Corticotropin-Releasing Factor Receptors Differentially Regulate Stress-Induced Tau Phosphorylation
Hyperphosphorylation of the microtubule-associated protein tau is a key event in the development of Alzheimer's disease (AD) neuropathology. Acute stress can induce hippocampal tau phosphorylation (tau-P) in rodents, but the mechanisms and pathogenic relevance of this response are unclear. Here, we find that hippocampal tau-P elicited by an acute emotional stressor, restraint, was not affected by preventing the stress-induced rise in glucocorticoids but was blocked by genetic or pharmacologic disruption of signaling through the type 1 corticotropin-releasing factor receptor (CRFR1). Conversely, these responses were exaggerated in CRFR2-deficient mice. Parallel CRFR dependence was seen in the stress-induced activation of specific tau kinases. Repeated stress exposure elicited cumulative effects on tau-P and its sequestration in an insoluble, and potentially pathogenic, form. These findings support differential regulatory roles for CRFRs in an AD-relevant form of neuronal plasticity and may link datasets documenting alterations in the CRF signaling system in AD and implicating chronic stress as a risk factor in age-related neurological disorders.
Neurobiology of Disease
Corticotropin-Releasing Factor Receptors Differentially
Regulate Stress-Induced Tau Phosphorylation
Robert A. Rissman,
and Paul E. Sawchenko
Laboratory of Neuronal Structure and Function and
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies
and Foundation for Medical Research, La Jolla, California 92037
Hyperphosphorylation of the microtubule-associated protein tau is a key event in the development of Alzheimer’s disease (AD) neuro-
pathology. Acute stress can induce hippocampal tau phosphorylation (tau-P) in rodents, but the mechanisms and pathogenic relevance
of this response are unclear. Here, we find that hippocampal tau-P elicited by an acute emotional stressor, restraint, was not affected by
preventing the stress-induced rise in glucocorticoids but was blocked by genetic or pharmacologic disruption of signaling through the
type 1 corticotropin-releasing factor receptor (CRFR1). Conversely, these responses were exaggerated in CRFR2-deficient mice. Parallel
CRFR dependence was seen in the stress-induced activation of specific tau kinases. Repeated stress exposure elicited cumulative effects
on tau-P and its sequestration in an insoluble, and potentially pathogenic, form. These findings support differential regulatory roles for
CRFRs in an AD-relevant form of neuronal plasticity and may link datasets documenting alterations in the CRF signaling system in AD
and implicating chronic stress as a risk factor in age-related neurological disorders.
Key words: Alzheimer’s disease; antalarmin corticotropin-releasing factor; corticotropin-releasing hormone; hippocampus; restraint
stress; tau phosphorylation
Alzheimer’s disease (AD) is defined neuropathologically by the
-amyloid plaques and neurofibrillary tangles
(NFTs), the latter consisting of hyperphosphorylated forms of
the microtubule-associated protein tau. Hyperphosphorylated
tau exhibits reduced ability to bind and stabilize microtubules
and can self-aggregate to form insoluble paired helical filaments
(PHFs), which comprise NFTs (Gustke et al., 1992; Bramblett et
al., 1993; Alonso et al., 1996). The incidence of NFTs is positively
correlated with cognitive deficit and neuronal loss in AD (Arria-
gada et al., 1992; Gomez-Isla et al., 1997), and the discovery that
mutations in the tau gene underlie autosomal dominant forms of
frontotemporal dementia suggests that pathological changes in
tau can serve as a principal cause of neurodegeneration and cog-
nitive impairment (Hutton et al., 1998; Poorkaj et al., 1998; Spill-
antini et al., 1998).
Exposure to a range of environmental insults, or stresses, can
activate tau kinases and induce tau phosphorylation (tau-P) in
the rodent CNS (Korneyev et al., 1995; Papasozomenos, 1996;
Korneyev, 1998; Yanagisawa et al., 1999; Planel et al., 2001, 2004;
Arendt et al., 2003; Feng et al., 2005). This effect has been re-
ported consistently in the hippocampal formation, a key struc-
ture in learning and memory, and the initial site of tau pathology
in AD (Braak and Braak, 1991). Although acute stress-induced
tau-P is reversible, the mechanisms that govern this phenomenon
are unknown, and it is unclear whether and how it may be man-
ifest under chronic stress conditions. Addressing these questions
may better define the elusive links between the stress axis and
AD-related pathogenic processes, as increased exposure and/or
sensitivity to stress in humans and rodent models confers in-
creased risk of dementia and AD neuropathology (Wilson et al.,
2003; Jeong et al., 2006).
Warranting consideration in this respect are glucocorticoids,
dominant stress hormones whose elevated levels in aging have
been linked to increased neuronal vulnerability in hippocampus
(Sapolsky et al., 1985, 1986). However, acute stress-induced
tau-P is reportedly unaffected in adrenalectomized mice (Kor-
neyev et al., 1995), suggesting that glucocorticoid secretion may
not be pivotally involved. Alternatively, the corticotropin-
releasing factor (CRF) signaling system plays an essential role in
initiating pituitary–adrenal responses to stress and has been im-
plicated as a transmitter/modulator in CNS systems that mediate
complementary autonomic and behavioral adjustments, earning
consideration as a general mediator/integrator of stress adapta-
tions (Chadwick et al., 1993). CRF and related ligands (urocort-
ins 1–3) exert their biological effects via two G-protein-coupled
receptors [CRF receptor 1 (CRFR1) and CRFR2] that are differ-
entially distributed in brain (Van Pett et al., 2000), and exert
convergent effects on a range of stress-related endpoints (Bale
and Vale, 2004). CRFR ligands can confer neuroprotection, in
Received Nov. 29, 2006; revised May 10, 2007; accepted May 11, 2007.
This work was supported by National Institutes of Health Grant DK026741, the Clayton Medical Research Foun-
dation, and the Ellison Medical Foundation and American Federation for Aging Research (R.A.R.). K.-F.L., W.V., and
P.E.S. are senior investigators of the Clayton Medical Research Foundation. We thank Drs. J. Serrats, D. Brown, Z. F.
