lay a pivurodegea broadof metal
er tissues,
etal ions,
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e brain has
for this was
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ve diseases
n Alzheimtc., is well-
already dehed, to the
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e (AD) and
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etal binding
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l ions seem
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at it seems
nt NDs?
Metal ions in amyloid aggregation: a
coordination chemistry perspective
Sylvestre Bonnet
[email protected]
Leiden Institute of Chemistry
Lorentz symposium on amyloid aggregation
Leiden, April 13th, 2015
Leiden University. The University to discover.
1
1
Preliminary remark
Chemistry
Physics
AD
CONCEPT
Biology
*
*
*
Why is in most of these NDs oxidative stress implicated?
And why do antioxidants, like vitamin E, have only limited effects as anti-ND drugs?
In addition to oxidative stress, aggregation of the key
proteins involved in the NDs is a common feature: is
there a link between the two processes?
What else can be done in terms of metal ions to combat
NDs like AD?
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2
In the following, we try to provide some fundamental considerations in order to answer the questions raised above.
Why Are Certain Metals Important for Living
Organisms?
Little abundance is not equal to unimportance: The metal
content of living organisms is normally lower than the most
abundant elements O, H, C and N. The most abundant
metals are the (earth)-alkalines Na, K, Mg, and Ca, which
contribute to 0.05 to 1.5 % of the body-weight. The other
metals are even less abundant (Table 1). Although some of
Metals and the brain
Table 1. Metal content for an average human of 70 kg[2] and brain.[3] [a]
Essential metals
Body content
[g per 70 kg]
Ca
K
Na
Mg
Fe
Zn
Cu
Mn
Mo
Co
1200
125
100
25
4
2.3
0.07
0.012
0.005
0.002
Brain content
[g per 1.5 kg]
0.5
0.15
0.006
Nonessential
Body
content [g]
Ti
Rb
Sr
Br
Al
0.7
0.7
0.3
0.3
0.06
[a] Only well-established essential and non-essential metals are listed.
Only some of the most abundant non-essential metals are listed.
A Bioinorganic View of Alzheimer!s Disease
CONCEPT
which is produced in form of
ATP in the respiratory chain.
The respiratory chain is an
overall redox reaction of
NADH (and succinate) with
oxygen (Figure 3).
The importance of the redox
active metal ions Fe and Cu is
immediately recognizable, as
three respiratory chain complexes I, II and IV, together
contain ten FeS clusters, five
hemes and three Cu centers.
This intricate machinery is used
to generate a proton gradient,
which is needed to drive the
formation of ATP by ATP synthase. In contrast, the chemical
reaction of NADH with O2 is
spontaneous, but produces only
heat (no ATP). Thus, a cell has
to make sure that the electrons
from NADH pass to O2 via the
respiratory chain to produce
ATP, and that no (or very
slowly) short cut occurs.
The reaction of NADH (like
other reductants, e.g., ascorbate, vitamin E, glutathione)
with molecular oxygen is slow,
Figure 1. Distribution of total Zn (above) and total Cu (bottom) in the hippocampus. (Reprinted with permisdue to the triplet ground state
sion from ref. [5], Copyright (2005) American Chemical Society.)
(i.e., two parallel electron spin)
of dioxygen. If O2 reacts as a
two-electron oxidant, the two
accepted electrons (or electron pair) should also have a parsite that modulates its reactivity[8] (Figure 2). Moreover, in
allel spin according to Pauli!s principle (i.e., also be a tripthe hippocampus, chelation of Zn has been shown to affect
episodic-like memory.[9]
let). Thus, a pair of electrons in a molecular orbital (like in
NADH or most other organic molecules) does not meet this
In contrast, for the potential labile Cu pool the function it
criterion. Or as Halliwell and Gutteridge put it:[11]
is not known. Very recently, You et al.[10] showed that amyloid-b (Ab) toxicity depends on the interaction of copper
“(The triplet ground state of O2)…imposes a restriction
ions, prion protein and NMDA receptors (Figure 2), suggeston electron transfer which tends to make O2 accept its
ing a neuromodulator role for Cu, similar to Zn. In terms of
electron one at the time, and contributes to explaining
Cu (and Fe), there is an important connection with another
why O2 reacts sluggishly with many non-radicals. Theomolecule to consider: oxygen.
retically, the complex organic compounds of the human
body should immediately combust in the O2 of the air
but the spin restriction and other factors slow this down,
The Oxygen Connection
fortunately!”
Right: Distribution of total Zn (above) and
total Cu
in the
hippocampus
these metals seem to be present
in (bottom)
a negligible
amount,
they
are essential; this means their absence is lethal, as is the
case for the 3 mg of Co. In contrast, other metals are present in higher amounts, like Ti (on average about 0.7 g), but
are considered to be non-essential.[2] Non-essential metals
are tolerated to a certain degree before they become toxic,
whereas the concentration of an essential metal has to be in
a relatively narrow range, too little or too much being deleterious for the organism. In other words, their concentration
has to be tightly controlled, and on different levels, that is,
University. The University to discover. 3
on the level of localization (body, organ,Leiden
cell, compartment.
etc.) and time.
As we have seen above, metal ions are particularly good in
One has also to note that when bioinorganic chemists talk
binding small molecules and in the case of the transition
The redox active metal ions, in particular Fe and Cu, can
metals, like Cu, Fe, Co and Mn for redox reactions. As such
accelerate (in a catalytic way) the reaction of reductants
about metals, they mean mostly metal ions, as the chemistry
it is understandable that the metal ions Cu and Fe play a
(like NADH, ascorbate) with oxygen by mediating electron
preponderant role in oxygen metabolism. Due to the high
transfer (NADH reduces metal, which in turn reduces O ).
is in aqueous solution, and there, metal ions are generally
oxygen consumption, that is, 20 %, of the brain (compared
Redox active metals can do this, because they can undergo
more stable and more soluble than elementary metals.
to 2 % of body-weight), the higher metal content makes
facile one-electron transfer reactions.
• Brain contains a large proportion of the body’s metal content
• Metal concentrations are low but their role is essential
H Verlag GmbH & Co. KGaA, Weinheim
2
sense. The high oxygen consumption is directly (but not exclusively) related to the high energy demand of the brain,
www.chemeurj.org
15911
Chem. Eur. J. 2012, 18, 15910 – 15920
As a consequence Cu and Fe not only dissipate energy,
but can also produce radicals by one-electron reduction (see
" 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
15913
General roles of metals in cells
Redox inert metal ions:
• carrying positive charges, maintaining
osmotic balance (Na+ and K+)
• structural elements (Mg2+, Zn2+) or serve
as messengers (Ca2+).
Redox-active transition metal ions:
• Mn+ + 1 e- ⇌ M(n-1)+
• electron transfer & storage
• catalysis
• (Lewis acid) catalysis (Mg2+, Zn2+)
Metal ions need to be controlled:
• do not cross membranes
• uptake necessary for metalloenzymes
• free metal ions are toxic
• controlling metal ions is realized through
coordination
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4
What is a metal complex?
kon
MP k⇆ M + P
Kd
off
Kd: thermodynamic dissociation
constant (-ΔG0)
kon,koff: rate constant for the
coordination and deco ordination
(ΔG≠on, ΔG≠off)
P can be a protein or peptide, a small
molecule (porphyrin, ionophore),
etc…
TS
G
ΔG≠off
ΔG0
ΔG≠on
M+P
MP
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5
Which residues can coordinate to metals?
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6
Controlling the metal balance in living cells
Metal transporter
e.g. CTR1 (copper)
Storage
e.g. Ferritin (iron)
Sensors
e.g. Zap1 (zinc)
transcriptional
regulation of Zn
levels
Metalloinsertase
e.g. Sco1 (inserts copper
into cytochrome c
oxidase)
Chaperone
e.g. Atx1 escorts
Cu to Gogli
Metalloenzyme
e.g. ferrochelatase
(iron)
New, Dalton Trans, 2012, DOI:10.1039/c2dt31933k
Leiden University. The University to discover.
7
An example: zinc homeostasis in neurons
efflux
uptake
et al., 2003, Eur. J. Pharmacol.)
Leiden University. The(Colvin
University
to discover.
8
Roles of synaptic zinc
• Modulation of glutamic
responses
• Modulation of GABA
responses
• Antagonism on Ca2+, K+ and
Na+ conductances
• higher local concentrations
than average [Zn] in cells or
in tissue (up to 300 µM)
Colvin et al., Eur. J. Pharmacol. 2003
& E.P. Huang, PNAS 1997
Leiden University. The University to discover.
9
The metal pool in cell
MP ⇆ M + P
high
Kd
MP ⇆ M + P
low
Kd
• For Zn2+, bound metal pool refers to ions bound to proteins with a Kd < 10−7 M
• For Zn2+, free pool implies Kd > 10−7 M (solvent-exposed sites at protein surface
• For Cu2+, « free » means Kd > 10-10 M
New, Dalton Trans, 2012, DOI:10.1039/c2dt31933k
Leiden University. The University to discover.
10
Why are « free » metal ions toxic?
1) The Irwing Willian Series
For high-spin complexes of the divalent ions of first-row
transition metals, the stability constant for the formation of a
complex follows the order
Mn2+ < Fe2+ < Co2+ < Ni2+ < Cu2+ > Zn2+
• Empirical rule
• Holds for a wide variety of ligand sets
• free Zn2+ and Cu2+ displace other metals in enzymes
Irving, Williams, J. Chem. Soc. 1953, 3192 and Gorelsky et al, Inorg. Chem. 2005, 44, 4947
Leiden University. The University to discover.
11
Why are free metal ions toxic?
2) Fenton chemistry
generation of toxic ROS by free metal ions
such as Cu2+, Cu+, Fe2+, or Fe3+
Fe3+ + H2O2 → Fe2+ + HOO• + H+
Fe2+ + H2O2 → Fe3+ + OH- + OH•
2+
Cu + ascorbate- → Cu+ + dehydroascorbate•
Cu+ + H2O2 → Cu2+ + OH- + OH•
Can Zn2+ generate ROS?
yes, by replacing other metals in their
protein, the other metal is released and
make ROS
Leiden University. The University to discover.
12
Amyloid-β in Alzheimer’s Disease
α-secretase
β- and γsectretase
APP
soluble Aβ
unordered
aggregates
« oligomers »
Amyloid
plaques
β sheet
not toxic
toxic
(ROS)
Degenerated
neuron
Healthy
neuron
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13
Alzheimer's disease and metals
• risk factors include exogenous Pb, Hg, Al exposure
• Zn content abnormally high in blood and hippocampus of AD
patients, but normal or low in cerebrospinal fluid or globally in
the brain
• total Cu levels generally depressed in AD brain or models
• metal homeostasis affected in AD
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14
Metals and plaque
Amyloid-β (βA)
high (mM) concentrations
of Cu, Zn, Fe
Zn and Cu enhance the aggregation of Aβ in vitro
Leiden University. The University to discover.
