The role of amyloid beta peptide variability toward progress of Alzheimer disease

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1 The role of amyloid beta peptide variability toward progress of Alzheimer disease Kerensa Broersen Brain Disorders and Therapeutics London, August 26 1

2 Alzheimer s disease Amyloid β peptide 2

3 Alzheimer s disease Amyloid β peptide Genetic preposition Neuroinflammation Tau Apolipoprotein E isoform Mitochondrial dysfunction Aging Environmental factors Hyperphosphorylated tau 3 3

4 The aggregation process of Aβ is characterized by intermediates that may have toxic activity Protofibrils Monomer Dimer Fibrils Oligomers Figure adapted from Kumar et al EMBO J (2011) 4

5 The aggregation process of Aβ is characterized by intermediates that may have toxic activity Species thought of as most toxic Protofibrils Monomer Dimer Fibrils Oligomers Figure adapted from Kumar et al EMBO J (2011) 5

6 Aβ variation result of heterogeneous γ-secretase activity Aβ37 Aβ38 Aβ40 Aβ42 Aβ43 Aβ46 Aβ49 6

7 Aβ variation result of heterogeneous γ-secretase activity 90% Aβ37 Aβ38 Aβ40 Aβ42 Aβ production in healthy aged subjects Aβ42: Aβ40 = 1:9 10% Aβ43 Aβ46 Aβ49 7

8 Aβ variation result of heterogeneous γ-secretase activity 90% Aβ37 Aβ38 Aβ40 Aβ42 10% Aβ production in healthy aged subjects Aβ42: Aβ40 = 1:9 Aβ43 Aβ46 Aβ49 Highly amyloidogenic 8

9 Aβ variation result of heterogeneous γ-secretase activity 90% Aβ37 Aβ38 Aβ40 Aβ42 10% Aβ production in healthy aged subjects Aβ42: Aβ40 = 1:9 Aβ43 Aβ46 Aβ49 Little amyloidogenic 9

10 Aβ variation result of heterogeneous γ-secretase activity 70% Aβ37 Aβ38 Aβ40 Aβ42 Aβ production in aged subjects with Alzheimer pathology 30% Aβ43 Aβ46 Aβ49 Aβ42:Aβ40 = 3:7 10

11 Aβ heterogeneity affects aggregation Aβ40 Aβ42 Aβ40, 15N-edited Aβ42, 15N-filtered 11 Kuperstein et al. EMBO J. 2010, Pauwels et al. JBC

12 Aβ heterogeneity affects toxicity Day 1-training (shock) 300 P< :7 Aβ42:Aβ40 ratio causes memory and learning impairment in mice 100 Passive avoidance response Enter latency (sec) Enter latency (seconds) Day 2-memory testing Training Testing Control Control Ratio 10:0 Aβ40 Ratio 9:1 1:9 Ratio 7:3 3:7 Ratio 0:10 Aβ42 12 Kuperstein et al., EMBO J. 2010

13 Aβ heterogeneity affects toxicity Microelectrode array: synaptic activity recordings in neuronal network grown on chip 60 recording electrodes Kuperstein et al. EMBO J

14 % initial rate Pathological mixtures of Aβ40 and Aβ42 induce acute synaptotoxicity Control Aβ40 Ratio 1:9 Ratio Control Aβ42 3:7 Ab1-40 Ratio 1:9 Ratio 3:7 Ab1-42 * * * ** Time of treatment, min ** * Kuperstein et al. EMBO J. 2010

15 Aβ variation result of heterogeneous γ-secretase activity Aβ37 Aβ38 Aβ40 Aβ42 Aβ43 Aβ46 Aβ49 15

16 Aβ peptide length affects aggregation rate Vandersteen et al (2012) J Biol Chem 287:

17 Tendency to form amyloid fibrils is independent of Aβ peptide length but morphological differences Aβ38 Aβ40 Aβ42 Vandersteen et al (2012) J Biol Chem 287: Aβ43 17

