Chemistry of Alzheimer’s Disease: Role of amyloid-beta, metal ions, and reactive oxygen species...

Chemistry of Alzheimer’s Disease:

Role of amyloid-beta, metal ions, and reactive oxygen species

Peter FallerLCC, Toulouse

[email protected]

Metals

BrainOxygen

Neuro-degeneration

Metaldisorder

OxidativeStress (ROS)

2% w/w of body

8% of Cu

Consumes20% of O2

Neurons in the brain

Neuron (nerve cell)

The nerve impulse. In the resting neuron, the interior of the axon membrane is negatively charged with respect to the exterior (A). As the action potential passes (B), the polarity is reversed. Then the outflow of K+ ions quickly restores normal polarity (C).

Synapse and Neurotransmitter

Nerve impulsecontinues

Molecular mechanism of learning

• Donald O. Hebb (1949) (Hebb’s rule):

« When an axon of a cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A’s efficiency, as one of the cells firing B, is increased »

Long term potentiation (LTP): a mechanism for establishing memory

(EPSPs excitatory postsynaptic potentials)

stimulation

FIGURE 53-3 An illustration of a synapse between the presynaptic and postsynaptic neurons. The glutamate released from presynaptic terminals activates both AMPA and NMDA receptors. While the AMPA receptor is responsible for basal synaptic transmission, the NMDA receptor acts like the volume controller regulating the efficacy of synaptic transmission. Synaptic transmission is enhanced if the NMDA receptor detects the co-activity of the presynaptic (release and binding of glutamate) and postsynaptic neuron (enough depolarization to expel Mg2+ from the channel pore). When such a coincidence event occurs, the NMDA receptor is activated, which opens the channel pore and allows Na+ and Ca2+ to rush in and K+ to rush out. The influx of Ca2+ then activates biochemical cascades that eventually strengthen the synapse. It is believed that some of these kinases bind directly to the C-terminus of the NR2B subunit, allowing efficient signal detection and amplification .

Long term Potentiation (LTP): a mechanism for establishing memory

Metals in the cell

Essential and toxic metal ions

health

toxicdeath

death

deficiency

concentration

phy

siol

ogic

al e

ffec

tEssentiel Toxique

(non-essentiel)

toxic

death

health

concentration

positiv

negativ

Biological system tries to keep the metal content constant (steady state)

General features: Metabolism of essential metals

Cell (microrganisms)

M M

ADP ATP

M MM

M M

MATP ADP

Sensor for Regulation

diffusionActive transport

Protein to stock metals

Different metals have different roles:

- e.g. Alcohol dehydrogenase : Zn(II) enzyme

- Cytochrome c oxydase Cu and heme for oxygen reduction

Make sure that the right metal goes to the right place!

Metal Specificity

How to make sure that the right metal ion goes to the right place?

1) thermodynamics:engeneer the site that it binds « specifically », i.e. prefentially the wanted metal-> coordination chemistry

2) kineticsspecific transporters/carriers called « metallo-chaperones » bring the right metal to the right place. Then the metals is well bound so that koff is very low

How to Reach Metal Specificity

Thermodynamic control

1 2

43

K1

K2

K4K3

1 2

43

+ metal ion

Si K2 >> K1,3,4 1 2

43

K1

K2

K4K3

Kinetic control

1 2

43 k4k3

k1 k2

Mn+ + Prot Mn+-Protk1

k-1

K1 = k1/k-1

For Cu kon diffusion controlledk1 = k2 = k3 = k4

1 2

43

1 2

43

« k1 >> k2,3,4 »

Thermodynamic versus Kinetic Control of Metal-binding

1 2

43

k4k3

k1 k2

parameters to optimize;

- size of the site (ion radius Ca2+: 100pm is larger than Mg2+ 72 pm)

- charge (metal-ligand most stable when neutral)

- number of ligands (Ca2+ 6-8 ligands; Cu+ 2-4)

- type of ligands (Pearsons model of hard/soft acid/base)

- geometry (Cu(II) likes square planar, Zn(II) tetrahedral, or pentacoordinated)

Thermodynamic control:

Chelate Effect

L L L M L

L M L

M +

M + 2 L

Association constant: monodentate < bidentate < tridentate etc.

