www.sciencemag.org/cgi/content/full/1180962/DC1

Supporting Online Material for

Amyloid-β Dynamics Are Regulated by Orexin and the Sleep-Wake Cycle

Jae-Eun Kang, Miranda M. Lim, Randall J. Bateman, James J. Lee, Liam P. Smyth, John R. Cirrito, Nobuhiro Fujiki, Seiji Nishino, David M. Holtzman

*To whom correspondence should be addressed. E-mail: holtzman@neuro.wustl.edu

Published 24 September 2009 on Science Express

DOI: 10.1126/science.1180962

This PDF file includes

Materials and Methods Figs. S1 to S4 References

MATERIALS AND METHODS Animals. All experimental procedures involving animals were performed in accordance

with guidelines established by the Animal Studies Committee at Washington University.

We bred Tg2576+/− hemizygous male mice (a generous gift from Dr. K. Ashe,

University of Minnesota) to C57BL6/SJL female mice (Taconic Farms) (1). Male

Tg2576+/− littermates at 3 months of age were used for the microdialysis experiments

and female Tg2576+/− littermates at 9 to 9.5 months of age were used for the chronic

sleep restriction experiment. APPswe/PS1dE9 male mice (The Jackson Laboratory) were

bred to B6/C3 female mice (The Jackson Laboratory) (2). Female APPswe/PS1dE9

littermates at 1.7 and 2.5 to 3 months of age were used for the chronic treatment of a dual

orexin receptor antagonist and the chronic sleep restriction experiments, respectively.

Three month old male C57BL6 mice were ordered from Charles River Laboratories and

utilized when they were 4 months old. Animals were raised and most experiments were

performed in a 12 hour dark and 12 hour light controlled room (lights on at 7 AM and

lights off at 7 PM). Some experiments were performed in a constant dim light controlled

room. Animals had access to food and water ad lib.

In vivo microdialysis. In vivo microdialysis to assess brain ISF Aβ1–x in the

hippocampus of freely moving mice was performed as previously described (3, 4). This

technique samples soluble molecules within the extracellular fluid that are smaller than

38 kDa, the molecular weight cutoff of the microdialysis probe membrane. Under

isoflurane volatile anesthetic, a guide cannula (MBR-style, Bioanalytical Systems) was

cemented into the left hippocampus (bregma −3.1 mm, 2.5 mm lateral to midline, and 1.2

mm below dura at a 12° angle). For intracerebroventricular (icv) infusion, a second guide

cannula was implanted into the lateral ventricle (bregma −0.2 mm, 1.0 mm lateral to

midline, and 2.5 mm below dura). After cannulation for microdialysis, mice recovered for

at least 2 weeks and demonstrated normal behaviors including sleep. After 2 weeks of

recovery, 2-mm microdialysis probes were inserted through the guide cannula so that the

membrane was contained entirely within the hippocampus (MBR2-10, 38 kDa MWCO

membrane, Bioanalytical Systems). Mice were transferred to microdialysis cages and

microdialysis started. Mice were habituated to the new environment for 3 days and

microdialysis samples during these 3 days were discarded. After 3 days, microdialysis

samples were saved and used for Aβ1–x analysis. Microdialysis perfusion buffer was

artificial CSF containing 0.15% bovine serum albumin that was filtered through a 0.1 μM

membrane. Flow rate was a constant 1.5 μl/min. For icv infusion, a drug or vehicle was

infused through the icv cannula a flow rate of 0.01μl/min for 6 or 24 hours. For reverse

microdialysis, a syringe containing artificial CSF was switched to a syringe containing a

drug dissolved in artificial CSF. The flow rate of the microdialysis fluid remained the

same during the drug infusion as pre- and post-infusion (1.5 μl/min). Samples were

collected every 60 min with a refrigerated fraction collector into polypropylene tubes and

assessed for Aβ1–x by ELISA at the completion of each experiment. Aβ1–x levels were

normalized for each mouse by averaging the concentration of all samples collected over 1

or 3 days before drug treatments and presenting each concentration as a percentage of

that mean. Murine ISF Aβx-40 levels were assessed using a similar protocol, except

longer sample times of 6 hr at a 0.5 μl/min flow rate were necessary to concentrate ISF

samples. To examine the effect of the light stimuli on murine ISF Aβx-40 fluctuation,

some of C57BL6 mice (n = 20) were maintained under constant dim light conditions for a

month and used for the microdialysis experiment which was also done under constant

dim light conditions. We measured endogenous circadian rhythms of C57BL6 mice by

monitoring running wheel activity under constant dim light conditions and used mice

which had normal endogenous circadian rhythms for the experiment. Following each

experiment, animals were sacrificed.

