Shinshu M ed J, 64⑷ :183〜194, 2016

Distribution of Transmissible Amyloid Proteins in the Liverwith Apolipoprotein A‑II Amyloidosis
Yingye LIU , Jinko SAWASHITA , Yaoyong WANG , Lin LI , Hiroki MIYAHARAXin DING , M u YANG and Keiichi HIGUCHI1) Department of Aging Biology, Institute of Pathogenesis and Disease Prevention,
Shinshu University Graduate School of Medicine
2) Department of Biomedical Sciences for Intractable Neurological Diseases, Institute for Biomedical
Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University
3) Fenyang Hospital Aﬃliated with Shanxi Medical University, Shanxi, China

In some amyloidoses, transmission by self‑propagating amyloid proteins plays a critical role in the
progression of the disease. Mouse senile amyloidosis is a disorder in which apolipoprotein A‑II (ApoA‑II)
deposits as amyloid ﬁbrils (AApoAII), and it might be transmitted by ingestion of those amyloid ﬁbrils.
Characterization of protein species responsible for transmission of mouse AApoAII amyloidosis should provide
valuable information. Here, we studied the distributions of ApoA‑II and inducing activities in liver fractions
that were insoluble or soluble and had diﬀerent degrees of amyloid deposition.ApoA‑II was mainly containedin the 3,000 x g pellet fractions regardless of the degree of amyloid deposition. The 3,000 x g pellet fraction
showed strong amyloid ﬁbril‑speciﬁc ﬂuorescence of ﬁbril‑bound thioﬂavin T and strong amyloidosis‑inducing
activity.Sonication of liver homogenate increased the proportion of ApoA‑II and inducing activity of the 100,000 x g pellet fraction.Weak inducing activity was found in the soluble fraction.We fractionated and isolated
multiple assemblies of AApoAII amyloid ﬁbrils by non‑denaturing polyacrylamide gel electrophoresis.ApoA‑
II proteins ranging from monomers to large oligomers had low amyloidosis inducing activity. These results
suggest that transmission of AApoAII amyloidosis may be primarily associated with the insoluble amyloid
ﬁbril structure. Shinshu Med J 64 : 183 ―194, 2016
(Received for publication February 9, 2016;accepted in revised form February 25, 2016)
Key words:mouse apolipoprotein A‑II,insoluble fraction,multiple assemblies of proteins,thioﬂavin T,
transmission of amyloidosis
Abbreviations:AApoAII,amyloid ﬁbril of apolipoprotein A‑II ;Aβ,amyloid β;AD,Alzheimerʼs disease;AI,amyloid
index ;ApoA‑II,apolipoprotein A‑II ;AS,amyloid score;DW,distilled water;PAGE,polyacrylamide gel electrophoresis;ThT, thioﬂavin T

Introduction

mally soluble proteins are deposited in tissues as
highly ordered,insoluble amyloid ﬁbrils .Currently,

Amyloidosis refers to a group of diseases caused

over 30 amyloid ﬁbril proteins are known to be

by structural disorders of proteins whereby nor-associated with amyloidoses , including amyloid
light chain amyloidosis, familial amyloid polyneur-

Corresponding author:Keiichi Higuchi

opathy and reactive amyloid A (AA) amyloidosis.

Department of Aging Biology, Institute of Pathogenesis

These are characterized by amyloid deposition inand Disease Prevention, Shinshu University Graduate
School of M edicine, 3‑1‑1 Asahi, M atsumoto, Nagano

multiple organs.In some types of amyloidosis,such

390‑8621, Japan

as Alzheimerʼs disease (AD) and prion diseases,

E‑mail:keiichih＠shinshu‑u.ac.jp

amyloid deposits are limited to the brain .Apolipo-

No. 4, 2016183

Liu.Sawashita.Wang et al.