Ex: EDTA (hexadentate)

Co-EDTA

Chelate Effect

Example: Complexes of Ni(II):

Ni2+ (aq) + 6 NH3 [Ni(NH3) 6] 2+ (aq) log K = 8.6

Ni2+ (aq) + 3 en [Ni(en)3] 2+ (aq) log K = 18.3

Mainly entropic effect

Ni(NH3) 6] 2+ (aq) + 3 en [Ni(en)3] 2+ (aq) + 6 NH3

log K = 9.7; ΔG° = -67kJ/mol, ΔH°= -12 kJ/mol; -TΔS° = -55 kJ/mol

CHIMIE FONDAMENTALE, Chottard, Depezay, Leroux

Metal-ions binding and pKa of ligands

MII-OH2 MII-OH

Metal ion pKa(2+)

No metal 14,0Ca 13,4Mn 11,1Cu 10,7Zn 10,0

+ M2+ MII- + H+

NH

HN

NH

N

No metal 6,0Co 4,6Ni 4,0Cu 3,8

Competition between metal ion and proton(low pH used to remove metal ion from ligand)

2++

« pKa »

2+ +

+ H+

In biology:

Bases: AcidsOxygen (hard) hard: Fe(III), Co(III), Ca(II)Nitrogen (intermediate) intermediate: Fe(II), Zn(II), Cu(II)Sulfur (soft) soft: Cu(I), Hg(II)

Hard acids prefer hard bases: more ionic bondSoft acids prefer soft bases: more covalent bond

Hard Lewis acids: weakly polarizable, small ionic radii, high positive charge, strongly solvated, empty orbitals in the valence shell and with high energy LUMOs.

Soft Lewis acids highly polarizable, large ionic radii, low positive charge, completely filled atomic orbitals and with low energy LUMOs.

Hard Lewis bases weakly polarizable, small ionic radii, strongly solvated, highly electronegative, high energy HOMO

Soft Lewis bases highly polarizable,large ionic radii, intermediate electronegativity, low energy HOMOs.

Concept of hard/soft acid/base (HSAB, Pearson)

Biological Ligands: amino acides (peptide/proteines)

Histidine

Méthionine

Cysteine

Selenocysteine

Tyrosine

aspartique acid

glutamique acidH

amino acid side chain pKa

Backbone : terminal amine pK ~8;, terminal COO- pKa ~4

H

Irving-Williams series

Stability constant (log K1) of divalent mtal ions

Problem: even coordination optimized for a specific metal There is the possibility that other ions binds stronger

Example of a thermodynamic contol: Calcium

Normally Ca2+ concentrations are high extracellularly (~2mM) and unbound Ca2+ is low in the cytosol (~10nM). Ca2+ influx is used for signalling (secondary messanger).

Upon entrance Ca2+ binds to proteins, e.g. calmodulinCa2+ Kd: 0.1 µM – 1µM

Ca-binding induces conformationalChange, and opens binding site for protein (red star) (Mg2+ Kd: ~1mM (intracellular free Mg2+ : 0.5 -1 mM))

Apo-Calmodulin Ca-Calmodulin

Asn

AspAsp

Glu

Thr

H2O

Ca-binding site

Example: Metallothionein

Metallothioneins are cysteine rich proteins binding metal ions, They are thought to be involved in metal metabolism (Zn and Cu) and in metal detoxification (Cd, Hg) normally they bind Zn(II) and Cu(I), but under high exposure to other metals, in particular Cd(II) and Hg(II) they will bind them as well.

Cysteines contain a thiol group, i.e. RSH. Metals bind to the thiolate(R-S-, deprotonated thiol)

R-SH + Mn+ [R-S-M] (n-1)+ + H+

General affinity of metal ions for thiolates (and metallothioneins):Zn(II) < Cd(II) <Cu(I) < Hg(II)

Example:Snail has 2 metallothioneins: HpCdMT and HpCuMT

Apparent Kd Cu ZnHpCdMT1 pM 30 pMHpCuMT0.1 fM 20 fM

Although HpCdMT binds Cu stronger than Zn, HpCdMT binds Zn in the cell!

