JNK regulates FoxO-dependent autophagy
Ping Xu,1Madhumita Das,1Judith Reilly,1and Roger J. Davis1,2,3
1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA;2Howard
Hughes Medical Institute, Worcester, Massachusetts 01605, USA
The cJun N-terminal kinase (JNK) signal transduction pathway is implicated in the regulation of neuronal
function. JNK is encoded by three genes that play partially redundant roles. Here we report the creation of mice
with targeted ablation of all three Jnk genes in neurons. Compound JNK-deficient neurons are dependent
on autophagy for survival. This autophagic response is caused by FoxO-induced expression of Bnip3 that displaces
the autophagic effector Beclin-1 from inactive Bcl-XL complexes. These data identify JNK as a potent negative
regulator of FoxO-dependent autophagy in neurons.
[Keywords: autophagy; Beclin 1; Bnip3; JNK; Neurons]
Supplemental material is available for this article.
Received August 19, 2010; revised version accepted January 6, 2011.
The cJun N-terminal kinases (JNKs) are encoded by three
genes (Davis 2000). Two of these genes (Jnk1 and Jnk2) are
expressed ubiquitously, while the Jnk3 gene is selectively
expressed in neurons (Gupta et al. 1996). Compound mu-
tation of these Jnk genes causes early embryonic lethality
in mice (Kuan et al. 1999; Sabapathy et al. 1999). Con-
sequently, studies of JNK deficiency in neurons have
focused on an analysis of mice with partial loss of JNK
(Davis 2000; Weston and Davis 2007). These studies have
demonstrated isoform-specific functions of JNK in neu-
rons (Brecht et al. 2005).
It is established that JNK plays an important role in the
phosphorylation of microtubule-associated proteins—
including Doublecortin (Gdalyahu et al. 2004), MAP1B
(Chang et al. 2003; Barnat et al. 2010), MAP2 (Chang
et al. 2003), the stathmin protein family of microtubule-
destabilizing proteins (Tararuk et al. 2006), and Tau
(Yoshida et al. 2004)—may influence microtubule func-
tion. This action of JNK is important for neurite forma-
tion. Thus, JNK contributes to bone morphogenic protein-
stimulated dendrite formation (Podkowa et al. 2010), the
structure of dendritic architecture (Coffey et al. 2000;
Bjorkblom et al. 2005), axodendritic length (Tararuk et al.
2006), and axonal regeneration (Barnat et al. 2010). More-
over, JNK can regulate kinesin-mediated fast axonal trans-
port on microtubules (Morfini et al. 2006, 2009) and
contributes to the regulation of synaptic plasticity (Chen
et al. 2005; Zhu et al. 2005; Li et al. 2007; Thomas et al.
2008). Together, these data demonstrate that JNK plays
a key role in the physiological regulation of neuronal
activity (Waetzig et al. 2006).
The JNK signaling pathway has also been implicated in
stress-induced apoptosis (Kuan et al. 1999; Tournier et al.
2000), including neuronal death in models of excitotox-
et al. 2007). This JNK-induced apoptotic response is medi-
ated, in part, by the expression and/or phosphorylation of
members of the Bcl2-related protein family (Weston and
Davis 2007; Hubner et al. 2008; Morel et al. 2009; Hubner
et al. 2010). These data indicate that JNK plays a critical
role during the injury response associated with neuro-
degeneration and stroke.
The dual role of JNK in mediating both physiological
responses (e.g., neurite development) and pathological
responses (e.g., neuronal injury) requires that the actions
of JNK are context-specific (Waetzig and Herdegen 2005).
These effects of JNK may be mediated by compartmen-
talization of specific pools of JNK in different subcellular
locations or within different signaling complexes (Coffey
et al. 2000). JNK may also cooperate with other signal
transduction pathways to generate context-specific re-
sponses (Lamb et al. 2003). However, the fundamental
role of JNK in neurons and the mechanisms that account
for these divergent biological responses to JNK signaling
remain poorly understood.
Studies of mice with deficiency of one Jnk gene have
provided a foundation for current knowledge of the role of
JNK in neurons. However, partial loss of JNK expression
represents a limitation of these studies because of re-
dundant functions of JNK isoforms (Tournier et al. 2000;
Jaeschke et al. 2006). Creation of a model of compound
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JNK deficiency is important because compound JNK de-
ficiency represents a more relevant model for understand-
ing the effects of pharmacological JNK inhibition than
deficiency of a single JNK isoform. JNK inhibitors have
been identified that may be useful for the treatment of
neurodegenerativediseases andstroke(Borselloetal. 2003;
Hirt et al. 2004; Repici et al. 2007; Carboni et al. 2008;
Esneault et al. 2008; Wiegler et al. 2008; Probst et al. 2011).
A model of neuronal compound JNK deficiency is required
to test whether the actions of these drugs are mediated by
loss of JNK function. Moreover, an experimental model of
into the physiological role of JNK in wild-type neurons.
The purpose of this study was to examine the proper-
ties of neurons with simultaneous ablation of the Jnk1,
Jnk2, and Jnk3 genes. We report the creation and charac-
terization of mice with triple deficiency of neuronal JNK
isoforms in vivo and in primary cultures in vitro.
Establishment of neurons with compound JNK
deficiency in vitro
To examine the function of JNK in neurons, we prepared
primary cerebellar granule neurons (CGNs) from mice
with conditional Jnk alleles. Cre-mediated deletion of
conditional Jnk resulted in neurons that lack expression
of JNK (Fig. 1A,B) and exhibit defects in the phosphory-
lation of the JNK substrates cJun (Davis 2000) and neuro-
filament heavy chain (Fig. 1C,D; Brownlees et al. 2000).