Human CSF collection. CSF was collected and analyzed from 20- to 50-year-old male

participants exactly as described (5, 6). Aβ1-40 levels were measured as described in

each sample (6).

Sleep monitoring. Implantation with electroencephalograph (EEG) and electromyogram

(EMG) electrodes for polysomnographic recordings was performed simultaneously with

microdialysis cannulation. For EEG recording, two stainless steel screw electrodes were

placed over the left frontal bone (bregma +1.0mm and −1.5mm lateral to midline) and the

right parietal bone (bregma −3.0mm and 2.5mm lateral to midline). A third EEG

electrode was placed on the cerebellum and served as ground. For EMG recording, two

wire electrodes were inserted in the neck musculature. Insulated leads from the EEG and

EMG electrodes were soldered to a mini-connector. After surgery, mice recovered for at

least 2 weeks. To determine sleep states, mice were transferred to recording cages and a

mini-connector was connected to flexible recording cables. Mice were habituated to the

recording cages for 3 days. Once the habituation period was completed, recording started

simultaneously with microdialysis sample collection and continued for 3-5 days. Signals

were displayed on a monitor and stored in a computer for the off-line analysis of sleep

states. EEG and EMG activities were assessed using a P511K A.C. Pre-amplifier (Grass-

Telefactor Instruments), digitized with a DigiData 1440A Data Acquisition System

(Molecular Devices), and recorded digitally using pClamp 10.2 (Molecular Devices).

EEG and EMG signals were filtered (EEG: high pass 1 Hz; low pass 30 Hz; EMG: high

pass 10 Hz; low pass 100 Hz) and then used to identify vigilance states. EEG/EMG

records were scored semiautomatically by using a sleep scoring software (SleepSign,

Kissei Comtec Co., LTD., Japan) into 10-sec epochs as Awake, REM, and NREM on the

basis of standard criteria of rodent sleep (7). This preliminary scoring was visually

inspected and corrected when appropriate.

Acute sleep deprivation. Sixteen mice were subjected to sleep deprivation, and the

experiments were carried out between 7:00 AM and 1:00 PM. Briefly, sleep was

disturbed by gently touching the animal's back with a soft paintbrush or with cage

rotation to maintain wakefulness as needed, as assessed by the typical EEG/EMG pattern

of non-REM or REM sleep. For 8 mice, αCRF9-41, an antagonist of CRF receptors, was

infused into the hippocampus by reverse microdialysis from 30 min before sleep

deprivation until the end of the light period.

Chronic sleep restriction. First, a smaller experiment was performed using six female

Tg2576+/− mice at 9 to 9.5 months of age were used for the sleep restriction

experiments, and eight female APPswe/PS1dE9 mice at 2.5 to 3 months of age were run

in parallel. Tg2576+/− mice typically do not start forming Aβ plaques until 10-12 months

of age (8). APPswe/PS1dE9 mice over-express both a mutated human presenilin-1

(PS1-dE9) and a chimeric mouse/human APP (Mo/hu APP695swe), and have been

shown to form Aβ plaques at a much earlier age, starting at approximately 3-4 months of

age (2). The mice were approximately 4 weeks older on the date of sacrifice. APP

transgenic mice were subjected to chronic sleep restriction for 21 days using the

platform-over-water method as previously described (9). Mice were randomly divided

between small platform and large platform groups and their genotypes blinded to the

experimenter. The small platform setup consisted of a closed PVC pipe measuring 3.4 cm

in diameter which was affixed to the bottom of a regular sized shoebox cage. The cage

was then filled with water to 1 cm below the surface of the PVC pipe. The large platform

setup was identical, with the exception of measuring 11.5 cm in diameter. Mice were

housed in a behavior room with controlled 12 hour light:dark cycle with lights on at