protein A‑II (ApoA‑II)is the second most abundantamyloidosis. Furthermore, we fractionated isolated

apolipoprotein in plasma high‑density lipoprotein

amyloid ﬁbrils into multiple ApoA‑II assemblies by(HDL) after apolipoprotein A‑I but the physiologi-

non‑denaturing polyacrylamide gel electrophoresis

cal functions of ApoA‑II are still unclear. The(PAGE). The fractions were then assessed for theirmolecular weight of ApoA‑II is 8.7 kDa. In mice,amyloidosis‑inducing activities. We selected the

ApoA‑II polymerizes into amyloid ﬁbrils (AApoAII)

liver for our investigation for the following reasons.

and deposits systemically (except in the brain)as a

First, the liver has the most severe AApoAII

function of age . Based on these ﬁndings, theamyloid deposition among organs and relatively

amyloid ﬁbrils of mouse senile amyloidosis werelarge amounts of amyloid ﬁbrils could be isolatedtermed “
AApoAII”.AApoAII is present universallyfrom the liver. Second, intravenous injection of

in aged mice of various strains.M oreover,a heredi-

amyloid ﬁbrils reproducibly induces amyloid deposi-tary form has been found in humans . Intriguingtion in the liver.

recent studies have suggested that the aggregates ofM aterials and Methods

misfolded ApoA‑II protein act as seeds that induce
conversion of the native ApoA‑II to ﬁbrillar

Animals

AApoAII assemblies.When young and amyloidosis‑

R1.P1‑Apoa2 is a congenic strain of mice thatnegative mice were injected with AApoAII amyloid

has the amyloidogenic c allele of the ApoA‑II geneﬁbrils extracted from various organs,feces or milk

(Apoa2 ) . R1.P1‑Apoa2of amyloid‑laden mice, AApoAII amyloidosis wasunder speciﬁc pathogen‑free conditions in the Divi-

readily induced . Thus, AApoAII amyloidosis

sion of Laboratory Animal Research, Department

could be transmitted through a mechanism similarof Life Science, Research Center for Human andto that of infectious prion diseases.

Environmental Science,Shinshu University.A com-mice were maintained

Infectious prions consist of multimers of prion

mercial diet (MF ; Oriental Yeast, Tokyo, Japan)

proteins diﬀering in size and solubility. They are

and tap water were available ad libitum.All experi-

resistant to proteinase K treatment and fragmenta-

ments were performed with the approval of thetion by chemical or physical methods

Committee for Animal Experiments of Shinshu.The mostinfectious units were reported to be much smaller
than the ﬁbrils . Similarly, Aβ species seem to

University.
Induction and detection of AApoAII amyloid

contain small and large oligomers, protoﬁbrils anddepositionﬁbrils .Furthermore,the sizes of the soluble pools

Amyloidosis was induced in 2‑month‑old femaleconsisting of small Aβ aggregates were found to

R1.P1‑Apoa2 mice by intravenous injection of 100

correlate highly with the severity and progression

μg AApoAII ﬁbrils. Two, 4, 7, 10, and 13 monthsof AD. It was found that the soluble and insol-

after injection, the treated mice were killed and auble fractions diﬀered in their induction of Aβ‑part of the liver was removed and stored at ‑70°C.

amyloid deposition in a mouse model of AD, andEach organ was ﬁxed in 10% neutral buﬀered for-small Aβ aggregates provided relatively moremalin, embedded in paraﬃn and cut into serial 4‑

molecular interfaces to promote eﬃcient propaga-

μm sections for detection of amyloid deposition.

tion compared with large aggregates or ﬁbrils .