These triple Jnk knockout (JNKTKO) neurons exhibited
altered morphology, including hypertrophy (Figs. 1E–G;
Supplemental Fig. S1). Immunofluorescence analysis us-
ing an antibody to Tau (data not shown) and Ankyin
The JNK signaling pathway is implicated in microtu-
bule stabilization and the regulation of axodendritic mor-
phology (Coffey et al. 2000; Chang et al. 2003; Bjorkblom
et al. 2005; Tararuk et al. 2006; Barnat et al. 2010). JNK
inhibition may therefore increase microtubule instability
and cause neurite retraction. Indeed, the JNKTKOneuronal
hypertrophy was associated with a reduction in the
number of dendrites (Fig. 1H; Supplemental Fig. S1). To
test whether JNKTKOneurons exhibited increased micro-
tubule instability, we examined the presence of stable
microtubules containing detyrosinated Tubulin by immu-
nofluorescence analysis (Schulze et al. 1987; Webster et al.
1987; Khawaja et al. 1988). Contrary to expectations, no
decrease in microtubules with detyrosinated Tubulin was
at 3 d of culture in vitro (DIV) and then examined at 10 DIV. (A) Genotype analysis of JNKTKOneurons. The floxed Jnk1 and deleted Jnk1
alleles are detected as 1095-base-pair (bp) and 395-bp PCR products, respectively. (B) Extracts prepared from JNKTKOneurons were examined
by immunoblot analysis using antibodies to JNK and a-Tubulin. (C) Control and JNKTKOneurons were examined at 10 DIV by immunoblot
analysis using antibodies to phospho-neurofilament H and a-Tubulin. (D) Control and JNKTKOneurons were examined by immunofluo-
rescence microscopy by staining with DAPI and antibodies to bIII tubulin and phospho-Ser63cJun. Bar, 20 mm. (E) Control and JNKTKO
neurons were examined by phase-contrast microscopy. Bar, 75 mm. (F) Control and JNKTKOneurons were stained with calcein-am ester and
examined by fluorescence microscopy. Bar, 65 mm. (G) Wild-type (control) and JNKTKOneurons were stained with Mitotracker Red at 10 DIV
and imaged by differential interference contrast (DIC) and fluorescence microscopy. Bar, 8 mm. (H) Control and JNKTKOneurons were
examined by immunofluorescence microscopy by staining with DAPI and antibodies to bIII tubulin and Ankyrin G. Bar, 20 mm.
Establishment of JNK-deficient neurons. Wild-type (control) and Jnk1LoxP/LoxPJnk2?/?Jnk3?/?CGNs were infected with Ad-cre
GENES & DEVELOPMENT311
(Supplemental Fig. S2). Together, these data confirm that
JNK regulates neuronal morphology, but the mechanism
may be only partially accounted for by altered microtu-
Comparison of control and JNKTKOneurons demon-
strated that JNK deficiency caused a marked increase in
life span during culture in vitro (Fig. 2A; Supplemental
Fig. S3A). To confirm that the loss of JNK activity in-
creased life span, we employed a chemical genetic strategy
using neurons prepared from mice with germline point
mutations that confer sensitivity of JNK to the prede-
signed small molecule drug 1NM-PP1 (Jaeschke et al.
2006). This chemical genetic analysis confirmed that
JNK inhibition caused both hypertrophy and increased
neuronal viability in vitro (Fig. 2B; Supplemental Fig. S3B).
A defect in transport might contribute to the axonal
Indeed, it is established that JNK acts as a negative reg-
ulator of kinesin-mediated fast axonal transport (Morfini
et al. 2006, 2009). These data suggest that JNKTKOneurons
may exhibit altered kinesin-mediated transport. We found
an accumulation of mitochondria (Fig.1G), synaptic vesi-
cles (Supplemental Figs. S4, S5), and lysosomes (Supple-
mental Fig. S6) in JNKTKOneurons. Live-cell imaging of
mitochondria demonstrated the presence of fast transport
in wild-type neurons, but mitochondria were immobile
in JNKTKOneurons (Supplemental Fig. S7). This loss of
transport in JNKTKOneurons contrasts with expectations
that JNK deficiency might increase transport (Morfini
et al. 2006, 2009). It is established that fast transport of
mitochondria is mediated by the conventional kinesin
KIF5b (Tanaka et al. 1998). However, no decrease in Kif5b
expression was detected in JNKTKOCGNs (Supplemental
Fig. S8). A more general defect in trafficking may therefore
account for the mislocalization of organelles in JNKTKO
Neuronal JNK deficiency causes increased
autophagy in vitro
Live-cell imaging indicated that the morphology of mito-
chondria in JNKTKOneurons was different than control
neurons (Fig. 1G). Electron microscopy confirmed that
JNKTKOmitochondria were larger than control mitochon-
dria (Supplemental Fig. S9). Numerous double-membrane
structures, morphologically similar to autophagosomes,
rons. The presence of large numbers of autophagosomes
in JNKTKOneurons suggests that these cells may exhibit
increased autophagy. Indeed, biochemical analysis dem-
onstrated that an increased amount of the autophagic
effector protein Atg8/LC3b was processed by conjugation
of phosphatidylethanolamine to the C terminus of the
LC3b-I form to create LC3b-II, which is tightly associated
with the autophagosomal membrane (Kabeya et al. 2004;
Sou et al. 2008) in JNKTKOneurons compared with control
neurons (Fig. 3A). Atg8/LC3b expression was increased in
JNKTKOneurons (Fig. 3A,E), and Atg8/LC3b was redistrib-
uted from a location primarily in the soma of control
neurons to the neurites of JNKTKOneurons (Fig. 3D). The
Atg8/LC3b immunofluoresence detected in JNKTKOneu-
localization to autophagosomal membranes. Moreover,
the p62/SQSTM1 protein, which directly binds the auto-
phagiceffectorAtg8/LC3 (Pankiv et al. 2007),wasdetected
in wild-type neurons but not in JNKTKOneurons (Fig. 3A).
The loss of p62/SQSTM1 suggests that autophagic flux
is increased in JNKTKOneurons compared with control
neurons (Klionsky et al. 2008). To confirm this conclusion,
we examined the effect of lysosomal inhibition on the
conversion of LC3b-I to LC3b-II. If the autophagic flux is
increased, blocking autophagy should lead to increased
accumulation of LC3b-II. Consistent with an increase in
autophagic flux, we found that inhibition of autophagy
caused a greater increase in LC3b-II in JNKTKOneurons
compared with control neurons (Supplemental Fig. S11).
