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Alzheimer’s Disease and Amyloid β -peptide polymerization: Structures and Strategies

Alzheimer’s Disease and Amyloid β -peptide polymerization: Structures and Strategies

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Alzheimer’s Disease and Amyloid β -peptide polymerization: Structures and Strategies

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  1. Alzheimer’s Disease and Amyloidβ-peptide polymerization: Structures and Strategies Lars Tjernberg Karolinska Institutet, KASPAC

  2. 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

  3. 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

  4. 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

  5. 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

  6. 4.8 Å 10-11Å 10-11Å Amyloid fibrils show a cross-β fibre diffractionpattern Peptide chain 4.8Å Fibril direction

  7. 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

  8. 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)

  9. 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

  10. 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

  11. 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

  12. 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

  13. Random coil β-sheet Soluble Insoluble NontoxicToxic Aβ polymerization Monomer Di/Trimer Oligomer Protofibril Fibril

  14. 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)

  15. 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

  16. 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

  17. 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

  18. -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!

  19. 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)

  20. -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

  21. 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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.

  28. 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

  29. 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

  30. 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

  31. Curcumin inhibits formation of Ab oligomers Yang, F. et al. J. Biol. Chem. 2005;280:5892-5901

  32. 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

  33. 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

  34. 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

  35. 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

  36. 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

  37. 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.

  38. 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

  39. C HQKLVFFAED Y N G K DEAFFVLKQH C Constrained peptides containing the HQKLVFFAED motif form fibrils

  40. 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

  41. The turn-motif is of importance for the secondary structure CD-spectra: • =KFFEYNGKKFFE => β-sheet • =KFFEAAAKKFFE => random

  42. The peptide KFFEYNGKKFFE forms a β-hairpin in solution B. Persson Molecular modeling in agreement with NMR data

  43. 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

  44. 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

  45. 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!

  46. 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

  47. 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

  48. 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.

  49. 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

  50. 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