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Martin Hof

Single molecule fluorescence clarifies the role of monosialoganglioside GM1 and sphingomyelin in the in-membrane oligomerization of β- amyloid. Martin Hof. Sugar head. ceramide. http://hof-fluorescence-group.weebly.com/. Lipid Topics

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Martin Hof

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  1. Single molecule fluorescence clarifies the role of monosialoganglioside GM1 and sphingomyelin in the in-membrane oligomerization of β-amyloid Martin Hof Sugar head ceramide http://hof-fluorescence-group.weebly.com/

  2. Lipid Topics • Size and character of ganglioside GM1 nano-clusters • Sphingomyelin/cholesterol nanoheterogeneities; influence of GM1 • In membrane oligomerisation of β-amyloid • Fluorescence Methods • Monte Carlo simulations of FluorescenceLifetime Imaging -FörsterResonanceEnergyTransfer • Calibration free Fluorescence Correlation Spectroscopy: Z-scan FCS • Fluorescence Correlation Cross Spectroscopy Šachl, Humpolíčková; Štefl, Johansson, Hof 2011, Biophys. J., 101, L60-L62. Amaro, Šachl, Jurkiewicz, Coutinho, Prieto, Hof Biophysical Journal. 107, 12 (2014), 2751-2760.

  3. Lipid Topics • Size and character of ganglioside GM1 nano-clusters • Sphingomyelin/cholesterol nanoheterogeneities; influence of GM1 • In membrane oligomerisation of β-amyloid • Fluorescence Methods • Monte Carlo simulations of FluorescenceLifetime Imaging -FörsterResonanceEnergyTransfer • Calibration free Fluorescence Correlation Spectroscopy: Z-scan FCS • Fluorescence Correlation Cross Spectroscopy Benda, Beneš, Mareček, Lhotský, Hermens, Hof Langmuir, 2003. 19(10) 7. Štefl, Kułakowska, Hof 2009, Biophysical Journal, 97(3): p. L1-L3 for 2-colour PIE Z-scan FCS

  4. Lipid Topics • Size and character of ganglioside GM1 nano-clusters • Sphingomyelin/cholesterol nanoheterogeneities; influence of GM1 • In membrane oligomerisation of β-amyloid • Fluorescence Methods • Monte Carlo simulations of FluorescenceLifetime Imaging -FörsterResonanceEnergyTransfer • Calibration free Fluorescence Correlation Spectroscopy: Z-scan FCS • Fluorescence Correlation Cross Spectroscopy Schwille, Meyer-Almes, Rigler, Biophys J 72 (1997) 1878-1886. Bacia, Kim, Schwille Nat Methods 3 (2006) 83-89

  5. Main theme and motivation In-membrane oligomerization of -amyloid (Aβ) monomers • Amyloidosis: • Haemodialysis (b2-microglobulin) • Type II diabetes (IAPP) • Alzheimer (b-amyloid) • Parkinson’s (a-synuclein) b-amyloidis a 39-to-43 a.a. peptide produced by secretase cleavage of amyloid-precursor peptide (APP) Early stage oligomers • memory impairment • disrupting synaptic signalling • neuron toxicity Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  6. Main theme and motivation In-membrane oligomerization of -amyloid (Aβ) monomers • Amyloidosis: • Haemodialysis (b2-microglobulin) • Type II diabetes (IAPP) • Alzheimer (b-amyloid) • Parkinson’s (a-synuclein) b-amyloidis a 39-to-43 a.a. peptide produced by secretase cleavage of amyloid-precursor peptide (APP) Early stage oligomers: What are the factors which catalyse or prevent oligomerisation? Membranes? Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  7. In-membrane oligomerization of -amyloid (Aβ) monomers • Cellular membrane can be a catalyst for the oligomerisation of Ab • Lipid specificity? GM1 was identified to seed oligomerisation Physiological relevance? • Concentration of AbnanoM [Ab] in µM range • GM1 in neuronal cells max 2-4% [Ganglioside GM1] above 20% • Domains in plasma membranes are,if existing at all,transient and < 20nm Liquid ordered phase (Lo) bilayers“raft models” Sugar head ceramide Kakio, Nishimoto, Yanagisawa, Kozutsumi, Matsuzaki Biochemistry 2002, 41, 7385; Yagi-Utsumi, Matsuo, Yanagisawa, Gekko, Kato, K. Int. J. Alzheimer's Dis. 2011, 2011, 925073. Yanagisawa, Odaka, Suzuki, Ihara, Nat. Med. 1995, 1, 1062.

