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Alzheimer’s Disease and Amyloid β -peptide polymerization: Structures and Strategies. Lars Tjernberg. Karolinska Institutet, KASPAC. Outline of the talk. A. Amyloid B. Amyloid β -peptide (A β ) as a target in Alzheimer’s disease C. Inhibition of A β polymerization

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alzheimer s disease and amyloid peptide polymerization structures and strategies

Alzheimer’s Disease and Amyloidβ-peptide polymerization: Structures and Strategies

Lars Tjernberg

Karolinska Institutet, KASPAC

slide2

Outline of the talk

A. Amyloid

B. Amyloid β-peptide (Aβ) as a target in Alzheimer’s disease

C. Inhibition of Aβ polymerization

D. A novel screen for Aβpolymerization inhibitors

amyloid
Amyloid
  • The term amyloid was first used in the 1850s to describe deposits that were stained with iodine
  • Thought to be composed of starch
  • Main component is protein
  • Thought to be amorphous
  • 1920s: Stains with Congo red =>
  • ordered structure
congo red stains amyloid
Congo Red stains amyloid
  • Stain tissue with Congo red
  • View under polarized light
  • Turn polarizer: red becomes green and vice versa
  • This phenomena is called birefringence
  • Indicates an ordered structure

Plaque core stained with Congo Red

amyloid is composed of protein fibrils

100 nm

Amyloid is composed of protein fibrils
  • Amyloid fibrils:
  • can be observed by electron microscopy (EM)
  • are ~8nm wide
  • can be isolated from tissue
  • can form from synthetic peptides
  • are resistant to proteolysis

EM by Johan Thyberg, KI

amyloid fibrils show a cross fibre diffraction pattern

4.8 Å

10-11Å

10-11Å

Amyloid fibrils show a cross-β fibre diffractionpattern

Peptide chain

4.8Å

Fibril direction

amyloid proteins
Amyloid proteins

Protein Disease .

Amyloid b-peptide Alzheimer’s disease

Gelsolin Finnish-type fam. amyloidosis

Islet amyloid polypep. Type II diabetes

Immunoglobulin l.c. Light-chain amyloidosis

Lysozyme Heriditary syst. amyloidosis

Medin Aortic medial amyloid

Serum amyloid A Secondary syst. amyloidosis

Transthyretin Senile syst. amyloidosis

Fam. Amyloid polyneuropathy

the amyloid plaque in alzheimer s disease
The amyloid plaque in Alzheimer’s disease
  • Core of Amyloid b-peptide (arrows)
  • Neurofibrillary tangles (Tau)
  • Dystrophic neurites
  • Activated microglia and astrocytes

D. Selkoe, Nature, 399, A23-31 (1999)

the amyloid b peptide a b

b

g

Ab

The Amyloid b-peptide, Ab

Lumen/

extracellular

  • Derived from the b-amyloid precursor protein (APP, ~700 residues transmembrane protein)
  • First cleavage by BACE
  • Second cleavage by -secretase (protein complex containing presenilin)
  • 40-42 residue peptide
  • Especially the longer variant (Aβ42) has a strong tendency to polymerize
  • Identified in1984 (Glenner & Wong)
  • Main component of the AD-plaque

1.

Membrane

Cytosol

2.

Cytosol

slide10

A: Ex vivo plaque core

B: In vitro plaque core

Amyloid plaque cores

Are composed of fibrils formed from the amyloid β-peptide (Aβ)

A

Several lines of evidence indicate that the polymerization process could be a drug target in Alzheimer’s disease

a is of importance in ad
Aβ is of importance in AD
  • Aβ plaques are always present in AD brain
  • Brain Aβ correlates with degree of dementia
  • Aβ becomes neurotoxic upon polymerization
  • All familial AD mutations => elevated levels of Aβ42
  • Extra copy of APP (Down’s syndrome) => early onset AD
  • Transgenic mice overexpressing Aβ develop AD-like
  • lesions and show impaired memory
the amyloid cascade