Together, these data demonstrate the presence of an active
autophagic response in JNKTKOneurons.
Autophagy may contribute to the increased survival
of JNKTKOneurons (Hara et al. 2006; Komatsu et al.
2006). Indeed, studies using a pharmacological inhibitor
culture in vitro. (A) Wild-type (control) and JNKTKOCGNs
infected without or with Ad-cre at 3 DIV were examined by
phase-contrast microscopy at 7–28 DIV. Bar, 45 mm. (B) Jnk1LoxP/LoxP
Jnk2M108G/M108GJnk3?/?CGNs were untreated (control) or infected
with Ad-cre at 3 DIVand with the drug 1-NM-PP1 (1 mM) at 7 DIV
(iJNK). The CGNs were examined using phase-contrast micros-
copy. Bar, 45 mm. (C) Wild-type (control) and JNKTKOCGNs in-
fected with Ad-cre at 3 DIV were incubated without and with
1 mM chloroquine (CQ) at 11 DIV and then examined by phase-
contrast microscopy at 12 DIV. Bar, 65 mm. Quantitative analysis
of neuronal viability is presented in Supplemental Figure S3.
Increased life span of JNK-deficient neurons during
Xu et al.
312GENES & DEVELOPMENT
demonstrated that autophagy was required for the in-
creased life span of JNKTKOneurons compared with
control neurons (Fig. 2C; Supplemental Fig. S3). More-
over, RNAi-mediated knockdown of the autophagic effec-
tor Beclin-1 caused decreased survival of JNKTKOneurons,
but not control neurons (Fig. 4). Together, these data dem-
onstrate that the survival of JNKTKOneurons depends on
TORC1 does not mediate the effects of JNK deficiency
on neuronal autophagy
The mTOR protein kinase complex TORC1 is a potent
negative regulator of autophagy (Guertin and Sabatini
2007). Decreased TORC1 activity in JNK-deficient neu-
rons may therefore account for the observed increase
in autophagy. To test TORC1 function, we examined the
phosphorylation of the TORC1 substrate pSer389p70S6K.
We found that JNK deficiency did not alter the phos-
phorylation of this TORC1 substrate in neurons (Sup-
plemental Fig. S12). These data demonstrate that JNK
deficiency regulates autophagy by a TORC1-independent
Increased autophagy in JNK-deficient neurons
is mediated by a FoxO1/Bnip3/Beclin-1 pathway
The finding that JNK deficiency in neurons triggers an
autophagic response (Fig. 3) was unexpected, because
studies of nonneuronal cells have implicated JNK in the
induction of autophagy (Yu et al. 2004; Ogata et al. 2006;
Wei et al. 2008) or as an effector of autophagy-associated
cell death (Yu et al. 2004; Shimizu et al. 2010). Indeed, we
found that autophagy caused by serum withdrawal was
compromised in compound mutant fibroblasts that lack
JNK expression (Supplemental Fig. S13). This finding mark-
edly contrasts with the effect of compound JNK deficiency
in neurons to induce spontaneous autophagy (Fig. 3).
These data indicate that the role of JNK in autophagy
suppression may be restricted to neurons.
To test whether the autophagic mediator Beclin-1 may
be relevant to autophagy caused by JNK deficiency in
DIV were harvested at 10 DIV to prepare protein extracts that were examined using antibodies to LC3b, p62/SQSTM1, and a-Tubulin.
(B) Extracts prepared from control and JNKTKOCGNs were examined by immunoblot analysis by probing with antibodies to Bcl-XL,
Bnip3, Beclin-1, and a-Tubulin. Coimmunoprecipitation assays were performed by immunoblot analysis of Bcl-XL immunoprecipitates.
(C) Extracts prepared from control and JNKTKOCGNs were examined by immunoblot (IB) analysis by probing with antibodies to AKT,
pSer308-AKT, pSer473-AKT, FoxO1, pSer246-FoxO1, and a-Tubulin. CDK2 activity was measured in an immunecomplex kinase assay
(KA) using Rb as the substrate. The relative CDK2 activity is indicated below. (D) Control and JNKTKOCGNs were stained with bIII-
Tubulin and LC3b antibodies and examined by fluorescence microscopy. Bar, 10 mm. (E) Gene expression in CGNs was examined by
quantitative RT–PCR analysis of mRNA and normalized to the amount of Gapdh mRNA in each sample (mean 6 SD; n = 3).
Statistically significant differences are indicated. (*) P < 0.05. (F) Control and JNKTKOCGNs were stained with DAPI and antibodies to
FoxO1 and bIII-Tubulin. The neurons were examined by fluorescence microscopy. The merged image represents colocalization of
FoxO1 with DAPI. Bar, 10 mm.
JNK deficiency in neurons causes increased autophagy. (A) Wild-type (control) and JNKTKOCGNs infected with Ad-cre at 3
GENES & DEVELOPMENT313
down of Beclin-1 expression. Knockdown of Beclin-1 sup-
pressed biochemical markers of autophagy in JNKTKO
neurons, including increased LC3b-II and decreased
p62/SQSTM1 (Fig. 4). These data demonstrate that
Beclin-1 may mediate the effects of JNK deficiency to
cause increased autophagy in neurons.
It is established that the JNK-regulated interaction of
Bcl2 with the BH3 domain of Beclin-1 may contribute to
autophagy (Wei et al. 2008). We therefore examined the
interaction of Beclin-1 with Bcl2 family proteins in neurons.