15
Role of the Zn2+ on plaque formation
ious groups
Level, ppm
2"
% 0.22
% 0.21
%bnormal
0.14
%etabolic
0.24
%ncentra0.17
%dhesive0.24
digestion
% 0.27
n supple%tion
0.08has
%tudy
0.21
that
%ognition
0.36
-matched
%
0.24
% 0.04
mice with knocked out Zntransporter (ZnT3): less plaques
2.4 %because
0.02
less Zn in the synapse
Cu2"
,a
2.6 % 0.05
2.2
% 0.05
behaved
indistinguishably,
with no plaque
detect- low respeonse curve for human A3 1-40 (1.6
Congo-red
2.5
0.03
able%aggregation
observedstained
in a peptide
so- p.M) un til the Zn concentration reached
2.4
% 0.05
lution
of 0.8 p.M and an 15 % aggregation 300 nM, corresponding to the saturation of
at
higher
peptide
concentrations.
high-affiinity binding (4). At Zn concentra2.6 % 0.08
next titrated the formation of >0.2-Cortex
tions abcNe 300 nM, corresponding to low2.6 We
% 0.03
pim A3 particles against increasing Zn con- affinity thinding (4), human A131 40 aggre2.4
%
0.09
centrations (Fig. 2B) and observed a shal- gated. In contrast, rat A 1 40 remained sta2.4 % 0.09
2.3 % 0.06
Fig. 1. Scatchard analysis of 65Zn binding to rat A3 -40. DisHippocampus
2.0
% 0.12
solved
peptides (1.2 nmol) were dot blotted onto
0.20-pm polyvinylidene
fluoride (PVDF) membrane (Pierce) and a competition
2.5
% 0.08
e effects analysis
was performed as described (4). Rat A11 40 and human CD
f Zn on Ad1 was
+/+car40 were
methoxy
ZnT3︎-/or each). There
no synthesized by solid-phase fluorenylZnT3︎
ll 40 in bonyl (FMOC) chemistry. Purification by reverse-phase highthose of pressure liquid chromatography and amino acid sequencing 03
Lee
et al, PNAS
2002,
7705
= plaques
Fig. 2.The regression
Reducedline
deposition
ofofamyloid
in hAPP":ZnT3!/! mouse
indicates
a KA
3.8 99,
tide (rat confirmed the synthesis.
of binding(A–D)
is 1 :1 Coronal
Although sections
the data points
for
of 24-month-old
female hAPP" mouse brains
d by rat F.M. Stoichiometrybrains.
the Scatchard curve
are slightly
suggestive
biphasic
curve,
!/!
16
Leiden
The University
to ,aTFL-Zn
discover.
yamyloid
fibers (asterisks
with
ZnT3"/"
(A and of
C)aor
ZnT3University.
(B aand D) genotype,
stained with
(A
biphasic iteration yields association constants of 2 and 9 A.M,
atence
brains.was which
seendo in
and
B) or Congo
red (C andseparate
D). Compared with ZnT3"/" mice that
0.7 had
not
justify
an
interpretation
of
physiologically
ich has
to be abnormal
behaved
indistinguishably,
with
no detect- plaques
curve
human(Co)
low respeinonse
A3 1-40
her
(1.6
agedbeen reported
TFL-ZnCongo
red-stained
cerebral
and
Bound
Zn2+ forcortex
fibers
data are
binding
fromand
(thesenumerous
the
aboveinScatchard
,rich
may bemossy
important
for sites
the metabolic
ablederived
observed
aggregation
a peptide sop.M) un til the Zn (PAM)
concentration reached
sequence
!/!
hippocampus
(Hi),
had markedly
reduced
number
analysis).
because
increased concentralution of 0.8
p.MZnT3
and an mice
15 % aggregation
300 nM,
the plaques.
ceA3
ofall
hippocampi
saturation of
to of
corresponding
in
dofof
(Bar $at100
"m.)
promoteoftheage
peptide's
higher
peptide concentrations.
high-affiinity binding (4). At Zn concentramonths
in adhesiveratZnresistance
and
and
to proteolytic
digestion
We next titrated the formation of >0.2- tions abcNe 300 nM, corresponding to low!/! females
titutions
the safety of and
oral Zn supple- pim A3 particles against increasing ZnBcon- affinity thinding (4), human A131 40 aggreT3
oreover,
tiontoinat
the at-risk
population has centrations (Fig. 2B) and observed1.0a shal- gated. In contrast, rat A 1 40 remained sta*>Arg
st
zinc
f luoresbrought
a study thatIt is possible that differences
0.9 in background strains affect the
vely
(9)]into question by
!/!
"/!
T3
or
ZnT3
strated
adverse
effects
on cognition
peptide's
phenotype
of transgenic
mice
hAPP. In fact, we have
0.8 expressing
Fig. 1. Scatchard
analysis of 65Zn
binding
to rat A3 -40. Dis2
±
D
subjects
but
not
in
age-matched
=L
solved peptides (1.2 nmol) were
%,
respectively,
in
dot5N blotted
poly"/!
"/"0.20-pm
cient
to
0.7-onto
foundvinylidene
that fluoride
the (PVDF)
hAPP
F1 mice
had higher plaque
ls (5).
CU:ZnT3
membrane
a competition
(Pierce)
and
c=
L..
mmunity
-V
mice
(Fig.
1E).the effects
r these
reasons,
we
V studied
analysis
was the
as described
Rat A11
performed
(4).0.6burden
than
original
Tg2576
transgenic
at 12 months of
40 and human mice
CD
ysiological
concentrations
of
Zn
on
binding
2+
fluorenyl methoxy carAd1 40 were synthesized by solid-phase
0.530%
total brain
b-% 17.9
% 25.7
vs.121.1
plaqueshighper 10 coronal sections,
ability of
of synthetic
human All 40age
in (213.7
bonyl (FMOC)
chemistry.
Purification
by
reverse-phase
histidine
0.4(26).
As seen
withto thoserespectively).
ion
and compared
its effects
of pressure liquid However,
chromatography and
amino
acid sequencing
for
data
analysis,
used
only
F1
manifest
03 we
U=
0.3confirmed
the
synthesis.
The regression line indicates a"/!
KA of 3.8
at-mouse
the peptide (rat
U- of were
"/! mice with the
in
totalspecies
zinc
derived offrom
binding crossing
is 1 :1 Although
the data points:ZnT3
for
Soluble Ad 1-40 is produced by animals
rat F.M. Stoichiometry
0.2- hAPP
Aβ
soluble
up
to
3.7
mM
in
cerebrospinal
fluid!
ofmanner
Zn
to (6); however,
the
Scatchard
curve
are
slightly
suggestive
of
a
biphasic
curve,
a
1-40
only• Human
in
nal
tissue
AD amyloid
identical strain
0.1
iteration backgrounds.
yields association constants
of 2 and 9 A.M,
petitive
ition
is not
a feature
0.7
•aged
[Znrat2+brains.
]>300 biphasic
nMdoinduce
formation
rast,
levels
ofofiron
not justify anamyloid
interpretation of
physiologically separate
myloidogenesis
occurs"/"
in other aged which
he
of
KA
Bound Zn2+ (PAM)
2+
sites
data
are
derived
binding
from
(these
the
above
Scatchard
rat, sequence
AβIncreased
to Zn !!!
!
"!" Mice.
1-40 immune
dntrast
female
als
with
human• Ad
to theZnT3
analysis).Ratio of Soluble!Insoluble A# in hAPP :ZnT3
log [Zn21
which
is strongly
conserved
nt
ages
(Table
1). in all log [Am]
analysis
we examined the effect of synaptic zinc
on levels of soluble
ted
animal species, except rat Next,
and
eals
The rat-mouse Ad substitutions
(8).only
!
.
The
levels
of
insoluble
and
insoluble
A
D BA!40 and A!42 were
c
.8
p.M),
1.0,
>Gly, Tyr-ePhe,
and His-*>Arg
at
0.9!/!
After
12 months
of
mice
at 12 and 18
substantially in hAPP":ZnT3
ons 5, 10, and 13, respectively reduced
(9)]
0.8 0.9
*
ncreased
c
rovery
to cause
change95in the peptide's
0.7 0.8a +
of a markedly.
2
±
=L
5N 0.6 0.7cochemical
properties sufficient to
f 24-month-old
matograc=
L..
r, on
peptide its relative immunity
85CD~ ~ ~ -V~CU~ ~ ~ 0.5 0.6V
inred
due,theformation.
ongo
revealed
Zn
yloid
Because
binding
0.4 0.5A 80Bioinorganic
bA3 All
(4).
man
is mediated
by histidine View of Alzheimer#s Disease
all
of 40which
also
0.3 0.4m3 EDTA|
ation
at AP3 of
may be expected to manifest
0.2Zinc
U0.375
2ddA).
contrast,
UZn-binding
in theBy properties.
0.1 0.2U 0
70affinity ofl Zn to
e
binding
c studied
(Fig.
2B)
showed
eptide
inthe
...
0.1 20
C.
40with
N,
(Fig. 1) in a 65Zn
0
40 60 80 100 120
65 competitive
in
age
(Fig.
the
+
predominant form at neutral pH for CuII and CuI are shown
system
used
toPutting
(4) 2D).
Time (minutes)
measure the KA
of Pieces Together: Ab + Metals + ROS
filtered
to human
nding
In contrast
All1 40..EDTA
to
tated
by counting
Cd 1i) Sn 1i) Ba i) Hggi) Pb9I)
Mg~ii) AltO1) Cai1i) Mn~1i) Fe2(I1) Co II) N~iI) Cu i) AD
Z(I1)
in Figure 7. Moreover, an original redox mechanism was
log
[Am]
[Zn21
log
ers.
The
n A,140 (4), the Scatchard analysis
ed.
Theto number
ble
peprat All2.140 of
reveals
binding
only
proposed for the redox cycling of Cu–Ab, in which a low
Effect
of
Zn
12
on
I-labeled
Fig.
and
rat
human,
D
into
>0.2-pm
human,
40
aggregation
particles.
Aw1
2+
2+
c
=
inding association
(KA in
3.8 p.M), is selective
ntially
reduced
Aβ1-40
binding
to Zn
and
Cuwere filtered (Spin-X, Costar)
particles
0.9 , on 0.2- pm
100 solutions
Stock
and ratidea
in water
peptide
(16 K.M)
Ad1 40evolved
Thehuman
general
during
recent
years is that the misstoichiometry.
":ZnT3
"/"
0.8
* populated “entatic-like state” is responsible for all redox cye1:1
ahAPP
shaln
cellulose
acetate
at
mM
to
100
NaCI
mM
and
20
at
7.4
700g,
with
brought
tris-HCI
95
or
without
pH
(buffer
1)
c
0.7 a +
e have observed that the recovery of
whereas the ground state is too sluggish (Figure 7).[47]
metabolism
ofchloride
metalsalts,
ions
inet al,
AD
is not
a and
general
large
" filtration
EDTA
incubated
(50"/!
p.M) or metal
then
minm
Bush
Science
1994,
265,
1464
filtered
(30
The
37CC),
againover(700g,
0.6 4 min).cling,
All 40 in
chromatogrannbetween
hAPP
85- (33) relative
CD~ ~ ~ ~ ~ ~ ~ 0.5 to the OD214
and
thein the:ZnT3
fraction
ofof
the
filtrate was
calculated
the ratio of
the
filtrate OD214
byrather
reduced
presence
Zn,A31
due,40ininofthemetal
load
or
lack
ions.
It
is
a
mislocalization
due
ent
with
previous
17
0.4
University
to discover.
p.M adhesiveness
of the unfiltered
data
areLeiden
means + University.
indicated
otherwise here.
SD, n = 3, unlessThe
(A)
to25increased
of A3sample.
(4). All[30]
80- points
0.3 m3 EDTA|
A31
to
imbalance.
ions(25bound
to Ab
inand
plaques
of
with ormetal
without Zn2
then filtered
FM) or EDTA
(50 KM)
through
entration
brains
ofProportion
female
etermine
whether
thean
aggregation
P, ofincubated75The
0.2Zinc
aalso
0.2 enhanced
filter, titrated
in the against peptide concentration. (B) Proportion of Af1 40 (1.6 p.M) filtered
through
Af31 40 was
0.1
fn human
male
This
arepm
part
of
the
misplaced
metalThe
ions,
generally
too high
U ex70 concentration.
l
of"/!
nce
0
0.2-pm
titrated
1251 -labeled
Zn2
Zn, wemice.
incubated
human AP1340
the peptide
inagainst
filter,
(34) (15,000
cpm) was
contrast,
20 40 60 80...100 120
nT3
mice
(P
#min with
s concentrations
for
added
to30unlabeled
filteredmetal
as described
counts
A11In40contrast,
(1.665pM) as a intracellularly,
tracer, incubated, andthe
tracellularly.
ion above.
con-0 The
Zn2+
on
Time
(minutes)
or EDTA
and then
filtered
in the
minute
filtrate and retained
onAltO1)
theCai1i)excised
filter were
measured by a y counter. Dashed lines
per 0.535)
mice
(P
$
Cu i) Z(I1) Cd 1i) Sn 1i) Ba i) Hggi) Pb9I)
Fe2(I1) Co II) N~iI)there
Mn~1i)words
f(25a FM)
0.8-through
centration
isphysiological
too low..EDTAplasma
InMg~ii) other
is an(C)imbalance,
olutions
0.2-pimthefilters.