Amyloid deposition was identiﬁed in Congo red‑

Sonication of large Aβ assemblies into numerous

stained sections by polarizing microscopy. The

smaller seeds increased the infective activity .

intensity of the AApoAII deposition in each organ

Here,we investigated the distribution of ApoA‑II

was determined using an amyloid score (AS) thatamong insoluble and soluble protein fractions fromwas graded from 0 to 4: grade 0, no AApoAII ;the livers of mice at diﬀerent stages of amyloidosis.

grade 1,a minute amount ;grade 2,small amounts;

We also examined their abilities to transmit

grade 3,a moderate amount and grade 4,extensive

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Distribution of transmissible ApoA‑II amyloid proteinsFig. 1

Flow chart for extraction of insoluble and soluble ApoA‑II fractions from the liver.Liver tissues
in PBS were processed with a homogenizer.In some cases,the tissues were further sonicated on ice.
Homogenates without or with sonication were centrifuged at 3,000 x g for 5 min at 4°
C.The pellets
were re‑suspended in PBS (3kG‑P and S‑3kG‑P without and with sonication, respectively). The
supernatants were centrifuged at 100,000 x g for 1 hr at 4°
C. The supernatants (Sol and S‑Sol
without and with sonication, respectively) and the pellets (100kG‑P and S‑100kG‑P without andwith sonication, respectively)were obtained for further analysis.

AApoAII deposits. M ore detailed criteria for eachdetermined with the BCA assay (Pierce BCA Pro-

grade in each organ were described previously .

tein Assay Kit, Thermo Scientiﬁc, Tokyo, Japan).

The amyloid index (AI) is the average of the AS

Western blot and electron microscopic analy-

scores in 7 organs (heart, liver, spleen, stomach,sissmall intestine,tongue and skin)and a parameter ofFractions were analyzed on Tris‑Tricine/SDS‑

the degree of AApoAII deposition in the whole

16.5% polyacrylamide gels (SDS‑PAGE). Afterbody.Amyloid ﬁbrils were assayed bya thioﬂavin T

electrophoresis, the 6 protein fractions were trans-(ThT)ﬂuorometric method .

ferred to polyvinylidene diﬂuoride (PVDF) mem-

Extraction of insoluble and soluble ApoA‑II

branes using a semi‑dry Western blot apparatus

fractions from the liver(Nihon Eido, Tokyo, Japan) at 150 mA for 1.5 hr.

Liver tissue was homogenized in phosphate

ApoA‑II was detected with rabbit anti‑ApoA‑II

buﬀered saline (PBS) with a homogenizer (Power

antiserum

Masher,Nippi,Tokyo,Japan).In some cases,it was

method with X‑ray ﬁlm . The density of the posi-

further sonicated 3 times on ice for 30 sec with antive band of ApoA‑II was quantitated using NIH

ultrasonic homogenizer VP‑5S (Tietech Co, Ltd,

Images Ver. 1.61. AApoAII ﬁbrils were puriﬁed

Tokyo,Japan)at a power level of 4 at 30 sec inter-from the liver of mice with heavy amyloid deposi-vals.The 3kG‑P,100kG‑P,Sol,S‑3kG‑P,S‑100kG‑

tions and were used as a standard to calculate the

P and S‑Sol fractions were obtained as shown inrelative content of ApoA‑II in the fractions. The

Fig. 1. The protein content of each fraction was

negatively stained samples were observed with a

No. 4, 2016

and the enhanced chemiluminescence185

Liu.Sawashita.Wang et al.

JEM ‑1400 (HC)electron microscope (JEOL,Tokyo,

C.Then the mixture was centrifuged at 3,000 x g forJapan) .

2 min at 4°
C, and the supernatants were harvested

Fractionation and isolation of amyloid ﬁbrils

and used for transmission experiments. The pellet

by non‑denaturing PAGE

and supernatant were used for Western blotting

Amyloid ﬁbrils were isolated from livers 2,4,7,10

with anti‑ApoA‑II antiserum to assess immuno‑

and 13 months after induction of amyloidosis .
AApoAII amyloid ﬁbrils were separated into 5 frac-depletion.
Statistical analysis

tions by non‑denaturing PAGE as explained below.

