Intraneuronal

Aβ

immunoreactivity

is

not

a

predictor

of

brain

amyloidosis-β

or

neurofibrillary

degeneration

Amyloid

β

-LRB-

Aβ

-RRB-

immunoreactivity

in

neurons

was

examined

in

brains

of

control

subjects

people

with

Down

syndrome

and

patients

with

sporadic

Alzheimer

's

disease

to

determine

if

intraneuronal

Aβ

immunoreactivity

is

an

early

manifestation

of

Alzheimer-type

pathology

leading

to

fibrillar

plaque

formation

and/or

neurofibrillary

degeneration

The

appearance

of

Aβ

immunoreactivity

in

neurons

in

infants

and

stable

neuron-type

specific

Aβ

immunoreactivity

in

a

majority

of

brain

structures

during

late

childhood

adulthood

and

normal

aging

does

not

support

this

hypothesis

The

absence

or

detection

of

only

traces

of

reaction

with

antibodies

against

aa

and

aa

of

Aβ

in

neurons

indicated

that

intraneuronal

Aβ

was

mainly

a

product

of

α

and

γ-secretases

-LRB-

-RRB-

The

presence

of

N-terminally

truncated

and

in

the

control

brains

was

confirmed

by

Western

blotting

and

the

identity

of

was

confirmed

by

mass

spectrometry

The

prevalence

of

products

of

α

and

γ

secretases

in

neurons

and

β

and

γ-secretases

in

plaques

argues

against

major

contribution

of

Aβ-immunopositive

material

detected

in

neuronal

soma

to

amyloid

deposit

in

plaques

The

strongest

intraneuronal

immunoreactivity

was

observed

in

structures

with

low

susceptibility

to

fibrillar

Aβ

deposition

neurofibrillary

degeneration

and

neuronal

loss

compared

to

areas

more

vulnerable

to

Alzheimer-type

pathology

These

observations

indicate

that

the

intraneuronal

Aβ

immunoreactivity

detected

in

this

study

is

not

a

predictor

of

brain

amyloidosis

or

neurofibrillary

degeneration

The

constant

level

of

Aβ

immunoreactivity

in

structures

free

from

neuronal

pathology

during

essentially

the

entire

life

span

suggests

that

intraneuronal

amino-terminally

truncated

Aβ

represents

a

product

of

normal

neuronal

metabolism

Introduction

Neurofibrillary

degeneration

and

brain

amyloidosis

with

deposition

of

fibrillar

amyloid

β

-LRB-

Aβ

-RRB-

in

plaques

are

diagnostic

features

of

Alzheimer

's

disease

-LRB-

AD

-RRB-

Intracellular

processing

of

amyloid

precursor

protein

-LRB-

APP

-RRB-

with

β

and

γ-secretases

generates

and

in

the

endoplasmic

reticulum

trans-Golgi

network

and

endosomal

lysosomal

system

-LSB-

-RSB-

An

amino-terminally

truncated

peptide

-LRB-

-RRB-

is

the

product

of

APP

cleavage

with

α

and

γ-secretase

Cells

may

produce

and

secrete

several

species

of

Aβ

including

and

-LSB-

-RSB-

Human

neurons

are

Aβ-immunoreactive

-LSB-

-RSB-

The

nature

distribution

and

role

of

intraneuronal

Aβ

are

the

subject

of

controversy

-LRB-

see

review

by

Takahashi

-LSB-

-RSB-

-RRB-

Intraneuronal

Aβ

immunoreactivity

has

been

localized

in

lipofuscin

deposits

-LSB-

-RSB-

cathepsin

D-positive

vesicles

of

lysosomal

origin

-LSB-

-RSB-

multivesicular

bodies

within

presynaptic

and

postsynaptic

compartments

-LSB-

-RSB-

and

intracellular

and

extracellular

neurofibrillary

tangles

-LRB-

NFTs

-RRB-

-LSB-

-RSB-

Several

studies

of

cytoplasmic

Aβ

immunoreactivity

in

neurons

and

glial

cells

in

the

human

brain

have

been

conducted

to

determine

the

properties

of

Aβ

in

human

neurons

and

its

role

in

fibrillar

plaque

formation

-LSB-

-RSB-

The

key

observation

has

been

the

absence

of

-LSB-

-RSB-

or

only

minimal

intraneuronal

Aβ

immunoreactivity

-LSB-

-RSB-

in

normal

brain

Therefore

the

appearance

of

or

an

increase

in

Aβ

immunoreactivity

has

been

suggested

as

a

sign

of

neuronal

pathology

-LSB-

-RSB-

leading

to

fibrillar

plaque

formation

in

the

brain

of

people

with

AD

-LSB-

-RSB-

Overexpression

of

APP

overproduction

of

Aβ

and

early

intracellular

accumulation

of

Aβ

-LSB-

-RSB-

have

been

considered

the

foundation

for

early

onset

of

AD

pathology

and

functional

deterioration

in

adults

with

Down

syndrome

-LRB-

DS

-RRB-

in

their

's

-LSB-

-RSB-

The

loss

of

Aβ

immunoreactivity

in

areas

of

plaque

formation

has

led

to

the

conclusion

that

neurons

release

intracellular

Aβ

which

initiates

a

seeding

process

leading

to

plaque

formation

-LSB-

-RSB-

According

to

Bahr

et

al.

-LSB-

-RSB-

the

death

of

neurons

a

prominent

feature

of

AD

is

associated

with

the

release

of

oligomerized

intracellular

into

the

surrounding

milieu

which

may

stimulate

the

production

of

amyloidogenic

fragments

of

APP

amplify

the

levels

of

intracellular

Aβ

in

neighboring

cells

and

act

as

a

nidus

for

the

deposition

of

secreted

Aβ

The

association

of

intracellular

with

intraneuronal

tangles

has

been

considered

an

indication

that

the

growing

concentration

of

may

contribute

to

NFT

formation

-LSB-

-RSB-

The

presence

of

altered

aspartyl

residues

in

intracellular

NFTs

has

been

interpreted

as

indicating

that

racemized

Aβ

peptides

are

involved

in

neurofibrillary

degeneration

-LSB-

-RSB-

The

pattern

of

intraneuronal

Aβ

immunoreactivity

observed

in

the

control

cohort

in

our

previous

study

-LSB-

-RSB-

was

in

conflict

with

the

hypothesis

that

Aβ

immunoreactivity

in

neurons

is

the

key

and

an

early

event

in

the

cascade

of

pathology

leading

to

AD

Therefore

the

aim

of

this

study

was

to

reexamine

the

hypothesis

that

intraneuronal

Aβ

immunoreactivity

is

an

early

manifestation

of

Alzheimer-type

pathology

leading

to

the

formation

of

fibrillar

plaque

and/or

neurofibrillary

degeneration

To

test

this

hypothesis

we

examined

patterns

of

Aβ

accumulation

in

the

human

brain

during

development

adulthood

and

aging

and

patterns

of

changes

in

the

amount

and

distribution

of

Aβ

in

neurons

in

people

with

DS

and

DS/AD

and

sporadic

AD

Materials

and

methods

Human

tissue

The

brains

of

individuals

were

examined

Control

brains

included

cases

from

months

to

years

of

age

-LRB-

and

infants

children

and

adults

from

to

years

of

age

males

and

females

-RRB-

DS

brains

included

cases

from

weeks

to

years

old

-LRB-

infants

that

were

weeks

and

and

months

old

and

adults

from

to

years

of

age

males

and

females

-RRB-

In

the

AD

cohort

the

brains

of

subjects

from

to

years

of

age

-LRB-

males

and

females

-RRB-

were

studied

Diagnostically

three

subjects

had

a

mild

cognitive

impairment

-LRB-

MCI

-RRB-

corresponding

to

global

deterioration

scale

-LRB-

GDS

-RRB-

stage

-LSB-

-RSB-

a

frequent

clinical

precursor

of

AD

GDS

stage

-LRB-

mild

AD

-RRB-

was

reported

for

three

subjects

stage

-LRB-

moderate

AD

-RRB-

for

three

subjects

stage

-LRB-

moderately

severe

AD

-RRB-

for

subjects

and

stage

-LRB-

severe

AD

-RRB-

for

subjects

One

brain

hemisphere

was

fixed

in

buffered

formalin

for

to

several

months

and

then

dissected

into

cm

thick

slabs

The

tissue

blocks

were

dehydrated

for

days

in

ethanol

then

days

in

ethanol

and

finally

week

in

ethanol

The

samples

were

infiltrated

with

polyethylene

glycol

-LRB-

PEG

-RRB-

-LRB-

Merck

-RRB-

for

days

-LRB-

two

changes

of

days

each

at

room

temperature

-RRB-

and

with

PEG

for

another

days

-LRB-

two

changes

of

days

each

at

°C

-RRB-

The

slabs

were

embedded

in

fresh

PEG

-LSB-

-RSB-

The

tissue

blocks

were

cut

serially

at

μm

thick

sections

The

immunoproperties

of

intracellular

Aβ

in

tissues

fixed

in

formalin

for

to

several

months

were

also

compared

with

immunostainings

of

sections

from

five

brains

-LRB-

two

DS

and

three

AD

-RRB-

prefixed

in

paraformaldehyde

for

h

dissected

into

cm

thick

slabs

fixed

in

paraformaldehyde

for

the

next

h

cryoprotected

with

and

solutions

of

sucrose

-LRB-

days

-RRB-

frozen

and

cut

serially

into

μm

thick

sections

The

methods

selected

for

this

study

were

approved

by

the

New

York

State

Institute

for

Basic

Research

-LRB-

IBR

-RRB-

Institutional

Review

Board

The

tissue

samples

were

provided

by

the

Silberstein

Institute

for

Aging

and

Dementia

at

New

York

University

the

Brain

Bank

at

IBR

and

the

University

of

Miami

Brain

and

Tissue

Bank

In

postmortem

examination

all

samples

were

identified

only

by

an

anonymous

case

number

and

tissue

was

examined

blind

to

clinical

and

demographic

information

Immunostaining

Several

antibodies

were

applied

to

serial

sections

to

evaluate

Aβ

immunoreactivity

in

cortical

and

subcortical

structures

the

cerebellum

and

brainstem

Monoclonal

antibodies

-LRB-

mAbs

-RRB-

and

were

used

for

characterization

of

the

amino-terminal

portion

of

Aβ

-LRB-

Table

-RRB-

mAb

recognizes

an

epitope

in

residues

of

Aβ

-LRB-

Signet

Laboratories

-RRB-

-LSB-

-RSB-

mAb

recognizes

an

epitope

in

residues

of

Aβ

-LRB-

Novocastra

Laboratories

Ltd

NCL-β-amyloid

-RRB-

The

central

portion

of

Aβ

was

detected

with

mAb

which

recognizes

an

epitope

in

residues

of

Aβ

-LSB-

-RSB-

The

carboxyl

terminus

of

Aβ

was

characterized

with

pAbs

purified

from

rabbit

serum

by

epitope-specific

affinity

chromatography

These

antibodies

react

with

Aβ

residues

-LRB-

Catalog

nr

-RRB-

and

-LRB-

Catalog

nr

BioSource

International

Inc.