Because it depends also on the concentration of metal ions available

Estimated fee [Zn] : ~10 pMEstimated free [Cu]: ~ 1 fM

Kd = [free M+] [unbound HpMT] ----------------------------------

[M+-HpMT]

HpCdMT [M+-CdHpMT] free M+ 1fM---------------- = ---------- = ------- = 0.001[unbound HpCdMT] Kd 1pM

Example:Although HpCdMT bindsCu stronger than Zn, due to theAvilability in a cell it willBind Zb (red triangle)

Question for training:

You have two chelators A and BIn line with Irving-Williams:Kd of A for Cu(II) 1µMKd of A for Zn(II) 10µMKd of B for Cu(II) 10µMKd of B for Zn)(II 20µM

Define Kd (dissociation constant) and Ka (association constant)

Tell which chelator binds which metal when you do the following mixtures1) 1mM A, 1mM B and 1mM Cu(II)2) 1mM A, 1mM B and 1mM Zn(II)3) 1mM A, 1mM Cu(II) and 1mM Zn(II)4) 1mM B, 1mM Cu(II) and 1mM Zn(II)5) 1mM A, 1mM B, 1mM Cu(II) and 1mM Zn(II)

Zinc in a classic cell: thermodynamic control?

Zinc(II) is buffered by proteins, small molecules (amino acids etc)Zn(II) proteins and enzymes take Zn(II) up from « free » Zn(II)


The concentration of Zn(II) in mamalian cells is controled by the transcription factor MTF1. In simple way: MTF1 is a Zn-sensor, i.e. if Zn is bound to MTF1, this meansthere is too much « free » Zn in the cell.

What is a transcription factor?

The dissociation constant of Zn to MTF-1 has not been exactly determined,but was estimated to be 30 pM (Berg and coworkers, Biochemsitry 2004, p5437)

Define dissociation constant

Assuming when half or more of the MTF-1 in a cell is bound to Zn(II), MTF-1 initiates the transcription of the protein metallothionein to bindthe excess Zn(II).

What is the « free » Zn-concentration at which this happens? Calculate.Make a general conclusion about the concentration of a « free » metaland the affinity of its sensor

Thermodynamic control

1 2

43

K1

K2

K4K3

1 2

43

+ metal ion

Si K2 >> K1,3,4 1 2

43

K1

K2

K4K3

Kinetic control

1 2

43 k4k3

k1 k2

Mn+ + Prot Mn+-Protk1

k-1

K1 = k1/k-1

For Cu kon diffusion controlledk1 = k2 = k3 = k4

1 2

43

1 2

43

« k1 >> k2,3,4 »

Thermodynamic versus Kinetic Control of Metal-binding

1 2

43

k4k3

k1 k2

Reedijk Platinum Metals Rev., 2008, 52, (1), 2–11

Kinetics: Rate exchange of ligands

Copper trafficking pathways in eukaryotes (kinetic control)

O'Halloran T V , Culotta V C J. Biol. Chem. 2000;275:25057-25060

©2000 by American Society for Biochemistry and Molecular Biology

Kd of Cu(I)-proteins (in cell) 10-15 to 10-18 M

With Kd = koff/kon and assumed kon diffusion controlled (fastest possible) koff 106 – 109 s-1, i.e. 11 days to 350 years

Cu(I) trafficking is under kinetic control

Proposed pathway for copper transfer from ATX1 to CCC2.

O'Halloran T V , Culotta V C J. Biol. Chem. 2000;275:25057-25060

©2000 by American Society for Biochemistry and Molecular Biology

Copper in a classic cell

Banci et al. Nature 2010


You want to be able to add a very strong and specific chelator for Zn(II)and Cu(I) into a cell,

1) How would you design a very strong (as strong or stronger thanproteins in the cell) and « specific » chelator for Zn(II) and Cu(I). Make a propostion.