The
indicate
normal
Zn concentrations.
and CSF
of A1340 (1.6
Proportion
der uptheto 80%op.M)
used
of the
available
pepfiltered
pm
with
a
0.2filter
after
incubation
various
metal
ions
The
atomic
through
(3 pM). into >0.2-pm particles.
2. Effect
of Zn on human, 12I-labeled
Fig. to
and rat Aw1 publicahuman,
40 aggregation
from
intracellular
extracellular.
Indeed,
a recent
regation
o age
aggregate
>0.2-p1m
numbers
of theparticles
metal species
indicated
below
each solutions
Effects
(25 pM)
or Costar)
EDTA on 0.2- pm
species.(16(D)K.M)
Zn2+filtered
Stockare
human
and rat Ad1
in waterofwere
in into
all
three
40 peptide
(Spin-X,
2A) (10).
tothe
be akinetics
appears
shal- ofcellulose
of There(50
sence
tion
shown
that acetate
in the
affected
by AD,
human
0.2-pm
onhas
Data
measured
40at aggregation
100 mM NaCI
byand
20 mM filtration.
at pH
7.4 points
700g,brain
with or without
brought toregion
tris-HCI
(buffer 1)are
KM)
AP1
"/"
t
in
ZnT3
mice
negative log-linear
EDTA (50 p.M) or metal chloride salts, incubated (30 minm 37CC), and then filtered again (700g, 4 min). The
solutions
n = 2.
meansrelation
+ SD, between
compared
to
brain
regions
or bythe
same
region
n estimated
Ad peptide concentration
the non-affected
in the filtrate
fraction of the A31 40
was calculated
the ratio
of the filtrate
OD214 (33) relative to the OD214
as
thein and
=
rtion of filterable
25
p.M
peptide
of
the
All
+
unfiltered
data
are
n
means
unless
indicated
otherwise
sample.
points
SD,
3,
265 * 2 there
SEPTEMBER
* VOL.subjects,
SCIENCE
1 465 here. (A)
1994 with
in
healthy
Cuor EDTA
present
aques,
greatly
Zn2 (25 FM)
Proportion of A31
incubated loosely
or withoutbound
(50 KM) and then filtered through
but even atwas
the lowest
concentration
P,is more
a 0.2 pm filter, titrated against peptide concentration. (B) Proportion of Af1 40 (1.6 p.M) filtered through
!/!
of
(0.8compared
the
p.M), >70%
human
with
a higher power
to
catalyze
production
ofhuman
ROS.
with
titrated
Zn2 the
concentration.
The 1251on
against
-labeled
cpm) was
AP1340 (34) (15,000
In contrast,
40 solution aggregated.
Fig. 3. 0.2-pm
Thetofilter,
effect
ofA11
synaptic
zinc
deficiency
gender-disparate
deposition
added
unlabeled
as a tracer,concentration
described above. The counts
40per
(1.6 pM)
incubated, and filtered asdid
uous
However,
total
Cu
content
protein
to be no
effectof
of Zn2+the
on amyloid
appearedreduction
of
plaques.
Data
denote
number
(mean
"
SEM,
5
each)
of
in
minute
the
filtrate
and
retained
on the
excised
filter
were
measured
any $
counter.
per
Dashed
lines
by
Zirah
et
al,
J.Biol.Chem.
2006,
281,
2151
with!/!
no aggregation
of a 0.8[31]
1-40'
the normal physiological
Zn concentrations.
ZnT3
isunder
and CSF
of A1340
plasmabrain
(C) Proportionmale
not
change.
congophilic
plaques
in 10 coronal
sections
of 12-month-old
and(1.6
solution mice
detected
eptide
the indicate
p.M) filtered
The atomic
through a 0.2- pm filter after incubation with various metal ions (3 pM).(1ZE9)
!/!
"
laques
conditions in
andZnT3
25%
only The
aggregation
female
hAPP
mice
indicated
ZnT3
Asterisks
represent
numbers
the
metalwith
aremislocalization
indicated
belowgenotypes.
each species.
Effects of Zn2+
(25 pM) orsigEDTA
species
metal
pools
in ofquestion
for
are(D)
likely
of (50difference
presencenificant
of human
4-[pM solution. In• the
kinetics
by 0.2-pm filtration. Data points are
KM) on the
AP1 40 aggregation
between
male
femalemeasured
(P # 0.05).
with
soluble
or Aβ
1−42 and
, human and rat
n =1−16
2. Cu
means
SD,Aβ
theAStudied
“labile”
pools
of +Zn
and
around
certain
synapses
dis- Glu11
I-40 solutions
-
-
N
Aβ aggregation in vitro in presence of Zn
E
100
N
CONCEPT
~.
-
~.
NEUROBIOLOGY
E
-
C.
The zinc and copper Aβ complexes
Zn
2+ and Zn2+
Cu
• 1:1 binding
265 * 2seem
SEPTEMBER
* VOL.pools
SCIENCE
1 465
cussed
above. with
Only
these
available
for binding
1994
His13
His6
PNAS
MayCu
28, and
2002 Zn
" vol.
99 " no. 11 " 7707
dissociation
constants
depend
on " for
to• the
Ab due to
its moderate
affinity
(metalII
I
pH, ionichave
strength,
loproteins
mostlyauthor
much higher affinities, see Table 2). His14 Figure 7. Top: model of the most populated Cu and Cu –Ab complex at
2+
Table 2. Dissociation constants of Ab and SOD in soluble and aggregated states.
Complex
CuII–Ab
CuI–Ab
ZnII–Ab
CuII in SOD
ZnII in SOD
CuI in SOD (yeast)
Kd apparent (pH 7.4, no buffer) [m]
monomer
aggregate
~ 1 ! 10!10
~ 5 ! 10!8 [33]
1–10 ! 10!6
6 ! 10!18 ref. [35]
1.4 ! 10!14 ref. [35]
0.23 or 6 ! 10!15 refs. [36, 37]
~ 1 ! 10!10 [32]
neutral pH. Bottom: the pathway between the two redox states of Cu–
Ab. A low populated state (only about 0.1 %) in equilibrium with the
predominant state. This state redox cycles very efficiently and is responsible for all the redox reaction.
Therapeutic Aspects and Strategies
Based on the mismetabolism of Cu in AD, given that an imbalance towards an increase in extracellular Cu is prone to
catalyze ROS, the increase in antioxidants of the reducingagent-type (ascorbate, vitamin E) to fight oxidative stress
18
be a double-edged
sword. These antioxidants can also
Leiden University. The University tomight
discover.
drive the production of ROS. This might be a reason why
It has been proposed that Ab plays a causative role in
vitamin E supplementation did not show clear-cut effects on
AD etiology. Ab is found aggregated as amyloid fibrils in
AD progression. There are studies that show beneficial efthe extracellular plaques of AD brains, but is present as
fects but also others in which vitamin E supplementation
soluble monomer in healthy subjects. Thus, aggregation of
was detrimental (for recent review, see ref. [48]). It seems
Ab is a critical step and intermediates (often called oligosafer to use antioxidants in combination with drugs targeting
ACHTUNGREmers) seem to be the most toxic species.[38, 39] These oligoand reducing the misplaced Cu (and Fe) pool. This would
1–10 ! 10!6 [34]
pink = slightly broadened. Ellipsoid code stands for the signal shifting: circle = no shift, small ellipsoid = slight shift, large ellipsoid = si
Scheme 2. (A) List of the Potential Binding Functions
Affected by CuII (from ref 26) or FeII (in bold, the
The zinc and copper
Aβ broadened
complexes
mostly
residues); (B) CuII Binding Site in Aβ
(component I) and Proposition of FeII Binding Site
(1ZE9)
Glu11
• Low binding constants!!!
Zn
→ helps oligomerization
His13
His6
but for other proteins this
is almost « free » metal
His14
• link between Aβ and ROS
production
• enhanced hydrophobicity
caused by partial charge
neutralization of Aβ3upon binding of Zn2+
binding was ruled out. (iv) Regarding the carboxy
they were all equivalently affected in the case of
component I, while in the FeII case mostly those
Glu3 and to a lesser extent that of Asp7 are broa
suggests that both COO! groups from Asp1 and Gl
to FeII, whereas all COO! groups were in equilib
CuII apical position. (v) The carbonyl functions o
predominantly broadened in the CuII case, those o
the three His being less affected. In the FeII cas
functions from Asp1 and His6 are significantly m
than those of Ala2 and His13 and His14. This may b
the simultaneous formation of two metallacycle
!NH2 (Asp1) and the other with the imidazole r
instead of only one in CuII!Aβ16 component I. (v
noting that almost all CO and CRHR positions
fragment are noticeably broadened by FeII, a fact
observed in the CuII case. This may indicate that co
FeII in the 1!6 N-terminal part of the Aβ pep
constraints on the backbone peptide.
As may not be anticipated based on the differ
nature of the two CuII (d9) and FeII (d6) ions, the bi
FeII and of CuII (component I) are very close, showin
differences that may however impact the aggrega
Differences are more significant with the metal center
in component II of the CuII!Aβ species and in
complex, the other reduced redox metal ion of impo
that ZnII is not discussed since no consensual data ar
the literature). Indeed, CuI binds linearly to two ou
imidazoles moieties of His residues.22,23 Regarding c
of the CuII!Aβ species, two main coordination
proposed in the literature: (i) the three imidazole r
CO function from the Ala2-Glu3 peptide bond
references therein) or (ii) the NH2 (Asp1), the d
amidyl from the Asp1-Ala2 bond, the CdO group
Glu3, and an imidazole ring from either His6, His13, o
20 and 26 and references therein). Hence, whatever
tion retained, difference with the FeII binding site
Note that contrary to what is observed for CuII, n
dence of FeII binding to Aβ was found near physiolog
is attributed to a lesser Lewis acidity of FeII compar
2+
Leiden University. The University to discover.
19
by FeII addition are the Asp1 and the three His. Among the three
remaining carboxylic acids, Glu3 is the one mostly influenced by
FeII. Regarding the CO from His, the one in position 6 is more
affected than those in positions 13 and 14.
’ DISCUSSION
As previously observed for CuII!Aβ16 complexes,26,27 FeII
binding
Which model?to Aβ16 is very dynamic and likely involves several
differently populated coordination modes of similar types. As a
consensus has been reached in the literature on the nature of the
hAβ1−16 DAEFRHDSGYEVHHQK
CuII coordination sphere in component I, a comparison (detailed
mAβ1−16 DAEFGHDSGFEVRHQK
below) of NMR data obtained on CuII!Aβ16 component I and
hAβ1−42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
FeII!Aβ16 was used to propose FeII binding site(s) in Aβ16.
mAβ1−42 DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
Note that since no steric constraints are exerted by the Aβ16
ligand, hexacoordination of the FeII ion has been assumed. The
NMR data are compared in Scheme 2a (and Scheme S2,
5
10 13
II
II
Supporting
His Information), and the corresponding Cu and Fe
binding sites are depicted in Scheme 2b. (i) The R position of
• no differences in spectroscopy, binding
affinity,
or ROS
Asp1
is affected
by production
both CuII and FeII, suggesting that the !NH2
between truncated and full-length hAβ
is peptides
bound to both metal centers. (ii) The imidazole rings of the
II
II
threecompared
His are allto
broadened
• in mouse: altered Zn2+- and Cu2+-binding
human in the presence of Cu or Fe . For the
II
component I, it has been proposed by other
• Aβ deposition is not a feature of agedCu
rat !Aβ16
brain
techniques that while His6 is always bound to CuII, His 13 and
• Cu2+ preferentially bound to mouse Aβ
II
• choice of the model is crucial… His14 are in equilibrium for one binding position. In the Fe
species, the same kind of His binding takes place as indicated by
Leiden University.
