AGGREGATES, FIBRILS AND AMYLOID DISEASES: INSIGHTS FROM STUDIES OF THE ABETA PEPTIDE

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1 AGGREGATES, FIBRILS AND AMYLOID DISEASES: INSIGHTS FROM STUDIES OF THE ABETA PEPTIDE Astrid Gräslund Department of Biochemistry and Biophysics Stockholm University Lorentz workshop, Leiden, April 13, 2015 Timeline: 1907 Alois Alzheimer named the disease (AD) 1984 Glenner & Wong identified the Aβ peptide in AD patients brain plaques (Glenner, G.G. and Wong C.W. Alzheimer s disease and Down s syndrome: sharing of a unique cerebrovascular fibril protein. Biochem. Biophys. Res. Comm. 122 (1984) ) 1992 Hardy & Higgins proposed the Amyloid Cascade Hypothesis. (Hardy, J.A. and Higgins G.A. Alzheimer s disease: The amyloid cascade hypothesis. Science 256 (1992) ) From the Hardy & Higgins paper 1992

2 A production by Amyloid Precursor Protein (APP) processing Apolipoprotein E α2 macroglobulin, Low density lipoprotein receptor related protein Insulin degrading enzyme Tanzi, R.E. and Bertram, L. Twenty years of the Alzheimer s disease amyloid hypothesis: a genetic perspective. Cell 120 (2005)

3 Selkoe, D.J. Preventing Alzheimer s disease. Science 337 (2012) Alzheimerology in 2020 Risk assessment at around age 50 and then every 10 years: History (emphasizing family history) and neurological exam Brief cognitive screen and neuropsychological testing Gene screen on AD risk chip (+ other familial dementias) Imaging Aβ scan, tau scan, MRI Blood Aβ antibody challenge : basal and evoked Aβ levels CSF assays for Aβ, tau, and other biomarkers Outcome: a numerical AD risk score From Holzman, D.M., Mandelkow, E., Selkoe, D.J. Alzheimer Disease in Cold Spring Har. Perspect. Med. 2012, 2:a011585

4 A new paradigm for managing AD based on the AD risk category into which a person falls Risk category 1. Presymptomatic subjects with no evidence of Aβ accumulation in the brain and high risk based on genetic, plasma, or CSF studies 2. Presymptomatic subjects with evidence of Aβ accumulation in the brain 3. Presymptomatic subjects with evidence of Aβ and tau/synuclein accumulation in the brain 4. Symptomatic subjects with evidence of Aβ and tau/synuclein accumulation in the brain Treatment 1. Aβ synthesis or oligomer inhibitor 2. Aβ synthesis or oligomer inhibitor Aβ vaccination (active or passive) 3. Aβ synthesis or oligomer inhibitor + Aβ vaccination (active or passive) Antitau/anti synuclein therapy 4. Aβ synthesis or oligomer inhibitor + Aβ vaccination (active or passive) Antitau/anti synuclein therapy Neuroprotective agents Symptomatic agents, e.g., cholinesterase inhibitors, memantine, other neurotransmitter modulators, other psychotropic treatments The Amyloid β (Aβ) peptide ca residues: DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40IA42 A (1-40) in aqueous solution is essentially unstructured. - or - secondary structures can be induced in two segments [appr. A (15-25) and A (29-35)] by interactions with certain molecules at certain ratios. Normal aggregation pathways and kinetics are affected by such interactions.

5 Monomer structural propensities and dynamics for A (1-40) Unstructured & mobile Unstructured & mobile Partly PII partly PII -strand -strand From NMR and CD studies of A in aqueous solution close to physiological conditions Danielsson et al. (2005) FEBS J. 272, Model of fibril structure Core of fibril Fibril axis Soluble segment essentially unstructured Potential Aβ aggregation process heterogeneous! disordered aggregate off-pathway β-hairpin β-structured oligomer mature fibril monomer disordered aggregate on-pathway pre-fibrillar β-structured aggregate Review: Wärmländer et al., ChemBioChem (2013) 14,

6 Structures of A in pdb (some of them) A 42 fibril (ssnmr) A 40 fibril (ssnmr) A 40 fibril (ssnmr) Lührs et al., PNAS (2005) 102: A 40 fibril from human brain tissue (ssnmr) Petkova et al., Biochemistry (2006) 45: Paravastu et al. PNAS (2008) 105: solution NMR: A 40 in complex with Affibody protein Lu et al., Cell (2013) 154: Hoyer et al. PNAS (2008) 105: Review: Abelein et al., (2014 ) J Biol Inorg Chem, 19: Thioflavin T (ThT) is a common probe for amyloid formation. It becomes highly fluorescent when bound to amyloid fibrils. Biancalana, M., Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 1804 (2010)

7 Structural model for fibril-tht interactions Cross-β structure of amyloid fibrils Major reflections in diffraction patterns of amyloid ThT binding in channel model to fibril-like β-sheets Cross-β structure a generic structure of amyloid fibrils Sunde, M., Blake, C. From globular to the fibrous state: protein structure and strucural conversion in amyloid formation. Quart. Rev. Biophys. 31 (1998) 1-39.