CA

USA

-RRB-

The

reactivity

towards

other

species

of

Aβ

peptides

was

eliminated

through

a

series

of

preabsorption

steps

Purified

rabbit

polyclonal

antibodies

and

specific

for

the

carboxyl

terminus

of

-LRB-

residues

-RRB-

were

also

used

-LSB-

-RSB-

Fibrillar

Aβ

was

detected

with

rabbit

polyclonal

antibody

produced

by

immunization

of

rabbits

with

fibrillar

Rabbit

antibodies

were

purified

according

to

the

protocol

described

by

Miller

et

al.

-LSB-

-RSB-

Table

monoclonal

and

rabbit

polyclonal

antibodies

used

for

aa

Signet

Laboratories

-LRB-

developed

at

IBR

-RRB-

aa

aa

IBR

-LRB-

Dr.

R.

Kascak

-RRB-

aa

aa

aa

-LRB-

Drs.

D.L.

Miller

and

P.D.

Mehta

-RRB-

aa

-LRB-

Drs.

D.L.

Miller

and

P.D.

Mehta

-RRB-

Aβ

-LRB-

Drs.

D.L.

Miller

and

P.D.

Mehta

-RRB-

aa

of

IBR

-LRB-

Dr.

R.

Kascsak

-RRB-

Monoclonal

antibody

reacts

with

Aβ

both

on

western

blots

and

in

formalin

fixed

material

-LSB-

-RSB-

mAb

binds

APP

on

western

blots

as

a

native

protein

but

does

not

immunoreact

with

APP

after

fixation

with

formalin

and

dehydration

We

have

also

shown

that

in

cultures

of

cells

overexpressing

APP

and

formalin

fixed

and

dehydrated

both

antibodies

and

do

not

detect

APP

but

they

detect

Aβ

-LSB-

-RSB-

Also

the

study

of

cultured

cells

with

elevated

levels

of

APP

and

C-terminal

fragments

of

APP

revealed

that

mAb

does

not

detect

APP

and

C-terminal

fragments

of

APP

by

immunocytochemistry

-LSB-

-RSB-

-LRB-

Table

-RRB-

Table

and

Aβn

concentration

-LRB-

pmol/g

-RRB-

in

temporal

cortexControl

case

#Aβn

aBrain

samples

were

homogenized

in

formic

acid

as

described

in

Materials

and

methods

Aliquots

of

the

extracts

were

neutralized

and

subjected

to

PAGE

and

immunobloting

bands

migrated

above

the

kDa

standards

and

bound

mAb

bands

migrated

near

the

kDa

standard

and

bound

or

but

did

not

bind

mAb

was

only

one

band

in

this

extract

Its

level

was

too

low

to

be

revealed

by

mAb

and

so

could

not

be

identified

as

or

The

endogenous

peroxidase

in

the

sections

was

blocked

with

hydrogen

peroxide

in

methanol

To

enhance

immunoreactivity

of

Aβ

sections

were

treated

with

formic

acid

for

min

-LSB-

-RSB-

The

sections

were

then

treated

with

fetal

bovine

serum

in

phosphate

buffer

solution

-LRB-

PBS

-RRB-

for

min

to

block

nonspecific

binding

The

antibodies

were

diluted

in

fetal

bovine

serum

in

PBS

and

were

incubated

with

sections

overnight

at

°C

The

sections

were

washed

and

treated

for

min

with

either

biotinylated

sheep

anti-mouse

IgG

antibody

or

biotinylated

donkey

anti-rabbit

IgG

antibody

diluted

The

sections

were

treated

with

an

extravidin

peroxidase

conjugate

-LRB-

-RRB-

for

h

and

the

product

of

reaction

was

visualized

with

diaminobenzidine

-LRB-

mg/ml

with

hydrogen

peroxide

in

PBS

-RRB-

After

immunostaining

the

sections

were

lightly

counterstained

with

cresyl

violet

Phosphorylated

tau

protein

of

neurofibrillary

tangles

was

detected

with

mAb

-LRB-

-RRB-

recognizes

an

epitope

in

residues

of

the

human

tau

sequence

-LSB-

-RSB-

To

obtain

optimum

staining

with

sections

were

treated

with

alkaline

phosphatase

-LRB-

Sigma

Type

VII-L

μg/ml

in

PBS

pH

-RRB-

-LSB-

-RSB-

A

three-point

classification

was

used

to

estimate

semi-quantitatively

the

difference

of

immunoreactivity

with

mAb

in

four

brain

structures

of

control

adult

subjects

The

amygdala

and

cornu

Ammonis

were

selected

as

structures

susceptible

to

amyloidosis

β

and

neurofibrillary

degeneration

The

lateral

geniculate

body

and

dentate

nucleus

were

selected

as

structures

resistant

to

AD

pathology

and

almost

free

of

plaques

and

NFTs

even

in

severe

AD

Grade

corresponded

to

weak

immunoreactivity

present

in

about

of

neurons

grade

corresponded

to

moderate

immunoreactivity

in

almost

all

neurons

and

grade

corresponded

to

strong

immunoreactivity

present

in

almost

all

neurons

in

a

given

brain

structure

Sections

from

brain

of

DS

subjects

were

used

to

determine

whether

intraneuronal

Aβ

immunoreactivity

differs

in

people

with

an

extra

copy

of

the

gene

encoding

APP

The

difference

between

intraneuronal

Aβ

and

amyloid

in

plaques

was

determined

with

antibodies

detecting

the

amino

and

carboxyl-terminus

and

the

middle

portion

of

Aβ

peptide

The

relationship

of

neurofibrillary

changes

to

Aβ

immunoreactivity

in

neurons

was

evaluated

in

brains

of

people

with

DS/AD

and

sporadic

AD

Partial

purification

and

identification

of

Aβ

peptides

Samples

of

the

brain

cortex

-LRB-

mg

-RRB-

from

control

males

that

were

and

years-old

were

dispersed

in

ml

of

formic

acid

by

brief

sonification

and

centrifuged

for

min

at

g.

Supernatants

were

dried

by

centrifugal

evaporation

the

residues

were

resuspended

in

ml

of

-LRB-

v/v

-RRB-

formic

acid

and

centrifuged

as

above

The

solutions

were

subjected

to

size-fractionation

on

a

cm

Pharmacia

column

equilibrated

with

formic

acid

as

previously

described

-LSB-

-RSB-

The

Aβ-containing

fractions

were

identified

by

Western

blotting

and

their

contents

were

quantified

by

photodensitometry

as

previously

described

-LSB-

-RSB-

The

Aβ-containing

fractions

were

dried

under

vacuum

and

were

re-dried

out

of

ammoniacal

methanol

to

neutralize

traces

of

formic

acid

To

solubilize

Aβ

peptides

each

residue

was

treated

with

μl

of

mM

ammonia

and

centrifuged

as

above

and

the

supernatant

liquid

was

adjusted

to

pH

with

Antibody

raised

to

using

the

previously

described

methods

-LSB-

-RSB-

was

purified

on

a

peptide

affinity

column

and

was

coupled

to

an

Aminolink

matrix

-LRB-

Pierce-Endogen

-RRB-

at

a

concentration

of

pmol

per

μl

of

settled

matrix

The

Aβ

isoforms

were

immuno-adsorbed

to

μl

of

during

a

h

incubation

Following

washes

with

PBS

and

water

the

Aβ

isoforms

were

eluted

with

μl

portions

of

trifluoroacetic

acid

in

acetonitrile

About

of

total

Aβ

peptides

present

in

the

cortex

samples

were

recovered

by

this

method

as

evaluated

by

Western

blotting

The

shape

and

size

of

the

Aβ

band

in

Western

blotting

of

the

size-fractionated

material

and

the

immuno-purified

peptide

were

the

same

which

suggested

that

the

process

did

not

selectively

enrich

any

of

the

Aβ

peptides

Mass

spectrometry

and

Western

blotting

The

peptide

preparation

isolated

by

immunoadsorption

on

was

dissolved

in

μl

of

-LRB-

v/v

-RRB-

formic

acid

and

approximately

was

loaded

on

a

Symmetry

nanoAcquity

column

-LRB-

μm

mm

-RRB-

as

six

sequential

μl

injections

Following

the

sixth

injection

the

peptides

were

resolved

on

an

Atlantis

nanoAcquity

column

-LRB-

μm

mm

-RRB-

with

a

min

gradient

of

to

acetonitrile

in

water

containing

formic

acid

at

flow

rate

of

μl/min

The

eluate

from

the

column

was

analyzed

directly

on

a

Qtof

Micro

-LRB-

Waters

Corp.

-RRB-

mass

spectrometer

equipped

with

a

nanoflow

electrospayer

and

scanned

for

m/z

at

s

intervals

An

external

lock

mass

standard

-LRB-

leucine

enkephalin

m/z

-RRB-

was

analyzed

at

s

intervals

through

a

separate

orthogonal

electrosprayer

The

data

were

processed

using

MassLynx

-LRB-

Waters

Corp.