No coimmunoprecipitation of Beclin-1 with Bcl2 was
detected in control neurons. However, Beclin-1 was found
this interaction was markedly suppressed in JNKTKOneu-
rons (Fig. 3B). The BH3 domain-binding activity of Bcl-XL
is negatively regulated by phosphorylation of Bcl-XL on
Ser62(Upreti et al. 2008), but no increase in Bcl-XL
phosphorylation was detected in JNKTKOneurons by im-
munoblot analysis with a phospho-specific antibody (data
not shown). An alternative mechanism must therefore
mediate the dissociation of Beclin-1. Release of Beclin-1
from Bcl-XL complexes could be mediated by competi-
tion with another BH3 domain protein. Indeed, we found
that JNKTKOneurons expressed increased amounts of
Bnip3, a BH3-only member of the Bcl2 protein family
(Fig. 3B). Coimmunoprecipitation analysis demonstrated
that the release of Beclin-1 from Bcl-XL complexes was
associated with increased interaction of Bcl-XL with
Bnip3 (Fig. 3B).
The Bnip3 gene is known to be a target of FoxO tran-
scription factors that also increase the expression of the
autophagy-related genes Atg8/Lc3b and Atg12 (Salih and
Brunet 2008). The increased expression of these genes in
JNKTKOneurons (Fig. 3A,B,D,E) suggests that JNK de-
ficiency leads to FoxO activation. Indeed, gene expression
analysis demonstrated increased FoxO1 mRNA and pro-
tein expression in JNKTKOneurons (Fig. 3C–E). To test
whether FoxO1 contributes to the increased autophagy
detected in JNKTKOneurons, we examined the effect
of RNAi-mediated knockdown of FoxO1. Knockdown of
FoxO1 in JNKTKOneurons caused decreased expression of
Bnip3 and Atg genes, suppressed the increase in LC3b-II
and the decrease in p62/SQSTM1, and caused decreased
neuronal survival (Fig. 5). These data demonstrate that
FoxO1 is required for the increased autophagy and sur-
vival of JNKTKOneurons.
Cytoplasmic sequestration is a major mechanism of
FoxO1 regulation by signal transduction pathways, in-
cluding AKT (Salih and Brunet 2008). We found a small
increase AKT phosphorylation on Thr308and Ser473in
JNKTKOneurons (Fig. 3C), indicating that AKT activity
may be moderately increased in JNKTKOneurons com-
pared with control neurons. Nevertheless, we found in-
creased nuclear localization of FoxO1 in JNKTKOneurons
compared with control neurons (Fig. 3F). This nuclear
redistribution of FoxO1 in JNKTKOneurons was associated
with increased phosphorylation of FoxO1 on Ser246
(Fig. 3C), a site that dominantly induces nuclear accumu-
protein kinases (CDKs) (Yuan et al. 2008). Abortive cell
cycle re-entry has been observed duringneurodegenerative
processes (Kim and Bonni 2008), including stroke (Kuan
et al. 2004). Indeed, we found that CDK2 was activated
in JNKTKOneurons compared with control neurons (Fig.
the phenotype of JNKTKOneurons, we examined the effect
of CDK inhibition on control and JNKTKOneurons. We
found that CDK inhibition suppressed the increase in
Bnip3 and FoxO1 expression detected in JNKTKOneu-
rons (Fig. 6A). Moreover, CDK inhibition suppressed the
autophagy-related increase in LC3b-II, decrease in p62/
SQSTM1, and survival of JNKTKOneurons compared
with control neurons (Fig. 6B–E). These data confirm
autophagy and survival of JNKTKOneurons. (A) Wild-type
(control) and Jnk1LoxP/LoxPJnk2?/?Jnk3?/?(JNKTKO) neurons
infected with Ad-cre at 3 DIV were transfected at 7 DIV with
Beclin-1 siRNA or control siRNA. The expression of Beclin-1
mRNAwas examined at 11 DIV by quantitative RT–PCR analysis
of mRNA and normalized to the amount of Gapdh mRNA in
each sample (mean 6 SD; n = 3). Statistically significant differ-
ences are indicated. (*) P < 0.05. (B) Control and JNKTKOneurons
transfected with scrambled sequence or Beclin-1 siRNA were
examined at 11 DIV by immunoblot analysis with antibodies to
LC3b, p62/SQSTM1, and a-Tubulin. (C) The survival of RNAi
transfected control and JNKTKOneurons at 11 DIV was quantitated
(mean 6 SD; n = 20). Statistically significant differences are
indicated. (*) P < 0.05.
Effect of RNAi-mediated knockdown of Beclin-1 on
Xu et al.
314 GENES & DEVELOPMENT
a role for CDK activity in the induction of autophagy and
survival by a FoxO1/Bnip3/Beclin-1 pathway in JNK-
Mice with compound JNK deficiency in neurons in vivo
We tested the effect of transgenic expression of Cre
recombinase in the brain of mice with floxed Jnk on
neuronal function in vivo. Initial studies using Nestin-Cre
mice demonstrated that triple JNK deficiency in neuronal
progenitor cells caused early embryonic death (data not
shown). Similarly, expression of Cre recombinase in a
more limited region of the brain (telencephalon) using
Foxg1-Cre transgenic mice also caused early embryonic
death (data not shown). The early death of these JNKTKO
mice precluded analysis of the effects of triple JNK
deficiency on the brain. We therefore examined the effect
of Cre expression in a subset of neurons that are non-
essential for mouse survival. A mouse strain with Cre
recombinase inserted in the Pcp2 gene expresses Cre
recombinase in cerebellar Purkinje cells (Barski et al.
2000). This Pcp2-Cre strain enabled the creation of viable
mice with triple neuronal deficiency of JNK1, JNK2, and
JNK3 (Fig. 7). Purkinje cell defects represent one cause of
cerebellar ataxia (Grusser-Cornehls and Baurle 2001), but
ataxia was not detected in mice with compound JNK-
deficient Purkinje cells that were examined (Figs. 7, 8).
This observation indicates that Purkinje cells can func-
tion without the JNK signaling pathway.
Immunocytochemistry analysis demonstrated the loss
of JNK protein in the Purkinje cell layer of the cerebellum
(Fig. 7A), and genotype analysis of cerebellar DNA led
to the identification of loss-of-function alleles of Jnk1,
Jnk2, and Jnk3 (Fig. 7B). The JNKTKOPurkinje cells exhi-
bited reduced dendritic arborization (Supplemental Fig.
