preventing the stress-induced rise in glucocorticoids but was blocked by genetic or pharmacologic disruption of signaling through the
Key words: Alzheimer’s disease; antalarmin corticotropin-releasing factor; corticotropin-releasing hormone; hippocampus; restraint
Alzheimer’s disease (AD) is defined neuropathologically by the
accumulation of ?-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
correlated with cognitive deficit and neuronal loss in AD (Arria-
gada et al., 1992; Gomez-Isla et al., 1997), and the discovery that
frontotemporal dementia suggests that pathological changes in
tau can serve as a principal cause of neurodegeneration and cog-
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;
Arendt et al., 2003; Feng et al., 2005). This effect has been re-
ported consistently in the hippocampal formation, a key struc-
in AD (Braak and Braak, 1991). Although acute stress-induced
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-
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
6552 • TheJournalofNeuroscience,June13,2007 • 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.
CRFR knock-out mice. Mutant mice and littermate wild-type (wt) con-
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
CRFR1?/?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
Institute Institutional Animal Care and Use Committee approved all
Adrenalectomy and corticosterone replacement. Twelve-week-old male
adrenalectomy (ADX) via bilateral incisions on the dorsolateral flanks
under isoflurane anesthesia. ADX mice received replacement corticoste-
rone (10 ?g/ml) in drinking water containing 0.9% saline immediately
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?/?and CRFR2?/?mice,
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.
After 7 d of recovery, 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).
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 mM Tris-HCl, pH 7.4,
0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1 mM Na3VO4, and 1 ?M okadaic acid). Before
homogenization, protease inhibitors PMSF, NaF (1 mM), 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
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
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
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
blots, T181, S199, S212, T231, S422(1:1000; Biosource, Camarillo, CA),
S202/T205(1:500; AT8; Pierce Biotechnology), and S396/404(1:1000;
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 S217,
S262, S356, and S409phosphorylated tau (Biosource) were also tested but
?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-
tase (40 mg/ml; Sigma-Aldrich), which eliminated detectable PHF-1
labeling in all experimental groups (data not shown). For assessment of
teins were used. This included total glycogen synthase kinase-3? (GSK-
3?; 1:2500; BD Biosciences, San Diego, CA), activated GSK-3? (pY216;
1:1000; BD Biosciences), inactive GSK-3? (pS9; 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
tein (MAP) kinases [extracellular signal-regulated kinases 1 and 2
protein phosphatase 2A (PP2A-c; 1:5000; BD Biosciences). ?-Actin (1:
2000; Sigma-Aldrich) was used as a control for protein loading.
Immunohistochemistry. Mice were perfused with 4% paraformalde-
micrometer-thick frozen sections were cut on a sliding microtome and
stored at ?20°C in cryoprotectant solution (20% glycerol and 30% eth-
free-floating sections containing hippocampus using Mouse-on-Mouse
Immunodetection Kit reagents (Vector Laboratories, Burlingame, CA)
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
Rissmanetal.•CRFReceptorsandTauPhosphorylationJ.Neurosci.,June13,2007 • 27(24):6552–6562 • 6553
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
[S181, S199, S202/T205(AT8), T212, T231, S396/404(PHF-1), and
S422] 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)
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
over 20–60 min after stress, and the 20 min time point was se-
lected for subsequent analyses.
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
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
responses at the AT8 and PHF-1 sites in hippocampal extracts
These results confirm that acute stress-induced tau-P is not de-
pendent on glucocorticoid secretion.
The CRF family of signaling molecules is broadly involved in
1993) and undergoes alterations early in AD progression (Davis
et al., 1999). However, the nature of its involvement in AD neu-
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?/?mice were
significantly greater than those of wt animals at the S181( p ?
0.01), S199( p ? 0.001), T212( p ? 0.05), T231( p ? 0.05), and
PHF-1 ( p ? 0.01) sites. Phosphorylation responses of CRFR2
null mice at the AT8 and S422sites 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
response to stress, which was attenuated and exaggerated in
CRFR1- and CRFR2-deleted animals, respectively (Fig. 4A). In
ies were seen primarily in the hilus (polymorph and subgranular
signal under basal conditions was low but rose immediately after stress to levels that were
intensity values of intact unstressed controls, reveals that intact and ADX animals manifest
differ significantly. **Differs significantly from intact, unstressed controls ( p ? 0.001); ns,
Role of glucocorticoids in stress-induced tau-P. Western blot analysis of hip-
6554 • J.Neurosci.,June13,2007 • 27(24):6552–6562Rissmanetal.•CRFReceptorsandTauPhosphorylation
observed PHF-1 positive mossy fibers and a band of punctate
layer. In Ammon’s Horn, dominant features included labeled
perikarya scattered mainly throughout the pyramidal layer and
engulfing the pyramidal cell layer and in stratum lacunosum-
moleculare. Radially oriented processes, some traceable to la-
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-
40 min after administration of CRF or saline vehicle (Fig. 4B).