Because the AI value is a nonlinear index, theAmyloid ﬁbrils in sample buﬀer (12% glycerol,62.5

values from diﬀerent groups of mice were comparedmM Tris pH 6.8, 0.02% bromophenol blue) wereby using the nonparametric Mann‑Whitney U‑test.

applied to premade non‑denaturing 5‑20% PAGE
gel(SDG‑571,Bio Craft,Tokyo,Japan)and separated by electrophoresis in 25 mM Tris pH 8.3,192 mM

Results
Amyloid deposition induced with AApoAII

glycine running buﬀer with 0.01% SDS at 2 mA for

amyloid ﬁbril injection

20 hr. The non‑denatured PAGE gels were cut into

We injected AApoAII ﬁbrils into 2‑month‑old

5 fractions as follows:I, monomers and dimers (6‑female R1.P1‑Apoa2 mice to induce amyloidosis.

15 kDa);II, trimers,tetramers,and pentamers (15‑

Two months after induction, amyloid deposition

49 kDa);III,small oligomers (49 ‑82 kDa);IV,large

was detectable in the whole body and it further

oligomers (82‑180 kDa),and V, 〜ﬁbrils (＞180 kDa).

increased after 4 and 7 months (Fig. 2A). M ice

The gels containing the 5 fractions were mincedsacriﬁced 10 and 13 months after injection had

with scissors and homogenized 3 times in cold

severe amyloid deposition. In the liver, similar

distilled water (DW) with a Polytron homogenizer

results were observed (Fig. 2B).

for 30 sec with 30 sec intervals, and then fractions

Fractionation of soluble and insoluble amyloid

were ultracentrifuged at 100,000 x g for 1 hr at 4°C.

ﬁbrils by centrifugation

Supernatants were transferred to new tubes.

We separated soluble and insoluble fractions fromTransmission activities of the various frac-liver homogenates (Fig.1).The 3kG‑P,100kG‑P,S‑

tions

3kG‑P and S‑100kG‑P fractions of livers collected

We assessed the amyloidosis‑inducing activity of

4,7,and 10 months after injection showed ApoA‑II

the 6 insoluble and soluble fractions of liver

bands (Fig. 3A).However,ApoA‑II bands were nothomogenates and the 5 fractions isolated fromobserved in any of the fractions 2 months after

amyloid ﬁbrils with non‑denaturing PAGE gels.

injection (data not shown).The content of ApoA‑II

Thus, each fraction containing 10μg of ApoA‑II

was determined by Western blot analysis. The

was injected into the tail vein of three 2‑month‑old

results showed that ApoA‑II was mainly contained

female R1.P1‑Apoa2

in the 3kG‑P fraction (Table 1). After sonication,

mice. After 2 months,

amyloid deposition was determined.

ApoA‑II contents were decreased in the S‑3kG‑P

Immuno‑depletion analysis of soluble fraction

fraction and increased in the S‑100kG‑P fraction.

Protein G‑Agarose beads were washed three

Interestingly, sonication of 4m mice liver

times in PBS at room temperature and incubatedhomogenates increased the contents of ApoA‑II infor 60 min with 10μl of anti‑ApoA‑II antiserum or

the S‑100kG‑P fraction to 58.9 % of total ApoA‑II.

normal rabbit serum, then washed three times in

Based on the ThT assay, the ﬂuorescence levels of

PBS at 4°
C. The soluble (Sol) fraction of 7m‑1

the amyloid ﬁbrils in 3kG‑P, 100kG‑P, S‑3kG‑P,

mouse was diluted to a ﬁnal concentration of 1 mg/

and S‑100kG‑P fractions did not diﬀer (Fig. 3B).

ml in DW. The diluted fractions were allowed to

We did not detect amyloid ﬁbril‑speciﬁc ﬂuorescen-react with antibody‑bead complexes overnight at 4°

ce in the Sol and S‑Sol fractions.