06:00 and off at 18:00 daily. Each day for 21 days, they were placed onto their

respective platforms at 11:00 and removed at 07:00 the following day. During the four

hours off platforms, they were returned to their home cages with familiar bedding. After

sleep restriction, all mice were tested in the Morris Water Maze for deficits in spatial

learning and memory retrieval. Both sleep-restricted and control mice did not

significantly differ in the acquisition of learning the hidden platform location. However,

memory retrieval tested 48 hours later during the probe trial showed that sleep-restricted

mice spent significantly less time and swam significantly less distance in the correct

quadrant compared to controls. Prior to the sleep restriction study, three mice underwent

a proof-of-principle experiment in which they had EEG/EMG electrodes implanted for

sleep recording as described earlier (10). These data demonstrated that mice did not show

evidence of non-REM or REM sleep while on the small platforms, but were able to sleep

during their normal sleep-wake cycle on the larger platforms. Following chronic sleep

restriction and water maze testing, mice were sacrificed and their brains immediately

removed and fresh-frozen on powdered dry ice. Brains were sectioned at 30 μm on a

cryostat, thaw-mounted onto Fisher Superfrost Plus slides, with 10 sets taken from the

olfactory bulbs through the cerebellum. Slides were stored at -80 °C until processing for

immunohistochemistry.

Next, a larger experiment was performed using twenty female mice of the

APPswe/PS1dE9 genotype at 3 to 3.5 months of age (n=9 large platforms, n=11 small

platforms). These mice were run through the exact same timeline and behavioral protocol

as the previous smaller experiment, including the Morris Water Maze. After sleep

restriction, mice were sacrificed and perfused with a PBS-heparin solution via cardiac

puncture, their brains removed, and post-fixed in 4% paraformaldehyde. After 24 hours,

brains were then transferred to a 30% sucrose solution for 2 days. Brains were then

sliced on a freezing microtome at 50 μm and stored at 4°C until processing for

immunohistochemistry.

Chronic orexin receptor blockade. Ten female mice of the APPswe/PS1dE9 genotype

at 2 to 2.5 months of age were administered daily intraperitoneal injections of

almorexant, the dual orexin receptor antagonist (Actelion Pharmaceuticals, Switzerland),

or vehicle. Almorexant was dissolved in 0.25% methylcellulose and pH brought to 2.3

with 1M HCl as previously described (11), and the vehicle solution consisted of 0.25%

methylcellulose dissolved in ddH20 alone. Each mouse was weighed prior to the start of

the experiment and given 100 mg/kg dose per weight, or approximately 0.15 mL total

volume. An equivalent volume of vehicle was injected into each control mouse. Mice

were given 6 injections per week for eight weeks in total. Following eight weeks of

injections, mice were sacrificed and their brains immediately removed. A razor blade was

used to transect each brain down the sagittal suture. Half the brain was immediately

fresh-frozen on powdered dry ice to be sectioned at 30 μm on a cryostat, thaw-mounted

onto Fisher Superfrost Plus slides, with 10 sets taken from the olfactory bulbs through the

cerebellum. Slides were stored at -80 °C until processing for immunohistochemistry.

Aβ immunohistochemistry and plaque quantification. Floating brain sections in Tris-

buffered saline (TBS) were processed for Aβ immunostaining as previously described

(12), using the m3D6-biotin antibody to Aβ (gift from Eli Lilly) at 1:4000 concentration.

Fresh-frozen cryostat sections on slides were defrosted to room temperature and

processed for Aβ immunostaining as previously described (12), with several

modifications for fresh-frozen tissue sections. Tissue was first fixed in 4%

paraformaldehyde for 30 minutes, followed by washes in Tris-buffered saline (TBS).