18 All tested Aβ peptide lengths form oligomers but with different structural properties. Recognition of oligomeric Aβ by A11 antibody. Vandersteen et al (2012) J Biol Chem 287:

19 Aβ peptide length affects cytotoxicity: unexpected toxicity of Aβ38 Cell titer blue cell viability assay of neuroblastoma SHSY-5Y cells by incubation with oligomeric Ab. Chavez-Gutierrez et al (2012) EMBO J 31:

20 Aβ peptide length affects cytotoxicity: unexpected toxicity of Aβ38 Cell titer blue cell viability assay of neuroblastoma SHSY-5Y cells by incubation with oligomeric Aβ. 20 Vandersteen et al (2012) J Biol Chem 287:

21 Aβ mutations 21

22 D23N (Iowa mutant) and E22K (Italian mutant) Aβ42 form antiparallel β-sheet fibrils Qiang et al (2012) PNAS Aβ Vandersteen et al (2012) FEBS Letters

Alzheimer s disease Cerebral amyloid angiopathy (CAA) CAA is a common clinical symptom of early-onset familial AD BRAIN Amyloid-beta (Aβ) plaques BLOOD Aβ deposits in

23 Alzheimer s disease Cerebral amyloid angiopathy (CAA) CAA is a common clinical symptom of early-onset familial AD BRAIN Amyloid-beta (Aβ) plaques BLOOD Aβ deposits in cerebral blood vessel walls Vessel walls prone to rupture Narrows lumina Intracerebral and subarachnoid bleeding Infarcts Periventricular oedema Bugiani et al

24 ThT fluorescence intensity of fibrils varies with mutation ThT fluorescence (a.u.) 6000 A 42 E22K A Time (h) Hubin et al. Cell Mol Life Sci

25 Wild type and E22K Aβ1-42 fibrils interact differently with conformation-dependent fluorescent probes (ThT and bis-ans) Bis-ANS fluorescence (a.u.) E22K WT Emission wavelength (nm) Bis-ANS: binds to hydrophobic patches LeVine H. (2002) Hubin et al. Cell Mol Life Sci

26 Wild type and E22K Aβ1-42 differ in solvent accessibility in the Aβ region comprising residues [20-34] 21d old Aβ fibrils D2O Pepsin Deuterated fibrils Proteolysis Dissolve fibrils to Aβ monomer Measure H-D exchange using ESI-MS H/D exchange - Mass Spectrometry with proteolytic fragmentation Hubin et al. Cell Mol Life Sci

27 Changes in secondary structure during Aβ aggregation can be monitored by ATR-FTIR Wild type Aβ1-42 Antiparallel oligomers 1630 cm-1 Absorbance (a.u.) A cm Time Oligomers h 0h 0.0 Monomer in TBS ph Wavenumber (cm-1) Cerf et al. Biochem J (2009); Sarroukh et al. BBA Biomembrane (2013) 27

28 Changes in secondary structure during Aβ aggregation can be monitored by ATR-FTIR Wild type Aβ1-42 Parallel fibrils Time Fibrils 1630 cm-1 Absorbance (a.u.) A d 7d h Oligomers h 0h 0.0 Monomer in TBS ph Wavenumber (cm-1) Cerf et al. Biochem J (2009); Sarroukh et al. BBA Biomembrane (2013) 28

29 ATR-FTIR reveals major change in secondary structure during transformation of wild type Aβ oligomers into fibrils Oligomers Anti-parallel β-sheet 1630 cm Parallel β-sheet Fibrils cm Absorbance (AU) Absorbance (AU) Wavenumber (cm-1) Cerf et al. Biochem J (2009); Sarroukh et al. BBA Biomembrane (2013) Wavenumber (cm-1) 29

30 A minor peak around 1695 cm-1, representative of antiparallel β sheets, persists during E22K Aβ1-42 aggregation Wild type Aβ1-42 Parallel fibrils Time Fibrils E22K Aβ1-42 Antiparallel fibrils Absorbance (a.u.) E22K A 1-42 Absorbance (a.u.) A d 21d 7d h Oligomers d 96h h 24h 0h 0h Monomer in TBS ph Wavenumber (cm-1) Cerf et al. Biochem J (2009); Sarroukh et al. BBA Biomembrane (2013) Wavenumber (cm-1) 30

