Alzheimer

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Alzheimers Disease and Amyloid ß-peptide
polymerization Structures and Strategies

• Lars Tjernberg

Karolinska Institutet, KASPAC
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Outline of the talk
A. Amyloid B. Amyloid ß-peptide (Aß) as a target
in Alzheimers disease C. Inhibition of Aß
polymerization D. A novel screen for Aß
polymerization inhibitors
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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 gt
• ordered structure

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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
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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
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Amyloid fibrils show a cross-ß fibre diffraction
pattern
Peptide chain
4.8Å
Fibril direction
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Amyloid proteins

• Protein Disease .
• Amyloid b-peptide Alzheimers 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

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The amyloid plaque in Alzheimers disease

• Core of Amyloid b-peptide (arrows)
• Neurofibrillary tangles (Tau)
• Dystrophic neurites
• Activated microglia and astrocytes

D. Selkoe, Nature, 399, A23-31 (1999)
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The Amyloid b-peptide, Ab
Lumen/ extracellular
1.

• 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 (Aß42) has a strong
  tendency to polymerize
• Identified in1984 (Glenner Wong)
• Main component of the AD-plaque

Membrane
Cytosol
2.
Cytosol
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Amyloid plaque cores
Are composed of fibrils formed from the amyloid
ß-peptide (Aß)
A
Several lines of evidence indicate that the
polymerization process could be a drug target in
Alzheimers disease
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Aß is of importance in AD

• Aß plaques are always present in AD brain
• Brain Aß correlates with degree of dementia
• Aß becomes neurotoxic upon polymerization
• All familial AD mutations gt elevated levels of
  Aß42
• Extra copy of APP (Downs syndrome) gt early
  onset AD
• Transgenic mice overexpressing Aß develop
  AD-like
• lesions and show impaired memory

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The amyloid cascade
FAD mutations APP, PS12, other
Aß-42
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Aß polymerization
Monomer Di/Trimer Oligomer Protofibril
Fibril
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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)

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Amyloid ß-peptide as a target in AD Aß becomes
neurotoxic upon polymerizationPossible regimes
for pharmacological intervention

• Inhibit production of Aß ß- and ?-secretase
  inhibitors
• Increase clearance Vaccination
• Inhibit Aß aggregation Small Aß-binding
  compounds capable of interfering with Aß-Aß
  interactions

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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
• Aß aggregation has no physiological function

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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
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?-Secretase

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

Specific inhibition of APP processing is
necessary!
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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 Aß-42
• Ibuprofen-treated APP mice show reduced Aß-42 in
  brain
• Clinical trials ongoing

S. WEGGEN et al. Nature 414, 212 - 216 (2001)
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?-Secretase inhibitors in Clinical trials

• Ibuprofen in phase III
• Flurizan in Phase III
• Selective amyloid lowering agent
• Reduced insoluble Aß in Tg mice and improved
  memory
• Well tolerated
• May decrease cognitive decline

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Antibodies against Aß slow cognitive decline in
Alzheimers disease
30 AD patients were injected with aggregated
Aß-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. Neuron 38,
547-554 (2003) Clears amyloid Patients still
produce antibodies
New trials ongoing
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Aß 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 Aß or short
  fragments
• The peptide can be conjugated to a carrier
  protein or micro-beads

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Inhibition of Aß polymerization

• Aß polymerization is thought to be
  nucleation-dependent
• Several different Aß species may be toxic
• Inhibit as early as possible
• No high resolution structure

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An important region for Aß-Aß bindingDAEFRHDSGY
EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA
Tjernberg et al. J. Biol. Chem. 2718545-8
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Aß 16-20 (KLVFF) is important for Aß-Aß binding
Truncated variants of the central binding
decapeptide were synthesized and incubated with
radio-labeled Aß. Peptides containing the KLVFF
sequence were found to bind Aß.
Biacore study The peptide AcKLVFFAAC was
immobilized on the sensorchip and Ab was
injected. Lower trace Cys
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Aß fibril formation can be inhibited by short
peptides
Synthetic Aß was incubated in buffer in the
absence (A) or in the presence (B) of a short
peptide (QKLVFFA). Tjernberg et al. J. Biol.
Chem. 2718545-8
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Pentapeptides composed of D-amino acids can
inhibit Aß fibril formation
Pentapeptides were synthesized on a membrane and
incubated w. radio-labelled KLVFF
Tjernberg et al. J. Biol. Chem. 27212601-5.
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Short Aß-fibril inhibitors have effect in vivo

• iAß5 blocks Aß-42 neurotoxicity in cell culture
  and fibril formation in rat brain
• iAß5 dissasembles fibrillar deposits in rat brain
  and prevents/reverses neuronal shrinkage
• E.M. Sigurdsson et al. Nat. Med. 4822-826 and J.
  Neuropathol. Exp. Neurol. 5911-17
• Modified molecule crosses BBB

Clinical trials Phase I gt no toxicity
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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)2805892-5901
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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 Aß
Yang, F. et al. J. Biol. Chem. 20052805892-5901
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Curcumin inhibits formation of Ab oligomers
Yang, F. et al. J. Biol. Chem. 20052805892-5901
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A small organic Aß-polymerization inhibitor in
clinical trials

• Glycosaminoglycans (GAGs) bind to Aß, promote
  fibril formation and 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
  Aß
• Phase III studies ongoing

P. Aisen et al. Neurology 2006 671757-1763
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Assays for Aß polymerization
ThT-binding is the most frequently used assay
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The ThT assay
A
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The ThT assay
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CD and FCS
Wanted A simple, rapid and sensitive assay
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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.
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HTS-Assay for Aß polymerization inhibitors
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Constrained peptides containing the HQKLVFFAED
motif form fibrils
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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.
200227743243-43246
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The turn-motif is of importance for the secondary
structure
CD-spectra

• KFFEYNGKKFFE gt ß-sheet
• KFFEAAAKKFFE gt random

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The peptide KFFEYNGKKFFE forms a ß-hairpin in
solution
B. Persson
Molecular modeling in agreement with NMR data
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HTS-Assay for Aß polymerization inhibitors
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FRET
F
lmax Donor
lmax Acceptor
l
Far away no transfer
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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!
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Curcumin gives dose dependent changes in
fluorescence spectrum
The probe was incubated 2h in the presence of 0,
1, 3 or 10 uM curcumin
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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
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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.
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CD-spectroscopy The probe shows decreased ß-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
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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
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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

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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