  8. Central question • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Create lipid bilayers with composition ofhigher physiological relevance • Lower GM1levels to2-4% • avoid Liquid ordered (Lo) phase • Lower Ab concentration to nanoM Single molecule fluorescence to follow the oligomerisation of Aβ monomers dye- Ab40 Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  9. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Create lipid bilayers with composition ofhigher physiological relevance • Are there ”nanodomains” • in these model membranes? GM1 phase coexistence 25% ld +lo Ffluid ld A.Smith and J.Freed, J.Phys.Chem.B (2009) 113

  10. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Create lipid bilayers with composition ofhigher physiological relevance Novel FRET approach for determining nanodomains between 2 nm - 50 nm “Monte Carlo Simulations of Fluorescence Lifetime Imaging Microscopy - Förster Resonance Energy Transfer (MC/FLIM-FRET)” Šachl, Humpolíčková; Štefl, Johansson, Hof 2011, Biophys. J., 101, L60 Amaro, Šachl, Jurkiewicz, Coutinho, Prieto, Hof Biophysical Journal. 107, 12 (2014), 2751 GM1 phase coexistence 25%

  11. Monte Carlo simulations of Fluorescence Lifetime Imaging -Förster Resonance Energy Transfer Foerster 1948 Annalen d. Physik 6. Folge, 2, 11355-75

  12. Monte Carlo simulations of Fluorescence Lifetime Imaging -Förster Resonance Energy Transfer Uniform DA distribution Non-uniform DA distribution • I. Donors accumulate in domains, acceptors are excluded I. Loura, Prieto. 2000. J. Phys.Chem. B. 104:6911–6919.

  13. Monte Carlo simulations of Fluorescence Lifetime Imaging -Förster Resonance Energy Transfer Uniform DA distribution Non-uniform DA distribution • I. Donors accumulate in domains, acceptors are excluded II. • II. Donors and acceptors accumulate in domains Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  14. Monte Carlo simulations of Fluorescence Lifetime Imaging -Förster Resonance Energy Transfer Uniform DA distribution Non-uniform DA distribution Monte Carlo simulations generate fluorescence decay • Optimized domain radius • total domain area Šachl, Humpolíčková; Štefl, Johansson, Hof 2011, Biophys. J., 101, L60-L62.

  15. Size and character of ganglioside GM1 nano-clusters Test system for the FLIM-FRET approach: GM1 clustering in model lipid bilayers POPC Evidence for GM1 clusters in POPC supported lipid bilayers by AFM Shi, Yang, Kataoka, Zhang, Diaz, Cremer, JACS 2007 129 (18) Sugar head ceramide Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  16. D A Size and character of ganglioside GM1 nano-clusters GM1 clustering in free standing lipid bilayers (GUV’s) II. Non-uniform DA distribution A-GM1 D-GM1 Sugar head • GM1 clusters in free standing bilayers have similar sizes as determined by AFM/SPB’s • Chol has no effect on clustering ceramide Šachl, Amaro, Aydogan, Koukalová, Mikhalyov, Boldyrev, Humpolícková, Hof 2015, BBA-Molecular Cell Research, 1853(4), 850-7

  17. D Size and character of ganglioside GM1 nano-clusters • FLIM-FRETand Monte Carlo Simulations I. Non-uniform DA distribution • No significant change in FRET • DiD is able to diffuse through domains • Z-scan FCS does not give evidence for Lo (liquid-ordered) character Sugar head D-GM1 DiD ceramide Šachl, Amaro, Aydogan, Koukalová, Mikhalyov, Boldyrev, Humpolícková, Hof 2015, BBA-Molecular Cell Research, 1853(4), 850-7

  18. D Size and character of ganglioside GM1 nano-clusters • FLIM-FRETand Monte Carlo Simulations I. Non-uniform DA distribution • GM1 nano-clusters are fluid Sugar head D-GM1 DiD ceramide Šachl, Amaro, Aydogan, Koukalová, Mikhalyov, Boldyrev, Humpolícková, Hof 2015, BBA-Molecular Cell Research, 1853(4), 850-7

  19. Create lipid bilayers with composition ofhigher physiological relevance • Are there ”nanodomains” • in these model membranes? GM1 phase coexistence 25% A.Smith and J.Freed, J.Phys.Chem.B (2009) 113