University to discover. 20
temperature-dependent
study. (iii) As in the CuII case, the
the 1HThe
Tyr10 residue was not significantly affected by FeII and thus its
’ CONCLUDING REMARKS
We reported for the first time a study of FeII coo
Aβ at the molecular scale and show that the bi
confined in the 1!16 N-terminal fragment of the Aβ
also tentatively proposed a structural binding mod
with the data presently available. During the course
9028
Ensemble
methods for measuring metal binding
Article
Biochemistry, Vol. 48, No. 20, 2009
dx.doi.org/10.1021/ic201233b |Inorg. Chem. 20
4391
)
)
• Tyr fluorescence: quenched upon
addingat close to 1.7 molar equiv for soluble Aβ(1-42) and
achieved
Cu2+ (0.2, 0.4,
0.6, 0.8, and 1.0 mol
eq.)
toequiv for fibrillar Aβ. This is below the 2 molar equiv of
1.5No.molar
4392 Biochemistry, Vol. 48,
20, 2009
Sarell et al.
50 µM Aβ1-42 in water at pH 7.4.
2+
andrespectively
indicates
requiredcomplex
to bind 1154molar
equiv
The EPR spectrum at pH histidine
5 for the Cu-Aβ(1-42)
G (14.4
mK), of
2.23,Cu
and 2.01,
(designated
• Fluorescenceunder
returns
withconditions
additions
2+set of signals typical
all four buffer
gives aof
single
complex II), as shown in Figure 3b. At pH 7.4, complex I
with an affinity comparable to
that
Cu will bind to Aβ(1-42)
excess glycine,
L-II histidine,
or NTA
or square pyramidal coordination
of type
Cu2+, square-planar
remains the more intense of the two under all four conditions,
as shown
in Figure 3a.
Regardless
of the buffer, theAgain,
A,
and
in water,
or HEPES buffer,
ca. 80% of
thenotably
affinity
canphosphate,
be calculated
using
that
of L-histidine.
• No differencegeometry,
was seen
between
monomeric
2+
g , and g^ values are 171 G (16.0 mK), 2.27, and 2.02, respectively
Cu forms complex I. At pH 9 (Figure 3c), the low- and
and fibrillar Aβ
and
K setsvalues
of histidine at pH 7.4
eq 2, this time using the K the
1-42.
(designated
complex I). At pH 7.4, a new set of hyperfine peaks a1high-field a2
of signals of complexes I and II are of comparable
nm returns with additions of molar equivalents of glycine (b), Lhistidine (c), or NTA (d). Monomeric Aβ(1-42) (b) and fibrillar
Aβ(1-42) (O) with 50 μM Cu2+ present. ΔF = F - Fo, and ΔFmax =
Fmax - Fo, where Fo is the fluorescence (307 nm) with addition of 1
molar equiv of Cu2+.
Cu2+ ions in a Cu(Gly)2 complex, coordinating via the amino
)
)
)
)
g , and g^ values of of histidine
can be observed at a higher field
intensity in water,
phosphate buffer, and
HEPES buffer. Howandwith
theA ,concentration
at half-maximal
quenching.
for ethylmorpholine buffer (EM), the low-field species
The histidine competition ever,
experiment
indicates an apparent
observed exclusively at pH 5 is considerably less intense than
high-field
species. The relative
intensities
dissociation constant of 6 the
pM
for monomeric
and
8 pMof the
fortwo complexes are shown in Figure 3 d-f. The pH dependence of the forfibrillar Aβ at pH 7.4. Again,
these values are similar to those
mation of complexes I and II is very similar for phosphate, water,
HEPES (Figure
3d,e) with
a midpoint of
pH ∼9;
calculated using NTA as theand
competing
ligand.
Although
there
ishowever,
the midpoint in EM buffer (Figure 3f) is lower at pH 8.0.
are within a single order of
some variation, the Kd values
This behavior is also observed for Aβ(1-28) as shown in
Figure
S3 of the Supporting Information. Comparison of EPR
magnitude, between 6 and 60
pM.
spectra for Aβ(1-28) and Aβ(1-42) shows 2+
Importantly, we have obtained
similar affinities of Cua closeforcorrespondence at all pH values. The midpoint of the transition for Aβ(1CD spectra
28) was pHmethod
∼9, similar to
that fornear-UV
Aβ(1-42), andCD
again, in EM
Aβ with a second independent
using
adding NTA to 50 µM Aβ1-28 and
buffer this was reduced to pH 8.0.
spectroscopy. These near-UV
experiments have an advantage
50 µM Cu2+. Inset shows
The effect of EM buffer on the midpoint of transition between
complexes
I and II was
unexpected as
EM has
very low affinity
normalized CD signal at 320 nm
in that they measure a CD signal
directly
associated
with
thea CuNTA
for Cu2+ ions. Buffers can have a temperature dependence to
with upon addition of NTA.
Aβ complex. Figure 2 shows
theA recent
CD study
spectrum
their pH.
has shownof
thatAβ(1-28)
the pH of some buffers
2+
FIGURE 1: Fluorescence spectra of monomeric and fibrillar Aβ(1as 1at
pH pH
unit between
room temperature
ions
7.4. We
have and
loaded with
1 molar equivcanofvary
Cuby as much
42) with Cu2+: glycine, L-histidine,
and University.
NTA competition.
(a) of Aβ(1-28) to
FIGUREThe
2: CD spectra
with discover.
Cu2+ assessed via NTA
10 K (liquid helium temperatures) (52) (53). This may well
21
Leiden
University
previously
shown
thetheCu-Aβ
Increasing molar equivalents
of NTA
added to that
50 μM although
account for
discrepanciescomplex
between EMdoes
buffer not
and the other
Increasing additions of Cu2+ (0.2, 0.4, 0.6, 0.8, and 1.0competition.
molar
equiv)
2+
Aβ(1-28) and 50 μM Cu causes a decrease in the intensity of the
buffers electronic
tested.
givetherise
to CD
bands
transitions, there is a
to 50 μM Aβ(1-42) in water at pH 7.4 causes quenching
CD signal atof
320 the
nm. The inset shows
normalized
CD signal
at 320 for d-d
2+
Peisach and Blumberg have shown that a combination of A
nm
of
the
Cu
-Aβ(1-28)
complex
with
increasing
molar
equivatyrosine fluorescence signal. The tyrosine fluorescencelent
signal
at 307
positive CD band at 320 and
nmg values
assigned
to Aβ-Cu
imidazole
additions of NTA.
can indicate
the ligand type
that coordinates the
charge transfer transitions (31). This CD band increases in
intensity with addition of Cu2+. Figure 2 shows that as the
competing ligand NTA is added the intensity of the band
decreases. A plot of ellipticity at 320 nm, shown as an inset,
indicates the band is reduced to half its maximal intensity only
after 0.75 molar equiv of NTA is added. The binding curve using
Ensemble methods for measuring metal binding
7026 Biochemistry, Vol. 39, No. 23, 2000
0)
d
yl
e
)
d
d
L
n
or
X
d
n
k
s.
n
y
k
Miura et al.
Karr et al.
The effects of Zn(II) binding are also seen for histidine
Raman bands. The 1570 cm-1 band of metal-free histidine
is diminished in the spectrum of insoluble aggregates, and
concomitantly a band at 1604 cm-1 gains intensity (Figure
1B). The intensity increase of the 1604 cm-1 band is likely
to be caused by a shift of the histidine band from 1570 to
A︎β1-40
1604 cm-1, where a phenylalanine band originally resides.
This interpretation is confirmed by the appearance of a
negative peak at 1570 cm-1 and a positive peak at 1604 cm-1
in the difference spectrum, (R ) 2) - (R ) 0) (Figure 1C).
Recently, we have found that the wavenumber of the C4dC5
stretch vibration of histidine is sensitive to the site of metal
binding: 1580 ( 10 cm-1 in the Nπ-metal form and 1600
( 6 cm-1 in the Nτ-metal form (24, 25). The positive peak
A︎β1-40
at 1604 cm-1 in the difference spectrum indicates that Zn(II) binds to the Nτ atom of histidine in the insoluble Zn(II)-Aβ
aggregate.
binding
of Zn(II)fibrils,
to theCu
histidine
2+ in Aβ(1-40)
2+ with soluble
Figure1-40
2. EPR
spectraThe
of Cu
2+ in in
atom mayorbe
key
peptide
aggregation.
NτAβ(1-40),
25 aµM
Custep
buffer.
Hyperfine
lines arising from the I )FIGURE 2: Raman spectra of insoluble aggregates and the soluble
63,65Cu nucleus are identified by the m value. Aβ(1-40)
3/2weak
fibrils (37complex of Cu(II)-Aβ1-40. (A) Insoluble aggregates precipitated
A
band becomes detectable at I 1555 cm-1 in the
µM initial
concentration)
were at
assembled
in the presence
of 48from a Cu(II)-Aβ
1-40 mixture solution (pH 7.4) at R ) 4. (B)
spectrum
of peptide
the insoluble
aggregate
R ) difference
2 (Figure
1B). between
No
spectroscopic
Cu2+ bound
to « free »
µM Cu2+, separated by centrifugation, and washed to remove excessSoluble Cu(II)-Aβ1-40 complex in the supernatant of the same
However,
the
difference
spectrum
(R
)
2)
(R
)
0)
does
2+
Cu2+: g| ) 2.26,A︎
A|β
) and
175 ( 1AG.Cu
Soluble bound
Aβ(1-40)
with
Cu2+ containsmixture solution. The assignments of Raman bands are denoted as
β
︎
fibrils
-1 (Figure
not50 exhibit
any50 peak
1555A|cm
2+: g ) 2.26,
µM Aβ and
µM Cuaround
) 174 ( 4 G. All1C),
samples arefollows: His(π), histidine bound to metal via Nπ; Am-, deproto|
suggesting
the150
same
under
the50%
envelope
in 100 mMthat
Tris,
mM band
NaCl,ispHburied
7.4, buffer
with
glycerol (v/v).nated amide bound to metal. For the others, see the caption to Figure
intensities of the spectra are normalized by using
T ) 10cm
K;-1modulation
10 G; power
band at amplitude
R ) 0 (Figure
1A).0.5 mW;1. The Raman
of EPR
the conditions:
histidine 1570
-1
4; frequency -1
gain
5
×
10
9.38
GHz;
time
constant
40.96
ms;
conversionthe 1447 cm C-H bend band as an internal intensity reference.
Possibly, the 1555 cm band is ascribed to Leiden
the anti22 of
University.
The
University
discover.
The structure of
the His(π)
side chain isto
shown
on the right side
time 40.96 ms; four or eight scans.
symmetric
stretching vibration of COO- groups of the
the figure.
peptide C-terminus, three aspartates, and 2+
three glutamates
been reported: approximately two Cu ions per precipitated
(21). When the concentration of Zn(II) is elevated from 13R
amide III band intensity. The amide I band is also expected
and essentially
soluble
peptide.
increases
(Figure
1D), Theseto decrease in intensity upon amide deprotonation, but the
) Aβ(1-40)
2 to 4, the intensity
at 1555 none
cm-1 per
stoichiometries
determined
UV-visatspectrophotometric
suggesting
that anwere
additional
bandbyemerges
this waveintensity rather increases compared to that in the insoluble
detection
of soluble
peptide
(Micro
BCAseen
assay)
and metalaggregate. This is probably because the intensity decrease
number.
A similar
intensity
increase
is also
at 1290
13 The ratio obtained by ICP-MS
-1. Both
acid analysisassociated with the deprotonation is covered by an intensity
the 1555 and 1290 cm-1 bandsand
are amino
characteristic
cmions.
here is derived
direct measurements
on fibrils. Aincrease on going from the aggregated state in the solid to
of reported
the deprotonated
form from
of histidine
(histidinate) whose
possible explanation
themetal
discrepancies
in Cu
:peptide
imidazolate
ring bridgesfor
two
ions through
Nπ2+
- and
Nτ- ratiosthe isolated state in solution.
ligation
and additional
metal
binding
at
could (27).
be theDeprotonation
existence of multiple
types of
peptide
precipitates,
The C4dC5 stretching band of the metal-free histidine at
17,18,27 In our case,1570 cm-1 is diminished in the Raman spectrum of the
histidine occur
at high Zn(II)
concentrations.
theaNcommon
occurrence
with amyloid
peptides.
