/ www.sciencexpress.org / 24 September 2009 / Page 1 / 10.1126/science.1180962
Amyloid- (A ) accumulation in the brain extracellular
space is a hallmark of Alzheimer’s disease (AD). The
factors regulating this process are only partly understood.
A aggregation is a concentration-dependent process that
is likely responsive to changes in brain interstitial fluid
(ISF) levels of A . Using in vivo microdialysis in mice, we
found that ISF A levels correlated with wakefulness. ISF
A levels also significantly increased during acute sleep
deprivation and during orexin infusion, but decreased
with infusion of a dual orexin receptor antagonist.
Chronic sleep restriction significantly increased, and a
dual orexin receptor antagonist decreased A plaque
formation in amyloid precursor protein transgenic mice.
Thus, the sleep-wake cycle and orexin may play a role in
the pathogenesis of AD.
Alzheimer’s disease (AD) is the most common cause of
dementia. The accumulation of the amyloid- (A) peptide in
the brain extracellular space is a critical event in the
pathogenesis of AD. A is produced by neurons and secreted
into the brain interstitial fluid (ISF). An initiating factor in
AD pathogenesis occurs when soluble, monomeric A
undergoes a conformational change and converts into forms
such as oligomers, protofibrils, and fibrils. The accumulation
of these forms of A is concentration-dependent and confers
toxicity (1). Elucidating factors that regulate soluble A
levels is important for understanding AD pathogenesis.
Synaptic activity regulates the release of A from neurons
into the ISF (2, 3). How ISF A is regulated by normal
physiology is poorly understood.
To investigate ISF A metabolism, we monitored
hippocampal A levels using in vivo microdialysis in both
wild-type mice and human APP transgenic (Tg2576) mice,
which express a mutated form of human amyloid precursor
protein (APP) (4). ISF A was assessed in Tg2576 mice at 3
months of age, several months earlier than A deposition
begins. We found diurnal variation of ISF A levels. A
levels were significantly increased during the dark period
compared to the light period (Fig. 1A). ISF A levels
fluctuated over a 24-hour period with mean levels during the
light period being ~75% of mean A levels during the dark
period (Fig. 1B). ISF A levels were significantly correlated
with the amount of time spent awake (Fig. 1, C and D).
Conversely, ISF A levels were negatively correlated with
the amount of time spent asleep. This negative correlation
was even stronger with non-REM sleep (fig. S1). Despite
fluctuations in ISF A levels, full-length APP, APP C-
terminal fragments, and A1-40 and A1-42 were not
significantly different in total tissue homogenates of
hippocampus between dark and light periods (fig. S2). Thus,
the pool of ISF A is likely to be independently regulated
from total intracellular and membrane-associated A.
Next, we asked if diurnal A fluctuation was also present
in C57BL6, wild-type mice. Similar to Tg2576 mice,
C57BL6 mice also showed a significant difference in ISF A
levels between dark and light phases, when samples were
pooled over longer periods of time (Fig. 1, E and F). Thus,
the diurnal variation in A is intrinsic to normal cellular
To determine the underlying mechanism of the diurnal
variation in ISF A levels, we tested whether the light
stimulus itself could affect ISF A levels. Using C57BL6
mice, we measured ISF A levels over 2 days under constant
dim light conditions. Diurnal fluctuations of ISF A still
occurred, as did normal sleep-wake behavior (Fig. 1, G and
H). Thus, ISF A fluctuations are linked to the sleep-wake
cycle and not to light or dark exposure.
To see whether the diurnal fluctuation of ISF A is present
in humans, we assessed cerebrospinal fluid (CSF) levels of
A in N=10 young healthy male volunteers via lumbar
catheters over a 33 hour period and found clear evidence of
diurnal fluctuation of A in the CSF. A levels increased
throughout the first day with a peak in the evening, then
decreased overnight, and again increased throughout the
second day (Fig. 1I).