2) What could be the difference between a such strong chelatorfor Zn(II) and Cu(I) in terms of the abilility to bind Zn(II) and Cu(I)In the cell? Will the Zn and Cu-chelator be equal efficient?

Metals in the brain

Becker et al. Anal Chem. 2005 77:3208-16

Zinc

Copper

Zn-Pools

Different Zn-pools: A) tightly coordinated (thermodynamicly and kineticly)

more or less existing in all cellse.g. catalytic site of enzymes (peptidase), structural site of proteins (super-oxide dismutase) Zn-fingers

only accessible to very strong chelators (and long incubaiton)

B) labile Zn-pool The “extra” Zn in the Zn-containing neurons (absent in other neurons and cells)

not so tightly bound accessible to complexation of chelators

How to Measure Zn in the Zn-Containing Neurons ?

Different Zn-pools: - tightly coordinated (e.g. catalytic site of enzymes, structural site of proteins) not accessible- labile Zn-pool (The “extra Zn in the Zn-containig neurons) not tightly bound accessible

Can be measured by fluorophores “specific” to Zn

Examples for Fluorescent Detector of Zn

There are many more known:Jiang & GuoCoord. Chem. Rev. 2004, 248, 205-29

Examples for Fluorescent Detector of Zn

Jiang & GuoCoord. Chem. Rev. 2004, 248, 205-29

A) 2-Me-TSQ

B) Ratiometric Zn-sensor: FluoZin-3

How to Bring a Chelator in a Cell?

Example zinquin:

Zinc homeostasis in neurons

(Colvin et al., 2003, Eur. J. Pharmacol.)

• Modulation of glutamic

responses

• Modulation of GABA

responses

• Antagonism on Ca2+, K+ and

Na+ conductances

• Probable role in disease-

associated neurodegeneration

(e.g. Alzheimer’s disease)

Roles of synaptic zinc

(Colvin et al., 2003, Eur. J. Pharmacol.)

Zinc-Release in the Synaptic Cleft upon StimulationQian & Noebels, J. Physiol. 2005

without ZnT-3 with ZnT-3

Fluorescence increase of Zn-sensor

Extracellular Zn-chelator Intracellular Zn chelatorCa-EDTA DEDTC diethyldithiocarbamate

Training Training before afterchelator chelator Addition addition


You study a process x in Zn-rich neurons, in which you suspect that the labile Zn-concentration is changing either extra or intracellularly.

Design an experiment, which allows you to conclude where (intra or extracellualrly ) the labile Zn concentration is changing

much less known than for zinc, but evidence accumulates that copperCan be released into synaptic cleft (like zinc)

Cu(I)

Cu(I) specific fluorescence based sensor for biological applications developed (Fahrni et al. ; Cheng et al. etc)

Spatial resolved X-ray absorption

Cu(II):

Problem: Cu(II) normally quenches fluorescence, thus difficult to design fluorescent Cu-chelators And difficult to measure a labile Cu(II) pool(if it exists)

What about Cu in the Brain?

Porphyrin-Fl is quenched: indicates Cu(II)? Release upon stimulation

Presynapse postsynapse

MT-3

APP

M-Aβagrégats

ZnT3

ZnMT-3Zn

Cu

CuATP7a

Aβ

Aβ

MT-3

Synaptic Copper and Zinc

Indicate that up to 15µM Cu can be released into synptic cleft

Metals and Oxygen

NADH NAD+

Em (V)

succinate fumarate

0

-0.5

+0.5

NADH/NAD+

QH2Q

CytCred

QH2Q

CytCox

CytC

QH2/Q

2H+ + 1/2O2/H2O

+1.0

complex I

complex IATPasecomplex V

complex II

complex IV

complex III

complex IV

2H+ + 1/2O2 H2O

4H+ 2H+ 2H+H+

ADP + P ATP

complex III

9 FeS

FMN

1 FeS3 heme

3 Cu2 heme

3 Cu2 heme

1 FeS3 heme

9 FeS

membrane

2e-

2e-

2e-

2e-

2e-2e-

Respiratory Chain: Metals and Oxygen

Halliwell & Gutteridge put it on page 24 [11]: “(The triplet ground state of O2)…imposes a restriction on electron transfer which tends to make O2 accept its electron one at the time, and contributes to explaining why O2 reacts sluggishly with many non-radicals. Theoretically, 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, fortunately!”