π atom of
In
contrast
to the
dramatic
aggregation
EM
confirms
that
the solid
samples induced
assayed by
bybinding
ICP-MS andsoluble Cu(II)-Aβ1-40 complex (Figure 2B) as in the case
of amino
Zn(II), acid
Cu(II)analysis
did not are
much
the solubility of Aβ1-40
of the insoluble aggregate (Figure 2A), indicating that all
Aβreduce
fibrils.
at neutral
pH.
Only
∼5
and
∼60%
of
Aβ
were
precipi2+
1-40
Spectroscopic Measurements on Cu Bound to Aβ(1-three histidine residues are bound by Cu(II). Although the
tated
at R )environment
2 and 4, respectively.
Figureor 2fibrillarcorresponding band of Cu(II)-bound histidine is not clearly
40).by
TheCu(II)
coordination
of Cu2+ in soluble
compares
spectra ofby
theEPR
insoluble
aggregate
andspectraseen owing to the overlap of phenylalanine bands at 1604
forms ofthe
AβRaman
was monitored
spectroscopy.
EPR
soluble complex of Cu(II)-Aβ1-40 prepared at R ) 4, pH
and 1586 cm-1, the spectrum shows a distinct band at 1275
collected at 10 K of soluble Aβ(1-40) with stoichiometric -1
7.4. The Raman spectrum
of the aggregate (Figure 2A) is
cm , which is assignable to the ring-breathing mode of
amounts
of Cu2+
show
Cu2+
EPR
spectraZn(II)-Aβ
with distinguishable
almost
identical
to
that
of
the
insoluble
histidine. The ring-breathing
mode of histidine is known to
1-40
Intra2+ IM) 3/2 nucleus
M
hyperfine
the 63,65Cuthat
I
aggregate
at Rlines
) 2 arising
(Figure from
1B), indicating
metal binding (mgain
intensity when a metal ion binds to the Nπ atom but
labels
in
Figure
2).
The
magnitudes
of
the
A
and
g
values
are
|
|
peptide
to the Nτ atom of histidine is common to the insoluble
not to the Nτ atom (24). The metal coordination of the Nπ
2+ center with mostly nitrogen donor
consistentNo
withimidazolate
a Type 2 Cu
aggregates.
bridge
is generated by Cu(II).
atom of histidine together with nitrogen atoms of deprotoatomsRaman
(Figurespectrum
2).16 Ourof results
are in Cu(II)-Aβ
agreement1-40
with thenated amides may be characteristic of the soluble Cu(II)The
the soluble
2+
previously
proposed
3N1O
coordination
environment
for
Cu
complex (Figure 2B) is different from that of the aggregate
Aβ1-40 complex.
bound2A).
to soluble
Aβ(1-40).
Thesoluble
major differences
the EPR pH Dependence of Metal-Induced Aggregation of Aβ1-16.
(Figure
In the spectrum
of8 the
complex, a in
new
-1. The intensity of the amide
strong
bandfor
appears
1417 cm
spectra
a Cu2+at center
ligated
to four N donor atoms ratherAβ1-40 consists of the N-terminal hydrophilic and C-terminal
IIIthan
band3N1O
at 1240
cm-1 is alsoare
segments that can be cleaved between Lys16
significantly
decreased.
These
coordination
in the magnitudes
of the
A| and ghydrophobic
|
spectral
features
are consistently
by deprotonation
M explained
values.
On average,
A| values
are slightly
higher and g| isand Leu17 by secretase (3). It is known
M that Aβ1-40
M
16significantly
aggregates near its isoelectric point (pI ) 5.3)
andslightly
metal-coordination
main-chain than
amide
When
lower for 4Nof
coordination
fornitrogens.
3N1O coordination.
amide
deprotonates,
thepresence
amide Iofmode
Whennitrogen
fibrils assembled
in the
Cu2+ (CdO
and glyceroleven in the absence of metal ions (17, 29-31), possibly due
stretch)
is replaced
by the in-phaseand
andwashed
out-of-phase
stretchare separated
by centrifugation
with buffer
that doesto hydrophobic interactions among the C-terminal segments
- (28). The 1417 cm-1 band of
of peptides. The N-terminal hydrophilic segment itself is
ing vibrations
of
CdO/C-N
2+
2+
Exchange
not contain
Cu , the EPR spectrum of Cu bound to fibrils is
the soluble Cu(II)-Aβ1-40 complex is assignable
expected to be soluble in water. Accordingly, if the Nto the innearly identical to that observed for Cu2+ with soluble Aβ(1terminal peptide fragment, Aβ1-16, aggregates in the presence
phase stretch, whereas the out-of-phase stretch (∼1610 cm-1)
The major
the spectra
forSince
the solubleof metal ions, the aggregation is attributable solely to metalis 40).
too weak
to be difference
detected inbetween
the Raman
spectrum.
2+ is in the g M
and
fibrillar
forms
of
Aβ(1-40)
containing
Cu
⊥
the amide III mode mainly involves NH bending (20-22),
peptide interactions. We have examined the aggregation of
region
of the spectrum.
of the spectrum
sensitiveAβ1-16 in the absence and presence of
M Zn(II) and Cu(II) (R
it is
reasonable
to assumeThis
deprotonation
reducesisthe
Mthatregion
to the sequence order of ligand donor atoms. Thus, even when
the donor atom composition is 3N1O in both samples, g⊥ varies
Dynamics of Metal-Amyloid-β
e
s
f
e
e
f
o
R
d
S
s.
(
d
f
h
d
e
1.
y
d.
Leiden University. The University to discover.
(27) Rochet, J.-C.; Lansbury, P. T., Jr. Curr. Opin. Struct. Biol. 2000, 20, 60.
23
CONCEPT
Metals + ROS +
predominant form at neutral pH for CuII and CuI are shown
in Figure 7. Moreover, an original redox mechanism was
proposed for the redox cycling of Cu–Ab, in which a low
populated “entatic-like state” is responsible for all redox cyrs is that the miscling, whereas the ground state is too sluggish (Figure 7).[47]
eneral large overislocalizationThere
due are two populations of copper Aβ complexes at neutral pH
to Ab in plaques
rally too high exe metal ion con~99.9%
is an imbalance,
a recent publicaaffected by AD,
the same region
bound Cu present
duction of ROS.
concentration did
Redox properties of the copper Aβ complexes
~0.1%
ization are likely
tain synapses dislable for binding
u and Zn (metales, see Table 2).
oluble and aggregatno buffer) [m]
aggregate
~ 1 ! 10!10 [32]
II
Figure 7. Top: model
of the University.
most populated
CuUniversity
and CuI–Abtocomplex
at 24
Leiden
The
discover.
neutral pH. Bottom: the pathway between the two redox states of Cu–
Ab. A low populated state (only about 0.1 %) in equilibrium with the
predominant state. This state redox cycles very efficiently and is responsible for all the redox reaction.
Therapeutic Aspects and Strategies
AD: putative role of copper and zinc
•
•
loss of Cu from bound pools, subsequent redistribution to
extracellular space, specifically to Aβ in the cerebrospinal
fluid, and eventually to serum
emerging consensus: copper and zinc relocate from
intracellular to extracellular stores, and from bound to
free pools
(Cu,Zn)bound → (Cu,Zn)"free" →
•
→
loss of protein-bound Cu may be either a cause or an effect
of neuron degeneration (e.g., following apoptotic events)
Leiden University. The University to discover.
25
Metals and Amyloid-β in Alzheimer’s Disease
No ROS
+
not
toxic
APP
Healthy
neuron
healthy brain
Zn
Cu
toxic
(ROS
with Cu)
Degenerated
neuron
Alzheimer brain
Leiden University. The University to discover.
26
P. Faller and C. Hureau
Where does synaptic free zinc and copper come from?
Normal conditions
• Zn transported by ZnT3 into
vesicles containing Glu
• Zn and Glu are expelled into
synaptic cleft upon activation
• Glu binds to the NMDA
receptor (NMDA-R) and
triggers Ca2+ influx into
postsynapse
• Aβ is cleaved from the amyloid
precursor protein (APP) by βand γ-secretase
• Zn2+ binds to NMDA-Receptors
at the postsynaptic terminal,
stimulating Cu2+ release (blue
spheres) into the synapse via
ATP7A
Figure 2. Zinc and copper at the synapse. Normal conditions (plain
Leiden
University
to discover.
arrows):
Zn University.
is transported byThe
the Zn
transporter 3 (ZnT3)
into vesicles27
containing the neurotransmitter glutamate (Glu). Zn and Glu are expelled into the synaptic cleft upon activation. Glu binds to the NMDA receptor (NMDA-R) and triggers Ca influx into the postsynapse. Ab is
cleaved from the amyloid precursor protein (APP) by b- and g-secretase.
Cu seems to be transported into the synaptic cleft by the translocation of
the Cu transporter (ATP7a) to the postsynaptic membrane, upon stimulation of the NMDA receptor. Cu might interact with the prion protein
(PrP) and modulate the putative regulation of NMDA-R by PrP. Alzheimer condition (dashed arrows): accumulation of Ab, Cu and Zn leads
to Ab aggregates, which can be toxic through different mechanisms (not
all shown), including an over-activation of the NMDA-R (maybe PrP
Figure 3. Respiratory chain in mitochondria. The metal centers heme
(Fe–porphyrin) and FeS (iron–sulfur clusters) are indicated. Top: spatial
model of complexes I–V embedded in the membrane. Bottom: the redox
potentials of the substrate (NADH) and the product (H2O), the compounds between the complexes and the range of redox potentials of the
complexes I, III and IV are indicated (vs. NHE). Electron transfer is indicated by the blue arrow (QH2 and Q: reduced and oxidized ubiquinone;
FeS: iron–sulfur cluster; CytC: cytochrome c).
The Nasty Intermediates
Cu and Fe not only dissipate energy from NADH or other
reductants, but they can also produce radicals, as they are
P. Faller and C. Hureau
Where does synaptic free zinc and copper come from?
Alzheimer condition
• accumulation of Aβ
• Cu2+ and Zn2+ leads to toxic
Aβ oligomers
• Over-activation of the
NMDA-R
• membrane destabilization (?)
• very high Ca2+ influx
• production of reactive
oxygen species (ROS)
• neuron apoptosis
Figure 2. Zinc and copper at the synapse. Normal conditions (plain
Leiden
University
to discover.
arrows):
Zn University.
is transported byThe
the Zn
transporter 3 (ZnT3)
into vesicles28
containing the neurotransmitter glutamate (Glu). Zn and Glu are expelled into the synaptic cleft upon activation. Glu binds to the NMDA receptor (NMDA-R) and triggers Ca influx into the postsynapse. Ab is
cleaved from the amyloid precursor protein (APP) by b- and g-secretase.
Cu seems to be transported into the synaptic cleft by the translocation of
the Cu transporter (ATP7a) to the postsynaptic membrane, upon stimulation of the NMDA receptor. Cu might interact with the prion protein
(PrP) and modulate the putative regulation of NMDA-R by PrP. Alzheimer condition (dashed arrows): accumulation of Ab, Cu and Zn leads
to Ab aggregates, which can be toxic through different mechanisms (not
all shown), including an over-activation of the NMDA-R (maybe PrP
mediated or modulated) resulting in very high Ca influx and production
of reactive oxygen species (ROS).
Other bioinorganic effect of Aβ amyloid
Demuro
mbrane Permeabilization by Soluble Amyloid Oligomers
below). Thus, metal metabolism has to be tightly controlled
in order to avoid these reactions. This is achieved by ensuraction
monomeric,
oligomeric,
fibrillar
ing that: 1) metal
ionsof(Fe,
Cu) are tightly
boundand
in proteins
forms
of Aβ1-42 at to
a constant
peptide
in a well-defined
coordination,
restrict their
reactivity to
2+
cytosolicside
freereactions,
Ca levelsand
in
their purpose concentration,
and to avoid on
unwanted
SH-SY5Y cells
2) free Fe and Cu concentrations are kept generally very
(in vitro model of neuronal function)
low, as their reactivity is quite unrestricted (not controlled).
