Supplementary information for

Blood-derived amyloid-β protein induces Alzheimer’s disease pathologies

Running title: Peripheral Aβ contributes to AD

Xian-Le Bu1,#, Yang Xiang1,#, Wang-Sheng Jin1, Jun Wang1, Lin-Lin Shen1, Zhi-Lin

Huang2, Kuan Zhang3, Yu-Hui Liu1, Fan Zeng1, Jian-Hui Liu4,5, Hao-Lun Sun1, Zhen-

Qian Zhuang1, Si-Han Chen1, Xiu-Qing Yao1, Brian Giunta6, Yi-Chu Shan4, Jun Tan7,

Xiao-Wei Chen3, Zhi-Fang Dong2, Hua-Dong Zhou1, Xin-Fu Zhou8, Weihong Song9,*,

Yan-Jiang Wang1,*

1 Department of Neurology and Centre for Clinical Neuroscience, Daping Hospital,

Third Military Medical University, Chongqing, China.

2 Ministry of Education Key Laboratory of Child Development and Disorders and

Chongqing Key Laboratory of Translational Medical Research in Cognitive

Development and Learning and Memory Disorders, Children’s Hospital of Chongqing

Medical University, Chongqing, China.

3 Brain Research Center, Third Military Medical University, Chongqing, China

4 National Chromatographic Research and Analysis Center, Key Lab of Separation

Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese

Academy of Sciences, Dalian, China.

5 University of Chinese Academy of Sciences, Beijing, China

6 Neuroimmunology Laboratory; 7 Rashid Laboratory for Developmental

Neurobiology, Silver Child Development Center, Department of Psychiatry and

Behavioral Neurosciences; Morsani College of Medicine, University of South Florida,

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Tampa, Florida, USA.

8 School of Pharmacy and Medical Sciences and Sansom Institute, University of

South Australia, Adelaide, Australia.

9 Townsend Family Laboratories, Department of Psychiatry, Brain Research Center,

The University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3,

Canada.

# These authors contributed equally to this work.

* To whom correspondence should be addressed: Dr. Yan-Jiang Wang, email:

yanjiang_wang@tmmu.edu.cn; and Dr. Weihong Song, email: weihong@mail.ubc.ca

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Tables of Contents

Supplementary Materials and Methods…………………………………….………3

References……………………………………………………………………..……12

Supplementary Figure S1-S2………………………………………….……….……14

Supplementary Materials and Methods

Two-photon intravital imaging of brain entry of human Aβ

Monomeric HiLyte Fluor 488-labeled human Aβ42 was dissolved in 1,1,1,3,3,3-

hexafluoro-2-propanol (Sigma-Aldrich, St. Louis, MO, USA) to 1 μg/μl and stored at

−20°C. It was then dissolved in PBS to obtain final dilutions of 0.1 μg/μl immediately

before injection. Blood vessels were labeled using Texas Red-dextran (70kDa,

5ug/ul). Ten-month-old female C57BL/6J mice received 20 μg of human Aβ42 Hilyte

Fluor 488 and 1mg Texas Red-dextran intravenously via the tail vein, and Aβ was

visualized with two-photon intravital imaging at 2 and 4 hours after injection.

The two-photon intravital microscopy was performed as described previously 1.

Briefly, the mouse was placed onto a warming plate (37–38 °C) and anesthetized by

inhalation of 1–1.5% isoflurane (RWD Life Science) in pure O2. The skin and soft

tissues were removed under a dissecting microscope after local application of

xylocaine. A custom-made recording chamber was then glued to the skull. A small

craniotomy (~1mm x 1 mm) on the parietal lobe (coordinates: Bregma −0.56 mm,

1.65mm lateral to midline) was made. The craniotomy was filled with 1–1.5% low-

melting-point agarose to minimize brain pulsations. After surgery, the mouse was

transferred into the recording apparatus and the anesthesia level was decreased to 0.5–

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0.8% isoflurane (breathing rate was around 120 breaths per minute). The recording

chamber was perfused with warm artificial cerebrospinal fluid (ACSF) containing (in

mM): 125 NaCl, 4.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 20

glucose, pH 7.4, when bubbled with 95% O2 and 5% CO2. The temperature of the

mouse was maintained in the range of 36.5–37.5 °C throughout the recording. Two-

photon imaging was performed with a custom-designed two-photon microscope

system based on 12 kHz resonant scanner (model “LotosScan 1.0”, Suzhou Institute

of Biomedical Engineering and Technology). Two-photon excitation light was

delivered by a mode-locked Ti:Sa laser (model “Mai-Tai DeepSee”, Spectra Physics),

and a x40/0.8 NA water-immersion objective (Nikon) was used for imaging.