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Distribution of transmissible ApoA‑II amyloid proteinsFig. 2Amyloid deposition after the induction of amyloidosis.The treated mice were killed after 2,4,7,
and 10 months (each, n＝3)and 13 months (n＝1)after induction of amyloidosis.(A)The degree ofamyloid deposition in the whole body was compared using the AI. (B) The grade of amyloid
deposition in the liver was compared with the AS.

Table 1

Protein and ApoA‑II contents in insoluble and soluble fractions without and with sonication(% of total protein and ApoA‑II)
Without sonication

Mouse

3kG‑P

100kG‑P

With sonication
3kG‑P

Sol

100kG‑PSol

Protein(%)

ApoAII(%)

Protein(%)

ApoAII(%)

Protein(%)

ApoAII(%)

Protein(%)

ApoAII(%)

Protein(%)

ApoAII(%)

Protein(%)

ApoAII(%)

342±08a184±21a474±16a161±15a

305±22a

535±09a343±77913

153±6987504±56a

141±58417

249 ±27589

600±50a344±44 941±39

110±3560±38545±11a327±16 775±135 177±73 225±135 49 6±68

a

10m‑1
10m‑2 438±28 981±11
10m‑3

53±04

19 ±11509 ±29a354±17 69 9 ±29

a

2m‑1
2m‑2
2m‑3
4m‑1
4m‑2
4m‑3
7m‑1
7m‑2
7m‑3

174±10 301±29

427±08The treated mice were killed after 2, 4, 7, and 10 months (each, n＝3)and the liver of each mouse was analyzed.
a :no bands were positive for anti‑ApoA‑II antiserum.
b :the 4m‑3 mouse lacked ApoA‑II positive bands and ﬁgures are means of two mice (4m‑1 and 4m‑2).

Next, we determined the amyloidosis‑inducing

activity was signiﬁcantly increased in the S‑100kG‑

activity in the fractions (Fig. 3C). Four fractions

P fractions of 10m‑1 mice. All the Sol and S‑Sol(3kG‑P, 100kG‑P, S‑3kG‑P and S‑100kG‑P) con-fractions except for Sol of 4m mice showed weaktaining 10μg of ApoA‑II protein and Sol and S‑

amyloidosis‑inducing activity.

Sol fractions (containing 10μg of protein) wereTo analyze the ultrastructure of the AApoAII in

injected into the tail veins of 2‑month‑old R1.P1‑

each fraction,we observed negatively stained ﬁbrils

Apoa2 mice (n＝3 mice per fraction). The 3kG‑Pusing electron microscopy (Fig. 4). We observed

and 100kG‑P fractions showed amyloidosis‑induc-very small amounts of amyloid ﬁbril‑like structures

ing activity similar to that of standard AApoAIIin 3kG‑P, 100kG‑P, S‑3kG‑P and S‑100kG‑P frac-ﬁbrils. After sonication, the amyloidosis‑inducing

tions of 4m‑1 mice. Abundant amyloid ﬁbrils were

No. 4, 2016187

Fig. 3

Liu.Sawashita.Wang et al.

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No. 4, 2016

Fig. 4

Fig. 3

ApoA‑II in the six soluble and insoluble fractions from the livers of mice.(A)Western blot analysis of the 6 fractions (5μg ofbars are 100 nm.

the livers of 4m‑1, 7m‑1, and 10m‑1 mice were observed by transmission electron microscopy.M agniﬁcation was X20,000.Scale

Transmission electron microscopic images of 6 negatively stained insoluble and soluble fractions of livers. Six fractions frompositive bands, ﬁgures are means of 2 mice.

the ThT binding assay and compared with puriﬁed AApoAII amyloid ﬁbrils. (C) Amyloidosis‑inducing activity of 6 fractions.
Values are means ± standard deviation (n＝3). , p＜0.05 vs. without sonication. Because the 4m‑3 mouse showed no ApoA‑II‑protein)obtained from the livers of 4m‑1, 7m‑1, 10m‑1 mice (Table 1) detected with anti‑ApoA‑II antiserum. Arrows indicate
ApoA‑II monomer,dimer,trimer.(B)The intensity of amyloid ﬁbril‑speciﬁc ﬂuorescence per μg of ApoA‑II was determined withDistribution of transmissible ApoA‑II amyloid proteins189

Liu.Sawashita.Wang et al.