Dioxygen: triplet ground state (two unpaired electrons)Organic molecules: mostly paired electrons

Why NADH does not react fast with oxygen?

Reductant (NADH) O2

slow electron transfer

Reductant Metal ion (Cu(II)) O2

Fast electron transfer

e-

e-

e-

O2 O2°- H2O2

H2O

°OHSOD

catalase GSH reductase

2 GSH

GS-SG

Mred Mox

H2O

reductant

DNA/RNA

protein

lipid

Mred Mox

reductant

NADPH

NADP+

GSH reductase

SOD

Reactive Oxygen Species (ROS)

Strong link between redox metal and oxygenRedox metals can be «good» or «bad»

So called reducing agents (ascorbate, glutathione etc) canbe prooxidants

Depends on the Coordination

O2 metabolismRedox

Oxidative Stressmetals(HO●, O2

●-…)(Cu, Fe, Mn..)

Conclusion

Tight link between redox metals and ox stress

Redox metals (e.g.Copper, iron) are ideal to abolish or produce radicals

Coordination of the metal ion defines reactivity

Reactive Oxygen Species (ROS)

O2 O2ˉ H2O2 HO ˉ + HO

2H2O

+ eˉ + eˉ+ eˉ + 2H+

Free or loosely bound redox-metals (e.g. Cu)

SOD (Cu,Zn)

Strong link between redox metal and oxygenRedox metals can be «good» or «bad»

Depends on the Coordination

+ eˉ + 2H+

CytC Oxidase (Cu,Fe)

Redox Metals and Reducing Agents

Reducing agents of organic molecule type (VitC, VitE, glutathion etc)Antioxidants like to give an electron

e.g. VitC: °R + VitC -> H-R + °VitC

But another possibility:

PROoxidant: R + VitC -> °R + °VitC (VitC as prooxidant)

Proxidant activity of VitC can be catalyzed by redox metals (often loosly bound)

Production of HO° by different Cu-Complexes

Coordination chemistry of Cu determines the amount of HO°

Under aerobic conditionsand with ascobate

Metal metabolism has to be tighly controlled

Guilloreau et al. ChemBioChem, (2007)

Alzheimer’s DiseaseAnd the Amyloid Cascade Hypothesis

Alzheimer’s Disease: Morphological HallmarksNeuronal deathAmyloid plaquesNeurofibrillary tangles

Important Factors in Alzheimer’s Disease (AD)

- Aggregation of the peptide amyloid-beta (Aβ)

- Hyperphosphorylation of the protein tau (neurofibrillary tangles)

- Genetic factors (mutations) increasing the risk of AD

- Diminution of acetylcholine concentration in the brain

- Role of metal ions

- Role of membranes

- Oxidative stress (production dreactive oxygen species (ROS) like OH°, H2O2, O2

-, NO°)- lipide peroxydation- protein oxydation- DNA/RNA adducts

- etc

Drugs on the market:

Approved by FDA:

Inhibitors of AcetylcholineraseDonepezil (Aricept ™) ENA-713 (Exelon ™) Galantamine (Reminyl ™) Tacrine (Cognex ™)

NMDA- receptor antagonist.Memantine (Namenda ™) Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie the degeneration of cholinergic cells. Memantine is a fast, voltage-dependent NMDA- receptor antagonist. It blocks the NMDA receptor in the presence of sustained release of low glutamate concentrations and thus attenuate NMDA receptor function.

Not approved by FDA, but medication sold over the counterAlpha-tocopherol (Vitamin E)Melatonin ???