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,
of the granule cell layer (Fig. 4B). In Ammon’s Horn, we ob-
pericellular labeling throughout the pyramidal cell layer and,
more sporadically, in the dendritic zone, with particular concen-
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-
demonstrates a similar dependence on CRFR integrity.
Interpretation of data derived from conventional knock-out an-
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 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.
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
responses of seven AD-relevant tau sites in hippocampal extracts from wt, CRFR1?/?, and
versely, CRFR2?/?mice showed normal basal levels of tau-P at all sites but exaggerated
Differential regulation of stress-induced tau-P by CRFR status. Phosphorylation
Rissmanetal.•CRFReceptorsandTauPhosphorylation J.Neurosci.,June13,2007 • 27(24):6552–6562 • 6555
CRFR dependence (Fig. 6). Several kinases examined, including
(pS9), or total (unphosphorylated) form of GSK-3?, the pT183/
Y185form of the 46 and 54 kDa c-Jun N-terminal protein kinases
(JNK46/54), and the pT202/Y204form of the mitogen-activated
protein kinases, ERK2, but not ERK1, displayed upregulation in
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
some or all of the effects of genotype on restraint-induced tau-P.
The activated (pY216) form of GSK-3?, implicated in phosphor-
in CRFR1?/?animals and was exaggerated in CRFR2?/?mice.
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
the AT8 and PHF-1 sites, although less-marked elevations of
phosphorylated GSK-3? and ERK2, and of p35 levels, in un-
stressed CRFR1?/?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
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
sequestered into insoluble cellular fractions (Iqbal et al., 1994).
We therefore used sequential fractionation in the absence (RAB
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
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
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
6556 • J.Neurosci.,June13,2007 • 27(24):6552–6562Rissmanetal.•CRFReceptorsandTauPhosphorylation
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.
Alterations in tau phosphatase activity have been implicated as
contributing to stress-induced tau-P (Planel et al., 2001, 2004,
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).
tions under any experimental condition (data not shown). In
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
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
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-
observed in CRFR2 mutants were paralleled by altered activities
of specific tau kinases. Furthermore, the demonstration that re-
for evidence implicating chronic stress as a risk factor in age-
related neurological disorders, including AD.
Tau normally binds and stabilizes neuronal microtubules, facili-
tating their roles in cellular structure, polarity, and transport
and promote cytoskeletal destabilization (Sengupta et al., 1998).
these aggregate into insoluble NFTs, a defining feature of AD
results in tau-P at AD-relevant sites defines one potential means
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-
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
the present findings suggest that the generality of stress-induced
reductions in body temperature (7–10°C), which can differen-
tially modulate tau kinase and phosphatase activities, leading
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-
conditions. **Differs significantly from unstressed controls ( p ? 0.001);†Differs from un-
Rissmanetal.•CRFReceptorsandTauPhosphorylationJ.Neurosci.,June13,2007 • 27(24):6552–6562 • 6557
tion by CRFR1, which is normally restrained by CRFR2-based
Complementary monitoring of the activation status of tau ki-
Its activity is stimulated or inhibited by phosphorylation at Y216
by very high basal phosphorylation levels in CRFR1?/?mice, which may relate to elevated
ERK1, were relatively unresponsive. Levels of cdk5 were stable across the conditions in force
fromwtcontrol( p?0.01);**p?0.001.†Differssignificantlyfromwtstresscondition( p?
using phosphoepitope-specific antibodies to interrogate stress- and/or genotype-dependent
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
phosphorylated) GSK3 are unresponsive over these conditions. Both phospho-JNK isoforms
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
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
6558 • J.Neurosci.,June13,2007 • 27(24):6552–6562Rissmanetal.•CRFReceptorsandTauPhosphorylation
and S9, 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-
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-
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 T231(Drewes et al., 1992), were relatively
unresponsive. Relative levels of cdk5, a major tau kinase active at
T231, AT8, and PHF-1 sites (Patrick et al., 1999), were also stable
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.
Two aspects of our findings warrant fur-
ther consideration. First, CRFR2?/?mice
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-
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
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
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-
signaling system as integrally involved.
CRF is best known as a hypothalamic neuropeptide that gov-
supporting a differential involvement of CRFRs, acting via specific tau kinases, in mediating acute stress-induced tau-P (red
in phosphorylated tau, a portion of which is sequestered in detergent-soluble fractions. It remains to be determined whether
Rissmanetal.•CRFReceptorsandTauPhosphorylationJ.Neurosci.,June13,2007 • 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;
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
Souza et al., 1986). However, this work predated the cloning and
characterization of CRFRs, CRF-BP, and additional ligands that
interact differentially with them.
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
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.
Ammon’s Horn and the hilus of the dentate gyrus, whereas
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-
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.
a transient and reversible phenomenon. As such, it would seem
well positioned to contribute to the rapid dendritic/synaptic re-
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-
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
mechanisms to modulate them.
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