Fig. 5

Immuno‑depletion analysis of soluble fractions. The Sol fraction of the 7m‑1 mouse was immuno‑
depleted with anti‑ApoA‑II antiserum or normal rabbit serum. (A)Western blot analysis of the pellet
and supernatant after immuno‑depletion. The intensity of 〜25 kDa protein band of supernatant was
reduced after immuno‑depletion with anti‑ApoA‑II antiserum (AII) compared with the band depletedwith normal rabbit serum (NS). Arrow indicates 〜25 kDa bands.(B)Amyloidosis‑inducing activity of
10μg of supernatant protein (n＝3 mice per fraction). , p＜0.05.

observed in the 3kG‑P fractions of 7m‑1 and 10m‑

Fractionation of amyloid ﬁbrils with non‑1 mice, but only small amounts of smaller amyloid

denaturing PAGE

ﬁbril‑like structures were found in the 100kG‑P

Four, 7, 10 and 13 months after the induction of

fractions of these mice.After sonication,shortened

amyloidosis,AApoAII ﬁbrils were isolated from theamyloid ﬁbrils were observed in the S‑100kG‑Plivers . We analyzed the extent of polymerization

fractions in 7m‑1 and 10m‑1 mice. In contrast, noof ApoA‑II in AApoAII ﬁbrils with non‑denaturing

amyloid ﬁbril‑like structures were found in either

PAGE. Speciﬁcally, the distribution of monomers,

the Sol or S‑Sol fractions.

dimers, trimers, tetramers, pentamers, small

Immuno‑depletion analysis of soluble fraction

oligomers, large oligomers and ﬁbrils was deter-

Soluble fraction of 7m‑1 mouse was immuno‑

mined.The distribution patterns of these assemblies

depleted with anti‑ApoA‑II antiserum (Fig. 5).

did not change with the progression of amyloidosis

Although ApoA‑II monomers could not be detected,(Fig. 6A).At 4,7 and 10 months,ﬁve fractions (I‑V)

bands with a molecular weight of 〜25 kDa were

were cut out from PAGE gels as judged by their

speciﬁcally detected with anti‑ApoA‑II antiserum.

molecular weights. The proteins were extracted

We tried immune‑depletion several times with anti‑

from the 5 gel fractions and assessed for amyloid

ApoA‑II antiserum independently and the intensity

ﬁbril‑speciﬁc ﬂuorescence with the ThT bindingof this protein band was reduced to 〜60% (0.64±0.

assay. The ﬂuorescence of each fraction was very

13, n＝4) of the band depleted with normal rabbit

low.Those results suggested that ApoA‑II proteins

serum, but did not disappear completely (Fig. 5A,

in each fraction did not have a β‑pleated structureright two lanes). We next determined the(Fig. 6B).To assess the amyloidosis‑inducing activ-

amyloidosis‑ inducing activity of immuno‑depleted

ity,fractions were injected into the tail veins of R1.

soluble fractions. The fractions immuno‑depleted

P1‑Apoa2 mice.All 5 of the fractions showed weakwith anti‑ApoA‑II antiserum showed weakertransmission activity (Fig. 6C).

amyloidosis‑inducing activity than that in a fraction depleted with normal rabbit serum (Fig. 5B).
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Distribution of transmissible ApoA‑II amyloid proteinsFig. 6