Amyloid- Dynamics Are Regulated by Orexin and the Sleep-Wake Cycle
Jae-Eun Kang,1 Miranda M. Lim,1 Randall J. Bateman,1,2,3 James J. Lee,1 Liam P. Smyth,1 John R. Cirrito,1,2 Nobuhiro Fujiki,4
Seiji Nishino,4 David M. Holtzman1,2,3,5*
1Department of Neurology, Washington University, St. Louis, MO 63110, USA. 2Hope Center for Neurological Disorders,
Washington University, St. Louis, MO 63110, USA. 3Alzheimer’s Disease Research Center, Washington University, St. Louis,
MO 63110, USA. 4Sleep and Circadian Neurobiology Laboratory, Department of Psychiatry and Behavioral Sciences, Stanford
University, Palo Alto, CA 94304, USA. 5Department of Developmental Biology, Washington University, St. Louis, MO 63110,
*To whom correspondence should be addressed. E-mail: email@example.com
/ www.sciencexpress.org / 24 September 2009 / Page 2 / 10.1126/science.1180962
Because A levels correlated with wakefulness, we asked
whether manipulating sleep behavior would alter ISF A
levels. Mice were forced into wakefulness for 6 hours at the
beginning of the second 12 hour light period when they
would naturally be asleep. During sleep deprivation (SD), ISF
A levels were significantly higher compared to ISF A
levels during the normal light period 24 hours previously
(Fig. 2, A to C). Following SD, mice spent more time
sleeping and had an immediate reduction in ISF A levels.
Thus, the state of wakefulness, and not time of day, is
associated with increased ISF A levels.
Restraint stress in Tg2576 mice can acutely increase ISF
A mediated by corticotropin releasing factor (CRF) (5).
CRF9–41, an antagonist of CRF receptors, was administered
by reverse microdialysis at the beginning of SD. In the
presence of the CRF receptor antagonist, ISF A levels were
still significantly higher compared to ISF A levels during
the normal light period 24 hours previously (Fig. 2, D to F).
The SD-induced increase in ISF A did not significantly
differ in the presence or absence of the CRF antagonist,
thereby excluding the CRF stress pathway as mechanism of
action for SD to increase A levels.
We next asked what molecular mechanism might mediate
the diurnal fluctuation of A levels. Orexin is a molecule that
regulates wakefulness and other physiological functions, and
is strongly implicated in narcolepsy/cataplexy and disorders
of sleep and arousal (6). Orexin release from hypothalamic
neurons shows a diurnal fluctuation similar to that of ISF A
(7). Orexin neurons project to the hippocampus where orexin
receptors are expressed and is the location where we
monitored ISF A (8). We asked if orexin administration
would modulate ISF A levels. Intracerebroventricular (icv)
infusion of orexin-A (1.5 pmole/hr) was given for 6 hours at
the beginning of the light period. This dose induces
wakefulness in rodents (9). During orexin infusion, ISF A
levels were significantly increased compared to ISF A levels
measured during the light period of the preceding day (Fig. 3,
A and B). Infusion of vehicle did not significantly affect ISF
A (fig. S3, A and B).
The orexin family (orexin-A and orexin-B) has two
receptor subtypes: orexin receptor 1 (OXR1) and orexin
receptor 2 (OXR2). We asked whether endogenous orexin
signaling via orexin receptors is involved in the diurnal
variation of A levels. We infused a dual orexin receptor
antagonist, almorexant, during in vivo microdialysis for ISF
A. Icv administration of almorexant for 24 hours suppressed
ISF A levels and abolished the natural diurnal variation of
A (Fig. 3, D and E). Removal of almorexant immediately
restored the diurnal rhythm in ISF A levels during the next
24 h period. Control icv infusions of vehicle did not affect
ISF A levels (fig. S3, C and D). Almorexant decreased the
total amount of time spent awake by approximately 10% (Fig.
3F). Thus, endogenous orexin signaling via orexin receptors
is required for the diurnal rhythm of ISF A levels.
Because sleep-wake behavior modulates ISF A levels, we
asked whether chronic sleep deprivation could ultimately
affect A plaque deposition in the brain. APP transgenic mice
of the APPswe/PS1dE9 genotype were subjected to chronic
sleep restriction for 20 hours daily for 21 days. Sleep-
restricted animals showed markedly greater A plaque
deposition compared to their age-matched littermate controls
(Fig. 4, A to G). We also observed significantly greater A
plaque burden using Tg2576 mice (fig. S4). We next asked
whether chronic orexin receptor blockade could decrease A
plaque deposition in APPswe/PS1dE9 mice at an age when
plaques are just forming. Systemic treatment with almorexant
once daily for 8 weeks significantly decreased A plaque
formation in several brain regions compared to vehicle-
treated age-matched control mice (Fig. 4H).