Analysis for brain uptake of 125I-Aβ40

Ten-month-old female C57BL/6J mice received a bolus injection with 6.9 MBq 125I-

Aβ in 0.3ml of lactate Ringer’s solution (6g/L NaCl, 0.2g/L CaCl2, 3.1g/L sodium

lactate, 0.3g/L KCl, pH 7.1) via the tail vein. Mice were sacrificed by overdose of

ketamine at 2h, 4h and 8h after injection (n = 6 per group). Brain samples were

collected and weighted. The radioactivity (CPM values) of brain samples was

measured with a wipe test counter (CAPRAC), and the resulting counts/min were

normalized per gram of brain.

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Microdialysis

The parameters for the microdialysis probes used for in vivo experiments were 220

µm OD membrane which is made of hydrophilic cellulose and does not absorb Aβ

(MBR-1-5 Brain, 35 kDa molecular weight cut-off; Bioanalytical Systems, West

Lafayette, USA). Guide implantation surgery was performed as previously described2,

3. Briefly, a separate group of ten-month-old C57BL/6J mice (n = 6) were

anaesthetized, and the skin was removed to expose the skull. Bore holes (0.75 mm)

were made above the left parietal cortex (bregma -3.1 mm, 2.4 mm lateral, -0.6 mm

relative to dura mater). MBR-5 guide cannulas were stereotactically inserted into the

cortex and cemented using binary dental cement. The solution circle system, including

a CMA 402 Syringe Pump, CMA 120 System for Freely Moving Animals and CMA

142 Microfraction Collector, was connected using Fluorinated Ethylene Propylene

(FEP) tubing. Human Aβ42 (American Peptide Company) was dissolved in PBS to 1

μg/μl. Ten-month-old female wild type mice received 100 μg of human Aβ42

intravenously via the tail vein. The constant flow rate was 1 μl/min. Microdialysis

samples were collected using a refrigerated fraction collector before intravenous

injection and at 2, 4 and 8 hours after injection.

Parabiosis

APPswe/PS1dE9 transgenic (Tg) mice were obtained from Jackson Laboratory (Bar

Harbor, MA, USA), and the mice were bred in the animal facility of Daping Hospital.

All mice husbandry procedures were approved by the Third Military Medical

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University Animal Welfare Committee. Female Wt and Tg mice were used in the

present study. Pairs of mice at 9 months of age were placed together in a cage for one

month to allow the mice to adapt to each other 4, 5. Female Wt mice and their age- and

weight-matched female Tg littermates were selected for parabiosis, including

parabiosis from 10 months of age to 12 months of age [pa(10-12m)Wt], parabiosis

from 10 months of age to 14 months of age [pa(10-14m)Wt], from 10 months of age

to 18 months of age [pa(10-18m)Wt] and from 10 months of age to 22 months of age

[pa(10-22m)Wt] (n = 6 per group). The age-matched female Wt mice without

parabiosis were used in parallel as controls (n = 6 per group). After parabiosis was

established between Wt and Tg mice, the circulation of the parabionts were connected

so that human Aβ species in the blood of the parabiotic Tg mice could be transported

into the peripheral circulation of the Wt mice. The parabiosis was performed as

previously described 6. Briefly, animals were anesthetized with ketamine (100 mg/kg),

xylazine (20 mg/kg), and acepromazine (3 mg/kg) and placed in a parallel orientation.

A left lateral incision was made on one mouse, whereas a right lateral incision was

made on the partner mouse, extending from the base of the ear toward the hip. The

incision included skin and muscle along the thorax and abdomen. The opposing

muscle layers of the two mice were joined with 5-0 silk sutures. The scapula of each

mouse was fixed together with 4-0 silk sutures. The corresponding dorsal and ventral

skin was sutured with 4-0 silk. After the surgery, the parabiotic mice were allowed to

recover in a warm and clean environment before being transferred into the husbandry

area. Prophylactic antibiotic treatment (enrofloxacin, 5 mg/kg) was started one day

prior to the surgery and continued for one week. All animals received analgesic/anti-

inflammatory treatment (acetylsalicylic acid 5 mg/kg) for two weeks. All experiments

that involved mice were approved by the Laboratory Animal Welfare and Ethics

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Committee of the Third Military Medical University (SYXK-PLA-20120031).

Tissue sampling

Mice were killed humanely with an overdose of 6% chloral hydrate (6 ml/kg). Blood

was sampled from the right atrium of the heart. The animals were perfused with 0.1%

NaNO2 in normal saline, and the brains were sampled and weighed. The right

hemisphere from each animal was fixed in 4% paraformaldehyde for histological

analysis, and the left hemisphere was frozen at -80°C for biochemical analysis.