S14). Immunofluorescence analysis using an antibody to
Calbindin D-28k indicated the presence of hypertrophic
Purkinje cell axons in deep cerebellar nuclei (DCN) (Fig.
7C). These hypertrophic axons were also identified in
sections of the JNKTKODCN stained with H&E (Fig. 7D),
by immunohistochemical staining with an antibody to
Calbindin D-28k (Fig. 7D), and staining using the Golgi
reagent (Supplemental Fig. S14). Staining with an anti-
body to GFAP demonstrated that the axonal hypertrophy
was associated with reactive gliosis (Fig. 7D). Electron
microscopy confirmed the hypertrophy of myelinated
Purkinje cell axons in the DCN of JNKTKOmice (Fig.
7E; Supplemental Fig. S15). Quantitative image analysis
demonstrated that the cross-sectional area of Purkinje
cell axons was significantly larger in the DCN of JNKTKO
mice compared with control mice (Fig. 7F). Fewer axonal
mitochondria and increased numbers of autophagosomes
on autophagy and survival of JNKTKOneurons. (A,B) Wild-
type (control) and Jnk1LoxP/LoxPJnk2?/?Jnk3?/?(JNKTKO)
neurons infected with Ad-cre at 3 DIV were transfected at
7 DIV with FoxO1 siRNA or control siRNA. The expres-
sion of FoxO1 mRNA (A) and Bnip3 mRNA (B) was
examined at 11 DIV by quantitative RT–PCR analysis of
mRNA and normalized to the amount of Gapdh mRNA
in each sample (mean 6 SD; n = 3). Statistically signifi-
cant differences are indicated. (*) P < 0.05. (C) Control and
JNKTKOneurons transfected with scrambled sequence or
FoxO1 siRNA were examined at 11 DIV by immunoblot
analysis with antibodies to LC3b, p62/SQSTM1, and
a-Tubulin. (D) RNAi transfected JNKTKOneurons were
examined at 11 DIV by quantitative RT–PCR analysis
of Atg3, Atg5, and Atg12 mRNA and normalized to the
amount of Gapdh mRNA in each sample (mean 6 SD;
n = 3). Statistically significant differences are indicated.
(*) P < 0.05. (E) The survival of RNAi transfected control
and JNKTKOneurons at 11 DIV was quantitated (mean 6
SD; n = 20). Statistically significant differences are
indicated. (*) P < 0.05.
Effect of RNAi-mediated knockdown of FoxO1
GENES & DEVELOPMENT315
were detected in JNKTKOmice compared with control
mice (Fig. 7F). In contrast, the size of both autophago-
somes and mitochondria were increased in JNKTKOmice
compared with control mice (Fig. 7F).
Neuronal JNK deficiency causes increased autophagy
The observation that compound JNK deficiency causes
increased autophagy in primary cultures of neurons in
vitro (Fig. 3) suggests that JNK may suppress neuronal
autophagy in vivo. To test this hypothesis, we examined
autophagy in mice with triple deficiency of JNK1, JNK2,
and JNK3 in Purkinje cells (Fig. 8). Electron microscopy
demonstrated that autophagy was influenced by com-
pound JNK deficiency because the size of axonal auto-
phagosomes in the DCN was significantly increased com-
pared with control mice (Fig. 7F). However, the altered
size of autophagosomes could be caused by either an in-
crease or a decrease in neuronal autophagy. We therefore
examined the amount of p62/SQSTM1 protein (which
directly binds the autophagic effector Atg8/LC3) (Pankiv
et al. 2007) in Purkinje cells by immunohistochemistry.
The p62/SQSTM1 protein was detected in the Purkinje
cell soma of control mice, but not in mice with com-
pound deficiency of JNK in Purkinje cells (Fig. 8A). This
loss of p62/SQSTM1 suggests that autophagic flux is in-
creased in JNKTKOneurons compared with control neu-
rons (Klionsky et al. 2008). The increased autophagy was
associated with nuclear phosphorylation of the transcrip-
tion factor FoxO1 on the activating site Ser246(Fig. 8A)
and increased expression of Bnip3 and Atg12 (Fig. 8B). The
amount of LC3b in the Purkinje cell soma was moder-
ately increased in compound JNK-deficient Purkinje cells
(Fig. 8B), but a large increase in LC3b was detected in
Purkinje cell axons within the DCN (Fig. 8C). Together,
these data indicate that the FoxO1–Bnip3 pathway that
Purkinje cells in vivo.
Studies of nonneuronal cells have implicated JNK in the
induction of autophagy (Yu et al. 2004; Ogata et al. 2006;
Wei et al. 2008). Indeed, we confirmed the conclusion
that JNK can contribute to increased autophagy by exam-
ining primary mouse embryonic fibroblasts (MEFs) with
compound JNK deficiency (Supplemental Fig. S13). The
mechanism of JNK-induced autophagy may be mediated
by phosphorylation of Bcl2 by JNK and the subsequent
release of the autophagic effector Beclin-1 (Wei et al. 2008).
Thesites ofJNKphosphorylationonBcl2 (Yamamoto etal.
et al. 2000; Upreti et al. 2008). This conservation suggests
that phosphorylation of Bcl2 and Bcl-XL is functionally
important. Phosphorylation of Bcl2 and Bcl-XL by JNK
JNKTKOneurons. (A,B) Wild-type (control) and Jnk1f/f
Jnk2?/?Jnk3?/?(JNKTKO) neurons infected with Ad-cre
at 3 DIV were treated without or with the CDK inhi-
bitor roscovitine (5 mM; iCDK) at 10 DIV. (A) The
neurons were examined by phase-contrast microscopy
at 11 DIV. Bar, 45 mm. (B) The number of viable neurons
was examined at 11 DIV (mean 6 SD; n = 20). Statisti-
cally significant differences are indicated. (*) P < 0.05. (C)
Control and JNKTKOneurons were examined after
treatment on 10 DIV with roscovitine (iCDK) for 8 h
by immunoblot analysis using antibodies to LC3b,
p62SQTM1, and a-Tubulin. (D) Control and JNKTKO
neurons were examined by immunofluorescence anal-
ysis after treatment with roscovitine (iCDK) for 8 h
using an antibody to LC3b. DNA was stained with
DAPI. Bar, 20 mm. (E) Control and JNKTKOneurons
wereexamined after treatment with roscovitine
(iCDK) for 8 h by quantitative RT–PCR analysis of
FoxO1 and Bnip3 mRNA and normalized to the amount
of Gapdh mRNA in each sample (mean 6 SD; n = 3).