Tissue was then quenched in 3% hydrogen peroxide solution for 10 minutes, washed

again in TBS, then incubated in 0.25% Triton-X solution for 30 minutes, followed by 3%

milk-Triton-X for 30 minutes. Finally the slides were incubated in primary antibody,

m3D6-biotin at 1:1000 overnight. The next day, the biotinylated antibody was amplified

using the Vectastain ABC kit (Vector Labs, CA) followed by visualization with DAB-

Nickel. Slides were dehydrated in ascending ethanols and coverslipped with Cytoseal.

Aβ immunostaining was visualized and images saved to file using the Hamamatsu

NanoZoomer brightfield microscope in the Alafi Neuroimaging Laboratory, Hope Center

for Neurological Disorders, Washington University. Aβ plaques were quantified in

several different brain regions, from two representative brain sections per animal, using

ImageJ (NIH freeware) by an experimenter blinded to groups. We attempted to quantify

as close to the same brain regions between animals as possible. Raw values were

recorded as a percentage of total area covered by plaques. Cortex was measured as the

region immediately dorsal to the dorsal hippocampus. In the smaller pilot experiment,

values are shown as absolute percentage of total area covered by plaques for each of the

two genotypes, Tg2576 and APPswe/PS1dE9 (Fig. S4A-B). Due to the low sample size,

data were combined for the two genotypes in Fig. S4C using values that were first

normalized to the average percent area within control animals for each genotype.

Student’s t-tests were then applied to the two brain regions of interest, the cortex and

hippocampus. In the next experiment with the larger cohort of APPswe/PS1dE9 mice,

values are shown as absolute percentage of total area covered by plaques. Two-tailed

Student’s t-tests were applied to all six brain regions of interest with Bonferroni

correction for the total number of comparisons (n=6, therefore P<0.00833).

Aβ detection. Microdialysis samples were analyzed for Aβ1–x using a denaturing,

sandwich ELISA specific for human Aβ1–x as previously described (3). Murine ISF

Aβx–40 was analyzed using a denaturing, sandwich ELISA with HJ2-biotin (generated

against Aβ 35-40) and HJ5.1 (generated against Aβ 13-28) mouse monoclonal antibodies.

Hippocampal tissue lysates were analyzed for Aβ1–40 and Aβ1–42 using a denaturing,

sandwich ELISA specific for human Aβ1–40 and Aβ1–42 as previously described (3).

Immunoblots. Hippocampal tissues were harvested at 1 AM (called the dark group) vs. 1

PM (called the light group). Western blots were performed as described (3). Briefly,

hippocampal tissues from 3 month-old Tg2576 mice were homogenized in RIPA buffer

containing the following: 150 mM NaCl, 50 mM Tris (pH 7.4), 0.5% deoxycholic acid,

0.1% SDS, 1% Triton X-100, 2.5 mM EDTA, and protease inhibitors. Western blotting

for full-length APP and APP-CTF was performed using 4-12% Bis-Tris NuPAGE gels

(Invitrogen, Carlsbad, CA) under reducing conditions with 10-30 μg of protein loaded per

lane. Nitrocellulose blots were probed with rabbit anti-APP-CTF (Zymed, San Francisco,

CA) followed by goat anti-rabbit conjugated to peroxidase (BioRad, Hercules, CA). The

same blots were probed with mouse anti-tubulin (Sigma, St. Louis, MO) as a loading

control protein. Bands were detected with Lumigen-TMA6 (Amersham, Piscataway, NJ)

and captured digitally using the Kodak ImageStation 440CF. Densitometry was

performed using the Kodak 1D Image Analysis software, and each band was normalized

to tubulin signal in each lane.

Drug treatment. αCRF9–41 peptide was purchased from Bachem (King of Prussia, PA).

One mg/ml stock solution was prepared in Milli-Q water and diluted in aCSF to final

concentration of 860 nM immediately before the experiments. 860 pmole of αCRF9–41

was administered directly into the hippocampus by reverse microdialysis over 12 hours.

Orexin-A was purchased from American Peptide (Sunnyvale, CA) and dissolved in

artificial cerebrospinal fluid (aCSF) at 3μg/ml. 1.5 pmole/hr of orexin-A or vehicle

(aCSF) was infused through the i.c.v. cannula at a flow rate of 0.03μl/min for 6 hours.