8 Kinetic studies of Aβ fibrillization. Synchronous measurements of Aβ(1-40) fibrillation by ThT fluorescence and CD. Slow random coil to β strand conversion. Transition occurs around 60 min. Conditions: 10 μm peptide, 10 mm Pi, ph 7.2, 37 C, mechanical agitation. Luo, J., Wärmländer, S., Gräslund, A., Abrahams, J.P. Alzheimer peptides aggregate into transient nanoglobules that nucleate fibrils. Biochemistry 53 (2014) Left panel: Relative intensities of NMR HSQC 15N-H crosspeaks after 0.5 hr (blue), 1.7 hr (green), 3.2 hr (cyan). Right panel: Parallel measurements of ThT fluorescence, CD and cell toxicity

9 Atomic Force Microscopy pictures taken during fibrillation. Samples taken from ThT assay, after (top) 20, 40, 57 min, (bottom) 71, 91, 340 min. ThT transition occurs around 60 min. Schematic view of fibrillation of Aβ(1-40) fibrillation pathways, involving monomers, prefibrillar oligomers (PFOs), fibrillar oligomers (FOs) and fibrils

10 Modulation of A aggregation Small molecules: Lacmoid Congo red Surfactants: SDS/LiDS Metal ions: Zn 2+ Cu 2+, Al 3+, Ca 2+ ph dependence / Li Membrane mimetics: + DHPC micelles DHPC/DMPC bicelles Small peptides Charge mirroring peptides Aggregation prone middle segment KLVFFA Small molecule aggregation modulators Lacmoid Congo red Reported small molecule compounds affecting A oligomerization and/or fibrillization: Necula et al., J Biol Chem (2007), 282,

11 Lacmoid inhibits A aggregation A alone 10:1 Lacmoid:A CD Lacmoid TEM Abelein et al., Biochemistry (2012) 51, Lacmoid prevents a -structure formation and thereby inhibits fibril formation NMR Signal Attenuation A alone A lac 1:1 A lac 1:2 A lac 1:4 1 H- 15 N-HSQC NMR spectra of 40 µm Aβ (1-40) with different concentrations of lacmoid at 3 C.

12 NMR Signal Attenuation suggests Chemical Exchange [lacmoid] Relative intensity lacmoid:a 1:1 (red) 2:1 (green), 4:1 (dark blue), 6:1 (orange), 10:1 (turquoise) residue Two site conformational exchange A,ω A, p A k ex B,ω B, p B Palmer III et al., Methods Enyzmol., 2001, 339, Mittermaier & Kay, Trends in Biochem Science, 2009, 34, Hansen et al., J Biomol NMR, 2008, 41, NMR visible invisible NMR relaxation dispersion experiments magnetization evolution time 1/2 Hz 15 N 180 2n Carr-Purcell-Meiboom-Gill (CPMG) pulse train scheme Information from relaxation dispersion: - structural information: Δ - thermodynamic information: - dynamic information: of the system: of the molecule: from two global fit parameters, and, and two resdiue-specific fit parameters, Δ and Palmer III et al., Methods Enyzmol., 2001, 339, Mittermaier & Kay, Trends in Biochem Science, 2009, 34, Hansen et al., J Biomol NMR, 2008, 41,

13 A Formation of transient dynamic co-aggregates 15 N CPMG relaxation dispersion profiles for selected residues at 3 C of pure A 40 (red) and 1:2 A 40 :lacmoid (green) and 3:10 A 40 :CR (blue). lacmoid Congo red Overall effect of chemcial exchange including both hydrophobic parts as well as the N-terminus. Absolute values of 15 N chemical shift changes from relaxation dispersion fitting for 1:2 A 40 :lacmoid (left) and 3:10 A 40 :CR molar ratios (right), respectively. Aggregation modulation by small molecules Exchange rate k ex Bound state population p B Estimated hydrodynamic radius Suggested major structural state of Aβ (from CD) Lacmoid 1000 ± 100 s ± 0.1 % 4.3 nm Mainly unstructured Congo red 1050 ± 150 s ± 0.4 % 2.8 nm More β-structure Lacmoid + k, 11 s -1 k amorphous material k 1000 s -1 Congo red + k, k 1050 s -1 k k coaggregated fibrils