-RRB-

software

including

Accurate

Mass

Measure

and

algorithms

Briefly

chromatograms

were

created

from

the

data

set

by

plotting

a

Da

window

around

the

m/z

's

of

interest

The

peaks

were

confirmed

to

have

the

component

of

interest

by

summing

the

mass

spectra

across

the

peak

determining

the

centroid

m/z

values

followed

by

deconvolution

and

transformation

to

the

accurate

mass

value

of

the

ion

The

resulting

ion

envelope

encompassing

mass

isomers

containing

zero

to

four

atoms

was

plotted

to

create

the

chromatogram

of

the

specific

peptide

For

the

ion

was

by

far

the

most

prevalent

and

its

ion

envelope

spanned

m/z

values

from

through

For

the

and

ions

were

the

most

prevalent

spanning

m/z

values

from

through

and

through

respectively

Western

blotting

was

performed

by

a

previously

described

method

which

allows

the

detection

of

sub-femtomol

quantities

of

or

-LSB-

-RSB-

Samples

and

appropriate

standards

were

subjected

to

PAGE

in

tris-tricine

gels

Rabbit

polyclonal

antibodies

-LRB-

raised

to

-RRB-

and

-LRB-

raised

to

-RRB-

were

used

to

detect

the

C-terminal

sequences

of

Aβ

isoforms

These

antibodies

are

highly

selective

for

their

targets

showed

no

cross-reactivity

with

a

excess

of

-LSB-

-RSB-

and

is

more

reactive

with

than

with

-LSB-

-RSB-

Monoclonal

antibody

-LSB-

-RSB-

was

used

to

detect

the

Aβ

sequence

-LSB-

-RSB-

The

blots

were

developed

wth

NBT

and

BCIP

and

the

bands

were

quantified

by

photodensitometry

-LSB-

-RSB-

Results

Age-associated

changes

of

Aβ

immunoreactivity

in

neurons

in

control

and

DS

brains

Infants

and

children

In

the

majority

of

the

brain

structures

examined

including

the

entorhinal

cortex

and

neocortex

amygdala

and

hippocampus

Aβ

immunoreactivity

was

present

in

neurons

of

the

and

month-old

normal

infant

brains

and

the

month-old

infant

diagnosed

with

DS

-LRB-

Fig.

-RRB-

At

this

age

Aβ-immunoreactivity

was

observed

in

only

about

of

the

neurons

However

fine

and

randomly

dispersed

Aβ-immunoreactive

granules

were

present

in

the

cytoplasm

in

almost

all

neurons

in

the

dentate

gyrus

In

infants

strong

Aβ-immunoreactivity

was

found

in

clusters

of

cytoplasmic

granules

in

almost

all

neurons

in

the

magnocellular

portion

of

the

lateral

geniculate

body

but

reaction

in

small

neurons

was

rare

and

weak

In

the

brains

of

and

year-old

children

about

half

of

neurons

in

the

cortex

and

basal

ganglia

were

Aβ-immunopositive

whereas

in

the

brain

of

the

year-old

child

the

percentage

of

immunoreactive

neurons

was

more

than

Fig.

Aβ

immunoreactivity

with

mAb

-LRB-

-RRB-

in

the

sector

dentate

gyrus

-LRB-

DG

-RRB-

amygdala

-LRB-

AMY

-RRB-

lateral

geniculate

body

-LRB-

LGB

-RRB-

and

cortex

-LRB-

COR

temporal

superior

gyrus

-RRB-

in

month-old

infant

with

DS

month-old

control

infant

and

and

year-old

control

subjects

-LRB-

C

-RRB-

reveals

age-associated

and

structure-specific

differences

A

few

immunopositive

neurons

are

present

in

sector

amygdala

and

cortex

in

infants

however

immunopositivity

is

detectable

in

the

majority

of

neurons

in

infant

dentate

gyrus

and

lateral

geniculate

body

In

young

adult

-LRB-

year-old

-RRB-

and

aged

subject

-LRB-

year-old

-RRB-

immunopositivity

is

present

in

the

majority

of

neurons

and

is

much

stronger

than

in

infants

Dense

deposits

are

present

in

almost

all

neurons

in

the

LGB

and

amygdala

and

dispersed

fine

granular

staining

is

present

in

the

majority

of

neurons

in

the

dentate

gyrus

Loose

granular

immunopositive

material

appears

in

about

of

neurons

in

sector

and

cortical

neurons

Adults

In

adults

Aβ

immunoreactivity

was

present

in

the

majority

of

the

neurons

but

the

amount

and

pattern

of

distribution

of

immunopositive

material

showed

a

broad

range

of

cell-type

and

brain

structure-specific

differences

Strong

Aβ

immunoreactivity

characterized

all

nuclei

in

the

amygdala

in

adults

with

the

strongest

reaction

in

the

lateral

and

ventral

subdivisions

-LRB-

mean

grade

SD

-RRB-

In

the

amygdala

numerous

Aβ-positive

cytoplasmic

granules

were

concentrated

at

one

pole

of

the

cell

and

formed

a

perinuclear

cap

In

the

cornu

Ammonis

-LRB-

CA

-RRB-

Aβ

immunoreactivity

showed

marked

sector-specific

differences

Strong

and

uniform

reaction

characterized

neurons

in

the

sector

and

moderate

reaction

was

observed

in

and

sectors

but

Aβ

immunoreactivity

in

neurons

in

the

sector

was

much

weaker

and

less

uniform

-LRB-

mean

grade

in

the

CA

SD

-RRB-

Apical

dendrites

in

pyramidal

neurons

in

the

CA

and

subiculum

proper

were

often

marked

with

rows

of

Aβ-immunoreactive

granules

Granule

cells

in

the

dentate

gyrus

contained

numerous

randomly

dispersed

Aβ-positive

granules

Strong

immunoreactivity

appeared

in

large

neurons

and

moderate

staining

appeared

in

small

neurons

of

the

caudate

nucleus

and

putamen

Reactions

in

neurons

in

the

thalamus

globus

pallidus

and

n.

accumbens

were

less

uniform

and

weaker

Aβ

immunoreactivity

in

cortical

pyramidal

neurons

was

stronger

and

more

common

than

in

cortical

granule

cells

In

the

cerebellum

a

moderate

amount

of

fine

granular

Aβ

immunoreactive

material

was

found

in

the

majority

of

Purkinje

cells

but

in

granule

cells

the

reaction

was

weak

and

was

present

only

in

a

minority

of

cells

Very

strong

Aβ

immunoreactivity

was

found

in

the

cytoplasm

in

all

neurons

in

the

LGB

and

dentate

nucleus

-LRB-

mean

grade

-RRB-

as

well

as

in

the

nucleus

olivaris

Aged

subjects

In

general

in

control

people

older

than

years

of

age

Aβ

immunoreactivity

was

reduced

in

about

of

neurons

of

the

amygdala

nucleus

basalis

of

Meynert

-LRB-

NBM

-RRB-

cornu

Ammonis

large

neurons

in

the

caudate-putamen

and

cortex

as

compared

to

control

adults

However

the

amount

and

pattern

of

Aβ

immunoreactivity

in

the

dentate

gyrus

LGB

dentate

nucleus

and

nucleus

olivaris

inferior

were

comparable

in

aged

subjects

and

normal

adults

In

addition

to

the

structure

and

age-associated

differences

in

neuronal

Aβ

immunoreactivity

there

were

interindividual

differences

between

people

of

the

same

age

and

gender

Strong

and

uniform

Aβ

immunoreactivity

was

found

in

of

the

control

subjects

moderate

and

uniform

in

and

weak

and

nonuniform

in

No

difference

between

control

and

DS

infants

-LRB-

Fig.

-RRB-

and

adult

with

DS

who

has

incipient

neurofibrillary

degeneration

-LRB-

years-old

-RRB-

was

detected

Intraneuronal

Aβ

immunoreactivity

in

control

brains

with

neurofibrillary

degeneration

and

in

DS/AD

or

sporadic

AD

In

the

control

group

mAb

revealed

neurofibrillary

degeneration

in

the

brains

of

all

persons

older

than

years

of

age

In

the

entorhinal

cortex

subiculum

and

amygdala

the

number

of

neurons

with

NFTs

increases

with

age

The

increase

in

material

was

paralleled

by

the

reduction

and

loss

of

cytoplasmic

Aβ

immunoreactivity

in

affected

neurons

in

the

older

subjects

All

subjects

with

DS

older

than

years-old

were

affected

with

neurofibrillary

degeneration

and

all

people

with

DS

older

than

years

of

age

had

developed

β

amyloidosis

The

increase

in

neurofibrillary

degeneration

neuronal

death

and

β

amyloidosis

was

associated

with

marked

reductions

of

intraneuronal

Aβ

immunoreactivity

in

the

entorhinal

cortex

-LRB-

Fig.

b

-RRB-

hippocampus

amygdala

NBM

and

neocortex

Fig.

of

neurofibrillary

tangles

-LRB-

arrowheads

mAb

a

-RRB-

in

neurons

in

the

second

layer

of

the

entorhinal

cortex

of

a

year-old

subject

with

DS/AD

is

associated

with

loss

of

cytoplasmic

Aβ

immunoreactivity

-LRB-

arrowheads

mAb

b

-RRB-

without

plaque

formation

in

islands

of

stellate

neurons

Amyloid

plaque

in

the

third

layer

-LRB-

asterisk

-RRB-

is

mAb

The

insula

of

a

subject

with

mild

AD

-LRB-

GDS

-RRB-

is

affected

by

early

and

severe

amyloidosis-β

Plaques

are

marked

with

arrowheads

-LRB-

c

-RRB-

mAb

material

is

present

in

both

plaques

-LRB-

asterisk

-RRB-

and

neurons

-LRB-

arrowheads

d

-RRB-

The

strongest

Aβ

immunoreactivity

is

present

in

almost

all

neurons

-LRB-

arrowheads

-RRB-

in

the

lateral

geniculate

body

-LRB-

low

and

high

magnification

AD

GDS

e

-RRB-

dentate

nucleus

-LRB-

DS/AD

GDS

year-old

f

-RRB-

and

nucleus

olivaris

inferior

-LRB-

DS/AD

GDS

years-old

g

-RRB-

however

amyloid

plaques

do

not

develop

in

these

structures

even

in

severe

AD

A

similar

pattern

of

reduction

of

Aβ

immunoreactivity

associated

with

neurofibrillary

degeneration

amyloidosis

β

and

neuronal

loss

was

found

in

people

with

MCI

and

subjects

with

sporadic

AD

-LRB-

GDS

stage

-RRB-

In

severe

AD

-LRB-

GDS

stage

-RRB-

almost

all

neurons

in

the

second

layer

of

the

entorhinal

cortex

were

affected

by

neurofibrillary

degeneration

detected

with

mAb

These

neurons

contained

only

traces

of

Aβ

immunoreactivity

or

were

free

of

cytoplasmic

Aβ

However

in

neocortex

the

reduction

of

intracellular

Aβ

immunoreactivity

was

less

pronounced

-LRB-

Fig.