31 The β-sheet index (1695/1630 intensities ratio) is proportional to the percentage of antiparallel arrangement of β-strands in a βsheet Anti-parallel β-sheet 1630 cm Parallel β-sheet Absorbance (AU) Absorbance (AU) cm High β-sheet index Wavenumber (cm-1) Low β-sheet index Wavenumber (cm-1) 31 Sarroukh R. et al (2010)

32 The β-sheet index (1695/1630 intensities ratio) is proportional to the percentage of antiparallel arrangement of β-strands in a βsheet Intensity (1695 cm -1 ) -index ratio = Intensity (1630 cm -1 ) Oligomers? E22K Aβ WT Aβ Time (h) Hubin et al. Cell Mol Life Sci

33 Centrifugation + Resuspension pellet Absorbance (a.u.) Running Stacking But: Fibrils and not remaining oligomers are responsible for observed antiparallel β-sheet conformation Total sample Pellet Wavelength (cm-1) Hubin et al. Cell Mol Life Sci

34 E22K Aβ1-42 fibrils can convert from antiparallel to parallel β-sheet structure in an acidic environment (switch from ph 7.4 to ph 2.0) -index ratio TBS ph mm HCl ph Time (h) Hubin et al. Cell Mol Life Sci

35 E22K Aβ1-42 fibrils can convert from antiparallel to parallel β-sheet structure in an acidic environment (switch from ph 7.4 to ph 2.0) -index ratio After 5d in TBS: switch to HCl TBS ph mm HCl ph Time (h) Hubin et al. Cell Mol Life Sci 2015 Protease resistant? Impact on BBB permeability? 35

36 Conclusions Physiologically relevant mixtures of Aβ peptides may behave differently from what you would expect based on their isolated characteristics. What is the perfect in vitro read-out for AD-related toxicity? Antiparallel β-sheet fibrils exist, are remarkably stable but can also be converted upon changing environmental conditions. 36

37 Acknowledgements Universiteit Twente Ellen Hubin Annelies Vandersteen Ine Segers Nolten Elske Cornelisse Universite Libre de Bruxelles Vincent Raussens Stephanie Deroo Rabia Sarroukh Erik Goortmaghtigh NIMR, UK Annalisa Pastore Geoff Kelly University of Sussex Louise Serpell Kyle Morris Thomas Williams Funding Vrije Universiteit Brussel Wim Jonckheere Kris Pauwels Katholieke Universiteit Leuven Frederic Rousseau Joost Schymkowitz Bart De Strooper Inna Kuperstein Iryna Benilova 37

38 E22K Aβ: more random coil and turn at the expense of β-structure. -index ratio 0.16 Absorbance (a.u.) E22K A 0.0 A Wavenumber (cm-1) Approx. 8 residues Aβ1-42 E22K Aβ1-42 β-structure α-helix 8 10 random turn

39 H/D exchange shows 20 % difference in accessibility of backbone amide hydrogens between wild type and E22K Aβ1-42 fibrils H D Amide II band -3 x 10 Absorbance N-H band area N-D band area cm

40 H/D exchange shows 20 % difference in accessibility of backbone amide hydrogens between wild type and E22K Aβ1-42 fibrils H D Unexchanged Amide (%) WT Aβ E22K Aβ Time (min)

41 H/D exchange monitored by ESI-MS and pepsin proteolysis reveals a higher solvent accessibility for the Italian mutant in the central region DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA K Peptide [1-19] [20-42] [35-42] WT E22K WT E22K WT E22K Backbone amide protons exchanged 14.0 ± ± ± ± ± ± 0.3 Protected NH/total 4/18 3.7/ / /22 3.9/7 3.6/7 41