Sequence of human Aβ in brain homogenate

Protein extracts were obtained from brain tissue lysed in RIPA buffer supplemented

with protease and phosphatase inhibitors. Aβ immunoprecipitation was performed

using 6E10 antibody by the protein G-Agarose. Glycine buffer (0.1M at pH 2.5) was

used to elute the antigen, and the elutes were then frozen-dried in vacuum. The

proteins were dissolved in 10mM NH4HCO3, denatured by 95℃ for 5min. Then they

were digested by Glu-C with the ratio of 1:25 (enzyme/protein, w/w) and incubated at

37℃ overnight. All the digests were stored at -20℃ before usage.

All the samples were analyzed on an Orbitrap Fusion Lumos (Thermo, CA) mass

spectrometer coupled with an EASY-nLC 1200 HPLC system (Thermo, CA). The

Buffer A (98% H2O+2%ACN+0.1% FA) and Buffer B (20% H2O+80 %ACN+0.1%

FA) were used for gradient separation. Peptides were separated on a home-made C18

column (1.9 μm, 150 mm×100 μm i.d., ReproSil-Pur C18-AQ [Dr. Maisch GmbH]) at

the flow rate of 600 nL/min. Gradient elution condition was 9-25%B (30 min), 25-

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50%B (41 min), 50-95%B (1 min) and 95%B (3 min). Peptides were detected in

Orbitrap Fusion Lumos mass spectrometer by means of data-dependent mode. Survey

scans were acquired from m/z 350 to 1500 at the resolution of 60,000 at m/z=200.

MS/MS scans within cycle time of 3s were obtained by higher energy collision

dissociation (HCD) and detected in Orbitrap with resolution of 15,000@200. The

collision energy was set as 30% and isolation window was 1.6 m/z. Precursors were

subject to dynamic exclusion of 18s.

Acquired raw files were searched in pFind (version 3.0.11) against the sequence of

human Aβ peptide. The variable modification was methionine oxidation. The enzyme

was Glu-C with three max missing cleavage sites. Peptide identification was based on

a search with mass tolerance of 20 ppm for both the precursor ion and the fragment

ion. The FDR was determined by searching a reverse database. An FDR of 0.01 for

spectra was required.

AD type pathologies

Coronal sections of the brain were cut and stored at 4°C as previously described 7. A

series of five equally spaced tissue sections (~1.3 mm apart) spanning the entire brain

were used for each type of stain. To determine whether peripheral Aβ entered and

deposited in the brain of Wt mice, brain sections were selected and stained with 6E10

(Mouse anti-Aβ antibody, Covance; Catalog number SIG-39300; 1:200), 4G8 (Mouse

anti-Aβ antibody, Sigma; Catalog number SIG-39200; 1:200), Congo red and

thioflavine-S as previously described 7. CAA was also detected with 1A4 (Rabbit anti-

mouse smooth muscle actin, Abcam; Catalog number ab5694; 1:200) and 6E10

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double immunofluorescence. For the intraneuronal Aβ staining, brain sections were

stained via double immunohistofluorescence for DAPI and A11 (Rabbit anti-Aβ

oligomers antibody, Abcam; Catalog number ab126892; 1:200). A series of sections

was also selected and stained via double immunohistofluorescence for 6E10 and

microglia (Rat anti-mouse CD45, Abcam; Catalog number ab10558; 1:200), 6E10 and

astrocytes (Chicken anti-mouse GFAP, Abcam; Catalog number ab4674; 1:200). For

the microhemorrhage staining, brain sections were stained for hemosiderin with 2%

potassium ferrocyanide in 2% hydrochloric acid for 15 min, followed by

counterstaining with 1% Neutral Red solution for 10 min at room temperature.

Microhemorrhage profiles were counted using a microscope, and the average number

of hemosiderin deposits per section was calculated. Immunostaining with antibodies

against NF200 (Mouse anti-NF200 antibody, Cell Signaling Technology; Catalog

number 2836S； 1:200) was used to evaluate neuronal axon damage. Quantification

was conducted by an investigator who was blinded to the group information of the

samples. The area fraction and/or average optical density (AOD) value was quantified

with ImageJ software.

ELISA assays

A two-step extraction method of frozen brain tissue from the left hemisphere was used

for Aβ analysis. First, the brain tissue was homogenized and sonicated in RIPA buffer

(Beyotime). Homogenates were centrifuged at 100,000 x g for 1 h at 4°C, and the

supernatant was collected and represented the “soluble” extract. The resulting pellet

was then homogenized in 200 μl of 70% formic acid (FA) and centrifuged again at

100,000 x g for 1 h at 4°C. The supernatant was collected and neutralized 1: 20 with 1

M Tris-HCI (pH = 11), and represented the “insoluble” extract. Human and mouse

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Aβ40 and Aβ42 levels were measured using ELISA kits (Covance and Invitrogen).