Yuan, and J. Radley for helpful discussions and K. Trulock and C. Arias for technical assistance. We also thank Dr. G.
Chrousos for providing antalarmin, Dr. R. Valentino for antalarmin protocols, Dr. P. Davies for the PHF-1 antibody,
and Dr. G. Drewes for permission to reproduce portions of his graphic incorporated in Figure 8.
Correspondence should be addressed to either Robert A. Rissman or Paul E. Sawchenko, Laboratory of Neuronal
Structure and Function, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037,
E-mail: email@example.com or firstname.lastname@example.org.
Copyright © 2007 Society for Neuroscience 0270-6474/07/276552-11$15.00/0
6552 • The Journal of Neuroscience, June 13, 2007 • 27(24):6552– 6562
vitro, by altering amyloid precursor protein processing and sup-
pressing tau kinases, and reduced central CRF expression has
been documented early in AD progression (Rehman, 2002;
Bayatti and Behl, 2005). Nevertheless, the nature of any associa-
tion between the CRF system and AD-related pathogenesis re-
mains to be elucidated. Here, we provide evidence to support a
push–pull involvement of CRFRs in regulating stress-induced
tau-P and tau kinase activity in murine hippocampus.
Materials and Methods
CRFR knock-out mice. Mutant mice and littermate wild-type (wt) con-
trols were bred from heterozygote breeder pairs of established lines back-
crossed to founder mice to achieve a pure C57BL/6 background (Smith et
al., 1998; Bale et al., 2000). Genotype was determined by PCR, and males
were used for experimentation at 15–22 weeks of age. Pregnant females
used for generating CRFR1-deficient mice received drinking water sup-
plemented with corticosterone (10
g/ml; Sigma-Aldrich, St. Louis,
MO) from embryonic day 12 to postnatal day 14 to prevent early mor-
tality as a result of pulmonary dysplasia (Smith et al., 1998). Because
mice exhibit adrenal cortical agenesis, experimental animals
were reinstated on corticosterone for 21 d before testing to allow the
normal nocturnal bias in appetitive behavior to approximate the circa-
dian fluctuation in circulating hormone levels. To assess effectiveness of
the replacement regimen, plasma corticosterone levels were determined
by RIA from blood samples collected when the mice were killed. The Salk
Institute Institutional Animal Care and Use Committee approved all
Adrenalectomy and corticosterone replacement. Twelve-week-old male
wt C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) underwent
adrenalectomy (ADX) via bilateral incisions on the dorsolateral flanks
under isoflurane anesthesia. ADX mice received replacement corticoste-
g/ml) in drinking water containing 0.9% saline immediately
after surgery. Animals were used in stress experiments 21 d after surgery.
Restraint stress. Acute restraint stress involved placing mice in venti-
lated 50 ml conical tubes for 30 min; repeated stress involved 14 consec-
utive daily exposures. Animals were killed at various intervals ranging
from 20 min to 24 h after stress. Control mice were handled comparably
but were not otherwise manipulated.
Intracerebroventricular injections. CRFR1
along with age-matched wt controls (n ⫽ 3/group), were anesthetized
with isoflurane and implanted stereotaxically with 26 ga guide cannulas
(Plastics One, Wallingford, CT) aimed to terminate above the lateral
ventricle. Cannulas were affixed to the skull with dental acrylic adhering
to jeweler’s screws partially driven into the skull and sealed with stylets.
After7dofrecovery, stylets were replaced with 33 ga injection cannulas,
and 2 h later the animals were remotely injected with 0.5
g of synthetic
mouse/human CRF in 2
l of saline, or vehicle alone, over ⬃1 min. To
approximate the time frame used in acute stress experiments, animals
were killed 40 min after intracerebroventricular injection and perfused
for immunohistochemistry, as described below. CRF was provided by
Dr. J. Rivier (Salk Institute).
In vivo pharmacology. The small-molecule CRFR1-selective antagonist
antalarmin (Webster et al., 1996) was administered (20 mg/kg, i.p. injec-
tion) 20 min before stress exposure. All animals were handled twice daily
for 28 d before experimentation and received daily mock intraperitoneal
injections to minimize stress of injection at testing. Antalarmin was sol-
ubilized in equal volumes of absolute ethanol and Cremaphor EL
(Sigma-Aldrich), as described previously (Webster et al., 1996; Pernar et
al., 2004). This stock solution was diluted in prewarmed (50°C) distilled
water and adjusted to a final concentration of 4 mg/ml immediately
Western blot analysis. Mice were anesthetized with sodium pentobar-
bital (40 mg/kg), which has been demonstrated to not influence tau-P
over the time frame used here (Papasozomenos, 1996). After sedation,
animals were decapitated, and the hippocampus was rapidly dissected
and frozen on dry ice. Hippocampal tissues were homogenized in radio-
immunoprecipitation assay (RIPA) buffer (50 m
M Tris-HCl, pH 7.4,
0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 150 m
M NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM Na
, and 1
M okadaic acid). Before
homogenization, protease inhibitors PMSF, NaF (1 m
M), aprotinin, leu-
peptin, and pepstatin (1
g/ml each) were added. RIPA fractions were
obtained by centrifuging twice at 40,000 ⫻ g for 20 min, and the super-
natant was collected. For analysis of tau solubility (repeated stress), se-
quential fractionation of RAB and RIPA extracts were performed as de-
scribed previously (Higuchi et al., 2002; Kraemer et al., 2003). In this
case, tissues were first homogenized in high-salt RAB (0.1
M MES, 0.75
NaCl, 1 m
M EGTA, and 0.5 mM MgSO
) and centrifuged at 40,000 ⫻ g for
40 min. The supernatant was collected (soluble RAB fraction), and pellets
were resuspended in RIPA buffer to obtain detergent-soluble fractions.