42 Conclusions & perspectives: is there a link between the underlying β-sheet orientation of Aβ fibrils and disease-associated pathology? - E22K Aβ42 (and D23N Aβ40 and Aβ42) arrange into anti-parallel β-sheet fibrils. - E22K fibrils are structurally different from WT Aβ42. - Antiparallel β-sheet fibrils from E22K Aβ42 can rapidly (< 30 min) converse into parallel by changing the ph. Work in progress relation with CAA: Do antiparallel β-sheet fibrils disintegrate the blood-brain barrier blood-brain-barrieron-a-chip technology. 42

$7 Å X-ray fiber diffraction two$ reflections but no differences in

$5 Å E22K Aβ1-42 X-ray fibre diffraction$

43 In our search for additional confirmation Aβ1-42 Equator ~ 9.6 Å Meridian ~ 4.7 Å X-ray fiber diffraction two reflections but no differences in structure. Meridian ~ 4.7 Å Equator ~ 9.5 Å E22K Aβ1-42 X-ray fibre diffraction in collaboration with L. Serpell (University of Sussex, UK), Morris KL & Serpell L (2012). 43

44 Fibrils from E22K and WT Aβ42 are similarly toxic to astrocytes (and neuroblastoma cells). WT Aβ42 fibrils E22K Aβ42 fibrils 44

45 Mature E22K Aβ1-42 fibrils can convert from antiparallel to parallel structure E22K swich 0,25 -index 0,20 0,15 0,10 0,05 0, time (h) 45

46 E22K Aβ1-42 shows sustained (SDS-stable) oligomer formation. E22K Aβ1-42 WT Aβ1-42 Fibrils Higher oligomers Tetramer Trimer Monomer SDS-PAGE/WB (antibody 6E10) 46

Quantitative analysis consists of deconvolution of the amide I band into its primary components, followed by a curve fitting α-helix and random coil turn β-sheet Parallel β-sheets: 1 peak at 1630

47 Quantitative analysis consists of deconvolution of the amide I band into its primary components, followed by a curve fitting α-helix and random coil turn β-sheet Parallel β-sheets: 1 peak at 1630 cm-1 Antiparallel β-sheets: peaks at 1630 cm-1 and 1695 cm-1 Absorbance Wavenumber (cm-1) Goormaghtigh E. et al (1999) 1600 Beta :73.46% Random :11.32% Alpha :7.68% Turns :7.54% cm

48 E22K Aβ1-42 shows sustained (SDS-stable) oligomer formation. E22K Aβ1-42 WT Aβ1-42 Native PAGE/WB (antibody 6E10) 48

49 Attenuated Total Reflectance (ATR) FTIR spectroscopy has the capability to identify functional groups (C=O, C-H, N-H,...) Sample (Aβ peptide) spread onto internal reflection element Evanescent wave Incident infrared beam ATR (diamond) crystal Reflected beam Detector Interferogram IR spectrum Fourier transformation 49

50 Thioflavin T (ThT) is less easily locked in its excited comformation in antiparallel fibrils, leading to a lower final ThT fluorescence intensity 6000 Ab42 WT Ab42 E22K ThT fluorescence (a.u.) 5000 WT Aβ E22K Aβ Time (h) 53

51 Amide I and Amide II bands contain secondary structure information Ferritin Concanavalin A Amide I C=O vibration 1700 Amide II: N-H bending 1600 Wavenumber (cm-1)

52 Aggregation of Aβ peptide is predicted to vary with peptide length TANGO is an algorithm designed to predict the aggregation propensity of peptide sequences. Vandersteen et al (2012) J Biol Chem 287:

53 Antiparallel β-sheet architecture of Italian mutant E22K Aβ1-42 fibrils Figure adapted from Masuda et al. Bioorg & Med Chem (2005), FDX: Louise Serpell, Sussex University, UK 58

Amyloid pathways and diseasemodifying treatments for AD: what changes are necessary in new trials? John Hardy

Amyloid pathways and diseasemodifying treatments for AD: what changes are necessary in new trials? John Hardy Reta Lila Weston Research Laboratories Department of Molecular Neuroscience UCL Institute of