For instance, intracellular free Cu is estimated to be in the
atto- to femtomolar range and Fe in the nm range[12] (but
see above, the particularity of Zn and Cu in certain synaptic
Cytosolic Ca2+ levels probed inclefts).
time by fluorescence imaging using a Ca2+
indicator, fluo-3-AM, that was pre-loaded into the cells prior to experiment
37 °C in 5% CO2, and the medium
!10,000) were plated in 35-mm
Corp.) and grown overnight. Loads accomplished by incubating with
salt solution) for 30 min at room
Hanks’ balanced salt solution and
o ensure complete hydrolysis. A
to load cells with calcein by incu-
ng system consisted of an inverted
d with a Leitz 16X objective. Flunm argon ion laser, and emitted
ed by a cooled CCD camera (Casse images (1 frame s$1) were capare package (Universal Imaging,
intensities were measured from
dual cells. Signals are expressed as
n fluorescence (%F) divided by the
nt (F). A small proportion (9%) of
ving low initial fluorescence and
se were excluded from analysis.
g a fixed aliquot (70 !l) of a diluted
mber (1-ml volume) directly above
the resulting concentration expen we pipetted the same volume of
amber and measured the resulting
ells relative to that of the initial,
ration yielded a dilution factor of
lating the effective concentrations
Downloaded from http://www.jbc.org/ at WALAEUS LIBRARY on February 16, 2015
d by
heir
ence
es to
omer
mers
om a
rved
h the
ubsen (6
orescells
and
on of
ined
each
reand
et al J. Biol. Chem. 2005, 280, 17294
What about Zn and ROS? ZnII is redox inert under physiological conditions, and hence cannot directly catalyze the
• massive Ca2+ uptake
triggered
by Aβ oxygen
oligomers
production
of reactive
species (ROS). However, inII
directly make
this is possible,
when Zn
can outcompete the bind• Aβ oligomers (not fibrils)
membranes
permeable
ing of a redox active metal ion to a protein. Then the redox
active metal is released and prone to catalyze ROS production. Considering the coordination chemistry of Cu, Fe, and
Leiden
The
University
to discover.
Mn,
this isUniversity.
most likely the
case
for FeII. Indeed
it has been29
proposed that ZnII could substitute for FeII-binding in APP
tween monomers, oligomers and fibrils, we express these concentra(amyloid
precursor protein).[13]
tions in units of !g ml . As a rough guide, a concentration
of 0.6 !g/ml
$1
A#42 corresponds to 200 nM monomer and !7 nM oligomer and 70 pM
fibrils.
RESULTS
15914
A#42 Oligomers, but Not Monomers or Fibrils, Increase Intracellular Free Ca2"—Homogeneous populations of monomeric, oligomeric, and fibrillar A# were prepared as described
above and characterized by size exclusion chromatography and
electron microscopy. The oligomeric preparation had an approximate molecular mass of 90 kDa, contained very little
material of lower molecular mass, and was comprised of
spherical vesicles with diameters of 2–5 nm (Fig. 1). The
monomeric preparation contained no detectable oligomeric
aggregates as analyzed by size exclusion chromatography
(Fig. 1). The morphology of the fibrillar preparations was as
published previously (34).
The actions of homogeneous monomeric, oligomeric, and
fibrillar preparations of soluble A#42 amyloid were examined
by adding aliquots of the samples to fluo-3-loaded SH-SY5Y
cells (Fig. 2). Fig. 2A illustrates images and corresponding
Ca2"-dependent fluorescence measurements in a representative cell. Applications of monomers or fibrils at final concentrations of 6 !g/ml evoked no detectable change in fluorescence,
whereas subsequent application of the same amount of oligomer evoked large and rapid (!5 s) increases in Ca2"-depend-
www.chemeurj.org
The Nasty Intermediates
Cu and Fe not only dissipate energy from NADH or other
reductants, but they can also produce radicals, as they are
good in one-electron reduction processes. Thus, Fe2 + and
Cu + can rapidly reduce O2, O2C!, or H2O2 in one-electron
processes and generate O2C!, H2O2 and HOC, respectively.
These products are part of the so-called reactive oxygen species (Figure 4).
In general, an efficient ROS production has to be metalcatalyzed or induced by light. As light does not penetrate
into most parts of the human body (in particular the brain),
in most places ROS production needs metal centers. Under
certain conditions a high ROS production is wanted, for instance in the defense reaction of phagocytes (like macrophages or neutrophils) against pathogens. Here ROS are
produced by metalloenzymes, like the chloroperoxidase or
NADPH oxidase, which contain a heme cofactor (Fe–porphyrin).[14]
In most cases, however, the production of high amounts
of ROS is not wanted, as they can degrade lipids, nucleic
acids and proteins. A canonical example of the deleterious
effects of ROS is the damage observed by irradiation of grays or X-rays. They are known to produce ROS (and other
radicals) and can be lethal at high doses. Nevertheless, ROS
production is a byproduct of several cellular processes, in
particular in the oxidative phosphorylation in mitochondria
(but also in P450, peroxisomes, etc.).[14] To limit the damage
of byproduct ROS, several defense systems are available,
like the enzymes catalase or superoxide dismutase, or some
! 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Currative strategies for inorganic chemists
erences in molecular weights be-
Figure 3. Respiratory chain in mitochondria. The metal centers heme
(Fe–porphyrin) and FeS (iron–sulfur clusters) are indicated. Top: spatial
model of complexes I–V embedded in the membrane. Bottom: the redox
potentials of the substrate (NADH) and the product (H2O), the compounds between the complexes and the range of redox potentials of the
complexes I, III and IV are indicated (vs. NHE). Electron transfer is indicated by the blue arrow (QH2 and Q: reduced and oxidized ubiquinone;
FeS: iron–sulfur cluster; CytC: cytochrome c).
• trap « free » metals
• re-dissolve Aβ plaque
• relocate metals from synaptic cleft into neurons
clioquinol
neutral, apolar
→ crosses membranes !!!
• Zn and Cu chelator (1:2 Kd ~10-9 and 10-10 M, respectively)
• used clinically in Japan in the 1960’s; overdoses of clioquinol may impair
metal homeostasis in the CNS
• reduces Aβ load in AD patients
Leiden University. The University to discover.
30
Chem. Eur. J. 2012, 18, 15910 – 15920
Take home message
« Large discrepancies have been reported between the findings obtained by different
research groups for the Aβ−membrane interaction. One reason for this is the
different initial states of Aβ in solution, that is, completely monomeric or already
partially aggregated. »
Matsuzaki Accounts Chem. Res. 2014, 47, 2397
• biometals influence / are influenced by Aβ aggregation
• coordination mode is highly flexible, pH and metal dependent
• Aβ binds essentially Cu2+, Zn2+; bound metals are « free »
• chose well your model of Aβ
• characterize well your starting state
Leiden University. The University to discover.
31
Bibliography
Review
pubs.acs.org/CR
dx.doi.org/10.1021/cr300009x | Chem. Rev. 2012, 112, 5193
Bioinorganic Chemistry of Alzheimer’s Disease
Kasper P. Kepp*
DTU Chemistry, Technical University of Denmark, DK 2800 Kongens Lyngby, Denmark
8. Methionine Synthase, Vitamin B12, and Homocysteine in Alzheimer’s Disease
5209
9. ALS and AD: Same Thing, but Different
5209
10. Exogenous Metal Exposure and Alzheimer’s
Disease
5210Reviews
10.1. Aluminum
5210
pubs.acs.org/acschemicalbiology
10.2. Cadmium
5211
10.3. Lead
5211
10.4. Mercury
5212
Natural
Chelators
against Alzheimer’s
Untangling Amyloid-β,11.
Tau,
andMetal
Metals
in Alzheimer’s
Disease
5212
Masha G. Savelieff,† Sanghyun Lee,†,§ Disease
Yuzhong Liu,†,‡ and Mi Hee Lim*,†,‡
12. Combining the Hypotheses: A Bioinorganic
†
‡
Life Sciences Institute and Department of Chemistry,
University
of Michigan,
Ann Arbor, Michigan 48109, United5215
States
View of
Alzheimer’s
Disease
12.1. Dysfunction of Proteins Involved in Metal
CONTENTS
Homeostasis:
5215
ABSTRACT: Protein misfolding and metal ion dyshomeostasis
are MT as an Example
believed
underlie numerous neurodegenerative
diseases, toward Apoptosis
12.2. Converging
5216
1. Introduction: Alzheimer’s Disease
from ato Chemincluding Alzheimer’s disease (AD). The pathological hallmark of
The Zinc Cascade: An Example of Metalist’s Point of View
5193 amyloid-β12.3.
AD is accumulation of misfolded
(Aβ) peptides and
Based
5217
1.1. Definitions and Symptoms hyperphosphorylated tau (ptau)
5193 proteins in the brain.
SinceEtiology
AD
etiology remains unclear, 5194
several hypotheses
haveFurther
emergedComments
to
12.4.
on the Pathogenesis
5221
1.2. Risk Factors
elucidate its pathological pathways. The 13.
amyloid
cascade hypothConcluding
Remarks: Ten Focal Points of Future
2. AD Pathogenesis: Three Current
Competing
esis, a leading hypothesis for AD development, advocates Aβ as the
DOI:
10.1002/chem.201202697
Research
5222
Hypotheses
5195
principal culprit. Additionally, evidence suggests that tau may
5224
Aβ and tau Author
have also Information
been shown to impact each other’s pathology either directly or
indirectly.
3. Amyloid Cascade Hypothesis contribute to AD pathology.
5195
Furthermore, metal ion dyshomeostasis
is associated
with these misfolded
Corresponding
Authorproteins. Metal interactions with Aβ and tau/ptau
5224 also
3.1. Production of Aβ
5195
influence their aggregation properties and neurotoxicity. Herein, we present current understanding on the roles of Aβ, tau, and
5224
3.2. Aβ Production−Clearance Imbalances
in AD
5196 on each ofNotes
metal ions, placing
equal emphasis
these proposed features, as well as their inter-relationships in AD pathogenesis.
Biography
5224
3.3. Structural Forms and Toxicity of Amyloids
5196
Acknowledgments
5224
4. Metal Ion Hypothesis
5196
lzheimer’s
disease (AD)5196
is the mostList
common
neuroAMYLOID
PRECURSOR PROTEIN (APP)
of Abbreviations
and
Acronyms
5224
4.1. The Justification of the Metal Ion
Hypothesis
degenerativeComdisease, accounting forReferences
60−80% of all
Aβ peptides are derived from APP, which is5224
expressed in
4.2. Coordination Structures of Aβ−Metal
dementias.1 Currently, it affects approximately
[a, b]5.4 million
various tissues and organs of the human body, including the
plexes
Peter Faller*[a, b]Americans
and Christelle
Hureau
and 24 million 5197
people worldwide, and these
3−7
APP,
which
consists
of
three
splice
variants
(APP695,
brain.
4.3. Coordination Structures numbers
of Aβ are
Sequence
expected to increase dramatically.1,2 AD cases
APP751, and APP770), is a type I membrane protein with one
are categorized as early onset5198
AD (EOAD)1.or INTRODUCTION:
late onset AD
Variations
ALZHEIMER’S
DISEASE
FROM
transmembrane
domain (TMD)
(Figure
1). TheA N-terminal
1,3,4
EOAD,
which
(LOAD),
with
65
years
of
age
as
the
cutoff.
4.4. The Affinities of Metal Ions for Aβ
5199
CHEMIST’S POINT
VIEW
domainOF
resides
in the extracellular space; the C-terminal
a fraction of5199
all cases, has a strong genetic
4.5. The Role of Zinc in AD only constitutes
that is sporadic, and Symptoms
component.1,3,4 Most cases are LOAD 1.1.