14 Three-state transition of Aβ induced by surfactant SDS Circular Dichroism: Unstructured monomer β-structure accompanied by loss of NMR signal Wahlström et al. (2008). FEBS J. 275, Far-UV CD spectrum of 150 µm Aβ in 10 mm sodium-phosphate buffer, ph 7.4, in 0 15 mm SDS at 28 C Abelein et al., J Biol Chem (2013) 288, SDS micelle Two α-helices induced in residues and Jarvet et al. (2007) J. Biomol. NMR 39, Aggegation kinetics monitored by ThT fluorescence / (time of halfcompletion of aggregation) without SDS 15.5 ± 2.2 h 0.9 mm SDS 1.9 ± 0.2 h 2.75 mm SDS 1.2 ± 0.2 h -structure is the most aggregation prone state Kinetic traces of 150 µm Aβ aggregation without (black) and in the presence of 0.9 (red), 2.75 (blue) and 12.5 mm (green) SDS monitored by ThT fluorescence at 28 C without agitation.

15 Structure of A -SDS co-aggregates by SAXS Pair distance distribution function p(r): SAXS data of 150 µm Aβ in the presence of 2.75 mm SDS at different time points SAXS model: Fitting parameters as a function of time: >350 Å r cyl ~ 75 Å (fixed diameter) p cyl 2r cyl ~ 60 Å p sph = 1- p cyl p cyl Time-dependent formation of fibrillar structures by TEM Above cmc TEM images of 150 µm Aβ incubated at 28 C without (A) and with 0.9 (B), 2.75 (C) and 10 mm (D) SDS.

16 Surfactant-A co-aggregates show dynamic exchange LiDS Lacmoid Congo red Exchange rate 1100 ± 150 s ± 100 s ± 150 s -1 k ex Bound state 1.0 ± 0.1 % 1.0 ± 0.1 % 3.2 ± 0.4 % population p B Estimated hydrodynamic radius Suggested major structural state of Aβ (from CD) 6.0 nm 4.3 nm 2.8 nm More β-structure Mainly unstructured More β-structure 150 µm A with and without 1.15 mm LiDS at 10 C and 700 MHz Hydrophobic parts interact with the hydrophobic part of surfactant-a co-aggregates Similar exchange dynamics system, yet different structural states of the coaggregated A. Conclusions: organic molecules and surfactants compounds + monomeric peptide dynamic co aggregates co aggregated material ~ 60 Å > 350 Å k, k LiDS/SDS k 1100 s 1 k [min 1 /h 1 ] [min 1 /h 1 ] Lacmoid + k, 11 s 1 k amorphous material k 1000 s 1 Congo red + k, k 1050 s 1 k k co aggregated fibrils

17 Some important questions: For Aβ amyloid formation: Effects of membranes? Metal ions? For Aβ in cell biology: Role of spreading between neurons (prion like)? (Nath, S., Agholme, L., Kurudenkandy, F.R., Granseth, B., Marcusson, J., Hallbeck, M. Spreading of neurodegenerative pathology via neuron to neuron transmission of beta amyloid. J. Neurosci. 32 (2012) 8767) For the role of Aβ in Alzheimer s disease: Role of Tau? Other mechanisms? What are the toxic mechanisms? From: Selkoe, D.J. Alzheimer s disease: genotypes, phenotypes and treatments. Science 275 (1997) : Suggested influencing factors: dietary cholesterol, metabolic disorders, ApoE lipoprotein, lowdensity lipoprotein receptors, insufficient lysosomal proteosomal and insulin degrading enzymes... Conclusion: at least four broad classes of AD drugs could be envisioned: (i) protease inhibitors that partially decrease the activities of the enzymes (β and γ secretase) that cleave Aβ from APP; (ii) compounds that bind to extracellular Aβ and prevent its aggregation into cytotoxic amyloid fibrils; (iii) brain specific anti inflammatory drugs that block the microglial activation, cytokine release, and acute phase response that occur in affected brain regions; and (iv) compounds such as antioxidants, neuronal calcium channel blockers, or antiapoptotic agents that interfere with the mechanisms of Aβ triggered neurotoxicicity Acknowledgements Cooperations: Axel Abelein, Jens Danielsson, Sebastian Wärmländer (Stockholm University) Jinghui Luo, Jan Pieter Abrahams (Leiden University) Surfactant A project: Jørn Kaspersen Søren Nielsen Grethe Jensen Gunna Christiansen Jan Skov Pedersen Daniel Otzen, Aarhus University Lacmoid/Congo red project: Christofer Lendel (The Royal Inst. of Technology, Stockholm ) Lisa Lang (Stockholm University) Christopher M. Dobson, Benedetta Bolognesi (Cambridge University)