Statistically significant differences are indicated. (*) P <
CDK activity is required for the viability of
Xu et al.
316 GENES & DEVELOPMENT
(Yamamoto et al. 1999; Kharbanda et al. 2000) and other
protein kinases (Tournier et al. 2001; Terrano et al. 2010)
may represent an important mechanism of autophagy
regulation (Wei et al. 2008). Indeed, the properties of JNK
as a stress-responsive kinase provide an elegant mecha-
nism for coupling stress exposure to the induction of
autophagy (Ogata et al. 2006).
The JNK signaling pathway suppresses
Studies of nonneuronal cells demonstrate that JNK is
markedly activated from a low basal state when cells are
exposed to stress (Davis 2000). However, JNK is regulated
very differently in neurons. JNK1 remains constitutively
activated under basal conditions, while JNK2 and JNK3
exhibit low basal activity and are stress-responsive (Coffey
et al. 2000, 2002). The proautophagy role of JNK in non-
neuronal cells has been reported to be mediated by JNK1
(Wei et al. 2008). It is therefore intriguing that JNK1 is
constitutively activated in neurons. Based on studies
of nonneuronal cells (Wei et al. 2008), the constitutive
activation of JNK1 in neurons should cause autophagy.
A mechanism must therefore exist to prevent autophagy
activation by constitutively activated JNK1 in neurons.
Although the mechanism is unclear, these considerations
indicate that neurons are refractory to the proautophagy
JNK1 signaling pathway that has been identified in non-
neuronal cells (Wei et al. 2008).
Our analysis of compound JNK-deficient neurons dem-
onstrates that JNK regulates neuronal autophagy. In con-
trast to the proautophagy role of JNK nonneuronal cells,
neuronal JNK acts to suppress autophagy. Loss of neuro-
nal JNK function causes engagement of a transcriptional
Jnk2?/?Jnk3?/?mice (JNKTKO) that express Cre recombinase selectively in cerebellar Purkinje cells were examined. (A) Sections of the
Purkinje cell layer of control and JNKTKOmice were examined by immunohistochemical staining with antibodies to JNK1/2. Bar, 100
mm. (B) Cerebellar DNA was examined by PCR analysis to detect Jnk1+(1550-bp), Jnk1LoxP(1095-bp), Jnk1D(395-bp), Jnk2+(400-bp),
Jnk2?(270-bp), Jnk3+(430-bp), and Jnk3?(250-bp) alleles. (C) Sections of the Purkinje cell layer and DCN of control and JNKTKOmice
were examined by immunofluorescence staining with an antibody to Calbindin D-28k. Bar, 40 mm. (D) Serial sections of the DCN of
control and JNKTKOmice were examined by staining with H&E and by immunohistochemical staining antibodies to Calbindin D-28k
and GFAP. Bar, 100 mm. (E) The myelinated axons in the DCN of control and JNKTKOmice were examined by transmission electron
microscopy. Bars: top panels, 2 mm; bottom panels 0.125mm. (F) The axon area and the number and area of autophagosomes and
mitochondria in the myelinated axons of control and JNKTKOmice were measured. The data are presented as mean 6 SEM of 20 axons
of three different mice per group. Statistically significant differences between control and JNKTKOmice are indicated. (*) P < 0.05.
Compound deficiency of JNK in neurons in vivo. Young adult (8-wk-old) Pcp2-Cre mice (control) and Pcp2-Cre Jnk1LoxP/LoxP
GENES & DEVELOPMENT317
program that leads to increased expression of autophagy-
related genes and the induction of an autophagic response
(Fig. 3). One consequence of autophagy induction caused
by JNK deficiency is improved neuronal survival (Figs. 2;
Supplemental Fig. S3).
JNK can act as a molecular switch that regulates
FoxO-induced autophagy and apoptosis
FoxO transcription factors are implicated in the induc-
tion of both cell death (apoptosis) and cell survival
(autophagy) responses (Salih and Brunet 2008). The re-
sults of this study identify JNK as a signaling molecule
that may contribute to the coordination of these diver-
gent responses to FoxO transcription factor activation.
FoxO activation in neurons leads to the expression of
the target gene Bim, a proapoptotic BH3-only protein, and
causes cell death (Gilley et al. 2003). JNK activation in
neurons promotes expression of Bim, most likely because
JNK-dependent AP-1 activity is required for Bim expres-
sion (Whitfield et al. 2001). Moreover, JNK phosphory-
lates Bim on an activating site (Hubner et al. 2008), and
also causes the release of Bim from complexes with the
anti-apoptotic Bcl2 family protein Mcl-1 (Morel et al.
2009). Together, these processes initiate JNK-dependent
apoptosis. JNK inhibition can therefore prevent neuronal
cell death. Indeed, small molecule inhibitors of JNK cause
neuroprotection in models of neurodegenerative disease
(Borsello et al. 2003; Hirt et al. 2004; Repici et al. 2007;
Carboni et al. 2008; Esneault et al. 2008; Wiegler et al.
2008; Probst et al. 2011).
Activation of FoxO transcription factors can also cause
increased expression of autophagy-related genes, includ-
ing Atg8/Lc3b, Atg12, and Bnip3 (Salih and Brunet 2008).
While JNK cooperates with FoxO to increase proapoptotic
Bim expression (Whitfield et al. 2001), JNK deficiency
prevents induction of Bim expression (Fig. 3E) and pro-
motes a survival response that is mediated by increased
FoxO-dependent expression of the autophagy-related
target genes Atg8/Lc3b, Atg12, and Bnip3 (Figs. 3E,
5B–D). Indeed, inhibition of autophagy in JNK-deficient
neurons causes rapid death (Figs. 2C, 4C). This neuronal
survival response is relevant to stroke models in which
neuronal death is mediated by a JNK-dependent mech-
anism (Kuan et al. 2003; Pirianov et al. 2007).