d

-RRB-

Both

in

persons

with

DS/AD

and

persons

with

sporadic

AD

the

percentage

of

Aβ

immunopositive

Purkinje

cells

was

reduced

by

about

compared

to

age-matched

control

subjects

The

majority

of

granule

cells

failed

to

manifest

Aβ

immunoreactivity

In

the

cerebellum

the

decrease

in

neuronal

Aβ

immunoreactivity

was

observed

in

the

almost

total

absence

of

intraneuronal

NFTs

In

contrast

to

these

patterns

of

reduction

in

Aβ

immunoreactivity

a

constant

level

of

strong

reaction

was

present

in

almost

all

neurons

in

LGB

dentate

nucleus

and

nucleus

olivaris

inferior

in

people

with

DS/AD

or

sporadic

AD

These

structures

were

almost

free

of

NFTs

and

amyloid

plaques

in

all

AD

subjects

including

those

with

severe

AD

-LRB-

Fig.

g

-RRB-

Immunoproperties

of

intraneuronal

Aβ

The

lack

of

Aβ

immunoreactivity

or

presence

of

very

few

cytoplasmic

grains

immunoreactive

with

antibodies

detecting

an

epitope

in

residues

-LRB-

mAb

-RRB-

and

-LRB-

mAb

-RRB-

and

the

presence

of

cytoplasmic

immunoreactivity

with

antibodies

detecting

-LRB-

mAb

-RRB-

-LRB-

pAb

-RRB-

and

-LRB-

pAbs

and

-RRB-

indicates

that

neurons

harbor

mainly

amino-terminally

truncated

Aβ

-LRB-

Fig.

b

c

d

e

f

-RRB-

Stronger

staining

of

intracellular

Aβ

with

pAb

-LRB-

residues

-RRB-

and

pAb

-LRB-

-RRB-

than

with

pAb

-LRB-

-RRB-

indicates

that

the

major

component

of

cytoplasmic

Aβ

in

neurons

is

Intraneuronal

Aβ

was

not

labeled

with

pAb

that

detects

fibrillar

amyloid

in

cored

plaques

and

in

vessel

wall

-LRB-

not

shown

-RRB-

The

pattern

of

immunostaining

of

Aβ

in

fibrillar

plaques

confirmed

that

parenchymal

plaques

contain

mainly

Fig.

a

f

shows

immunoreactivity

of

intraneuronal

Aβ

in

the

sector

of

a

year-old

control

subject

No

reaction

in

neurons

stained

with

mAb

-LRB-

aa

-RRB-

-LRB-

a

-RRB-

and

mAb

-LRB-

aa

-RRB-

-LRB-

b

-RRB-

indicates

that

in

the

majority

of

neurons

the

amino-terminal

portion

of

Aβ

is

not

detectable

Immunoreactivity

of

intraneuronal

Aβ

is

shown

in

sections

stained

with

mAb

-LRB-

aa

-RRB-

-LRB-

c

-RRB-

pAb

-LRB-

aa

-RRB-

-LRB-

d

-RRB-

pAb

-LRB-

aa

-RRB-

-LRB-

e

-RRB-

and

pAb

-LRB-

aa

-RRB-

-LRB-

f

-RRB-

In

the

brain

of

subjects

with

AD

some

extracellular

ghost

tangles

contain

full

length

Aβ

peptides

immunoreactive

with

antibodies

-LRB-

g

-RRB-

-LRB-

h

-RRB-

and

pAb

-LRB-

i

-RRB-

Aβ

immunoreactivity

in

ghost

tangles

While

neuronal

neurofibrillary

changes

were

associated

with

loss

of

cytoplasmic

Aβ

immunoreactivity

some

ghost

tangles

-LRB-

extracellular

NFTs

-RRB-

were

Aβ-positive

-LRB-

Fig.

h

i

-RRB-

In

the

sporadic

AD

group

among

examined

subjects

Aβ-positive

ghost

tangles

were

found

in

brains

-LRB-

-RRB-

The

proportion

of

subjects

with

Aβ-reactive

ghost

tangles

increased

from

in

people

with

MCI

to

in

people

with

moderately

severe

AD

and

to

in

people

with

severe

AD

Aβ-positive

ghost

tangles

were

found

in

of

these

subjects

in

the

entorhinal

cortex

in

in

the

in

the

amygdala

in

the

and

in

the

subiculum

proper

and

in

the

and

temporal

cortex

Of

AD

subjects

with

Aβ-positive

ghost

tangles

they

were

numerous

in

moderate

in

number

in

and

were

rare

in

of

the

subjects

In

of

brains

of

persons

with

DS

-LRB-

-RRB-

Aβ-immunoreactive

ghost

tangles

were

found

in

the

entorhinal

cortex

amygdala

and

and

sectors

The

youngest

subject

with

positive

staining

was

years

of

age

at

demise

Aβ-positive

ghost

tangles

were

found

in

the

brains

of

all

DS

subjects

from

to

years

old

Of

DS

subjects

in

whom

Aβ-immunoreactive

ghost

tangles

were

present

they

were

numerous

in

moderate

in

number

in

and

rare

in

of

subjects

In

the

control

cases

no

Aβ-immunoreactive

ghost

tangles

were

observed

in

spite

of

the

presence

of

neurofibrillary

changes

in

all

subjects

older

than

years

of

age

Aβ-positive

ghost

tangles

were

strongly

immunoreactive

with

mAbs

-LRB-

residues

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

and

pAbs

-LRB-

-RRB-

and

-LRB-

-RRB-

The

reaction

with

pAb

-LRB-

-RRB-

was

weak

Aβ

isoforms

could

be

detected

on

Western

blots

of

crude

control

brain

extracts

Antibody

revealed

bands

containing

the

sequence

-LRB-

Fig.

lane

-RRB-

whereas

antibody

revealed

bands

containing

the

C-terminal

sequence

-LRB-

Fig.

lane

-RRB-

Immunoreactivity

with

mAb

-LRB-

Fig.

lane

-RRB-

unequivocally

showed

that

the

upper

band

contained

Aβ

peptide

sequences

possessing

amino

acid

residues

This

antibody

does

not

bind

to

even

at

a

higher

concentration

-LRB-

Fig.

lane

-RRB-

We

found

that

the

large

amounts

of

other

proteins

in

the

extracts

affected

the

electrophoretic

migration

rates

and

band

shapes

of

the

peptides

so

that

their

migration

rates

did

not

unambiguously

distinguish

from

Using

synthetic

Aβ

standards

we

showed

that

substances

in

a

crude

brain

extract

retarded

the

migration

of

so

that

it

migrated

at

the

rate

of

-LRB-

in

the

absence

of

brain

extract

-RRB-

Fig.

isoforms

in

extracts

from

control

brain

revealed

by

immunoblotting

A

formic

acid

extract

of

cerebral

cortex

from

brain

was

prepared

as

described

in

the

methods

section

A

portion

of

the

dried

extract

was

dissolved

in

PAGE

sample

buffer

and

a

μl

aliquot

containing

μg

of

protein

-LRB-

equivalent

to

μg

of

tissue

-RRB-

was

applied

to

the

lanes

developed

with

and

Because

mAb

is

much

less

sensitive

than

or

the

formic

acid

extract

was

fractionated

by

size-exclusion

as

described

in

the

methods

section

The

fraction

containing

peptides

in

the

mass

range

Da

was

concentrated

and

an

aliquot

containing

peptides

from

mg

of

tissue

was

applied

to

the

lane

developed

with

The

extracts

were

subjected

to

PAGE

and

blotting

along

with

synthetic

Aβ

standards

and

molecular

mass

markers

as

described

in

the

text

The

blots

were

developed

with

antibody

-LRB-

to

the

C-terminus

of

-RRB-

-LRB-

to

the

C-terminus

of

-RRB-

or

mAb

-LRB-

to

-RRB-

Lane

fmol

lane

fmol

lane

fmol

lane

fmol

Molecular

mass

markers

denoted

on

the

right-hand

margin

are

insulin

single

chains

and

aprotinin

Peptides

with

a

defined

C-terminus

may

have

distinct

N-termini

hence

we

operationally

define

the

peptides

as

those

that

bind

mAb

and

migrate

between

the

and

kDa

standards

Monoclonal

antibody

binds

to

Aβ

residues

therefore

may

contain

peptides

whose

N-terminal

residues

are

We

define

the

peptides

as

those

that

migrate

near

the

kDa

standard

and

do

not

bind

The

levels

of

peptides

in

three

normal

brain

cortex

samples

were

between

and

pmol/g

of

and

between

and

pmol/g

and

the

levels

of

peptides

were

between

and

pmol/g

of

and

between

and

pmol/g

Although

these

amounts

are

readily

detectable

they

are

orders

of

magnitude

below

the

Aβ

levels

in

an

AD

brain

sample

that

we

analyzed

-LRB-

not

shown

-RRB-

Detection

of

by

mass

spectrometry

We

immunoprecipitated

fmol

of

Aβ

peptides

from

brain

and

characterized

both

the

chromatographic

behavior

and

the

masses

of

the

peptides

by

mass

spectrometry

using

the

coupled

capillary

HPLC-MS

system

As

shown

in

Fig.

the

identity

of

the

Aβ

peptide

from

the

brain

was

confirmed

by

comparison

to

synthetic

which

eluted

at

the

same

time

and

displayed

the

same

m/z

profile

Deconvolution

and

transformation

of

the

ion

spectrum

to

the

monoisotopic

mass

gave

mass

values

of

Da

-LRB-

brain

sample

-RRB-

and

Da

-LRB-

synthetic

-RRB-

compared

to

the

theoretical

value

of

Da

-LRB-

ppm

and

ppm

errors

respectively

-RRB-

We

also

observed

ion

profiles

consistent

with

and

ions

in

the

mass

spectra

The

presence

of

detergent

in

the

sample

and

the

complexity

of

the

chromatogram

and

spectra

complicated

the

identification

of

other

Aβ

species

Fig.