Source: Alzheimer research forum http://www.alzforum.org/new/

http://www.alzforum.org/dis/tre/drt/donepezil.asp

http://www.alzforum.org/dis/tre/drt/ena713.asp

http://www.alzforum.org/dis/tre/drt/galantamine.asp

http://www.alzforum.org/dis/tre/drt/tacrine.asp

http://www.alzforum.org/dis/tre/drc/detail.asp?id=51

healthy brain

Healthyneuron

Alzheimer brain

soluble Aaggregates

Amyloidplaques

Degeneratedneuron

toxic(ROS) not toxic

Amyloid- in Alzheimer’s Disease

APP

α-secretaseβ- and γ-sectretase

D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A

Form Aβ42 (42 amino acids)

Amyloid-beta (Aβ) peptides

hydrophylic hydrophobic

D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V

Form Aβ40 (40 amino acids)

Metal binding site

Native Aβ peptides

Nielsen Methods Enzym. (1999) 309: 491

Aggregation of Amyloid-beta (Aβ)

Structure in water: random coil (Zagorski et al. JACS 126; 1992 (2004))

Aβ monomer

15’

1h

24h

micelle environment:Alpha-helical

Beta-sheet

?

? Craik et al. Biochem. 1998, 11064

Riek et al. PNAS 2005

Metals in Alzheimer’s Disease

Role of Metals in the Aggregation of the peptide -amyloid

Amyloid- (A)mM concentrationsof Cu, Zn, Fe

Evidence for a Role of Metals in Amyloid-β Aggregation

Some examples: - mM concentrations of metals in the ßA-plaques- metal homeostasis affected in AD- Zn and Cu enhance the aggregation of ß-A in vitro- metal chelator clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) reduce plaques in mice model clinical trials in phase II

- mice with knocked out Zn-transporter (ZnT3): less plaques because less

Zn in the synapse

I

Cl

HO

N1

clioquinol8

5

ZnZnZn

ZnZnZn

Zn

Zn

A

A plaques

In healthy conditions: redox metal metabolism is very well regulatedConcentration, compartimentation, transport, excretion etc(by transport proteins, sequestering proteins, chaperons etc.)

Deregulation of metal metabolism in Alzheimer’s disease Oxidative stress

Questions: - Is this deregulation of metals an early event (important) or late event (less

important) ? - What type of deregulation occurs?- Can we fix that with metal chelators ? (Some Cu(II) chelators entered clinical

phase II studies)

Metals are involved in Alzheimer’s disease

+ Zn Cu

APP

healthy brain

Healthyneuron

Alzheimer brain

Degeneratedneuron

toxic(ROSwith Cu)

not toxic

Metals and Amyloid- in Alzheimer’s Disease

No ROS

Cu and Zn binding supposed only to occur in Alzheimer’sCu promotes neurodegeneration of Aβ, Zn rather protects

+ Zn Cu

APP

healthy brain

Healthyneuron

Alzheimer brain

Degeneratedneuron

toxic(ROSwith Cu)


No ROS


1

not toxic

+ Zn Cu

APP

healthy brain

Healthyneuron

Alzheimer brain

Degeneratedneuron

toxic(ROSwith Cu)


No ROS


2

not toxic

+ Zn Cu

APP

healthy brain

Healthyneuron

Alzheimer brain

Degeneratedneuron

toxic(ROSwith Cu)


No ROS


3not toxic

+ Zn Cu

APP

healthy brain

Healthyneuron

Alzheimer brain

Degeneratedneuron

toxic(ROSwith Cu)


No ROS


4

Chelator(native, therapeutic)

not toxic

Dynamics of Metal-Amyloid-β

1. Intramolecular

2. Intermolecular

M M

M

M

M

M

M

M

NMR study of Cu(II) interaction with Ab

+H3N

O

O-

O

HN

O

HN

O

O-

O

NH

O

N

NH

HN

O

O-

O

NH

O

O-

O

NH

O

N

NH

HN

O

N

NH

NH

O-

O

Asp1 Ala2 Glu3 His6 His13 His14Glu11Asp7 Lys16

NH3+

pH 6.5pH 8.7

NMR study of Cu(II) interaction with Aβ : 13C data

III

Hureau, C.; Coppel, Y. et al. Angew. Chem. Int. Ed. 2009, 48 (50), 9522-9525.