Together, these data demonstrate that cross-talk be-
tween the FoxO and JNK signaling pathways leads to
neuronal death. In contrast, loss of JNK promotes FoxO-
induced survival mediated by increased autophagy. JNK
therefore acts as a molecular switch that defines the
physiological consequence of FoxO activation in neurons.
JNK is implicated in the induction of autophagy in
nonneuronal cells. However, JNK1 is constitutively acti-
vated in neurons, and these cells are refractory to JNK-
induced autophagy. Instead, JNK acts to suppress auto-
phagy in neurons by inhibiting FoxO-induced expression
of autophagy-related genes (e.g., Atg8/Lc3b, Atg12, and
Bnip3) and increasing the expression of proapoptotic
genes (e.g., Bim). JNK inhibition causes neuroprotection
that is mediated by loss of proapoptotic gene expression
and increased autophagy.
Materials and methods
We described Jnk1?/?mice (Dong et al. 1998), Jnk1LoxP/LoxPmice
(Das et al. 2007), Jnk2?/?mice (Yang et al. 1998), Jnk2M108G/M108G
mice (Jaeschke et al. 2006), and Jnk3?/?mice (Yang et al. 1997).
increased autophagy. Young adult (8-wk-old) Pcp2-Cre mice
(control) and Pcp2-Cre Jnk1LoxP/LoxPJnk2?/?Jnk3?/?mice
(JNKTKO) that express Cre recombinase selectively in cerebellar
Purkinje cells were examined. (A) Sections of the Purkinje cell
layer of Pcp2-Cre mice (control) and Pcp2-Cre Jnk1LoxP/LoxP
Jnk2?/?Jnk3?/?mice (JNKTKO) were examined by immunohis-
tochemical staining with antibodies to pSer246-FoxO1 and p62/
SQSTM1. Bar, 100 mm. (B) Sections of the Purkinje cell layer
of control and JNKTKOmice were stained with antibodies (to
Bnip3, LC3b, Atg12, and Calbidin-D28k) and examined by
fluorescence microscopy. Bar, 20 mm. (C) Sections of the DCN
of control and JNKTKOmice were stained with antibodies
(to Calbindin D-28k and LC3b) and examined by fluorescence
microscopy. Bar, 10 mm.
Compound JNK deficiency in Purkinje cells causes
Xu et al.
318 GENES & DEVELOPMENT
B6.129-Tg(Pcp2-cre)2Mpin/J mice (Barski et al. 2000), B6.Cg-
Tg(Nes-cre)1Kln/J mice (Tronche et al. 1999), and B6.129P2(Cg)-
Foxg1tm1(cre)Skm/J mice (Eagleson et al. 2007) were obtained from
The Jackson Laboratories. These mice were backcrossed to the
C57BL/6J strain (Jackson Laboratories) and were housed in a facil-
Care. The animal studies were approved by the Institutional
Animal Care and Use Committee of the University of Massachu-
setts Medical School.
Genomic DNA was examined by PCR analysis using primers to
identify wild-type and Jnk1?(Dong et al. 1998), Jnk1LoxP(Das
et al. 2007), Jnk1D(Das et al. 2007), Jnk2?(Yang et al. 1998),
Jnk2M108G(Jaeschke et al. 2006), Jnk3?(Yang et al. 1997), and
Cre+(Das et al. 2007) alleles.
Wild-type and Jnk1?/?Jnk2?/?MEFs (Tournier et al. 2000) were
cultured in Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal calf serum (Invitrogen). Primary cultures of
CGNs were prepared from postnatal day 6 mice (Kennedy et al.
2007). The CGNs were cultured 2 d in vitro with neurobasal
medium containing B27 supplements, 1% glutamine, 1% peni-
cillin/streptomycin, 25 mM glucose, and 25 mM KCl; seeded in
poly-D-lysine/laminin-coated chamber slides (Becton Dickenson)
or dishes (MatTek); and then infected with adenovirus-Cre
(Ad5CMVCre; ;100 multiplicity of infection) (Gene Transfer
Vector Core, University of Iowa) each day for 3 d. RNAi trans-
fection studies were performed using the PepMute siRNA trans-
fection reagent (SignaGen Laboratories) with 20 nM siRNA
(NM_019584 or NM_019739, Dharmacon RNA Technologies) at
7 d of culture in vivo (DIV) and again at 8 DIV. Some cultures
were treated with 1 mM 1-naphthylmethyl-4-amino-1-tert-butyl-
chem), 1 mM chloroquine (Sigma), or 5 mM roscovitine (LC
Laboratories). Neurons were also stained with calcein-am
ester (Calbiochem) and imaged by confocal fluorescence mi-
croscopy with a Leica SP2 instrument (Kennedy et al. 2007).
The expression of mRNA was examined by quantitative PCR
analysis using a 7500 Fast Real-Time PCR machine (Applied
Biosystems). TaqMan assays were used to quantitate Atg3
(Mm00471287_m1), Atg5 (Mm00504340_m1), Atg7 (Mm00512209_
m1), Atg8/Lc3b (Mm00782868_m1), Atg12 (Mm00503201_m1),
Beclin-1 (Mm01265461_m1), Bim (Mm01975020_s1), Bnip3
(Mm01275601_g1), FoxO1 (Mm00490672_M1), Gapdh
(4352339E), Kif3a (Mm00492876_m1), Kif5a (Mm00515258_m1),
Kif5b (Mm00515276_m1), and Kif5c (Mm00515265_m1) (Applied
Biosystems). The relative mRNA expression was normalized by
measurement of the amount of Gapdh mRNA in each sample
using TaqMan assays (Applied Biosystems).