elution

profile

of

Samples

from

-LRB-

a

-RRB-

control

brain

and

-LRB-

b

-RRB-

synthetic

were

prepared

and

analyzed

by

LC-MS

as

described

in

Materials

and

methods

The

chromatograms

show

only

the

MS

ion

counts

for

the

M

H

ions

of

mass

isomers

containing

zero

to

four

atoms

-LRB-

m/z

-RRB-

The

ion

intensities

determined

at

s

intervals

were

summed

background

subtracted

and

smoothed

The

y-axis

scale

is

ions

per

second

full

scale

-LRB-

-RRB-

The

identification

of

in

the

peaks

at

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

minutes

was

demonstrated

by

the

characteristic

ion

envelope

obtained

from

the

centroid

accurate

mass

spectra

for

the

M

H

ions

from

each

peak

-LRB-

to

min

-RRB-

shown

in

the

insets

Discussion

Aβ

immunoreactivity

in

neurons

in

normal

brain

Previous

studies

either

did

not

find

intracellular

Aβ

immunoreactivity

in

brains

of

control

subjects

-LSB-

-RSB-

or

found

only

punctate

Aβ

immunoreactivity

in

a

minority

-LRB-

-RRB-

of

examined

adults

-LSB-

-RSB-

Therefore

it

was

proposed

that

the

accumulation

of

intraneuronal

Aβ

is

involved

in

early

AD

pathology

-LSB-

-RSB-

However

this

study

of

control

cases

from

several

months

to

years

of

age

demonstrates

that

intraneuronal

Aβ

immunoreactivity

appears

in

the

first

year

of

life

increases

in

childhood

stabilizes

in

the

second

decade

of

life

and

remains

high

throughout

adulthood

Detection

of

stable

intracellular

Aβ

immunoreactivity

in

neurons

in

control

cases

throughout

the

entire

life

may

indicate

that

Aβ-immunoreactive

material

in

the

cell

body

reflects

normal

neuronal

metabolism

and

is

not

neuronal

pathology

The

observation

that

intraneuronal

Aβ

in

control

DS/AD

and

sporadic

AD

cases

was

almost

exclusively

amino-terminally

truncated

confirmed

and

extended

the

findings

of

the

Mori

et

al.

-LSB-

-RSB-

study

of

people

with

DS

and

the

Sergeant

et

al.

-LSB-

-RSB-

study

showing

that

amino-truncated

Aβ

peptides

represent

more

than

of

all

Aβ

species

in

subjects

with

preclinical

AD

Aβ

peptides

with

N-terminal

deletions

exhibit

enhanced

peptide

aggregation

relative

to

full-length

species

-LSB-

-RSB-

and

retain

the

neurotoxicity

and

β-sheet

structure

It

was

hypothesized

that

peptides

may

initiate

and/or

accelerate

plaque

formation

perhaps

by

acting

as

nucleating

centers

that

seed

the

subsequent

deposition

of

relatively

less

amyloidogenic

but

apparently

more

abundant

full-length

Aβ

-LSB-

-RSB-

Gouras

et

al.

-LSB-

-RSB-

considered

intracellular

accumulation

to

be

an

early

event

leading

to

neuronal

dysfunction

However

the

appearance

of

intraneuronal

Aβ

immunoreactivity

in

the

first

year

of

life

and

the

stable

and

strong

immunoreactivity

throughout

adulthood

in

the

absence

of

morphological

signs

of

cell

injury

or

degeneration

suggests

that

Aβ

detected

in

neurons

with

applied

antibodies

does

not

adversely

affect

cell

structure

This

lack

of

fibrillization

and

toxicity

may

indicate

that

these

Aβ

species

remain

in

inert

form

Possibly

this

inert

state

is

maintained

by

binding

with

blockers

of

fibrillization

and

toxicity

The

transport

of

Aβ

within

cytoplasmic

vesicles

or

vacuoles

-LSB-

-RSB-

might

be

another

factor

preventing

the

expression

of

their

cytotoxic

activity

The

detected

intraneuronal

Aβ

appears

to

be

the

physiological

metabolite

with

unknown

function

Higher

levels

of

secreted

APP

and

nonamyloidogenic

secreted

APP

and

lower

levels

of

Aβ

in

children

with

severely

autistic

behavior

and

aggression

compared

with

controls

-LSB-

-RSB-

may

indicate

that

modifications

of

APP

processing

and

potentially

Aβ

trafficking

are

clinically

significant

in

absence

of

neurodegeneration

or

neuronal

loss

The

very

strong

Aβ

immunoreactivity

in

the

LGB

nucleus

olivaris

inferior

and

dentate

nucleus

during

the

entire

course

of

human

adulthood

and

the

absence

of

or

minimal

amyloid

load

in

very

severe

AD

oppose

the

hypothesis

that

strong

intraneuronal

Aβ

immunoreactivity

is

a

predictor

of

fibrillar

plaque

formation

Convergent

findings

in

tg

mice

have

been

reported

by

Chui

et

al.

-LSB-

-RSB-

This

study

suggests

also

that

the

intensity

of

intraneuronal

Aβ

immunoreactivity

is

not

a

predictor

of

neurofibrillary

degeneration

The

strongest

Aβ

immunoreactivity

was

observed

in

neurons

in

the

LGB

nucleus

olivaris

inferior

and

dentate

nucleus

which

remain

free

of

neurofibrillary

degeneration

in

very

severe

AD

The

moderate

Aβ

immunoreactivity

in

the

second

layer

of

the

entorhinal

cortex

and

pyramidal

neurons

in

the

observed

in

this

study

and

the

early

onset

of

NFTs

observed

in

these

neuronal

populations

-LSB-

-RSB-

also

question

the

link

between

Aβ

immunoreactivity

and

susceptibility

to

neurofibrillary

degeneration

The

lack

of

pathological

changes

in

brain

structures

with

the

strongest

intraneuronal

Aβ

immunoreactivity

during

the

entire

human

lifespan

may

indicate

that

amino-terminally

truncated

Aβ

is

a

product

of

cell

metabolism

that

does

not

-LRB-

-RRB-

limit

cell

survival

-LRB-

-RRB-

predispose

to

fibrillar

Aβ

deposition

in

plaques

nor

-LRB-

-RRB-

cause

neurofibrillary

degeneration

Reduction

of

intraneuronal

Aβ

immunoreactivity

in

DS/AD

and

sporadic

AD

In

this

study

the

strongest

intraneuronal

Aβ

immunoreactivity

was

found

in

control

cases

compared

to

those

with

AD

and

in

DS/AD

Significant

weaker

Aβ

immunoreactivity

in

neurons

in

people

with

DS/AD

and

sporadic

AD

appear

to

be

the

result

of

AD

pathology

including

neurofibrillary

degeneration

and

neuronal

loss

In

the

memory

systems

of

persons

with

DS/AD

or

sporadic

AD

neurofibrillary

degeneration

is

the

major

cause

of

neuronal

loss

and

therefore

the

major

factor

contributing

to

the

reduction

of

intraneuronal

Aβ

Over

the

course

of

years

an

AD

patient

loses

of

the

neurons

in

the

and

in

the

sector

and

in

the

subiculum

-LSB-

-RSB-

People

with

DS/AD

older

than

years

of

age

are

affected

by

severe

loss

of

neurons

including

loss

in

the

second

layer

of

the

entorhinal

cortex

in

the

sector

in

the

subiculum

proper

and

in

the

amygdala

-LSB-

-RSB-

Large

portions

of

surviving

neurons

in

the

brains

of

people

with

AD

and

DS/AD

are

affected

with

neurofibrillary

changes

yet

they

show

little

or

no

Aβ

immunoreactivity

These

changes

explain

in

part

the

loss

of

Aβ

immunoreactivity

in

neurons

in

the

plaque

perimeter

as

noted

in

persons

with

DS/AD

-LSB-

-RSB-

The

lower

intraneuronal

Aβ

immunoreactivity

observed

in

people

with

sporadic

AD

or

DS/AD

may

also

reflect

a

shift

in

APP

processing

This

shift

would

be

from

amino-terminally

truncated

Aβ

accumulation

in

cell

cytoplasm

in

normal

brain

to

APP

processing

with

enhanced

generation

and

secretion

of

in

people

with

AD

Accumulation

of

in

ghost

tangles

Intracellular

and

extracellular

NFTs

have

been

shown

to

be

Aβ

immunoreactive

-LSB-

-RSB-

However

studies

of

purified

PHF

revealed

the

absence

of

beta-pleated

sheet

conformation

or

any

other

characteristic

of

an

amyloid

-LSB-

-RSB-

It

was

proposed

that

the

staining

of

intraneuronal

PHF

with

antibodies

to

amyloid

is

due

to

the

proximity

of

Aβ

molecules

to

the

PHF

in

the

cytoplasm

of

neurons

-LSB-

-RSB-

Deposition

of

Aβ

within

extracellular

NFTs

has

been

considered

a

secondary

event

-LSB-

-RSB-

Strong

immunoreactivity

of

extracellular

NFTs

with

antibodies

-LRB-

residues

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

could

be

an

indicator

of

the

presence

of

full

length

of

Aβ

in

extracellular

space

The

weak

reaction

of

ghost

tangles

with

pAb

-LRB-

-RRB-

may

suggest

a

lower

concentration

of

this

form

of

Aβ

in

the

extracellular

space

An

increase

in

the

percentage

of

Aβ-positive

ghost

tangles

in

people

with

progressing

AD

appears

to

reflect

the

increase

in

the

percentage

of

neurons

dying

due

to

neurofibrillary

degeneration

and

an

increasing

concentration

of

full

length

Aβ

in

the

extracellular

space

Spatial

and

temporal

separation

of

plaques

and

Aβ-positive

ghost

tangles

indicates

that

ghost

tangles

may

bind

Aβ

but

do

not

initiate

fibrillar

plaque

formation

Interindividual

differences

in

intraneuronal

Aβ

immunoreactivity

Intraneuronal

Aβ

immunoreactivity

revealed

some

interindividual

differences

Gouras

et

al.