4.6. The Role of Copper in ADalthough some genetic markers
5200 exist which Definitions
increase the
1,3,4
4.7. The Role of Calcium in ADpredisposition to develop AD.
5201
AD-afflicted
brains exhibit
Alzheimer’s
disease (AD)1−3 is the most common form of
in size, a reduction
in glucose
4.8. The Role of Iron in AD traits such as an overall decrease
5202
dementia
(estimated ∼50−60% of all cases), associated with
uptake AD
indicative
of diminished
neuronal activity/density, and
4.9. The Quest for Metal-Chelating
drugs
5203
loss
of memory (in particular episodic memory), cognitive
the presence of dense senile plaques (SP) and neurofibrillary
5. Oxidative Stress and Alzheimer’s
Disease
5204
decline,
and physical disability, ultimately
tangles (NFT), which contain aggregates of
amyloid-βand
(Aβ)behavioral
5.1. Reactive Oxygen and Nitrogen
5204 tau leading
to death.4−6 It is the sixth most common cause of
peptidesSpecies
and hyperphosphorylated
(ptau) proteins,
5.2. The Role of Oxidative Stress
in AD 3−7 As such, AD is5205
classified as adeath
proteinin
misfolding
respectively.
the US according to the Alzheimer’s Association, and
there is no cure for AD; present
5.3. Links between Oxidative disease.
StressCurrently,
and Other
more therapeutic
than 5 million Americans suffered from the disease in
1,3,4,8
AD
strategies only alleviate or treat
symptoms.
Pathogenic Events
5205
2011,Although
with prevalence
growing steadily.7 A large body of recent
pathology is relatively well understood, disease etiology is still
Figure 1. herein,
(Top) Schematic
representation
of APP
its cleavage by
6. Metallothioneins and Alzheimer’s
Disease
5206
research,
to be reviewed
has put
the AD
fieldandinto
uncertain. A fundamental understanding of
disease causing
α-, β-, and γ-secretases. APP cleavage by α-secretase releases soluble
6.1. Structure, Expression, andagents
Roles
of Metalcontact
withforbioinorganic
chemistry,
and
thisby γ-secretase
review generates
will either
is necessary
to develop diagnostics and
therapeutics
fragment (sAPPα);
subsequent
cleavage
lothioneins
Aβ(17−40)
or Aβ(17−42)
and AICD. Alternatively,
if β- and γpreventing or curing AD. The5206
volume of research
in AD
vast
attempt
toispresent
the
growing
role of bioinorganic
chemistry
secretases perform the cleavage of APP, soluble sAPPβ and Aβ
due to the mounting urgency
for a cure. in
In AD
this review,
we
6.2. Specific Functions of Metallothioneins
5206
research, with(mainly,
a particular
emphasis
on zinc
homeostasis.
Aβ(1−40/42))
are formed.
(Bottom)
Amino acid sequence
introduce in a tutorial
format an
overview of misfolded proteins
6.3. Investigated Roles of Metallothioneins
in AD
5207
Aβ(1−42): black, flanking criteria
APP residues;
red, putative
Cu2+-binding
The two main ofhistopathological
for
AD are
tau/ptau) and their potential involvement in
residues; blue,
hydrophilic
residues; peptides
green, hydrophobic
7. Metabolism, Aging, Diabetes,(i.e.,
andAβ,Alzheimer’s
observations
of extracellular
deposits
of fibrillar
called residues;
neuropathogenesis of AD in the context of the
amyloid cascade
underlined, self-recognition region. The color code illustrates the
Disease
5208the possible
senileroleplaques
widespread
intraneuronal
fibrillar
tangles.
and tau hypotheses. Furthermore,
of metaland ofbipolar
nature of Aβ with a hydrophilic N-terminus and a hydrophobic
7.1. Metabolism, Aging, and AD
5208 is presented, as well as AβC-terminus. Starting from the N-terminus, arrows indicate cleavage
ions in Aβ and tau/ptau pathologies
sites by β-, α-, and γ-secretases, respectively.
or ptau-mediated
and miscompart7.2. Zinc: A Link between Diabetes
and AD?metal ion dyshomeostasis
5208
Chemistry a European Journal 2009
A Bioinorganic View of Alzheimer!s Disease: When Misplaced Metal Ions
(Re)direct the Electrons to the Wrong Target
■
A
Leiden University. The University to discover.
32
Received:
12, 2012
mentalization. Due to the continued uncertainty
in the January
root
Published:
July 13, 2012
cause of AD, this review notably describes these
three proposed
Received: November 28, 2012
factors (Aβ, tau/ptau, and metals) in equal measure and their
Accepted: February 27, 2013
Published:
March 18, 2013| Chem. Rev. 2012, 112, 5193−5239
onset and progression.
5193
dx.doi.org/10.1021/cr300009x
inter-connections
in AD
© 2012 American
Chemical Society
Acknowledgements
© 2013 American Chemical Society
856
dx.doi.org/10.1021/cb400080f | ACS Chem. Biol. 2013, 8, 856−865
Peter Faller
Laboratoire de Chimie de Coordination
CNRS Toulouse, France
15910
! 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 15910 – 15920
Martina Huber
Leiden Institute of Physics
Leiden University, The Netherlands
Leiden University. The University to discover.
33
Inorganic Chemistry
ARTICLE
nic Chemistry
ARTICLE
increased from 278 to 298 K, peaks of the Fe -responsive residues
Scheme 1. Schematic
Representation of the Most Affected !(CHn)! Positions in Aβ16a
become broader. This is attributed to PRE effect. However, when
II
e, temperature induces strong modification in the broadserved on His Hε and Hδ and Asp1 HR and Hβ protons.
ing has mainly two origins: PRE (paramagnetism relaxaancement) due to the high-spin FeII (d6, S = 2) and
exchange. (i) PRE diminishes with distance by a power of
ll mainly affect atoms in close vicinity of the metal center,
o what is observed in the CuII case.26 In the case of a
al binding as encountered in the present system, broadexpected to increase with an increase in temperature.37,38
inked to the increase of koff at higher temperature.37,38
more, motion of the paramagnetic center with respect to
nd will also reduce the pseudocontact shift. This is the
hy only very small chemical shifts are observed, which are
e to chemical exchange more than to paramagnetism. (ii)
e between two chemically different states of apo- and
ptides (conformations, protonation states...) will also
oms in metal center coordination as detected by 1H
r the diamagnetic CuI 22 and ZnII or CdII.39 In that case,
ng is expected to decrease with an increase in temperar the FeII!Aβ16 system, a acombination of PRE and of
exchange is observed. Indeed, when the temperature is
the temperature is increased from 298 to 318 K (above 318 K the
sample evolves significantly), FeII-responsive features tend to
become sharper again, in line with the chemical exchange effect
becoming preponderant over the PRE effect. A very interesting
point is that two residue families could be distinguished: the first
one (His13 and His14) for which the peak narrowing upon
temperature increase between 298 and 318 K is strong, and the
second one (His6 and Asp1) for which it is less important. This
strongly suggests that the two broadening effects impact differently the His13-His14 diad compared to Asp1 and His6 residues.
This is tentatively attributed to either a stronger PRE effect on
Asp1 and His6 residues and/or a slower chemical exchange for
Asp1-His6 fragment compared to the rest of the peptide, including
His13 and His14.
13
C and 2D Experiments. To complete the 1H NMR data,
13
C and 2D data were recorded at 298 K and in the presence of
0.3 equiv of FeII. These conditions lead to the most effective
discrimination between peaks undergoing different broadening
amplitude. Figure 5 shows the impact of FeII addition to the 13C
signals. The Asp1 and to a lesser extent the Asp7 and Glu
COO!13C nuclei are broadened. His6 and Asp1 13CO fully
vanish, while those of Ala2, Phe4, His13, and His14 are less
affected but still strongly broadened. Regarding CR, those of
Leiden
University.
University
Asp1
and to a lesser
extent of Ala2 andThe
Phe4 are
broadened whileto
those of the three His residues are shifted but not affected by
II broadening. The C and C atoms
II are less affected with the
β
γ
13
C mostly broadened
exception of the Asp1 Cβ. The aromatic
II
are those of the three His with a slightly weaker broadening
observed for Cε (Figure S6, Supporting
II Information).
Figure 6 shows that the Hδ!Cδ (7.0 ppm; 117 ppm), Hε!Cε
(7.8 ppm; 136 ppm), and Hβ!Cβ (3.1 ppm; 28 ppm) correlation
peaks of the His residues disappeared after addition of 0.3 equiv
of FeII, while the His HR!CR (4.5 ppm; 53 ppm) correlation
peaks are shifted. The Asp1 HR!CR (4.1 ppm; 51 ppm), Hβ!Cβ
(2.7 ppm; 39 ppm), Glu3 Hγ!Cγ (2.2 ppm; 34 ppm), and Ala
HR!CR (4.3 ppm; 50 ppm) correlation peaks also disappeared
after addition of 0.3 equiv of FeII.
Information collected from the 1H, 13C, and 2D NMR data
COO! (A), CO (B), CR (C), and Cβ,γ (D) regions of the
(see also Figures S7 and S8, Supporting Information) is gathered
NMR spectra of Aβ16 peptide in 0.2 M phosphate buffer/D2O
II
in Scheme 1, in which the broadening and shift of relevant (CH)
spectrum in each panel) and in the presence of 0.3 equiv of Fe
trum in each panel), pH 7.2, T = 298 K, ν = 128.5 MHz. Bousejra-ElGarah
positions are et
recapitulated.
At 298 2011,
K, the residues
mainly affected
al Inorg Chem
50, 9024
Thank you for your attention !
The color code is as follow: black = disappeared, red = highly broadened, green = broadened, orange-yellow = moderately broadened, pale
pink = slightly broadened. Ellipsoid code stands for the signal shifting: circle = no shift, small ellipsoid = slight shift, large ellipsoid = significant shift.
Scheme 2. (A) List of the Potential Binding Functionsdiscover.
Affected by Cu (from ref 26) or Fe (in bold, the
mostly broadened residues); (B) Cu Binding Site in Aβ
(component I) and Proposition of Fe Binding Site
34
And iron?
2D 1H-13C
HSQC NMR
Aβ1-16
+0.3 eq. Fe2+
2D 1H!13C HSQC of 5 mM Aβ16 peptide (black) and 2 mM Aβ16 peptide in presence of 0.3 equiv of FeII (red) in 0.2 M phosphate buffer/
H 7.2, T = 298 K, ν = 500 MHz. (Left) Aromatic regions, (middle) (CR ; HR) regions, and (right) (Cβ,γ ; Hβ,γ) regions.
• binding site
located in 1-16 first amino acids
by FeII rapid
addition
are
the Asp1
and
thethe
three
His. Among
theand
three
2+ in
9027
• problem:
oxidation
of Fe
presence
of Aβ
remaining
carboxylic
acids,
Glu3
is
the
one
mostly
influenced
by
subsequent
precipitation
the CO from
His,-4the
one in position 6 is more
FeII. Regarding
• binding
probably weaker
(Kd ~10
M?)
those controlling
in positions machinery
13 and 14. !!!
• Fe affected
involvedthan
in ROS
dx.doi.org/10.1021/ic201233b |Inorg. Chem. 2011, 50, 9024–9030
•
•
•
•
’ DISCUSSION Leiden University. The University to discover.
As previously observed for CuII!Aβ16 complexes,26,27 FeII
binding to Aβ16 is very dynamic and likely involves several
differently populated coordination modes of similar types. As a
consensus has been reached in the literature on the nature of the
CuII coordination sphere in component I, a comparison (detailed
below) of NMR data obtained on CuII!Aβ16 component I and
FeII!Aβ16 was used to propose FeII binding site(s) in Aβ16.
