating both, p38 and, to a lesser extent,
JNK with speci®c or synergistic functions. Thus,
ASK1 triggers neurite extension in PC12 cells
with downstream phosphorylation of JNK and
p38; on the other hand, ASK1 is a crucial
modulator of NGF withdrawal-induced apoptosis
in PC12 cells and superior cervical ganglia with
activated c-Jun/JNK-axis and p38 (Kanamoto et
al., 2000). In contrast to ASK1, the Akt kinase
(also called protein kinase B) which acts downstream of phosphatidylinositol-3-kinase (PI3-K),
distinctly inhibits the activation of JNK and c-
c-Jun/AP-1 and neurodegeneration
T Herdegen and V Waetzig
Jun and protects against neuronal apoptosis
(Levresse et al., 2000).
The synergistic and antagonistic interplay of
di€erent signal cascades is nicely exempli®ed in
trigeminal neurinoma cells (Marushige and Marushige, 1999): apoptosis was rapidly induced
when the activation of JNK was coupled with
the inhibition of PI3-K/Akt, and the induction
was further enhanced by parallel inhibition of
ERK; rapid apoptosis occurred when the JNK
activation was coupled with p38 inhibition, and
this was potentiated by inhibiting PI3-K/Akt; the
concomitant inhibition of ERK and activation of
Akt induced apoptosis without JNK activation,
even though with a considerable delay (Marushige and Marushige, 1999).
Conclusions
Neurodegenerative events with regenerative e€orts and/
or apoptotic destruction induce AP-1 proteins in highly
complex temporospatial patterns. In terms of neurodegeneration, we have to di€erentiate between AP-1
functions in neuronal cells and in non-neuronal cells
which in turn harmfully a€ect neurons. Considering
the complex interplay of various reinforcing and/or
antagonizing activators and inhibitors of AP-1 func-
tions in the intra- and extracellular compartments, only
few ®ndings can be attributed to AP-1 proteins and
their upstream activators de®ning a role for degenerative and regenerative e€orts. In the majority of
experiments and observations, the AP-1 transcription
factors play their part in the orchestration of the ®ne
functional tuning underlying the neuronal plasticity.
Besides clearly de®ned neuronal responses exclusively or mainly triggered by c-Jun and the JNKs
upstream, c-Jun and JNKs might act as stand-by
molecules which help to implement decisions as
conditio sine qua non. The potentially relevant
physiological functions of the c-Jun/JNK-axis might
limit the use of antagonists as a novel strategy for the
treatment of neurological disorders.
2435
Abbreviations
AP-1: activator protein 1 (abbreviation for both, proteins
and DNA binding sites); ASK 1: apoptosis signal-regulating kinase 1; CEP-1347: Cephalon compound 1347; IEGs:
immediate-early genes; IR: immunoreactivity; JIPs: JNKinteracting proteins; JNKs: c-Jun N-terminal kinases;
JSAP1: JNK-associated protein 1; MKKs: MAP kinase
kinases; MKPs: MAP kinase phosphatases; MLKs: mixedlineage kinases; P13-K: phosphatidylinositol-3-kinase;
SAPKs: stress activated protein kinases; tPA: tissue
plasminogen activator; PAI-1: plasminogen-activator
inhibitor 1.
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