Protein concentrations were determined using a BCA Protein Assay Kit
(Pierce Biotechnology, Rockford, IL). Proteins were then boiled in sam-
ple buffer containing SDS, BME, and glycerol at 95°C for 5 min. Six
micrograms of protein were then separated by 12% SDS-PAGE. Proteins
were transferred to nitrocellulose membrane (0.2
m; Bio-Rad, Her-
cules, CA) and incubated in primary antibodies diluted in 5% milk-
PBS-T overnight at 4°C. Primary antibodies were detected with either
anti-mouse or -rabbit horseradish peroxidase-linked secondary antibod-
ies (1:1000; EMD Biosciences, La Jolla, CA) and developed with an en-
hanced chemiluminescence Western blot detection kit (Supersignal
West Pico; Pierce Biotechnology). Background subtraction was per-
formed, and quantitative band intensity readings were obtained using
NIH Image software.
Antibodies. Well characterized phospho-specific antibodies were used
for detection of phosphorylated residues on mouse tau. For Western
(1:1000; Biosource, Camarillo, CA),
(1:500; AT8; Pierce Biotechnology), and S
PHF-1; gift from Dr. P. Davies, Albert Einstein College of Medicine,
Bronx, NY). These antibodies were chosen based on their ability to re-
solve target bands at the appropriate molecular weight for phosphory-
lated tau (i.e., ⬃50–75 kDa). Phospho-specific antibodies against S
, and S
phosphorylated tau (Biosource) were also tested but
were found to resolve only very high-molecular-weight target bands (i.e.,
⬎100 kDa) and were therefore excluded from the analysis. Antibody
PHF-1 (1:500) was used for detection of phosphorylated tau in immu-
nohistochemical analyses. Specificity of PHF-1 in mouse tissue was con-
firmed by pretreating sections from stressed mice with alkaline phospha-
tase (40 mg/ml; Sigma-Aldrich), which eliminated detectable PHF-1
labeling in all experimental groups (data not shown). For assessment of
tau kinases, specific antibodies to phosphorylation sites or activator pro-
teins were used. This included total glycogen synthase kinase-3
; 1:2500; BD Biosciences, San Diego, CA), activated GSK-3
1:1000; BD Biosciences), inactive GSK-3
; 1:1000; Cell Signaling
Technology, Danvers, MA), cyclin-dependent kinase 5 (cdk5; 1:1000;
EMD Biosciences), cdk5 activator proteins, p25 and p35 (1:1000; Santa
Cruz Biotechnology, Santa Cruz, CA), phosphorylated c-Jun-N-terminal
kinase (JNK; 1:1000; Cell Signaling Technology), mitogen-activated pro-
tein (MAP) kinases [extracellular signal-regulated kinases 1 and 2
(ERK1/2); 1:500; Cell Signaling Technology], and the catalytic subunit of
protein phosphatase 2A (PP2A-c; 1:5000; BD Biosciences).
2000; Sigma-Aldrich) was used as a control for protein loading.
Immunohistochemistry. Mice were perfused with 4% paraformalde-
hyde as described previously (Bittencourt and Sawchenko, 2000). Thirty-
micrometer-thick frozen sections were cut on a sliding microtome and
stored at ⫺20°C in cryoprotectant solution (20% glycerol and 30% eth-
ylene glycol in 0.1
M phosphate buffer). PHF-1 was used to detect tau-P in
free-floating sections containing hippocampus using Mouse-on-Mouse
Immunodetection Kit reagents (Vector Laboratories, Burlingame, CA)
to avoid detection of endogenous mouse Ig. Endogenous peroxidase was
quenched with 0.3% hydrogen peroxide, followed by 1% sodium boro-
hydride to reduce free aldehydes. Reaction product was developed using
a nickel-enhanced glucose oxidase method (Shu et al., 1988).
Statistical analyses. Integrated intensity readings from Western blots
were analyzed using either a one- or two-way ANOVA using Prism4
software (GraphPad, San Diego, CA). Resultant data were plotted on bar
graphs, with data expressed as mean ⫾ SEM percentage of control values.
Rissman et al. • CRF Receptors and Tau Phosphorylation J. Neurosci., June 13, 2007 • 27(24):6552– 6562 • 6553
Time course of tau-P after acute restraint stress
We initially determined whether increased tau-P was observable
in response to acute restraint, an acknowledged “emotional”
stressor (Sawchenko et al., 2000). Western analysis was used to
examine tau-P at several AD-relevant N- and C-terminal sites
] in hippocampal extracts from C57BL/6 mice killed at vari
ous intervals after a single 30 min episode of restraint stress.
Relative to basal (nonstressed) values, all sites exhibited signifi-
cant increases in phosphorylation that were apparent at the ter-
mination of stress (0 min), with peak elevations (2- to 10-fold)
achieved 20 – 40 min later and sustained through 60 min (Fig. 1).
By 90 min, levels were reduced to or near those of unstressed
controls. These results demonstrate that a representative emo-
tional stressor induces rapid and reversible increases in tau-P at
multiple AD-relevant sites. Increments in tau-P were quite stable
over 20– 60 min after stress, and the 20 min time point was se-
lected for subsequent analyses.
Glucocorticoid involvement in stress-induced
Because of the dominant role of glucocorticoids in mediating
stress effects and their implication in neuronal loss (Sapolsky et
al., 1985, 1986) and pathology in mouse models of AD (Green et
al., 2006), we tested whether stress-induced tau-P was dependent
on stress-induced glucocorticoid secretion. Although a previous
study used immunoassay and found no effect of ADX on PHF-1
reactivity after acute cold water stress (Korneyev et al., 1995),
phosphatase inhibitors were not used, and only soluble fractions
of tau protein were examined. We examined stress-induced tau-P
responses at the AT8 and PHF-1 sites in hippocampal extracts
from ADX and control mice exposed to acute restraint stress (Fig.
2). Robust stress-induced tau-P responses were observed in ADX
mice that did not significantly differ from those of intact controls
at either the AT8 or PHF-1 phosphorylation sites (each p ⬎ 0.10).
These results confirm that acute stress-induced tau-P is not de-
pendent on glucocorticoid secretion.