Note that
since nochelators
steric constraints
exertedAD?
by the Aβ16
Natural
for are
curing
ligand, hexacoordination of the FeII ion has been assumed. The
NMR data are compared in Scheme 2a (and Scheme S2,
Metallothioneins
Supporting Information), and the corresponding CuII and FeII
MTbinding
function: Zn
transport
and homeostasis,
protection
heavy
metals, freeof
sites
are depicted
in Scheme
2b.against
(i) The
R position
II
II
copper, oxidative stress
Asp1 is affected by both Cu and Fe , suggesting that the !NH2
MT up-regulated when [Zn2+]free increases
is bound to both metal centers. (ii) The imidazoleMPACs:
rings of the
MTs may constitute a “gold standard” for the rational design of new
or FeII. For
the
three
His are
all broadened
in the
presence
of CuinIIexchange
extracts
Cu(II) from the
Cu−Aβ40
complexes
for Zn(II)
• Zn−MT-3
II
!Aβ16
component
I, it has
beenindicating
proposed
bytoxicother
Cu
interaction
renders the amyloids
nontoxic,
that the
• MT-3
amyloid oligomers
or precursors
the toxicbound
oligomers
Cu(II)13
or Zn(II)
and
techniques
that while
His6 istoalways
to contain
CuII, His
can reduce
neuro-degeneration
mouse hippocampus
• MT-3 are
His14
in equilibrium
for onein binding
position. In the FeII
species, the same kind of His binding takes place as indicated by
Curcumin
the
the 1H temperature-dependent study. (iii) As in the CuII incase,
incidence of AD among people in their 70s was about 4−5 times smaller
India
residue
was not significantly
by FeII and thus its
thanTyr10
in the US;
curry consumption
correlates withaffected
this tendency
• curcumin was found to protect against Aβ-induced cognitive deficits
• competitive 1:1 MPAC function is unlikely as Kd too low
• 10 patents for curcumin derivatives against AD
Leiden University. The University to discover.
35
binding was ruled out. (iv) Regarding the carboxylate residues,
they were all equivalently affected in the case of CuII!Aβ16
component I, while in the FeII case mostly those of Asp1 and
Glu3 and to a lesser extent that of Asp7 are broadened. This
suggests that both COO! groups from Asp1 and Glu3 are bound
to FeII, whereas all COO! groups were in equilibrium for the
CuII apical position. (v) The carbonyl functions of Asp1 were
predominantly broadened in the CuII case, those of Ala2 and of
the three His being less affected. In the FeII case, both CO
functions from Asp1 and His6 are significantly more affected
than those of Ala2 and His13 and His14. This may be in line with
the simultaneous formation of two metallacycles, one with
!NH2 (Asp1) and the other with the imidazole ring of His6,
instead of only one in CuII!Aβ16 component I. (vi) It is worth
noting that almost all CO and CRHR positions in the 1!6
fragment are noticeably broadened by FeII, a fact that was not
observed in the CuII case. This may indicate that confinement of
FeII in the 1!6 N-terminal part of the Aβ peptide induces
constraints on the backbone peptide.
As may not be anticipated based on the different chemical
nature of the two CuII (d9) and FeII (d6) ions, the binding sites of
FeII and of CuII (component I) are very close, showing only subtle
differences that may however impact the aggregation process.
Differences are more significant with the metal center binding sites
in component II of the CuII!Aβ species and in the CuI!Aβ
complex, the other reduced redox metal ion of importance (note
that ZnII is not discussed since no consensual data are reported in
the literature). Indeed, CuI binds linearly to two out of the three
imidazoles moieties of His residues.22,23 Regarding component II
of the CuII!Aβ species, two main coordination spheres are
proposed in the literature: (i) the three imidazole rings and the
CO function from the Ala2-Glu3 peptide bond (ref 40 and
references therein) or (ii) the NH2 (Asp1), the deprotonated
amidyl from the Asp1-Ala2 bond, the CdO group from Ala2Glu3, and an imidazole ring from either His6, His13, or His14 (refs
20 and 26 and references therein). Hence, whatever the proposition retained, difference with the FeII binding site is important.
Note that contrary to what is observed for CuII, no pH dependence of FeII binding to Aβ was found near physiological pH. This
is attributed to a lesser Lewis acidity of FeII compared to CuII.
’ CONCLUDING REMARKS
We reported for the first time a study of FeII coordination to
Aβ at the molecular scale and show that the binding site is
confined in the 1!16 N-terminal fragment of the Aβ peptide. We
also tentatively proposed a structural binding model consistent
with the data presently available. During the course of our study,
9028
36
dx.doi.org/10.1021/ic201233b |Inorg. Chem. 2011, 50, 9024–9030
Metal chelators for curing AD?
• danger associated with chelators: narrow therapeutic window, ie Kd
range that binds the target without stripping metal ions from
vital enzymes
• effective "metal−protein-attenuating compounds" (MPAC) should
have a Kd of ∼10−10 M to release Cu2+ from Aβ, or 10−8 M to strip
Zn2+, but should not strip Cu2+ or Zn2+ from systemic sites (e.g.,
10−12 M for serum albumin)
• other targets could be APP Zn and Cu binding sites (Kd ~10-6 and
10-8 M), or lowering free metal pool (Kd>10-7 M) like MT
Leiden University. The University to discover.
37
Aβ and membranes
Accounts
of Chemical
Matsuzaki Accounts
Chem.
Res. 2014,Research
47, 2397
Article
Notes
The authors declare no competing financial interest.
Biography
Katsumi Matsuzaki obtained his Ph D. in 1992 from Kyoto University
when he was an Assistant Professor there. He was appointed as an
Associate Professor at Kyoto University in 1997 and has been a full
Professor of Biophysical Chemistry at Graduate School of
Pharmaceutical Sciences, Kyoto University, since 2003.
GM1
■
Figure 6. Schematic representation of the proposed formation of toxic
• several forms of Αβ fibrils
amyloid fibrils by Aβ on GM1 clusters. Aβ is generated from the
• role of membranes in fibril
formation
proteolytic
cleavage of APP by β- and γ-secretases. When GM1
molecules do not form clusters, Aβ does not interact with neuronal
• Αβ fibrils triggered by clusters
of monosialoganglioside GM1
membranes. Aβ specifically binds to a GM1 cluster, changing its
conformation from a random coil to an α-helix-rich structure. Helical
species and aggregated β-sheets (∼15 mer) coexist at Aβ/GM1 ratios
Leiden
University.
The
University
to discover.
between ∼0.013
and ∼0.044.
However,
this β-structure
is stable and
38
does not form larger aggregates. The β-structure is converted to a
second, seed-prone β-structure at Aβ/GM1 values above ∼0.044. The
seed recruits monomers from the aqueous phase to form toxic amyloid
fibrils that may contain antiparallel β-sheets. In contrast, amyloid fibrils
formed in aqueous solution are less toxic and have parallel β-sheets.
progresses further and the Aβ/GM1 ratio exceeds ∼0.044, the
β-structure is converted to a second, seed-prone β-structure.
The seed recruits monomers from the aqueous phase to form
toxic amyloid fibrils that may contain antiparallel β-sheets. In
contrast, amyloid fibrils formed in aqueous solution are less
toxic and have parallel β-sheets.
An important conclusion of our study is that membranes
containing GM1 clusters not only accelerate the aggregation of
Aβ but also generate amyloid fibrils with potent cytotoxicity
and unique structures. The inhibition of this aggregation
cascade could be a promising strategy for the development of
AD-modulating drugs. For example, compounds that specifically bind to GM1 clusters can block the cascade at an early
stage. The α-helix-to-β-sheet conformational transition can be
inhibited by molecules that recognize the α-helical form.
Chemicals that bind and break amyloid fibrils could also reduce
cytotoxicity. We have already identified several candidate
molecules.76 The driving forces for Aβ binding, the detailed
structures of intermediate species and the final amyloid, and the
mechanism underlying cytotoxicity will be elucidated in future
studies. Furthermore, effects of pH, endosomal lipids, and
membrane curvature on the fibril formation by Aβ will be
interesting subjects of research because the GM1-bound Aβ has
been suggested to form in endosomes.77
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
Funding
This work was supported in part by The Research Funding for
Longevity Sciences (25-19) from National Center for Geriatrics
and Gerentology (NCGC), Japan.
2402
REFERENCES
(1) Selkoe, D. J. Alzheimer’s disease: Genes, proteins, and therapy.
Physiol. Rev. 2001, 81, 741−766.
(2) Citron, M. Alzheimer’s disease: Treatments in discovery and
development. Nat. Neurosci. 2002, 5, 1055−1057.
(3) Iwatsubo, T.; Odaka, A.; Suzuki, N.; Mizusawa, H.; Nukina, N.;
Ihara, Y. Visualization of Aβ 42(43) and Aβ 40 in senile plaques with
end-specific Aβ monoclonals: Evidence that an initially deposited
species is Aβ 42(43). Neuron 1994, 13, 45−53.
(4) Naslund, J.; Haroutunian, V.; Mohs, R.; Davis, K. L.; Davies, P.;
Greengard, P.; Buxbaum, J. D. Correlation between elevated levels of
amyloid β-peptide in the brain and cognitive decline. JAMA, J. Am.
Med. Assoc. 2000, 283, 1571−1577.
(5) Seubert, P.; Vigo-Pelfrey, C.; Esch, F.; Lee, M.; Dovey, H.; Davis,
D.; Sinha, S.; Schlossmacher, M.; Whaley, J.; Swindlehurst, C.;
McCormack, R.; Wolfert, R.; Selkoe, J.; Lieberburg, I.; Schenk, D.
Isolation and quantification of soluble Alzheimer’s β-peptide from
biological fluids. Nature 1992, 359, 325−327.
(6) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: The amyloid
cascade hypothesis. Science 1992, 256, 184−185.
(7) O’Nuallain, B.; Shivaprasad, S.; Kheterpal, I.; Wetzel, R.
Thermodynamics of Aβ(1−40) amyloid fibril elongation. Biochemistry
2005, 44, 10709−12718.
(8) Mawuenyega, K. G.; Sigurdson, W.; Ovod, V.; Munsell, L.;
Kasten, T.; Morris, J. C.; Yarasheski, K. E.; Bateman, R. J. Decreased
clearance of CNS β-amyloid in Alzheimer’s disease. Science 2010, 330,
1774.
(9) Lambert, M. P.; Barlow, A. K.; Chromy, B. A.; Edwards, C.;
Freed, R.; Liosatos, M.; Morgan, T. E.; Rozovsky, I.; Trommer, B.;
Viola, K. L.; Wals, P.; Zhang, C.; Finch, C. E.; Krafft, G. A.; Klein, W.
L. Diffusible, nonfibrillar ligands derived from Aβ1−42 are potent
central nervous system neurotoxins. Proc. Natl. Acad. Sci. U. S. A. 1998,
95, 6448−6453.
(10) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; dParadis, M.;
Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi, R.
E. Rapid induction of Alzheimer Aβ amyloid formation by zinc. Science
1994, 265, 1464−1467.
(11) Matsuzaki, K. Physicochemical interactions of amyloid β-peptide
with lipid bilayers. Biochim. Biophys. Acta 2007, 1768, 1935−1942.
(12) Kotler, S. A.; Walsh, P.; Brender, J. R.; Ramamoorthy, A.
Differences between amyloid-β aggregation in solution and on the
membrane: insights into elucidation of the mechanistic details of
Alzheimer’s disease. Chem. Soc. Rev. 2014, DOI: http//dx.doi.org/
10.1039/C3CS60431D.
(13) Sciacca, M. F.; Kotler, S. A.; Brender, J. R.; Chen, J.; Lee, D. K.;
Ramamoorthy, A. Two-step mechanism of membrane disruption by
Aβ through membrane fragmentation and pore formation. Biophys. J.
2012, 103, 702−710.
(14) Butterfield, S. M.; Lashuel, H. A. Amyloidogenic proteinmembrane interactions: mechanistic insight from model systems.
Angew. Chem., Int. Ed. 2010, 49, 5628−5654.
(15) Brender, J. R.; Salamekh, S.; Ramamoorthy, A. Membrane
disruption and early events in the aggregation of the diabetes related
peptide IAPP from a molecular perspective. Acc. Chem. Res. 2012, 45,
454−462.
dx.doi.org/10.1021/ar500127z | Acc. Chem. Res. 2014, 47, 2397−2404