Concentrations of proinflammatory cytokines, including IL-1β, IL-6, IFN-γ and TNF-

α, were measured in brain extracts by ELISA according to the manufacturer’s

instructions (4A Biotech). The total protein concentrations were determined using a

PierceTM BCA protein assay kit (Thermo Scientific). The levels of Aβ and

proinflammatory cytokines were normalized to the protein concentration of the

samples.

Western blotting

Proteins in the animal brain homogenate were extracted with RIPA buffer. Samples

were loaded onto SDS-PAGE (4-10-15% acrylamide) gels. Separated proteins were

transferred to nitrocellulose membranes. The blots were probed with the following

antibodies: anti-Aβ (6E10, Covance; Catalog number SIG-39300) which recognizes

both human Aβ and APP, anti-mouse Aβ (Biolegend; Catalog number 805801), anti-

phosphorylated-tau antibodies including anti-pS199 (Abcam; Catalog number

ab81268) and anti-pS396 (Signalway; Catalog number 11102), anti-total tau (Tau5,

Millipore; Catalog number MAB361), and anti-β-actin (Sigma-Aldrich). The

membranes were incubated with IRDye 800CW secondary antibodies (Li-COR) and

scanned using an Odyssey fluorescent scanner. The band density was normalized to β-

actin during the analysis.

Electrophysiology

Mice were deeply anesthetized with 30% chloral hydrate (3 ml/kg, i.p.) and

transcardially perfused with N-methyl-D-glucamine (NMDG) artificial cerebral spinal

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fluid (ACSF) prior to decapitation as described previously 8. Brains were rapidly

dissected from the skull and placed for sectioning in ice-cold cutting solution (NMDG

ACSF) aerated with 95% O2 and 5% CO2. NMDG ACSF contained (in mM): NMDG

93, HCl 93, KCl 2.5, NaH2PO4 1.2, CaCl2 0.5, MgSO4 10, NaHCO3 30, HEPES 20,

Na-ascorbate 5.0, Na-pyruvate 3.0, Thiourea 2.0, NAC 12, and D-glucose 25, pH 7.3.

Acute coronal hippocampal slices (400-μm thick) were sectioned from the middle

third of the hippocampus using a vibratome (VT1000S, Leica Microsystems,

Bannockburn, IL, USA) in cutting solution. The slices were then incubated in

oxygenated HEPES ACSF for 1 h at 30°C. HEPES ACSF contained the following (in

mM): NaCl 92, KCl 2.5, NaH2PO4 1.2, CaCl2 0.5, MgSO4 10, NaHCO3 30, HEPES

20, Na-ascorbate 5.0, Na-pyruvate 3.0, Thiourea 2.0, NAC 12, and 25 D-glucose, pH

7.3. Subsequently, the slices were gently transferred into a recording chamber filled

with normal ACSF. The fEPSP evoked by stimulation of the Schaffer

collateral/commissural pathways were recorded in the hippocampus with pipettes (1–

2 MΩ) filled with ACSF. Test fEPSPs were evoked at a frequency of 0.033 Hz and at

a stimulus intensity that was adjusted to approximately 50% of the intensity that

elicited the maximal response. After a 20-min stable baseline, LTP was induced by

high-frequency stimulation (HFS, 100 pulses at 100 Hz). All recordings were

conducted at room temperature (approximately 25°C) using a Multiclamp EPC 10

amplifier (HEKA Electronics, Lambrecht/Pfalz, Germany).

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Statistical analysis

All data are presented as the mean ± SEM. Students t-test and Mann-Whitney U test

were used for the comparison of two groups where appreciate. One-way analysis of

variance and Tukey’s test was used for the comparison of multiple groups with equal

variances. Kruskal-Wallis test was used for the comparison of multiple groups with

unequal variances. Two-sided P values less than 0.05 were defined as statistically

significant. All analyses were carried out using GraphPad Prism, version 6.0

(GraphPad Software, USA).

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Supplementary Figures

Supplementary Figure 1. Mouse Aβ (mAβ) in transgenic AD mice (Tg), wild type

(Wt) mice and parabiotic Wt mice. (a) Biochemical quantification of mouse Aβ40 and

Aβ42 levels in soluble and insoluble brain extracts of parabiotic Wt mice (n = 6 for

each group, mean ±s.e.m., students t-test, *P<0.05, **P<0.01). (b) 6E10, thioflavine-

S, congo red and mouse Aβ specific antibody staining found no Aβ plaque in the

brains of 36 months (36m) Wt mice. Scale bar = 200 um.

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Supplementary Figure 2. Protein sequencing of synthetic human Aβ (a) and human

Aβ in the brain homogenates of transgenic AD mice (b). No specific human Aβ

sequence was found in wild type mice.

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