CRFRs differentially regulate stress-induced
The CRF family of signaling molecules is broadly involved in
physiological and behavioral responses to stress (Chadwick et al.,
1993) and undergoes alterations early in AD progression (Davis
et al., 1999). However, the nature of its involvement in AD neu-
ropathology is unclear. We next investigated the role of CRFRs in
acute stress-induced tau phosphorylation using mice deficient in
CRFR1 (Smith et al., 1998) or CRFR2 (Bale et al., 2000). Western
analyses indicated a tendency for CRFR1
mice to exhibit
higher basal levels of tau-P than unstressed wt animals at several
sites, although this difference was statistically reliable only at the
AT8 epitope ( p ⬍ 0.01) (Fig. 3). More importantly, CRFR1-
deficient mice failed to exhibit significant increases in tau-P at
any site at 20 min after stress, compared with age-matched wt
controls. In contrast, CRFR2 knock-outs displayed normal basal
levels of tau-P ( p ⬎ 0.05 vs wt) but showed robust responses to
acute stress that commonly exceeded those seen in wt animals.
Specifically, phosphorylation responses of CRFR2
significantly greater than those of wt animals at the S
( p ⬍
( p ⬍ 0.001), T
( p ⬍ 0.05), T
( p ⬍ 0.05), and
PHF-1 ( p ⬍ 0.01) sites. Phosphorylation responses of CRFR2
null mice at the AT8 and S
sites did not differ significantly
from those of wt stressed animals ( p ⬎ 0.05).
To probe the localization of tau-P responses, immunohisto-
chemical methods were used to examine the distribution of
PHF-1 reactivity and its stress and CRFR dependence. Immuno-
labeling results were highly compatible with biochemical data in
showing prominent upregulation of PHF-1 staining in wt mice in
response to stress, which was attenuated and exaggerated in
CRFR1- and CRFR2-deleted animals, respectively (Fig. 4A). In
the dentate gyrus of stressed wt animals, PHF-1 positive cell bod-
ies were seen primarily in the hilus (polymorph and subgranular
regions), but also in deep aspects of the granule cell layer. We also
Figure 1. Time course of stress-induced tau phosphorylation. Western blots of hippocampal
extracts probed for tau-P at select AD-relevant sites in unstressed controls (C) and mice killed 0,
signal under basal conditions was low but rose immediately after stress to levels that were
markedly increased (2- to 10-fold) over control levels through 60 min and diminished thereaf-
ter. The 20 min poststress time point was adopted for use in subsequent experiments.
was used as a loading control.
Figure 2. Role of glucocorticoids in stress-induced tau-P. Western blot analysis of hip-
pocampal tau-P at the AT8 and PHF-1 sites under control (C) and acute stress (S) conditions in
intensity values of intact unstressed controls, reveals that intact and ADX animals manifest
comparably robust stress-induced increments in tau-P at each site, whose magnitude did not
differ significantly. **Differs significantly from intact, unstressed controls ( p ⬍ 0.001); ns,
nonsignificant ( p ⬎ 0.05). n ⫽ 3 mice per condition.
-Actin was used as a loading control.
6554 • J. Neurosci., June 13, 2007 • 27(24):6552– 6562 Rissman et al. • CRF Receptors and Tau Phosphorylation
observed PHF-1 positive mossy fibers and a band of punctate
(presumably axonal) elements in the inner third of the molecular
layer. In Ammon’s Horn, dominant features included labeled
perikarya scattered mainly throughout the pyramidal layer and
proximal dendritic zones and bands of axon terminal-like puncta
engulfing the pyramidal cell layer and in stratum lacunosum-
moleculare. Radially oriented processes, some traceable to la-
beled cell bodies and presumably representing dendritic labeling,
were seen in stressed wt and CRFR2
animals. Alterations in
immunostaining as a function of stress and genotype were man-
ifest as differences in the number and/or intensity of labeled ele-
ments, with no discernible differences in distribution.
To determine whether CRF is capable of independently elic-
iting hippocampal tau-P, wt and CRFR-deficient mice were im-
planted with lateral ventricular cannulas for intracerebroventric-
ular injection. Resultant PHF-1 immunoreactivity was examined
40 min after administration of CRF or saline vehicle (Fig. 4 B).
The general pattern of results was similar to that observed in
response to stress, in that wt and CRFR2
mice treated with
peptide displayed robust increases in hippocampal tau-P,
whereas labeling in CRFR1
animals was comparable with the
low level seen in saline-injected controls. In the dentate gyrus,
PHF-1 positive cell bodies were seen in the hilus and deep aspects
of the granule cell layer (Fig. 4 B). In Ammon’s Horn, we ob-
served PHF-1 labeling in mossy fibers and in the form of punctate
pericellular labeling throughout the pyramidal cell layer and,
more sporadically, in the dendritic zone, with particular concen-
tration at the septal pole of the hippocampus (data not shown). In
contrast to stress effects, however, we did not observe labeling of
pyramidal neurons or their processes after intracerebroventricu-
lar CRF injections. These findings indicate that central CRF ad-
ministration at least partially recapitulates the effects of stress and
demonstrates a similar dependence on CRFR integrity.
Effects of pharmacologic blockade of CRFR1
Interpretation of data derived from conventional knock-out an-
imals may be complicated by developmental or indirect effects of
lifelong lack of expression of the targeted gene. This is particularly
true of CRFR1-deficient mice, which exhibit chronically im-
paired pituitary–adrenal function (Smith et al., 1998). Despite
efforts to mitigate such effects by steroid replacement perinatally
and immediately before experimentation (see Materials and
Methods), confidence in the assertion of a regulatory role for
CRFR1 in this context would be bolstered if the effects were
maintained in the face of acute disruption of receptor function.
We therefore assessed the effect of antalarmin, a small-molecule,
selective CRFR1 antagonist (Webster et al., 1996), on basal and
stress-induced tau-P. Neither antalarmin nor the administration
vehicle significantly altered basal levels of tau-P at the AT8 or
PHF-1 sites, relative to untreated controls (Fig. 5). However, an-
talarmin treatment prevented stress-induced increments in
phosphorylation at both sites (lanes 5– 6; each p ⬎ 0.10 vs un-
treated controls). Phosphorylation responses in stressed, vehicle-
treated animals were comparable with those of stressed, un-
treated controls and significantly elevated over vehicle control
levels ( p ⬍ 0.01). These findings support a specific involvement
of CRFR1 signaling in stress-induced tau-P.