Major form at pH 7.4 (pure at pH 6.5) Minor form at pH 7.4 (pure at pH 9)

NMR and EPR study of CuII-Amyloid-13C-NMR (and 2D 13C-1H experiments) in solution, EPR (pulsed and ENDOR) on specifically isotopically labeled Aβ1-16

Hureau et al. Angew. Chem. 2009

Very dynamic, equilibrium between different coordination modes

mouseD-A-E-F-G-H-D-S-G-F-E-V-R-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A

Mouse:- No Aβ aggregation in brain- Less toxic to cells- Less aggregation in vitro (+/- Cu(II)) - Cu(II) binds differently to human and mouse

Murine Amyloid-beta (Aβ) peptides

humanD-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A

Comparison mouse/rat and human Aβ

Difference in Cu(II)-binding of mouse and human?

mouseD-A-E-F-G-H-D-S-G-F-E-V-R-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A

Mouse:- No Aβ aggregation in brain- Less toxic to cells- Less aggregation in vitro (+/- Cu(II)) - Cu(II) binds differently to human and mouse

Murine Amyloid-beta (Aβ) peptides

humanD-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A

Comparison mouse/rat and human Aβ

Which replacement of amino acid(s) is responsibme for the different Cu(II)-binding?

-> Replacement of Arg to Gly in human Aβ at position 5 induces mouse like Cu(II) binding (spectroscopic techniques: CD and EPR) -> sufficient

Human Aβ Mouse Aβ

Cu(II)added

No Cu(II)

13C Nuclear Magnetic Resonance (NMR) of Aβ and Cu(II)-Aβ

Cu(II)-coordination is different for human and mouse Aβ

Human

Mouse

pKa 7.7

pKa 6.2

Model of Cu(II)-binding to human and mouse Aβ ?

Eury, et al. Angew. Chem. 2011

Human

Mouse

pKa 7.7

pKa 6.2

Predominant forms of Cu(II)-binding to human and mouse Aβ at phys. pH?


Comparison of human and mouse Cu(II)-Aβ:What is the consequence of the different Cu(II) coordination?

1) Different affinity: Cu(II): mouse Aβ 3 x stronger than human Aβ

2) Redox activity: mouse Cu(II)-Aβ: lower redox activity

-> generates less ROS


O2 O2 ¯ H2O2 HO ¯ + HO

Cu+ Cu2+

Reductant(ascorbate)



Cu+ Cu2+ Cu+ Cu2+

Transgenic mice as Alzheimer’s model:

Transgenic miceExpress human and mouse Aβ:Cu(II) preferentially bound to mouseLess aggregation, less ROS production

HumansExpress only human Aβ:Cu(II) bound to humanMore aggregation, more ROS production

Limitation of transgenic mouse as AD model?

Amyloid plaques in AD model mice bind less metals than human (Leskovjan et al. 2009)


Presynapse postsynapse

MT-3

APP

M-Aβagrégats

ZnT3

ZnMT-3Zn

Cu

CuATP7a

Aβ

Aβ

MT-3

Copper, Zinc and Abeta in Alzheimer’s

- Deregulation of metal ions modulate Abeta toxicity- Could affect LTP (memory) and lead to neuronal death

- Still not clear who triggers whom (Abeta and metals)

Source: wikipedia

Metalsdysfunction

• One can find from time to time publications, in which the authors try to identify the metal that is bound to a certain protein under physiological conditions. The reason that they do not know the identity of the metal is that they started from the protein gene and gene analysis proposed a metalloprotein (e.g. by the identification of a metal-binding motive in the sequence). Then they overexpress the protein in a bacterium, purify it and measure the dissociation constant of the complexes of Cu(II), Zn(II), Fe(III), Co(II), Mn(II), Ca(II), K(I),Na(I),Mg(II) with that apo-protein (apo: demetallated protein). Then they conclude that the metal ion that has the highest affinity is the physiological bound one.

• What do you think about this strategy?

• Can you propose an alternative method to confirm the identity of the metal in the metalloprotein?

QUESTION:

Chemistry of Alzheimer’s Disease: Role of amyloid-beta, metal ions, and reactive oxygen species...

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