Non‑denaturing PAGE analyses of AApoAII amyloid ﬁbrils. (A) AApoAII amyloid ﬁbrils wereisolated from the livers of 4m‑1, 7m‑1 and 10m‑1 and 13m‑1 mice. AApoAII amyloid ﬁbrils wereanalyzed with non‑denatured PAGE and Western blot analysis.The distribution of monomers,dimers,
trimers, tetramers, pentamers, small oligomers, large oligomers and ﬁbril was determined. Fraction I(monomers and dimers),II (trimers,tetramers and pentamers),III (small oligomers),IV (large oligomers)
and V (ﬁbrils)were cut out and the proteins were extracted from the gels.(B)The intensity of amyloid
ﬁbril‑speciﬁc ﬂuorescence per μg of protein in each fraction isolated from 4m‑1,7m‑1 and 10m‑1 mice
was determined by the ThT binding assay. (C) Amyloidosis‑inducing activity of each fraction (10μgprotein)was determined (n＝3 mice per fraction).Figures in parentheses represent number of amyloid‑
positive mice/number of mice examined.

Discussion

distribution and transmissibility of AApoAII in insoluble and soluble fractions of the liver in which

In the studies of brain samples of AD patients,thediﬀerent amounts of amyloid were deposited. Six

size of the soluble pools consisting of small Aβ

fractions were isolated from the liver by homogen-aggregates was highly correlated with the severity

ization and centrifugation (Table 1). Determinationand progression of AD . Thus, we studied theof ApoA‑II contents in each fraction revealed thatNo. 4, 2016191

Liu.Sawashita.Wang et al.

over 90% of ApoA‑II was contained in the 3kG‑P

thereby modify the structure of ApoA‑II amyloid

fractions of the liver homogenates regardless ofprotein. We did not observe the eﬀects of progres-whether deposition was light (4 months after induc-sion of amyloid deposition in the liver on the bio-tion) or heavy (10 months after injection). After

chemical characteristics including transmission

sonication, the ApoA‑II content was decreased in

activity of amyloid ﬁbrils.

the S‑3kG‑P fraction and increased in the S‑100kG‑

All the Sol and S‑Sol fractions showed weakP fraction. Abundant amyloid ﬁbrils were observed

amyloidosis inducing activity (Fig. 3C). We did notin the 3kG‑P fractions. However, they were rarely

detect ApoA‑II proteins in our Western blot analy-

found in 100kG‑P fractions. After sonication, shor

sis nor did we ﬁnd any amyloid ﬁbril‑like structures

tened amyloid ﬁbrils were observed in the S‑100kG‑

in electron microscopic examination of the Sol andP fractions. Sonication might break AApoAII

S‑Sol fractions (Fig. 4).In the mouse model of AD,

amyloid ﬁbrils in the 3kG‑P fraction and fragment-very small amounts of soluble Aβ with higher

ed ﬁbrils might be fractionated into the S‑100kG‑P

amyloidosis‑inducing‑activity might mediate thefraction. Amyloidosis‑inducing activity of the S‑

spread of Aβ amyloidosis . We detected bands

100kG‑P fraction from the 10m‑1 mouse wasreactive with anti‑ApoA‑II antiserum in the Sol

signiﬁcantly higher than that of the 100kG‑P frac-

fraction (Fig. 5), in immuno‑depletion analysis, wetion (Fig. 3C).It was reported that sonicated prions

estimated a molecular weight of 〜25 kDa. This 〜

and Aβﬁbrils in brain homogenates had increased

25 kDa band was also detected in Sol and S‑Sol

seeding potentials after fragmentation of ﬁbrils into

fractions of 7m and 10m mice (Fig. 3A). Reduction

smaller,soluble species by increasing the active siteof this band by immuno‑depletion with ApoA‑IIof ﬁbrils

antiserum decreased amyloidosis‑inducing activity.

.