Modulation of tau kinase activity by stress and CRFRs
To identify potential mediators of acute stress-induced tau-P, we
used antibodies specific to active and inactive states of kinases
implicated in tau-P in the same tissue extracts used above to
interrogate changes in stress-induced kinase activation and their
Figure 3. Differential regulation of stress-induced tau-P by CRFR status. Phosphorylation
responses of seven AD-relevant tau sites in hippocampal extracts from wt, CRFR1
mice killed at 20 min after acute restraint stress (S) or no treatment (C). Quantitative
analysis revealed that wt animals displayed the expected increases in tau-P after stress at all
animals displayed elevated basal levels of phosphorylation at the AT8 and
PHF-1 sites but did not manifest significantly increased responses after stress at any site. Con-
mice showed normal basal levels of tau-P at all sites but exaggerated
responses to stress at five of the seven epitopes assayed. Data are presented as mean ⫾ SEM
percentageofwtcontrolvalues.*Differssignificantlyfromunstressedwtcondition( p ⬍ 0.01);
**p ⬍ 0.001;
differs from wt stressed group ( p ⬍ 0.05). n ⫽ 3 mice per condition.
was used as a loading control.
Rissman et al. • CRF Receptors and Tau Phosphorylation J. Neurosci., June 13, 2007 • 27(24):6552– 6562 • 6555
CRFR dependence (Fig. 6). Several kinases examined, including
the active (phosphorylated at Y
but not the inactive
), or total (unphosphorylated) form of GSK-3
, the pT
form of the 46 and 54 kDa c-Jun N-terminal protein kinases
(JNK46/54), and the pT
form of the mitogen-activated
protein kinases, ERK2, but not ERK1, displayed upregulation in
response to acute restraint. The time courses of stress effects seen
on these kinases were similar to those shown in Figure 1 for
stress-induced tau-P (data not shown). Relative levels of cdk5
were unchanged from steady state over the poststress intervals
examined, but one of its regulatory proteins, p35, was robustly
upregulated. We were unable to reproducibly detect the p35-
truncated product, p25.
When tested in hippocampal extracts from wt and knock-out
mice, each of the stress-responsive kinase forms or regulators also
exhibited modulation as a function of CRFR status that mirrored
some or all of the effects of genotype on restraint-induced tau-P.
The activated (pY
) form of GSK-3
, implicated in phosphor
ylating tau at S
, and PHF-1 sites, was most similar in
that the stress-induced increment seen in wt mice was not evident
animals and was exaggerated in CRFR2
Phosphorylation responses of both JNK isoforms were also sig-
nificantly greater in CRFR2 knock-outs than in wt controls ( p ⬍
0.05). These kinases also exhibited pronounced elevations in
basal phosphorylation in CRFR1-deficient mice, whose magni-
tude rivaled or exceeded stress-induced levels in wt mice. This
may relate to the elevated tau-P levels seen under this condition at
the AT8 and PHF-1 sites, although less-marked elevations of
and ERK2, and of p35 levels, in un-
mice may also contribute in this regard.
Overall, these results identify several tau kinases as potential ef-
fectors of CRFR-dependent effects of acute emotional stress on
Tau phosphorylation in response to repeated stress
Because past and present data characterize acute stress-induced
tau-P as a transient phenomenon, its relevance to neuropathol-
ogy may be questioned. Data from animal models and AD brain
have demonstrated that NFTs and other manifestations of tau
pathology are dependent on aberrantly phosphorylated tau being
sequestered into insoluble cellular fractions (Iqbal et al., 1994).
We therefore used sequential fractionation in the absence (RAB
buffer; see Materials and Methods) and presence (RIPA buffer) of
detergents to compare the persistence and solubility of phos-
phorylated tau in animals subjected to acute versus repeated (14
consecutive daily exposures) restraint stress (Fig. 7). Groups of
mice in each condition were killed 20 min or 24 h after their final
or only stress episode. Results from the acute stress condition
replicated findings detailed above in showing increased phos-
phorylation at the AT8 and PHF-1 sites 20 min after stress. At
24 h after stress, relative levels of tau-P were indistinguishable
from unstressed animals (lane 3). In terms of solubility, phos-
phorylated tau induced by acute stress was detected only in the
soluble fraction; that is, no additional signal was evident after
Figure 4. Cellular localization of stress- and peptide-induced tau-P. A, Immunoperoxidase staining for PHF-1 immunoreactivity in the dentate gyrus (top) and CA1 field (bottom) of mouse
hippocampus as a function of stress status and genotype. Phosphorylation is localized to distinct perikaryal/dendritic and axonal elements (see Results) and, importantly, varies with stress exposure
and CRFR status in a manner identical to that seen by Western analysis (Fig. 3). B, PHF-1 in the dentate gyrus of mice injected intracerebroventricularly with synthetic CRF. Tau-P varied as a function
of genotype in a manner similar to that seen in stressed animals (A). gr, Granule cell layer; mol, molecular layer; pyr, pyramidal layer; slm, stratum lacunosum-moleculare; sr, stratum radiatum.
6556 • J. Neurosci., June 13, 2007 • 27(24):6552– 6562 Rissman et al. • CRF Receptors and Tau Phosphorylation
further extraction with detergent (lanes 6 – 8). In contrast, under
repeated stress conditions, comparably elevated AT8 and PHF-1
signal were present in soluble fractions at both 20 min and 24 h
after the final restraint episode. In addition, extraction of
detergent-soluble proteins (RIPA) revealed significant occur-
rence of phosphorylated tau at both time points (lanes 9 –10).
These results suggest that repeated stress leads to chronic eleva-
tions in phosphorylated tau and a shift in its disposition toward
more insoluble, and potentially pathogenic, forms.
Phosphatase involvement in restraint-induced
Alterations in tau phosphatase activity have been implicated as
contributing to stress-induced tau-P (Planel et al., 2001, 2004,
2007). To determine whether similar changes are associated with
acute or repeated restraint stress, we analyzed the same extracts
used in the preceding analysis (Fig. 7), for alterations in the cat-
alytic subunit of the dominant tau phosphatase, PP2A (PP2A-c).