-LSB-

-RSB-

observed

increased

apo-E

immunoreactivity

in

neurons

and

suggested

that

apo

E

might

be

a

powerful

modifier

of

intraneuronal

Aβ

accumulation

Neuron-generated

apo-E

may

bind

to

neuronal

Aβ

and

affect

both

its

secretory

pathway

and

its

storage

in

the

cell

cytoplasm

The

lack

of

apo-E

in

the

offspring

of

βAPP

tg

mice

crossed

with

apo-E

knockouts

reduces

significantly

the

amyloid

load

-LSB-

-RSB-

Chronic

inflammation

is

another

factor

that

may

influence

amyloid

formation

-LSB-

-RSB-

In

the

human

brain

there

appear

to

be

several

pools

of

Aβ

in

equilibrium

-LSB-

-RSB-

In

this

study

we

demonstrate

by

the

immunocytochemical

method

abundant

intraneuronal

amino-terminally

truncated

with

minor

presence

of

the

intraneuronal

The

presence

of

the

peptide

in

control

cortex

samples

was

confirmed

by

mass

spectrometry

However

in

extracts

from

cerebral

cortex

we

identified

by

Western

blotting

the

peptides

that

were

amino-terminally

truncated

-LRB-

-RRB-

and

full

length

in

similar

quantities

We

also

detected

both

the

peptide

and

the

full

length

peptide

although

the

levels

of

the

latter

were

or

times

lower

than

those

of

the

amino-terminally

truncated

one

The

differences

between

the

Aβ

peptides

detected

in

the

brain

by

the

immunocytochemical

and

biochemical

methods

suggest

that

the

Aβ

peptides

accumulated

intraneuronally

are

mainly

those

amino-terminally

truncated

while

the

full

length

peptides

are

mainly

dispersed

in

extracellular

space

and

hence

are

not

detected

by

the

immunocytochemical

method

as

intracellular

deposits

Deposition

of

full

length

Aβ

in

ghost

tangles

and

the

increase

of

Aβ-positive

ghost

tangles

could

be

considered

an

indicator

of

the

presence

of

in

extracellular

space

and

an

increase

of

this

extracellular

form

of

Aβ

in

advanced

stages

of

AD

It

appears

that

the

immunocytochemical

methods

applied

in

this

and

other

studies

-LSB-

-RSB-

do

not

monitor

the

neuronal

secretory

pathway

of

APP

processing

leading

to

normal

secretion

of

and

Stern

et

al.

-LSB-

-RSB-

have

shown

that

full

length

APP

is

extremely

sensitive

to

fixation

methods

and

its

immunogenicity

could

be

easily

destroyed

The

possibly

enhanced

secretion

of

these

peptides

in

AD

that

may

result

in

fibrillar

Aβ

deposition

in

plaques

also

is

not

detected

The

lack

of

detection

or

poor

detectability

of

the

product

of

β

and

γ-secretases

in

autopsy

material

might

be

the

result

of

tissue

preservation

method

the

minute

amount

of

that

might

be

secreted

without

long-term

storage

the

prevalence

of

amino-terminally

truncated

Aβ

masking

the

detection

of

or

the

masking

of

the

amino-terminal

epitopes

by

interactions

with

other

proteins

One

may

speculate

that

deposition

of

amino-terminally

truncated

Aβ

might

be

up

or

down-regulated

by

physiological

and

pathological

factors

independently

of

any

changes

in

the

secretory

pathway

This

study

suggests

that

neuron-type

and

brain-structure-specific

patterns

of

intraneuronal

Aβ

immunoreactivity

established

in

teenagers

and

maintained

at

a

constant

level

during

adulthood

and

aging

reflect

normal

cell

metabolism

rather

than

pathological

changes

Reduction

and

loss

of

immunoreactivity

in

neurons

with

neurofibrillary

changes

appears

to

be

a

response

to

the

progression

of

pathological

changes

in

the

neuron

The

absence

of

a

link

between

the

age

at

onset

and

the

progression

of

accumulation

of

intraneuronal

Aβ-positive

material

in

control

subjects

with

the

fibrillar

plaque

formation

or

neurofibrillary

degeneration

in

people

with

AD

indicates

that

the

form

of

intraneuronal

Aβ

detected

by

applied

methods

does

not

predict

plaque

formation

or

neurofibrillary

degeneration

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

intraneuronal amyloid-β
alzheimer’s disease


The

muscle

protein

dysferlin

accumulates

in

the

Alzheimer

brain

Dysferlin

is

a

transmembrane

protein

that

is

highly

expressed

in

muscle

Dysferlin

mutations

cause

limb-girdle

dystrophy

type

Miyoshi

myopathy

and

distal

anterior

compartment

myopathy

Dysferlin

has

also

been

described

in

neural

tissue

We

studied

dysferlin

distribution

in

the

brains

of

patients

with

Alzheimer

disease

-LRB-

AD

-RRB-

and

controls

Twelve

brains

staged

using

the

Clinical

Dementia

Rating

were

examined

AD

cases

-LRB-

mean

age

years

and

mean

disease

duration

years

-RRB-

and

age-matched

controls

-LRB-

mean

age

years

-RRB-

Dysferlin

is

a

cytoplasmic

protein

in

the

pyramidal

neurons

of

normal

and

AD

brains

In

addition

there

were

dysferlin-positive

dystrophic

neurites

within

Aβ

plaques

in

the

AD

brain

distinct

from

tau-positive

neurites

Western

blots

of

total

brain

protein

-LRB-

RIPA

-RRB-

and

sequential

extraction

buffers

-LRB-

high

salt

high

salt/Triton

SDS

and

formic

acid

-RRB-

of

increasing

protein

extraction

strength

were

performed

to

examine

solubility

state

In

RIPA

fractions

dysferlin

was

seen

as

kDa

bands

in

normal

and

AD

brains

In

serial

extractions

there

was

a

shift

of

dysferlin

from

soluble

phase

in

high

salt/Triton

to

the

more

insoluble

SDS

fraction

in

AD

Dysferlin

is

a

new

protein

described

in

the

AD

brain

that

accumulates

in

association

with

neuritic

plaques

In

muscle

dysferlin

plays

a

role

in

the

repair

of

muscle

membrane

damage

The

accumulation

of

dysferlin

in

the

AD

brain

may

be

related

to

the

inability

of

neurons

to

repair

damage

due

to

Aβ

deposits

accumulating

in

the

AD

brain

Introduction

Aggregation

of

neuronal

proteins

is

a

common

mechanistic

theme

in

neurodegenerative

diseases

-LSB-

-RSB-

Whether

due

to

point

mutations

or

post-translational

modification

normally

soluble

proteins

are

altered

and

form

insoluble

fibrillar

aggregates

-LSB-

-RSB-

Alzheimer

's

disease

-LRB-

AD

-RRB-

the

most

common

form

of

dementia

is

characterized

pathologically

by

abundant

diffuse

and

neuritic

plaques

throughout

most

cortical

regions

Amyloid

β-protein

-LRB-

Aβ

-RRB-

is

a

amino

acid

peptide

cleaved

from

a

larger

precursor

protein

-LRB-

amyloid

precursor

protein

or

APP

-RRB-

that

deposits

extracellularly

as

senile

plaques

either

as

loose

nonfibrillar

diffuse

plaques

or

as

a

more

compacted

fibrillar

form

with

dystrophic

neurites

coursing

through

the

plaque

-LSB-

-RSB-

Other

aggregated

proteins

found

in

the

AD

brain

include

the

microtubule-associated

protein

tau

-LSB-

-RSB-

as

neurofibrillary

tangles

and

the

presynaptic

protein

α-synuclein

-LSB-

-RSB-

in

the

form

of

Lewy

bodies

The

alteration

deposition

and

aggregation

of

amyloid

proteins

may

be

an

ill-fated

response

to

the

disease

process

or

themselves

lead

to

a

cascade

of

cellular

responses

ultimately

resulting

in

neuronal

dysfunction

and

death

Nonbrain

protein

aggregates

have

been

described

in

muscle

disease

For

example

amyloid-like

deposits

have

been

described

in

inclusion

body

myositis

-LSB-

-RSB-

Other

protein

aggregates

found

in

muscle

disease

include

desmin-related

myopathies

actinopathies

and

myosinopathies

-LSB-

-RSB-

Aggregates

containing

these

proteins

have

not

been

described

in

brain

disease

Another

muscle

protein

associated

with

myopathy

is

dysferlin

Dysferlin

is

a

plasma

membrane

and

cytoplasmic

vesicle-associated

protein

implicated

in

three

adult-onset

muscle

diseases

-LRB-

limb-girdle

dystrophy

type

Miyoshi

myopathy

and

distal

anterior

compartment

myopathy

-RRB-

-LSB-

-RSB-

Although

described

in

peripheral

nerve

-LSB-

-RSB-

and

demonstrated

by

Western

blot

in

human

and

mouse

brain

-LSB-

-RSB-

there

is

little

known

about

the

function

of

dysferlin

in

the

brain

or

its

role

in

neurodegenerative

disease

We

describe

the

pattern

of

dysferlin

expression

in

the

normal

and

AD

brain

and

examine

dysferlin

accumulation

in

association

with

amyloid

deposition

in

the

brains

of

AD

patients

Materials

and

methods

Case

materials

Case

materials

were

derived

from

research

participants

in

a

longitudinal

study

of

healthy

aging

and

dementia

who

were

studied

postmortem

The

diagnostic

criteria

are

consistent

with

probable

AD

reported

by

the

National

Institute

of

Neurological

and

Communicative

Disorders

and

Stroke

and

Alzheimer

's

Disease

and

Related

Disorders

Association

-LSB-

-RSB-

and

confirmed

by

autopsy

to

meet

high

probability

of

AD

according

to

National

Institute

of

Aging-Reagan

criteria

-LSB-

-RSB-

The

clinical

dementia

rating

-LRB-

CDR

-RRB-

was

used

to

determine

the

presence

or

absence

of

dementia

and

stage

its

severity

-LSB-

-RSB-

The

global

CDR

is

derived

by

a

synthesis

of

individual

ratings

in

six

cognitive

and

functional

categories

where

CDR

indicates

no

dementia

and

CDR

or

corresponds

to

very

mild

mild

moderate

or

severe

dementia

-LSB-

-RSB-

The

CDR

is

closely

correlated

with

the

presence

of

dementia

pathology

at

autopsy

-LSB-

-RSB-

The

Washington

University

Human

Studies

Committee

approved

all

procedures

Twelve

brains

were

examined

Three

cases

were

rated

as

nondemented

-LRB-

CDR

-RRB-

with

a

mean

age

of

years

Nine

AD

brains

were

examined

three

each

of

mild

-LRB-

CDR

-RRB-

moderate

-LRB-

CDR

-RRB-

and

severe

-LRB-

CDR

-RRB-

The

mean

age

of

the

demented

cases

was

years

with

mean

disease

duration

of

years

-LRB-

Table

-RRB-

Control

cases

were

all

Braak

stage

I

or

II

-LSB-

-RSB-

Sampling

during

postmortem

exam

was

done

from

different

cortical

subcortical

and

brainstem

regions

Paraffin-embedded

sample

for

this

study

were

taken

from

midfrontal

cingulate

and

entorhinal

regions

three

sections

were

stained

for

each

antibody

with

appropriate

controls

-LSB-

-RSB-

Biochemistry

was

performed

on

midfrontal

gyrus

adjacent

to

the

section

taken

for

immunohistochemistry

for

each

case

Table

characteristics

and

findings

at

postmortem

examinationCaseAge

-LRB-

years

-RRB-

SexCDRDisease

duration

-LRB-

years

-RRB-

Brain

wt

-LRB-

g

-RRB-

Braak

scoresCERAD

criteriaNIA

Reagan

IIA

IA

IA

VICDefiniteHigh

Immunohistochemistry

Immunohistochemistry

was

done

as

previously

described

-LSB-

-RSB-

Briefly

serial

μm

sections

of

paraformaldehyde-fixed

paraffin-embedded

tissue

were

prepared

and

incubated

overnight

at

°C

with

antibodies

against

dysferlin

-LRB-

Novacastra

Laboratories

-RRB-

tau

-LRB-

gift

from

J.