Almorexant, a dual orexin receptor antagonist (Actelion Pharmaceuticals), was dissolved

in DMSO at 12.7 mg/ml and 13.9 nmole/hr of the drug or vehicle (DMSO) was infused

through the icv cannula a flow rate of 0.01 μl/min for 24 hours.

Statistical Analysis. Data in figures represent mean ± SEM. All statistical analysis was

performed using Prism version 5 for Windows (GraphPad). Statistical analysis for the

microdialysis experiments was performed using a Student’s t-test, or one-way ANOVA

with Dunnett’s post-hoc multiple comparison test. Correlations between ISF Aβ levels

and the amount of minutes awake or sleeping per hour were analyzed using the Pearson

correlation. Statistical analysis for Aβ plaque quantification was performed using two-

tailed Student’s t-tests and Bonferroni corrections for the number of comparisons, as

described above. Values were noted as significant if p ≤ 0.05, unless noted otherwise in

the text.

10

SUPPLEMENTARY FIGURES

Figure S1. Negative correlation between ISF A levels and the amount of time in different sleep states

(total sleep and non-REM sleep) in 3 month old Tg2576 mice. (A) There was a significant negative

correlation between ISF A levels and the minutes of total sleep (non-REM sleep and REM sleep) per

hour (r = −0.53, ***P < 0.0001, n = 7). (B) There was a significant negative correlation between ISF

A levels and the minutes of NREM sleep per hour (r = −0.55, ***P < 0.0001, n = 7). Because ISF

Aβ could only be measured hourly and REM sleep is only ~10 % of sleep time that occurs each hour,

we could not attempt to correlate REM sleep time and ISF Aβ levels. NREM = non-rapid eye

movement.

11

Figure S2. Tissue Aβ1-40, Aβ1-42 and APP levels in the hippocampus during dark vs. light periods in 3

month old Tg2576 mice. (A, B) As determined by ELISA, there were no significant differences in the

concentration of either Aβ40 or Aβ42 in guanidine-soluble fractions (n = 6 per each group) in the dark

vs. the light period. (C, D) The same tissue homogenates were also assessed for the levels of full-

length APP (FL-APP), APP β-C-terminal fragment (CTF), and APP α-CTF. There were no significant

differences between the levels of FL-APP, β-CTF, and α-CTF in the dark vs. light group (n = 6 per

each group). Each band was normalized to the amount of α-tubulin in each lane. Data represent mean ±

SEM.

12

Figure S3. The effect of vehicle infusions on ISF Aβ variation in 3 month old Tg2576 mice. (A) After

24 hr of baseline measurement, aCSF was infused i.c.v. for 6 hours from the beginning of the light

period (n = 8). Orexin-A peptide was dissolved in aCSF. (B) Infusion of aCSF did not significantly

alter the diurnal variation of ISF Aβ levels. (C) After 24 hr of baseline measurement, DMSO was

infused i.c.v. for 24 hours from the beginning of the dark period (n = 6). The dual orexin receptor

antagonist, almorexant, was dissolved in DMSO. (D) Infusion of DMSO did not significantly affect the

diurnal rhythmicity of ISF Aβ levels. Data represent mean ± SEM. NoT = no treatment; d = day.

13

Figure S4. Aβ plaque deposition after chronic sleep restriction in both Tg2576 and APPswe/PS1dE9

transgenic mice. (A) Tg2576 female mice show significantly greater Aβ plaque area in the cortex after

sleep restriction compared to large platform controls (*P<0.05, n=4 per group). (B) APPswe/PS1dE9

female mice show significantly greater Aβ plaque area in the cortex and hippocampus after sleep

restriction compared to large platform controls (*P<0.05, n=3 per group). (C) Values were normalized

to average plaque load in control mice of each genotype and expressed as fold-change over baseline for

the combined genotypes (*P<0.05, n=7 per group). Underneath, representative micrographs of Aβ

plaque deposition are shown from Tg2576 mice within (D) control cortex, (E) sleep restricted cortex,

(F) control hippocampus, and (G) sleep restricted hippocampus. Scale bar = 100 m.

14

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