We found no evidence of PP2A-c in detergent-soluble RIPA frac-
tions under any experimental condition (data not shown). In
soluble RAB fractions, relative levels of PP2A-c from hippocampi
of acute stressed mice did not differ reliably from controls (lanes
1–3) but were significantly elevated at both 20 min ( p ⬍ 0.001)
and 24 h ( p ⬍ 0.01; lanes 4 –5; see supplemental Fig. 1, available
at www.jneurosci.org as supplemental material) after repeated
stress. Although additional characterization is needed, these find-
ings identify PP2A as a potential contributor to alterations in
tau-P under repeated stress conditions.
Our findings extend the range of insults that provoke tau-P to
include a representative emotional stressor and provide evidence
on its mechanism (Fig. 8). We fail to implicate stress-induced
glucocorticoid secretion in this phenomenon but provide data
from pharmacologic and genetic manipulation to support a dif-
ferential involvement of CRFRs. The abrogation of stress-
induced tau-P in CRFR1-deficient animals and the enhancement
observed in CRFR2 mutants were paralleled by altered activities
of specific tau kinases. Furthermore, the demonstration that re-
peated stress exerts cumulative increases in tau-P and results in its
sequestration in insoluble forms defines a possible underpinning
for evidence implicating chronic stress as a risk factor in age-
related neurological disorders, including AD.
Relationship to previous studies
Tau normally binds and stabilizes neuronal microtubules, facili-
tating their roles in cellular structure, polarity, and transport
(Stamer et al., 2002). Phosphorylation can disrupt these activities
and promote cytoskeletal destabilization (Sengupta et al., 1998).
Aberrantly phosphorylated forms of tau aggregate into PHFs, and
these aggregate into insoluble NFTs, a defining feature of AD
(Kopke et al., 1993). In this light, the observation that acute stress
results in tau-P at AD-relevant sites defines one potential means
by which stress exposure may translate into neuropathology. This
effect has been elicited by a range of strenuous challenges, includ-
ing heat shock (Papasozomenos, 1996), starvation (Yanagisawa
et al., 1999; Planel et al., 2001), forced swimming in cold water
(Korneyev et al., 1995; Okawa et al., 2003; Feng et al., 2005; Yo-
shida et al., 2006), glucoprivation (Planel et al., 2004), ether in-
halation (Ikeda et al., 2007), and hibernation (Arendt et al., 2003;
Hartig et al., 2007). Two broad categories of stress models are
now commonly recognized. These may be termed “physiologi-
cal” and “emotional” and are distinguished by the sensory mo-
dalities that register the challenges, the patterns of neuronal acti-
vation they induce in brain, the extent to which they invoke
affective responses, and the circuitry that mediates adaptive re-
sponses to them (Sawchenko et al., 1996; Watts, 1996; Herman
and Cullinan, 1997; Dayas et al., 2001). Whereas the stressors
shown previously to elicit tau-P fall mainly or exclusively in the
physiological category, restraint is a prototypic emotional stres-
sor. Because established models of anxiety, fear, and social stress
share key features with restraint such as a capacity to engage a
stereotyped set of interconnected limbic forebrain cell groups
(Duncan et al., 1996; Campeau et al., 1997; Martinez et al., 2002),
the present findings suggest that the generality of stress-induced
tau-P may extend into the realm of stresses encountered in every-
Several challenges that elicit tau-P are associated with marked
reductions in body temperature (7–10°C), which can differen-
tially modulate tau kinase and phosphatase activities, leading
Planel et al. (2001, 2004) to hypothesize that hypothermia may be
a common underlying mechanism. Restraint also results in re-
duced core temperature, although of lesser magnitude (0.5–2°C)
(Clement et al., 1989; Turek and Ryabinin, 2005; Meijer et al.,
2006). It remains to be determined whether the correlation be-
tween body temperature and tau-P noted in other paradigms
(Planel et al., 2007) extends to emotional stressors.
Whereas human tau is normally phosphorylated at 2–3 mol/
mol of protein, PHF-tau from AD brain is hyperphosphorylated
at a 7–10 molar ratio (Kopke et al., 1993). Murine tau can be
phosphorylated and form PHFs in vitro (Kampers et al., 1999).
We find that restraint provokes tau-P at each of seven epitopes
examined, all but one of whose responses is differentially modu-
lated by CRFR status. Coupled with the finding that central CRF
administration stimulates PHF-1 phosphorylation in a CRFR1-
dependent manner, we suggest a mechanism involving media-
Figure 5. CRFR1 antagonist blocks stress-induced tau-P. Quantitative Western blot analysis
oftau-PattheAT8andPHF-1sitesin hippocampal extracts from micepretreatedwithvehicle or
the small-molecule CRFR1 antagonist antalarmin and subjected to acute stress or no additional
treatment,expressedas a percentage of levels in untreated controls (C). At each site, antagonist
treatment did not affect basal levels of phosphorylation but blocked the stress-induced incre-
ment. The vehicle used for drug administration had no significant effect under basal or stress
conditions. **Differs significantly from unstressed controls ( p ⬍ 0.001);
Differs from un
treated stressed group ( p ⬍ 0.05). n ⫽ 3 percondition.
-Actin was used as aloading control.
Rissman et al. • CRF Receptors and Tau Phosphorylation J. Neurosci., June 13, 2007 • 27(24):6552– 6562 • 6557
tion by CRFR1, which is normally restrained by CRFR2-based
Complementary monitoring of the activation status of tau ki-
nases identified several candidate effectors. GSK-3
has been im-
plicated in catalyzing tau-P at S
, and, to a lesser
, and confers PHF-like changes (Liu et al., 2003).