Trojanowski

-RRB-

and

amyloid

β-protein

-LRB-

Signet

-RRB-

Sections

were

developed

by

avidin

biotin

complex

method

-LRB-

Vector

Laboratories

-RRB-

for

immunohistochemistry

with

DAB

-LRB-

brown

-RRB-

Vector

Red

-LRB-

pink

-RRB-

and

Vector

SG

-LRB-

blue

-RRB-

as

chromogens

Alexa-Red

and

Green

-LRB-

Jackson

Laboratories

-RRB-

antibodies

were

used

for

immunofluorescence

Sections

were

examined

with

a

Nikon

Research

microscope

and

images

were

digitized

and

viewed

with

AnalySIS

analytic

software

-LRB-

Soft

Imaging

Systems

GmbH

-RRB-

Western

blots

Western

blotting

and

biochemical

fractionation

were

performed

as

described

previously

-LSB-

-RSB-

Briefly

two

sections

of

gray

matter

-LRB-

g

-RRB-

from

the

midfrontal

cortex

of

AD

and

normal

age-matched

control

brains

were

dissected

from

underlying

white

matter

After

dissection

gray

matter

was

divided

into

two

samples

-LRB-

Fig.

-RRB-

One

sample

was

homogenized

in

RIPA

-LRB-

mM

NaCl

SDS

sodium

deoxycholate

mM

Tris

pH

mM

NaF

mM

EGTA

levamisole

mM

-RRB-

to

estimate

total

dysferlin

The

other

sample

was

homogenized

and

subjected

to

sequential

extraction

buffers

of

increasing

protein

extraction

strength

For

each

buffer

pellets

were

washed

twice

and

supernatants

pooled

Samples

were

first

homogenized

in

ml/gram

of

tissue

of

high-salt

-LRB-

HS

-RRB-

buffer

-LRB-

mmol/l

Tris

pH

mmol/l

NaCl

mmol/l

NaF

mmol/l

ethylenediaminetetraacetic

acid

with

protease

inhibitors

-RRB-

and

centrifuged

at

g

for

min

The

pellets

were

re-extracted

and

the

supernatants

were

pooled

The

pellets

were

sequentially

extracted

twice

with

ml/gram

of

HS

Triton

-LRB-

HS/T

-RRB-

and

once

with

ml/gram

sodium

dodecyl

sulfate

-LRB-

SDS

-RRB-

sample

buffer

-LRB-

SDS

sucrose

mmol/l

Tris

pH

mmol/l

ethylenediaminetetraacetic

acid

mmol/l

dithiothreitol

-RRB-

The

pellets

were

extracted

with

ml/g

formic

acid

-LRB-

FA

-RRB-

and

disrupted

with

two

sequential

sonication

bursts

FA

was

evaporated

in

a

vacuum

centrifuge

-LRB-

Eppendorf

-RRB-

SDS

sample

buffer

-LRB-

ml/g

-RRB-

was

added

to

the

dried

pellets

followed

by

vigorous

vortex

and

the

pH

was

adjusted

with

NaOH

SDS

sample

buffer

was

added

to

the

HS

and

HS/Triton

fraction

and

all

of

the

samples

were

boiled

for

min

The

FA

samples

were

centrifuged

at

g

for

min

to

remove

insoluble

debris

Five

microliters

of

each

fraction

was

loaded

in

separate

lanes

for

SDS-polyacrylamide

gel

electrophoresis

followed

by

Western

blot

analysis

with

antibodies

again

dysferlin

The

immune

complexes

were

visualized

with

the

use

of

the

ECL

Plus

kit

-LRB-

Amersham

-RRB-

according

to

the

manufacturer

's

protocol

The

autoradiographs

obtained

were

scanned

and

the

band

intensity

quantified

utilizing

TotalLab

software

-LRB-

Nonlinear

Dynamics

Newcastle

on

Tyne

UK

-RRB-

Statistical

analyses

were

performed

by

analysis

of

variance

using

SPSS

statistical

software

Fig.

representation

of

sequential

protein

extraction

buffers

Samples

are

divided

into

equal

portions

The

first

sample

is

homogenized

in

RIPA

buffer

and

blotted

to

estimate

total

dysferlin

levels

The

other

sample

is

homogenized

in

sequential

extraction

buffers

of

increasing

protein

extraction

strength

The

first

extraction

is

in

high-salt

-LRB-

HS

-RRB-

buffer

The

supernatants

are

saved

and

the

pellet

is

extracted

with

HS

Triton

-LRB-

HS/T

-RRB-

buffer

After

saving

the

supernatants

the

resultant

pellet

is

homogenized

in

sodium

dodecyl

sulfate

-LRB-

SDS

-RRB-

sample

buffer

Resulting

pellets

are

finally

extracted

with

formic

acid

-LRB-

FA

-RRB-

Supernatants

are

blotted

to

estimate

differing

levels

of

dysferlin

solubility

with

the

HS

fraction

representing

the

most

soluble

phase

and

the

FA

fraction

representing

the

most

insoluble

phase

Results

Localization

of

dysferlin

in

normal

-LRB-

CDR

-RRB-

brain

Microscopic

evaluation

of

the

dysferlin

staining

in

the

normal

-LRB-

CDR

-RRB-

brain

localized

expression

to

the

cytoplasm

of

large

pyramidal

neurons

of

layers

III

V

and

VI

in

the

neocortex

-LRB-

Fig.

-RRB-

layer

II

neurons

of

the

entorhinal

cortex

CA

neurons

in

the

hippocampus

and

large

projection

neurons

in

the

amygdala

-LRB-

not

shown

-RRB-

Dysferlin

was

not

localized

to

astrocytes

endothelial

cells

or

microglia

-LRB-

arrowheads

in

Fig.

-RRB-

Little

to

no

dysferlin

neuritic

staining

was

seen

in

control

cases

-LRB-

Braak

stage

I

or

II

-RRB-

Fig.

of

dysferlin

in

brains

of

nondemented

subjects

a

b

Demonstrates

the

localization

of

dysferlin

immunoreactivity

in

the

brain

There

is

a

neuronal

expression

of

dysferlin

localized

in

cytoplasm

of

large

pyramidal

-LRB-

layers

III

V

VI

-RRB-

neurons

in

the

frontal

cortex

at

low

power

-LRB-

a

-RRB-

Little

to

no

neuritic

staining

was

detected

Dysferlin

expression

is

confined

to

neurons

-LRB-

arrow

DAB

brown

in

b

-RRB-

and

is

not

seen

in

glial

cells

stained

with

GFAP

-LRB-

small

arrowheads

Vector

Red

pink

in

b

-RRB-

Magnification

of

-LRB-

a

-RRB-

is

and

the

scale

bar

in

-LRB-

a

-RRB-

μm

Magnification

of

-LRB-

b

-RRB-

is

and

the

scale

bar

in

-LRB-

b

-RRB-

μm

Dysferlin

in

the

AD

brain

Dysferlin

neuritic

accumulation

is

found

throughout

the

AD

brain

and

in

double-label

studies

it

is

localized

in

Aβ

neuritic

plaques

in

the

neocortex

-LRB-

Fig.

-RRB-

Double-labeled

studies

demonstrate

co-localization

of

dysferlin

-LRB-

brown

in

Fig.

-RRB-

and

Aβ

-LRB-

pink

in

Fig.

-RRB-

in

the

hippocampus

Fig.

of

dysferlin

with

amyloid

pathology

in

the

AD

brain

a

b

Demonstrates

the

location

of

the

dysferlin

accumulation

in

the

AD

brain

with

double-labeled

studies

a

Diffuse

dysferlin

accumulation

-LRB-

DAB

brown

-RRB-

and

Aβ

-LRB-

DAB-nickel

black

-RRB-

in

the

frontal

cortex

Using

VectorRed

-LRB-

pink

Vector

Laboratories

-RRB-

as

the

chromogen

for

Aβ

and

DAB

-LRB-

brown

Vector

Laboratories

-RRB-

as

the

chromogen

for

dysferlin

the

pathologic

deposition

is

localized

as

neuritic

pathology

in

Aβ

neuritic

plaques

in

the

neocortex

hippocampus

-LRB-

b

-RRB-

Magnification

of

-LRB-

a

-RRB-

is

and

the

scale

bar

in

-LRB-

a

-RRB-

μm

Magnification

of

-LRB-

b

-RRB-

is

and

the

scale

bar

in

-LRB-

b

-RRB-

μm

Dysferlin

neuritic

pathology

occurs

in

conjunction

with

tau

neuritic

pathology

In

Fig.

we

demonstrate

that

neurites

in

plaques

may

comprise

tau

dysferlin

or

both

Triple-labeled

studies

of

midfrontal

-LRB-

Fig.

-RRB-

cingulate

-LRB-

Fig.

-RRB-

and

entorhinal

-LRB-

Fig.