Aβ binding proteins (ACT, ApoE, other)

metal ions,

pH, other

Aggregation

Plaques

Inflammation

Tau fibrils

Calcium levels

ROS

Neuronal damage, AD

The amyloid cascade

FAD mutations: APP, PS1&2, other

Aβ-42

a polymerization

Random coil β-sheet

Soluble Insoluble

NontoxicToxic

Aβ polymerization

Monomer Di/Trimer Oligomer Protofibril Fibril

toxic a b species
Toxic Ab species
  • Ab-fibrils are toxic
  • Lorenzo et al. PNAS 91: 12243-12247 (1994)
  • Protofibrils are toxic
  • C. Nilsberth et al. Nat. Neurosci.4: 887-893 (2001)
  • Diffusable aggregates are toxic
  • M. P. Lambert et al. PNAS 95: 6448-6453 (1998)
slide15
Amyloid β-peptide as a target in ADAβ becomes neurotoxic upon polymerizationPossible regimes for pharmacological intervention:
  • Inhibit production of Aβ: β- and -secretase inhibitors
  • Increase clearance: Vaccination
  • Inhibit Aβ aggregation: Small Aβ-binding compounds capable of interfering with Aβ-Aβ interactions
anti amyloid strategies
Anti-amyloid strategies
  • b-secretase:

+ No severe phenotype in knock-out mice

- Difficult to develop inhibtors due to large active site

g-secretase:

+ Several examples of efficient inhibitors

  • Many different substrates besides APP i.e. Notch
  • - Knock-out lethal in mice
  • Vaccination
  • + Vaccination can reduce amyloid burden
  • Clinical trials stopped due to side effects
  • Aggregation inhibitors
  • + Aβ aggregation has no physiological function
slide17

Crystal structure of BACE with inhibitor

Eight residue transition state inhibitor

Ki = 1.6 nM

Hong Science 2000

+ KO mice viable

- Large binding pocket, difficult to find good inhibitors

slide18

-Secretase

  • Is composed of (at least) four transmembrane proteins
  • Mediates the final catalytic step in the processing of APP (C99) into Aβ
  • Is a potential drug target in Alzheimer’s disease
  • Has other substrates, e.g. Notch

Specific inhibition of APP processing is necessary!

slide19

Nonsteroidal anti-inflammatory drugs(NSAIDs)

  • There is an inflammatory component in AD
  • Large studies have shown that some none steroid anti-inflammatory drugs (NSAIDs) decrease the incidence of AD
  • The mechanism behind this effect is unknown, but might be due to lower levels of Aβ-42
  • Ibuprofen-treated APP mice show reduced Aβ-42 in brain
  • Clinical trials ongoing

S. WEGGEN et al. Nature 414, 212 - 216 (2001)

secretase inhibitors in clinical trials
-Secretase inhibitors in Clinical trials
  • Ibuprofen in phase III
  • Flurizan in Phase III
  • Selective amyloid lowering agent
  • Reduced insoluble Aβ in Tg mice and improved memory
  • Well tolerated
  • May decrease cognitive decline
antibodies against a slow cognitive decline in alzheimer s disease
Antibodies against Aβ slow cognitive decline in Alzheimer’s disease

30 AD patients were injected with aggregated Aβ-42

20 of them generated antibodies

Patients who generated antibodies showed less decline of cognitive functions and activities of daily living

Trial stopped since several patients got meningoencephalitis

C. Hock et al. Neuron38, 547-554 (2003)

Clears amyloid

Patients still produce antibodies

New trials ongoing

slide22

New vaccination strategy

A fragment of Aβ is attached to

small beads

The immune system responds by producing antibodies direct to Aβ

The antibodies bind and sequester Aβ

Professor Bengt Winblad, Karolinska University Hospital, Huddinge

Aβ immunotherapy for treatment of AD

  • There are several trials ongoing
  • Both passive and active immunization are evaluated
  • Passive immunization might increase cerebral amyloid angiopathy (CAA)
  • The antigen can be full length Aβ or short fragments
  • The peptide can be conjugated to a carrier protein or micro-beads
inhibition of a polymerization
Inhibition of Aβ polymerization
  • Aβ polymerization is thought to be nucleation-dependent
  • Several different Aβ species may be toxic
  • Inhibit as early as possible
  • No high resolution structure
an important region for a a binding daefrhdsgy evhhq klvff aedvgsnkga iiglmvggvv ia

1. Synthesize short peptide sequences corresponding to segments of Aβ on a membrane

  • 2. Incubate membrane with labeled full-length Aβ
  • 3. Quantify binding
  • The region Aβ9-22 is important for Aβ-Aβ binding
An important region for Aβ-Aβ bindingDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA

Tjernberg et al. J. Biol. Chem. 271:8545-8

a 16 20 klvff is important for a a binding

Radio-labeled KKLVFF was used to probe the KLVFF bindingsite

Aβ 16-20 (KLVFF) is important for Aβ-Aβ binding

Truncated variants of the central binding decapeptide were synthesized and incubated with radio-labeled Aβ.