Herein, we demonstrated diurnal variation in A levels in
the brain of awake and behaving animals. Perturbations in
both orexin signaling and the sleep-wake cycle had acute
effects upon A dynamics. Furthermore, chronic sleep
restriction accelerates A plaque burden, while enhancing
sleep via orexin receptor blockade markedly inhibits A
One factor that influences A levels is synaptic activity.
Periods of wakefulness are associated with a net increase in
synaptic strength, and periods of sleep are associated with a
net decrease in synaptic strength (10-12). Differences in
synaptic activity between sleep and wake states, specifically
via orexin signaling, may underlie the dynamic fluctuations in
ISF A levels.
How might changes in hourly ISF A levels contribute to
eventual A plaque deposition? Recent work with a gamma
secretase inhibitor has shown that changes in ISF A levels as
little as 20% blocks plaque formation and growth over weeks
(13). Thus, behavioral and pharmacological manipulations of
wakefulness that resulted in changes in ISF A of 20-25%
likely caused the observed changes in A accumulation.
Sleep is a complex behavioral state whose ultimate
functions remain poorly understood. Sleep disturbances, in
addition to being prominent in neurodegenerative diseases
(14), could exacerbate a fundamental process leading to
neurodegeneration, and optimization of sleep time could
potentially inhibit aggregation of toxic proteins and slow the
progression of AD.
References and Notes
1. D. J. Selkoe, Nat Cell Biol 6, 1054 (Nov, 2004).
2. F. Kamenetz et al., Neuron 37, 925 (Mar 27, 2003).
3. J. R. Cirrito et al., Neuron 48, 913 (Dec 22, 2005).
4. K. Hsiao et al., Science 274, 99 (1996).
5. J. E. Kang, J. R. Cirrito, H. Dong, J. G. Csernansky, D. M.
Holtzman, Proc Natl Acad Sci U S A 104, 10673 (Jun 19,
6. T. S. Kilduff et al., J Neurosci 28, 11814 (Nov 12, 2008).
7. Y. Yoshida et al., Eur J Neurosci 14, 1075 (Oct, 2001).
8. C. Peyron et al., J Neurosci 18, 9996 (Dec 1, 1998).
9. Z. L. Huang et al., Proc Natl Acad Sci U S A 98, 9965
(Aug 14, 2001).
10. V. V. Vyazovskiy, C. Cirelli, M. Pfister-Genskow, U.
Faraguna, G. Tononi, Nat Neurosci 11, 200 (Feb, 2008).
11. G. F. Gilestro, G. Tononi, C. Cirelli, Science 324, 109
(Apr 3, 2009).
12. J. M. Donlea, N. Ramanan, P. J. Shaw, Science 324, 105
(Apr 3, 2009).
13. P. Yan et al., J Neurosci 29, 10706 (Aug 26, 2009).
14. J. F. Gagnon, D. Petit, V. Latreille, J. Montplaisir, Curr
Pharm Des 14, 3430 (2008).
15. We thank E. D. Herzog and G.M. Freeman Jr. for
assistance and P. J. Shaw for discussion. This work was
supported by NIH grants AG025824, AG030946,
NS065667, AG029524, MH072525, Neuroscience
Blueprint Center Core Grant P30 NS057105, Cure
Alzheimer’s Fund, Alzheimer’s Association Zenith
Award, and Eli Lilly.
/ www.sciencexpress.org / 24 September 2009 / Page 3 / 10.1126/science.1180962
Supporting Online Material
Materials and Methods
Figs. S1 to S4
24 August 2009; accepted 11 September 2009
Published online 24 September 2009;
Include this information when citing this paper.
Fig. 1. Diurnal rhythm of ISF A levels in the hippocampus
of mice and CSF A levels in human subjects. (A) ISF human
A levels expressed as a percentage of basal ISF A levels
over 6 light-dark periods in Tg2576 mice (n = 8). Total
number of minutes spent awake per hour in the same mice. (B
and C) Mean ISF A levels were 24.4% higher (***P <
0.0001, n = 8) and the number of minutes awake were 167
minutes higher (**P = 0.007, n = 7) during dark vs. light
periods. (D) ISF A levels correlate with the number of
minutes awake per hour (r = 0.53, ***P < 0.0001, n = 7). (E
and F) Mean ISF murine A levels and minutes awake over 2
days in C57BL6 mice. Under 12 hr dark/12 hr light
conditions, ISF murine A levels were 18.5% higher (***P <
0.0001, n = 10) and the number of minutes awake was 223
minutes higher (***P =0.0001, n = 5) during the dark
periods. (G and H) Under constant dim light conditions, ISF
A levels were 22.7% higher (*P = 0.05, n = 10) and the
number of minutes awake was 114 minutes higher (***P
=0.0003, n = 5) during the typical hours for the dark phase.