-RRB-

cortex

illustrate

the

relationship

of

tau

-LRB-

pink

arrowheads

-RRB-

and

dysferlin

-LRB-

brown

arrows

-RRB-

within

an

Aβ

plaque

-LRB-

blue

-RRB-

Double-labeled

immunofluorescent

studies

-LRB-

Fig.

f

-RRB-

demonstrate

that

tau

-LRB-

green

in

-RRB-

and

dysferlin

-LRB-

red

in

Fig.

-RRB-

have

both

distinct

and

overlapping

-LRB-

yellow

in

Fig.

-RRB-

epitopes

in

neuritic

plaques

Dysferlin

does

not

appear

to

co-localize

with

the

more

abundant

tau

dystrophic

neurites

Fig.

of

dysferlin

with

tau

pathology

in

the

AD

brain

a

f

Demonstrates

the

location

of

the

dysferlin

and

tau

accumulation

in

the

AD

brain

with

triple-labeled

immunohistochemistry

studies

-LRB-

a

c

-RRB-

and

double-labeled

immunofluorescence

studies

-LRB-

d

f

-RRB-

a

Midfrontal

b

cingulate

and

c

entorhinal

demonstrate

tau

-LRB-

Vector

Red

pink

arrowheads

-RRB-

dysferlin

-LRB-

DAB

brown

arrows

-RRB-

and

Aβ

-LRB-

Vector

SG

blue

-RRB-

Note

the

distinction

between

tau-positive

and

dysferlin-positive

structures

Double-labeled

immunofluorescence

studies

demonstrated

neurites

within

amyloid

plaques

Neurites

may

comprise

tau

-LRB-

green

d

-RRB-

dysferlin

-LRB-

red

e

-RRB-

or

overlapping

epitopes

-LRB-

yellow

in

merged

image

of

f

-RRB-

Magnification

of

-LRB-

a

f

-RRB-

is

and

the

scale

bar

in

-LRB-

d

-RRB-

μm

Biochemistry

Western

blot

analysis

following

RIPA

extraction

demonstrated

the

presence

of

dysferlin

as

kDa

bands

in

all

brains

examined

-LRB-

Fig.

-RRB-

however

there

was

a

decrease

in

the

total

dysferlin

content

in

CDR

-LRB-

severe

AD

-RRB-

brains

compared

to

CDR

-LRB-

nondemented

-RRB-

There

is

a

progressive

decrease

in

total

dysferlin

with

progressive

disease

-LRB-

r

P

-RRB-

Western

blot

analysis

following

serial

extraction

of

proteins

demonstrated

a

change

in

the

solubility

state

of

dysferlin

across

the

spectrum

of

dementia

-LRB-

Fig.

-RRB-

No

dysferlin

could

be

demonstrated

in

the

HS

fraction

In

contrast

to

the

control

CDR

brains

however

there

was

a

significant

reduction

of

dysferlin

in

the

HS/T

fraction

in

the

demented

cases

-LRB-

CDR

and

-RRB-

and

was

undetectable

at

the

CDR

level

of

severity

-LRB-

F

P

-RRB-

In

the

SDS-soluble

fraction

from

the

demented

cases

there

was

an

accumulation

of

kDa

bands

with

dementia

progression

that

is

a

more

insoluble

form

of

dysferlin

was

found

in

severe

CDR

brains

compared

to

milder

stages

of

dementia

-LRB-

CDR

-RRB-

or

in

nondemented

individuals

-LRB-

F

P

-RRB-

Dysferlin

did

not

aggregate

or

accumulate

in

the

FA-soluble

fractions

of

the

control

or

AD

brains

Fig.

Blot

analysis

of

dysferlin

in

nondemented

and

AD

brains

a

Nondemented

-LRB-

CDR

-RRB-

and

AD

brains

-LRB-

mild

CDR

moderate

CDR

and

severe

CDR

-RRB-

were

extracted

in

RIPA

as

described

in

the

Materials

and

methods

Five

microliters

of

each

extraction

were

loaded

into

separate

lanes

of

a

SDS-polyacrylamide

gel

electrophoresis

and

after

transfer

to

nitrocellulose

membranes

were

probed

with

antibodies

against

dysferlin

The

blot

demonstrates

the

presence

of

dysferlin

in

both

normal

and

AD

brains

with

declining

levels

of

protein

across

disease

progression

Band

intensities

were

quantified

and

compared

in

a

bar

graph

There

is

a

linear

decrease

in

total

dysferlin

associated

with

disease

progression

-LRB-

r

P

-RRB-

b

The

same

brains

were

sequentially

extracted

with

HS

HS/T

SDS

and

FA

as

described

in

the

Materials

and

methods

section

as

loaded

onto

SDS-polyacrylamide

gel

electrophoresis

as

described

earlier

Dysferlin

was

not

seen

in

either

the

HS

or

FA

fractions

Dysferlin

was

demonstrated

prominently

in

HS/T

fraction

in

the

CDR

brains

with

a

significant

reduction

-LRB-

F

P

-RRB-

of

dysferlin

in

the

HS/T

fraction

in

the

demented

cases

-LRB-

and

-RRB-

and

was

nearly

undetectable

in

severe

AD

-LRB-

CDR

-RRB-

In

contrast

the

SDS

fraction

representing

insoluble

phases

of

dysferlin

demonstrated

significant

increases

-LRB-

F

P

-RRB-

in

the

kDa

bands

as

dementia

progressed

from

mild

-LRB-

CDR

-RRB-

to

severe

-LRB-

CDR

-RRB-

Discussion

Dysferlin

is

a

new

protein

described

in

the

AD

brain

associated

with

neuritic

plaques

Dysferlin

is

widely

expressed

in

the

brain

and

is

found

in

intracytoplasmic

compartments

in

pyramidal

neurons

in

normal

brains

Dysferlin

begins

to

accumulate

in

dystrophic

neurites

in

the

AD

brain

at

mild

stages

of

dementia

-LRB-

CDR

-RRB-

Dysferlin

is

found

at

all

stages

of

disease

with

greater

accumulation

in

more

advanced

disease

-LRB-

CDR

-RRB-

Significantly

our

Western

blot

analysis

demonstrated

dysferlin

in

RIPA-extracted

fraction

in

all

cases

however

in

the

serial

extracted

fractions

an

alteration

in

the

solubility

state

of

dysferlin

was

found

in

demented

cases

-LRB-

CDR

-RRB-

compared

with

nondemented

cases

This

coincides

with

the

finding

of

neuritic

pathology

by

immunohistochemistry

The

HS/T

fraction

represents

the

transmembrane

cytoskeletal

fraction

of

normal

dysferlin

and

decreases

with

advanced

stages

of

dementia

This

may

represent

the

loss

of

membrane

integrity

and

alteration

in

cytoskeleton

structure

In

contrast

the

SDS-soluble

fraction

represents

more

insoluble

forms

of

the

protein

suggesting

aggregation

of

dysferlin

may

be

related

to

disease

progression

This

alteration

of

solubility

and

accumulation

is

seen

in

other

neurodegenerative

diseases

-LRB-

e.g.

Parkinson

's

disease

multiple

system

atrophy

dementia

with

Lewy

bodies

-RRB-

-LSB-

-RSB-

Dysferlin

is

a

member

of

ferlin

family

homologous

to

protein

in

C.

elegans

-LSB-

-RSB-

It

is

highly

expressed

in

muscle

but

little

is

known

about

the

role

of

the

protein

in

the

brain

Several

members

of

the

ferlin

family

have

been

mapped

Dysferlin

is

found

on

chromosome

-LSB-

-RSB-

and

deficiency

is

associated

with

three

clinical

phenotypes

Limb-Girdle

muscular

dystrophy

type

Miyoshi

myopathy

and

distal

anterior

compartment

myopathy

-LSB-

-RSB-

Another

member

of

the

ferlin

family

Otoferlin

is

mapped

to

chromosome

and

is

associated

with

autosomal

recessive

nonsyndromic

deafness

-LSB-

-RSB-

Two

other

members

of

the

ferlin

family

have

been

described

Myoferlin

-LRB-

-RRB-

-LSB-

-RSB-

and

-LRB-

-RRB-

-LSB-

-RSB-

Myoferlin

is

a

type

II

transmembrane

protein

upregulated

in

Duchenne

muscular

dystrophy

and

may

have

a

role

in

muscle

regeneration

and

repair

-LSB-

-RSB-

The

function

of

is

unknown

Dysferlin

is

thought

to

play

a

role

in

muscle

membrane

maintenance

and

repair

-LSB-

-RSB-

In

C.

elegans

the

protein

plays

a

role

in

vesicle

trafficking

and

membrane

fusion

and

can

bind

to

phospholipids

in

a

calcium-dependent

manner

-LSB-

-RSB-

In

dysferlin

null-mice

muscle

cells

are

defective

in

repairing

membrane

disruptions

leading

to

degeneration

-LSB-

-RSB-

If

dysferlin

has

a

similar

membrane

repair

function

in

neurons

then

it

is

possible

that

the

deposition

of

dysferlin

in

AD

may

be

related

to

the

inability

of

neurons

to

repair

damage

due

to

accumulating

Aβ

pathology

throughout

the

progression

of

AD

The

findings

reported

suggest

that

there

may

be

common

mechanisms

of

membrane

repair

in

degenerative

diseases

of

muscle

-LRB-

e.g.

dystrophinopathies

and

sarcoglycanopathies

-RRB-

and

brain

-LRB-

e.g.

AD

-RRB-

The

aggregation

of

normally

soluble

proteins

in

fibrillar

lesions

is

the

neuropathologic

hallmark

of

many

neurodegenerative

diseases

Whether

the

aggregates

or

their

precursors

are

the

actual

toxic

species

is

still

under

debate

-LSB-

-RSB-

however

it

is

likely

that

the

conversion

of

proteins

from

a

soluble

to

insoluble

state

impairs

the

long-term

viability

of

neurons

In

addition

because

dysferlin

appears

to

accumulate

in

conjunction

with

Aβ

deposition

and

disease

progression

it

may

be

able

to

serve

as

a

marker

of

disease

progression

Continued

efforts

aimed

at

understanding

abnormalities

in

the

misfolding

of

protein

leading

to

neurodegenerative

disease

such

as

Aβ

and

tau

or

proteins

associated

with

aggregation

such

as

dysferlin

will

provide

insights

into

disease

mechanisms

underlying

neurological

disorders

characterized

by

abundant

filamentous

lesions

O O O B O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O

alzheimer’s disease
amyloid β-protein
tau protein
protein aggregation
neurodegeneration