Peptides containing the KLVFF sequence were found to bind Aβ.

Biacore study

The peptide AcKLVFFAAC was immobilized on the sensorchip and Ab was injected.

Lower trace: Cys

a fibril formation can be inhibited by short peptides
Aβ fibril formation can be inhibited by short peptides

Synthetic Aβ was incubated in buffer in the absence (A)

or in the presence (B) of a short peptide (QKLVFFA).

Tjernberg et al. J. Biol. Chem. 271:8545-8

pentapeptides composed of d amino acids can inhibit a fibril formation

Sequences similar to a reversed Aβ 16-20 sequence show strong binding

Aβ was incubated with D-peptides:

A=none, B=yyrrl,

C=lflrr, D=yfllr

Pentapeptides composed of D-amino acids can inhibit Aβ fibril formation

Pentapeptides were synthesized on a membrane and incubated w. radio-labelled KLVFF

Tjernberg et al. J. Biol. Chem.272:12601-5.

slide28

Saline iAβ5P

Partial decrease of amyloid deposition

Escape latency

Chacon et al.Molecular Psychiatry (2004) 9; 953-961.

iAβ5P improves working memory performance

Short Aβ-fibril inhibitors have effect in vivo

  • iAβ5 blocks Aβ-42 neurotoxicity in cell culture and fibril formation in rat brain
  • iAβ5 dissasembles fibrillar deposits in rat brain and prevents/reverses neuronal shrinkage
  • E.M. Sigurdsson et al. Nat. Med. 4:822-826 and J. Neuropathol. Exp. Neurol. 59:11-17
  • Modified molecule crosses BBB

Clinical trials Phase I => no toxicity

slide29

Curcumin, a small organic compound can affect amyloid in vivo

Curcumin crosses the BBB and binds to plaques

Yang, et al. J. Biol. Chem. (2005);280:5892-5901

slide30

Curcumin suppresses amyloid accumulation in aged APP transgenic mice

Tg2576 mice were placed on chow supplemented w. curcumin at 17 months of age and brains were removed after 22 months

A-C = control

D-F = Curcumin

G. Image analysis of plaque burden

H. ELISA measurement of guanidine- soluble Aβ

Yang, F. et al. J. Biol. Chem. 2005;280:5892-5901

slide31

Curcumin inhibits formation of

Ab oligomers

Yang, F. et al. J. Biol. Chem. 2005;280:5892-5901

a small organic a polymerization inhibitor in clinical trials
A small organic Aβ-polymerization inhibitor in clinical trials
  • Glycosaminoglycans (GAGs) bind to Aβ, promote fibril formationand are present in amyloid deposits
  • Low molecular weight GAG mimetics bind to Ab and inhibits fibril formation
  • APP tg mice treated with 3-amino-1-propanesulfonic acid (3APS) show reduced amyloid burden
  • Phase II: 3APS is safe, tolerated and reduces CSF Aβ
  • Phase III studies ongoing

P. Aisen et al. Neurology 2006; 67:1757-1763

assays for a polymerization

Assay

Dye binding, Thioflavin T (ThT) and other

Circular dichroism (CD) spectroscopy

Light scattering

Fluorescence correlation spectroscopy (FCS)

Atomic force or electron microscopy (AFM or EM)

Principle

ThT shifts fluorescence spectrum upon binding

Aβsecondary structure changes upon polymerization

Particles scatter light

The diffusion time changes upon polymerization

Visualization of the aggregates

Assays for Aβ polymerization

ThT-binding is the most frequently used assay

slide34

The ThT assay

C

ThT

Turbidity

Aβ was incubated +/- the Collagenous Alzheimer Amyloid plaque Component (CLAC) Söderberg et al. FEBS J. 272: 2231-36

D

B

ThT

Extensive aggregation => ThT assay gives false low values

Preformed Aβ fibrils were incubated +/- CLAC

A

slide35

Short Aβ peptides were incubated and assayed for ThT binding. The nonapeptide QKLVFFAED showed the highest ThT signal.

The samples were subjected to electron microscopy.

A) HQKLVFFAED

B) HHQKLVFFAE

C) QKLVFFAED

Tjernberg et al. J. Biol. Chem. 274:12619-25

ThT can give false positive results

The ThT assay

slide36

CD 217 nm (β)

2 min

40 min

CD 200 nm (random)

80 µM Aβ was incubated 90 min in TBS (open symbols) or

“CD buffer” (filled symbols)

Inset: Data was recorded up to 20 h and the signal at 217 (β-sheet) and at 200 nm (random coil) was plotted.K Berndt

80 µM Aβ was incubated in the presence of rhodamine labeled Aβ

Upper panel: Fluorescence intensity vs time.