Its activity is stimulated or inhibited by phosphorylation at Y
werealso stress responsive, and more so inCRFR2 knock-outs; these kinases were distinguished
by very high basal phosphorylation levels in CRFR1
mice, which may relate to elevated
tau-P observed in this condition at the AT8 site (see Fig. 3). Levels of phospho-ERKs, particularly
ERK1, were relatively unresponsive. Levels of cdk5 were stable across the conditions in force
here,butits p35 activator protein was strongly upregulated by stress inwt andCRFR2
Data are presented as mean ⫾ SEM percentage of unstressed wt values. *Differs significantly
from wt control ( p ⬍ 0.01); **p ⬍ 0.001.
Differs significantly from wt stress condition ( p ⬍
-Actin was used as a loading control.
Figure 6. Modulation of tau kinase activity by stress and CRFR status. Western blot analyses
using phosphoepitope-specific antibodies to interrogate stress- and/or genotype-dependent
variations in the activation state of GSK-3, JNK46/54, and ERK1/2; relative levels of cdk5 and its
activator protein, p35, were also evaluated. The behavior of each kinase group recapitulates
aspects of the general pattern of tau-P responses in the same design. Thus, the activated
) form of GSK-3
is upregulated by stress in wt mice, and thisresponse is blocked CRFR1-
deficient animals and exaggerated in CRFR2 mutants. The inhibitory (pS
) form and total (un
phosphorylated) GSK3 are unresponsive over these conditions. Both phospho-JNK isoforms
Figure 7. Repeated stress results in chronic elevations and reduced solubility of phosphor-
ylated tau. Western analysis of hippocampal tau-P at the PHF-1 and AT8 sites of mice killed 20
min or 24 h after 30 min acute restraint or 14 consecutive daily exposures. Analysis was per-
formed on both soluble and detergent-soluble fractions. Under acute stress (AS) conditions,
tau-P is transient and contained wholly within the soluble fraction, whereas repeated stress
(RS) results in elevated levels of tau-P at both time points and the appearance of a significant
portion of phospho-tau in the detergent-soluble fraction. These data suggest that the effects of
repeatedemotional stress on tau-P are cumulative andassociatedwith increased sequestration
in the cellular fraction known to contain the bulk of PHFs in the AD brain. Note that for this
analysis, data are expressed as mean ⫾ SEM optical density, rather than as a percentage of
controlvalues, to reflect thelackof detectable signal indetergent-solubleextracts under control
conditions. *Differs significantly from control ( p ⬍ 0.01); **p ⬍ 0.001.
-Actin was used as a
loading control. C, Control.
6558 • J. Neurosci., June 13, 2007 • 27(24):6552– 6562 Rissman et al. • CRF Receptors and Tau Phosphorylation
, respectively (Cohen and Frame, 2001). We found stress-
induced increments in activated GSK-3
whose time course and
genotype dependence paralleled tau-P responses, without signif-
icant variation in the inhibitory form (cf. Okawa et al., 2003) or
total (unphosphorylated) GSK3 levels. The signaling intermedi-
ates that link CRFR ligand binding to alterations in GSK3 activity
are unclear. Though commonly associated with a Gs–cAMP–
protein kinase A mechanism, other signaling pathways can be
activated downstream of CRFRs by different G-proteins in a cell
type- and ligand-dependent manner (Grammatopoulos and
Chrousos, 2002; Arzt and Holsboer, 2006; Hillhouse and Gram-
matopoulos, 2006). Contributing to the lack of clarity is uncer-
tainty as to the proximate mechanism of tyrosine phosphoryla-
tion of GSK, with some reports identifying this as an
autophosphorylation event (Cole et al., 2004) and others impli-
cating distinct tyrosine kinases (Pyk2 and Fyn) in this regard (Lee
et al., 1998; Hartigan et al., 2001).
Similar stress- and genotype-dependent changes were ob-
served in levels of activated JNK, implicated in tau-P at the AT8
and PHF-1 epitopes (Atzori et al., 2001). High basal levels of
phospho-JNKs in CRFR1
mice paralleled, and may explain,
elevated AT8 and PHF-1 phosphorylation in this genotype. The
MAP kinases ERK1 and ERK2, which can target all tau sites ex-
amined here except T
(Drewes et al., 1992), were relatively
unresponsive. Relative levels of cdk5, a major tau kinase active at
, AT8, and PHF-1 sites (Patrick et al., 1999), were also stable
across conditions, but one of its activator proteins, p35, exhibited
strong CRFR-dependent stress responsiveness. Consistent with
previous findings (Okawa et al., 2003), we did not detect the
truncated p35 product, p25, which is a more potent cdk5 activa-
tor (Patrick et al., 1999).
Alterations in the activity of tau phosphatases, notably PP2A,
have also been implicated in stress-induced tau-P (Planel et al.,
2001, 2004). Here, we find that repeated
stress increases relative levels of the cata-
lytic subunit of PP2A, a finding that has
been associated with diminished enzy-
matic activity and potent autoregulation
(Baharians and Schonthal, 1998; Planel et
al., 2001). Our failure to discern an acute
stress effect on PP2A-c levels is not neces-
sarily indicative of a lack of phosphatase
involvement under these conditions.