Cell extracts were prepared using Triton lysis buffer (20 mM Tris
at pH 7.4, 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM
EDTA, 25 mM b-glycerophosphate, 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin and
leupeptin). Extracts (20–50 mg of protein) were examined by pro-
tein immunoblot analysis by probing with antibodies to LC3b
(Novus Biologicals); p62/SQSTM1; CDK-2 (Santa Cruz Biotech-
nology); AKT, pSer308-AKT, pSer473-AKT, Beclin-1, and Bcl-XL;
pThr389-S6Kand S6K (Cell Signaling); JNK1/2 (BD Biosciences
Pharmingen); phospho-neurofilament H (SMI-31R, Covance);
phospho-serine, Bnip3, and FoxO1 (Abcam); and a-Tubulin (Sigma).
The antibody to pSer246-FoxO1 was provided by Dr Azad Bonni
(Yuan et al. 2008). Immunecomplexes were detected by enhanced
chemiluminescence (NEN). Immunoblot analysis of immunopre-
cipitates was performed using the One-Step Complete Immuno-
precipitation-Western kit (Genescript Corp.).
Protein kinase assays
CDK2 activity was measured in an in vitro kinase assay using
Rb-C fusion protein (Cell Signaling) as the substrate, and was
quantitated using a PhosphorImager (Molecular Dynamics).
Primary CGNs were fixed by incubation with 4% paraformal-
dehyde for 1 h at room temperature and were permeabilized by
incubation with 90% methanol containing 5% acetic acid for
5 min at ?20°C. The slides were then blocked with 1% skim
milk in phosphate-buffered saline (PBS) for 1 h at room temper-
ature and incubated with antibodies to phospho-Ser63-cJun
(Cell Signaling), detyrosinated Tubulin, Synaptophysin, and
Tau (Chemicon); Ankyrin G and Lamp-1 (Santa Cruz Biotechnol-
ogy); Snap25 and FoxO1 (Abcam); LC3b (Novus Biologicals); and
bIII-Tubulin (Covance) in PBS supplemented with 1% skim milk
overnight at 4°C. Secondary antibodies were conjugated with
Alexa Fluor 488 or 546 (Molecular Probes) for 1 h at room
temperature. CGNs were loaded with 100 nM MitoTracker Red
(Molecular Probes) for 15 min at 37°C. All washed slides were
mounted with VectaShield mounting medium with DAPI (Vector
Laboratories) and were examined with a Leica SP2 laser-scanning
confocal fluorescence microscope.
Time-lapse fluorescence microscopy
The CGNs were cultured 12 d in vitro in poly-D-lysine/laminin-
coated 35-mm glass-bottom microwell dishes (MatTek) and
incubated with 100 nM MitoTracker Green (Molecular Probes)
for 3 min. Time-lapse fluorescence microscopy of CGN cells was
performed using a NikonTE2000-E2microscopewith a Yokogawa
CSU10b spinning-disk confocal scan head and custom laser
launch, acoustical optical tunable filter (NEOS), and relay optics
(Solamere Technology Group). Multiwavelength confocalZ-series
were acquired with a Nikon 603 Plan Apo oil objective (NA = 1.4)
and a QImaging Rolera MGi camera using the digitizer with
electron multiplication gain. Metamorph software controlled the
microscope hardware and image acquisition. The frames were
collected every 3 secs with an exposure time of 100 msec.
Cells and tissue were fixed with 1.25% glutaraldehyde for30 min
at room temperature and with 2.5% gluteraldehyde in cacody-
latebuffer for14 hat4°C.The cells werethen post-fixed with 1%
(w/v) osmium tetraoxide in PBS, dehydrated, and embedded in Lx
112/Araldite 502 epoxy resin. Ultrathin sections were mounted
on copper support grids in serial order, contrasted with lead
citrate and uranyl acetate, and examined on a Philips CM 10
transmission electron microscope (Gangwani et al. 2005). Quan-
titation of electron micrographs was performed by image anal-
ysis using the program AxioVision release 4.5 (Zeiss).
GENES & DEVELOPMENT 319
Immunohistochemical and immunofluorescence analysis
of tissue sections
Perfusion fixation of mice was performed using PBS supple-
mented with 4% (w/v) paraformaldehyde. Fixed tissues (24 h at
4°C) were processed and embedded in paraffin, and 4-mm sections
were prepared. These sections were stained with antibodies to
JNK1/2 (BD Biosciences Pharmingen), p62/SQSTM1 (Abnova), or
pSer246-FOXO1 (Yuan et al. 2008) using indirect immunoperox-
idase detection (Xu et al. 1998). Sections were also stained by
immunofluorescence after paraffin removal using antigen re-
trieval with antigen unmasking solution (Vector Laboratories)
and microwave irradiation. The sections were subsequently
blocked with 0.4% Triton X-100, 10% goat serum, 150 mM NaCl,
and 10 mM Tris-HCl (pH 7.4). Sections were incubated with
antibodies to Calbindin D-28k (Sigma), Bnip3 and Atg12 (Cell
Signaling), or LC3b (Novus Biologicals) for 12 h at 4°C and
washed. Immunecomplexes were detected by incubation with
secondary antibodies conjugated to Alexa Fluor 488 or 546
(Molecular Probes) for 1 h at 25°C. The slides were washed and
mounted with VectaShield mounting medium with DAPI (Vector
Laboratories) and examined with a Leica SP2 laser-scanning
confocal fluorescence microscope. Frozen sections (100 mm) of
the cerebellum were processed using the Rapid Golgi stain kit (FD
Differences between groups were examined for statistical signif-
icance using the Student’s test or analysis of variance (ANOVA)
with the Fisher’s test.
We thank Azad Bonni for providing the pSer246-FoxO1 antibody;
Tammy Barrett, Vicky Benoit, and Jian-Hua Liu for expert
technical assistance; and Kathy Gemme for administrative assis-
tance. These studies were supported by a grant from the National
Institutes of Health (NS054948). Core facilities at the University
of Massachusetts used by these studies were supported by the
NIDDK Diabetes and Endocrinology Research Center (DK32520).
R.J.D. is an Investigator of the Howard Hughes Medical Institute.
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