Lower panel: The corresponding intensity autocorrelation function.

A Pramanik

CD and FCS

Wanted: A simple, rapid and sensitive assay

a b oligomers can be detected by specific antibody
Ab oligomers can be detected by specific antibody

A-B:Single neuron labeled with oligomerspecific antibody Fluorescent (A)

or peroxidase-conjugated (B)secondary antibody

C: Dot blot analysis of soluble Ab in AD and control brain.

hts assay for a polymerization inhibitors

123456

1. Find Aβ-Aβinteracting motif

2. Design a peptide that mimics these interactions and label it with a donor and an acceptor (fluorescence resonance energy transfer, FRET)

3. Incubate with compound library and read fluorescence

HTS-Assay for Aβ polymerization inhibitors
slide39

C

HQKLVFFAED

Y

N

G

K

DEAFFVLKQH

C

Constrained peptides containing the HQKLVFFAED motif form fibrils

slide40

Amyloid fibril formation from a tetrapeptide, KFFE

EM

Congo Red

Model of a beta-sheet composed of four antiparallel KFFE peptides

Tjernberg, L. et al. J. Biol. Chem. 2002;277:43243-43246

the turn motif is of importance for the secondary structure
The turn-motif is of importance for the secondary structure

CD-spectra:

  • =KFFEYNGKKFFE => β-sheet
  • =KFFEAAAKKFFE => random
slide42

The peptide KFFEYNGKKFFE forms a β-hairpin in solution

B. Persson

Molecular modeling in agreement with NMR data

hts assay for a polymerization inhibitors43

123456

1. Find Aβ-Aβinteracting motif

K L V F F

K L V F F A E

Y

N

2. Design a peptide that mimics these interactions and label it with a donor (W) and an acceptor(dansyl) (fluorescence resonance energy transfer, FRET)

G

K

E A F F V L K

3. Incubate with compound library and read fluorescence

HTS-Assay for Aβ polymerization inhibitors
slide44

9 M urea

TBS

A probe containing 2 KLVFFAE motifs, Trp (donor) and dansyl (acceptor) was synthesized and emission scans were recorded

Close; energy transfer

FRET

F

lmax Donor

lmax Acceptor

l

Far away; no transfer

urea denaturation of inhibitor probes
Urea denaturation of inhibitor probes

The acceptor and donor fluorescence was measured at different urea concentrations and the ratio calculated

These probes could be useful for screening!

slide46

10

3

0

1

1

0

3

10

Curcumin gives dose dependent changes in fluorescence spectrum

The probe was incubated 2h in the presence of 0, 1, 3 or 10 uM curcumin

slide47

The probe shows decreased acceptor fluorescence in the presence of curcumin

The probe (0.5 uM) was incubated in the presence of curcumin (0, 0.25, 0.5, 0.75, 1.0, 1.5 or 2uM)

The inhibitor interacts with the probe

slide48

Buffer

Curcumin

Curcumin does not absorb the light emitted from the acceptor

A solution of curcumin (2.5 uM) was placed in the emission path, probe concentration 0.5 uM.

slide49

200 220 240 nm

Unordered conformation

Curcumin

β-sheet

CD-spectroscopy

The probe shows decreased β-sheet in the presence of curcumin

The probe (10 µM) was incubated in the absence (blue trace) or in the presence (green trace, t = 0, red trace, t = 30 min) of 36 µM curcumin

slide50

SEC - fluorescence detection

The probe was dissolved in 50% acetonitrile and injected onto a SEC column. The signal at 520 nm was recorded (acceptor emission).

Broken line: direct excitation of acceptor (350 nm)

Full line: excitation of donor (290 nm)

Conclusion: The probe is a monomer and shows intramolecular FRET

acknowledgements
Acknowledgements

Karolinska Institutet:

Lars Terenius Rudolf Rigler Johan Thyberg

The Picower Institute for Medical Research:

David Callaway

Swedish University of Agricultural Sciences:

Jan Johansson

Dainippon Sumitomo Pharma

conclusions
Conclusions
  • Amyloid is bad
  • The amyloid b-peptide is important in AD
  • The amyloid b-peptide can be a therapeutic target in AD
  • There are drugs aimed at lowering Ab-levels in clinical trials
  • Still more to learn