Issues of interpretation
Two aspects of our findings warrant fur-
ther consideration. First, CRFR2
displayed exaggerated responses to stress
at five of seven phosphorylation sites and
in three activated kinases. Although these
data support an interaction with CRFR1
in regulating tau-P, whether they repre-
sent convergent or parallel effects is uncer-
tain. In preliminary studies, we find that
mice deficient in both CRFRs fail to man-
ifest acute restraint-induced tau-P, sup-
porting some degree of interdependence
(R. A. Rissman and P. E. Sawchenko, un-
published observations). This interpreta-
tion is consistent with evidence that CRF,
but not CRFR1 expression or binding, is
upregulated in brain regions of CRFR2-
deficient mice, and that CRFR1 antago-
nists can normalize aspects of the behavioral phenotype of these
mutants (Kishimoto et al., 2000; Bale and Vale, 2003). These
results suggest that exaggerated stress-induced tau-P of CRFR2
mutants does not result from alterations in CRFR1 expression or
distribution. Second, although the consistent lack of stress re-
sponsiveness of CRFR1
mice suggests a target for interven
tion in tau pathologies, this is offset by a tendency toward in-
creased basal tau-P (AT8 and PHF-1 sites) and activated kinase
(notably JNK) expression in CRFR1 mutants. In addition, the
need to supplement CRFR1-deficient mice with corticosterone
during the perinatal period complicates interpretation by expos-
ing animals to higher glucocorticoid levels during the stress-
hyporesponsive period (Baram et al., 1997) and the switch in the
dominant isoform of tau that occurs at this time (Kosik et al.,
1989). It is noteworthy in both respects that acute pharmacolog-
ical interference with CRFR1 blocked restraint-induced incre-
ments in tau-P without affecting basal levels, suggesting that the
elevated baselines seen in knock-outs may result from chronic
CRF system involvement in AD
Exposure to stress can impact learning, memory, and hippocam-
pal morphology and function, and may confer increased risk of
AD (Wilson et al., 2003). Attention has focused on glucocorti-
coids as likely mediators of such effects, because increased circu-
lating levels of these hormones in aging have been linked to a
range of adverse effects in hippocampus (Sapolsky et al., 1985,
1986). Nevertheless, the present findings are consistent with
those of Korneyev et al. (1995) in demonstrating that stress-
induced tau-P proceeds unabated in animals lacking the capacity
to mount a glucocorticoid response, implicating instead the CRF
signaling system as integrally involved.
CRF is best known as a hypothalamic neuropeptide that gov-
Figure8. CRFRinvolvement in stress-inducedtauphosphorylation.Schematic summary of the progression of eventsleadingto
neurofibrillary tangle formation in AD, adapted with permission from Drewes (2004). Indicated on this are the present findings
supporting a differential involvement of CRFRs, acting via specific tau kinases, in mediating acute stress-induced tau-P (red
circles). Although acute stress effects are transient and readily reversible, repeated stress exposure produces cumulative increases
in phosphorylated tau, a portion of which is sequestered in detergent-soluble fractions. It remains to be determined whether
long-termincreases in stress exposureand/orsensitivity may result indevelopmentofthe paired helicalfilamentsandtangles that
represent a defining feature of AD neuropathology.
Rissman et al. • CRF Receptors and Tau Phosphorylation J. Neurosci., June 13, 2007 • 27(24):6552– 6562 • 6559
erns the endocrine arm of the stress response (Vale et al., 1981).
Aspects of its central distribution and actions suggest a general
involvement in integrating hormonal, autonomic, and behav-
ioral adaptations (Chadwick et al., 1993), motivating consider-
ation of a role for this system in stress-induced tau-P. Alteration
in CRF immunoreactivity is a prominent neurochemical change
occurring early in AD progression (Davis et al., 1999), in brain
regions vulnerable to AD neuropathology (Powers et al., 1987;
Raadsheer et al., 1995; Pedersen et al., 2002; Rehman, 2002;
Swaab et al., 2005). It has been suggested that these changes result
from sequestration of peptide by a specific binding protein (CRF-
BP) (Behan et al., 1995), which can reversibly neutralize CRF
bioactivity. Accordingly, competitive antagonists of CRF-BP ac-
tion enhance performance on a range of learning and memory
tasks (Behan et al., 1995). In addition, marked increases in CRF
binding have been described in cortical areas of the AD brain (De
Souza et al., 1986). However, this work predated the cloning and
characterization of CRFRs, CRF-BP, and additional ligands that
interact differentially with them.
CRFR1-deficient mice exhibit impaired hormonal and behav-
ioral responses to stress, whereas CRFR2 knock-outs are hyper-
responsive on many of the same measures (Smith et al., 1998;
Timpl et al., 1998; Bale et al., 2000; Coste et al., 2000; Kishimoto
et al., 2000). Subsequent work indicates that CRFR interactions
are not necessarily starkly differential but has generally supported
convergent influences of the two receptor mechanisms on many
stress-related endpoints (Bale and Vale, 2004). The circuitry that
provides for their interaction the present context is unclear.
CRFR1 mRNA is expressed prominently in the pyramidal layer of
Ammon’s Horn and the hilus of the dentate gyrus, whereas
CRFR2 transcripts are weakly expressed throughout the principal
cell layers of both structures (Van Pett et al., 2000). Unfortu-
nately, the lack of validated antisera has precluded histochemical
characterization of receptor protein disposition. Regarding li-
gands, extrinsic CRF-containing inputs to hippocampus have not
been described, leaving local interneurons (Chen et al., 2004) and
the CSF (Arborelius et al., 1999) as possible sources for delivering
CRF to hippocampal CRFRs. Among the urocortins, only a
sparse urocortin 1-immunoreactive input to hippocampus has
been documented (Bittencourt and Sawchenko, 2000). The ap-
parent paucity of CRFR2 ligands in hippocampal afferents sug-
gests that their hypothesized interaction with CRFR1-dependent
mechanisms may occur outside the hippocampus.
Effects of repeated stress
Acute stress-induced tau-P has been consistently characterized as
a transient and reversible phenomenon. As such, it would seem
well positioned to contribute to the rapid dendritic/synaptic re-
modeling seen in the hippocampus in response to stress (Fuchs et
al., 2006), but its pathogenic relevance has remained uncertain
(see Fig. 8). The present finding that repeated stress exposure
results in sustained elevations in tau-P, a portion of which is
sequestered in a detergent-soluble form, supports such a poten-
tial, because the bulk of dispersed PHFs from the AD brain reside
in this same fraction (Iqbal et al., 1984; Rubenstein et al., 1986). It
remains to be determined whether protracted stress exposure can
lead to PHF/NFT pathology in animal models. We think it more
likely that stress-induced alterations in tau-P and solubility may
enhance the vulnerability of affected neurons to other experien-
tial or genetic factors that play on tau dynamics. This view is
coarsely analogous to the “endangerment” hypothesis to explain
the role of glucocorticoids in the age-related impairment of hip-
pocampal function (Sapolsky et al., 1985, 1986). Studies per-
formed over a lifespan, or in aging, will be required to fully ap-
preciate the contribution of stress exposure and/or sensitivity to
tau-related neuropathologies and the capacity of CRFR signaling
mechanisms to modulate them.
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