(I) CSF A140 levels from human subjects expressed as a
percentage of basal CSF A140 levels over 33 hours (n = 10).
Mean peak CSF A140 levels (black bar) at 8-10 PM were
27.6% higher than mean trough CSF A levels (gray bar) at
9-10 AM (112.3 ± 6% vs. 84.7 ± 6% respectively, P = 0.004).
Data shown are means ± SEM.
Fig. 2. Acute sleep deprivation alters ISF A diurnal rhythm
independently of CRF receptor signaling in Tg2576 mice. (A)
Mice underwent acute sleep deprivation (SD – grey dashed
line) for 6 hours at the beginning of the light period. This
prevented the normal decrease in ISF A levels that occurs
during this period (n = 8). (B) Mean ISF A levels during SD
were 16.8% higher compared to those during the light period
24 hours earlier (black dashed line, P = 0.05, n = 8). (C)
Animals spent 126 more minutes awake during SD (***P <
0.0001, n = 5). (D) Mice underwent acute SD (grey dashed
line) at the beginning of the light period. 860 pmoles of
CRF9-41 was infused into the hippocampus from 30 min
before SD until the end of the light period (n = 8). (E) Mean
ISF A levels during SD with CRF9-41 infusion were 33.7%
higher compared to those during the light period 24 hours
earlier (black dashed line, P = 0.01, n = 8). (F) Mice spent
136 more minutes awake during SD with CRF9-41 infusion
(***P < 0.0001, n = 5). Data represent means ± SEM. SD =
Fig. 3. Effects of orexin and a dual orexin receptor antagonist
on ISF A levels in Tg2576 mice. (A) After 24 hr of baseline
measurement, 1.5 pmole/hr of orexin was infused icv for 6
hours at the beginning of the light period. This sustained ISF
A levels from the dark period, and kept the mice awake
longer. (B and C) Infusion with orexin increased A levels
duirng the light period and thereby abolished the normal 20%
difference between the dark and light period (*P = 0.01, n =
7). Orexin increased the amount of minutes awake by 163
minutes (**P = 0.009, n = 5), compared to that during the
light period 24 hours previously. (D) After 24 hr of baseline
measurement, 13.9 nmole/hr of almorexant was infused icv
for 24 hr from the beginning of the dark period (n = 8). This
continued to suppress ISF A levels from the light period. (E)
Mean ISF A levels differed by 29% between the dark and
light period during the control days, whereas there was no
difference between the dark and light period during the 24
hour infusion of almorexant (**P = 0.001, n = 8). (F) During
almorexant treatment, the number of minutes spent awake
was decreased by 108 minutes over the 24 hour period
compared to control days (**P = 0.005, n = 13). Data
represent means ± SEM. NoT = no treatment; d = day.
Fig. 4. A plaque deposition after chronic sleep restriction
and chronic orexin receptor blockade in APPswe/PS1dE9
transgenic mice (A) Mice that underwent chronic sleep
restriction for 21 days showed significantly greater A plaque
deposition in multiple subregions of the cortex compared to
age-matched control mice (**P < 0.0008 , *P < 0.008, n = 9-
11 per group, using Bonferroni-adjusted P < 0.0083 for
multiple t-tests. For hippocampus, P < 0.009. Representative
photomicrographs of A plaques are shown in (B) control
and (C) sleep restricted olfactory bulb (D) control and (E)
sleep restricted piriform cortex, (F) control and (G) sleep
restricted entorhinal cortex. (H) Mice treated with daily i.p.
injections of almorexant for 8 weeks showed significantly
less A plaque deposition in multiple subregions of the cortex
compared to age-matched vehicle controls (**P < 0.0008 , *P
< 0.008 n = 5 per group, using Bonferroni-adjusted P <
0.0083 for multiple t-tests. For cingulate cortex and
hippocampus, P < 0.009. Scale bar = 200 m. OB = olfactory
bulb, Cing = cingulate cortex, Piri = piriform cortex, Ent =
entorhinal cortex, Ctx = cortex (immediately dorsal to dorsal
hippocampus), and HC = hippocampus.
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