The ThT binding assay quantitates AApoAII

We will characterize these bands in future studies.

amyloid ﬁbrils with high sensitivity andIn studies of prion disease, it was found thatspeciﬁcity .In our previous work,manydenaturing

prions existed in a range of sizes and thattreatments partially disrupted amyloid ﬁbril struc-nonﬁbrillar small particles were the most infectious

ture and there was a distinct correlation between

agents .We used non‑denaturing PAGE to charac-

the ﬂuorescence determined with the ThT binding

terize multiple ApoA‑II assemblies of AApoAII

assay and amyloidosis‑inducing activity. Those

amyloid ﬁbrils that had been extracted from theresults suggested that the structure of amyloid

livers. The AApoAII amyloid ﬁbrils containedﬁbrils might be important for induction ofmultiple AApoAII assemblies from monomers toamyloidosis .The ﬂuorescence levels in ThT bind-ﬁbrils. We cut out sections (fractions I‑V)from theing assays did not diﬀer among 3kG‑P,100kG‑P,S‑

non‑denaturing PAGE (Fig.6).Wefound that ApoA‑

3kG‑P and S‑100kG‑P fractions and were about the

II assemblies in fractions I‑IV had very small

same as puriﬁed AApoAII ﬁbrils (Fig. 3B). The

amounts of amyloid ﬁbril‑speciﬁc ﬂuorescence andmolecular structure to which ThT binds is still notthat they had only slight activity for amyloid induc-clear. Thus, we do not know whether ApoA‑IItion. Thus, the speciﬁc amyloidosis‑inducing activ-

proteins in 3kG‑P,100kG‑P,S‑3kG‑P and S‑100kG‑

ity per ApoA‑II monomer of fractions I‑IV was low

P fractions showed similar ﬂuorescence and had thecompared with puriﬁed AApoAII ﬁbrils.In general,same molecular structure, although the electron

fraction V was very diﬃcult to extract from themicroscopic structures were clearly diﬀerent.

gels due to their high molecular weights. It is pos-

We had speculated that the composition ofsible that only degraded ﬁbrils were extracted andamyloid‑associated molecules such as apolipo-

did not show high inducing activity. From these

protein E and heparin sulfate proteoglycan mightﬁndings, we postulate that the partially puriﬁedchange during the progression of amyloidosis andAApoAII amyloid ﬁbrils isolated from the livers

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Distribution of transmissible ApoA‑II amyloid proteins

contained multiple ApoA‑II assemblies from mono-quite important inducers in the transmission ofmers to large oligomers.However,these assemblies

AApoAII amyloidosis.

had only a slight β‑pleated structure and a low

Declaration of Interest

amyloidosis‑inducing activity.We will characterizethese multiple assemblies using other isolation

The authors declare no conﬂicts of interest.This
research was supported by Grants‑in‑Aid formethods in future studies.
The results above suggest that multiple ApoA‑II

Scientiﬁc Research (B) 23390093 and 26293084, andassemblies exist as insoluble molecules.Their char-

Challenging Exploratory Research 26670152 fromacteristics, including amyloidosis‑inducing activity,

the M inistry of Education,Culture,Sports,Science,

do not change with the progression of amyloidosis.

and Technology of Japan and by grants from theWe cannot deny the possibility that very small

Intractable Disease Division, the M inistry ofamounts of soluble ApoA‑II with higher amyloidosis‑

Health, Labor, and Welfare to the Research Com-

inducing activity mediate the transmission ofmittees for Amyloidosis. We thank Drs. Kiyoshi

amyloidosis. However, we have found AApoAII

M atsumoto, Yoshizawa Takahiro and Ms. Kayoamyloid ﬁbrils in feces, milk, muscle and saliva ,Suzuki (Research Center for Human and Environ-and the ﬁbrils possessed amyloidosis‑inducing

mental Science, Shinshu University)for the care ofactivity. Thus, we believe that amyloid ﬁbrils aremice and technical assistance.

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（2016. 2. 9 received ; 2016. 2.25 accepted）

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