  20. D A Sphingomyelin/cholesterol nano-heterogeneities • FLIM-FRETand Monte Carlo Simulations II. Non-uniform DA distribution A-GM1 D-GM1 Please note: Mikhalyov, Gretskaya, Johansson, Chem. Phys. Lipids 2009, 159, 38. Mikhalyov, Molotkovsky, J. G. Russian Journal of Bioorg. Chem.2003, 29, 168….Headgroup labeled GM1

  21. D A Sphingomyelin/cholesterol nanoheterogeneities; influence of GM1 • FLIM-FRETand Monte Carlo Simulations II. Non-uniform DA distribution Sugar head A-GM1 D-GM1 ceramide Amaro, Šachl, Aydogan, Mikhalyov, Vácha, Hof, Angewandte Chemie, 2016 55 9411-9415

  22. Sphingomyelin/cholesterol nanoheterogeneities; influence of GM1 • FRET: GM1 accumulates in “domains”, DiD diffuses through • 2-colour Z-scan FCS usingGM1 and DiD • Solid state wideline and high resolution magic angle spinning NMR Nano-heterogeneities are still fluid and disordered, even with addition of GM1 Amaro, Šachl, Aydogan, Mikhalyov, Vácha, Hof, Angewandte Chemie, 2016 55 9411-9415; paper with alternative labels and NMR is in preparation

  23. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Create lipid bilayers with composition ofhigher physiological relevance • Model system with nano-heterogeneities that are disorderd and dynamiccompared to Lo domains, even with addition of GM1 GM1 phase coexistence Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  24. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? Single molecule fluorescence to follow the oligomerisation of Aβ40 monomers at low nM concentrations dye- Ab40 Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  25. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Mobility via 2-colour Z-scan FCS • DiD(1:200 000) • g-Aβ4012nM solution • (fraction of 1:4000 max) • Aβis bound to bilayer and senses membrane fluidity Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  26. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Mobility via 2-colour Z-scan FCS • DiD(1:200 000) • g-GM1 (1:200 000) • g-Aβ4012nM solution • (fraction of 1:4000 max) Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  27. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Mobility via 2-colour Z-scan FCS • Diffusion of Aβ slows down with time > oligomerisation? Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  28. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Oligomer formation via FCCS • 12 nM solution (1:1) of g- / r-Ab40 dye- Ab40 dye- Ab40 • g- / r- : HiLyte™ Fluor dyes Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  29. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Oligomer formation via FCCS • Spontaneous oligomerisation of Aβ40 !! Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  30. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Oligomer formation via FCCS CCF • Sphingomyelin triggers the oligomerisation of Aβ40 Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  31. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Oligomer formation via FCCS CCF • Sphingomyelin triggers the oligomerisation of Aβ40 Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  32. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Mobility via 2-colour Z-scan FCS • DiD(1:200 000) • g-GM1 (1:200 000) • g-Aβ4012nM solution • (fraction of 1:4000 max) Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  33. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Oligomer formation via FCCS • GM1 counteracts effect of SPMOligomerisation of Aβ40 is prevented!! Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  34. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Mobility via 2-colour Z-scan FCS • At physiological levels, GM1does not seed formation of Aβ40 oligomers Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  35. In-membrane oligomerization of -amyloid (Aβ) monomers • Does GM1 seed Aβ40 oligomerisation • under more physiological conditions? • Physiological levels of GM1 do not seedoligomer formation. • Sphingomyelin is a key trigger of oligomerisation • Molecular evidence for GM1 as inhibitor of oligomer formation Amaro, Šachl, Aydogan, Mikhalyov, Vácha, Hof, Angewandte Chemie, 2016 55 9411-9415

  36. In-membrane oligomerization of -amyloid (Aβ) monomers • All-atom Molecular Dynamic simulations • -Sphingomyelinpromotes change of conformation of Ab40 > higher aggregation propensity • - specific binding of Ab40 to headgroup ofGM1 sequestersthe peptide thus counteracting the effect of sphingomyelin Amaro, Šachl, Aydogan, Mikhalyov, Vácha, Hof, Angewandte Chemie, 2016 55 9411-9415

  37. Our molecular insights showing that • a) Sphingomyelin triggers oligomerisation • b) GM1 inhibits oligomer formation • clarify the role of GM1 in Aβ40 oligomerisation • support experimental results on the protective role of GM1 in cell cultures and rat models of Alzheimer’s disease • might be connected to decrease of GM1 levels with aging a disease progression Department of Biophysical Chemistry J. Heyrovský Institute of Physical Chemistry, A.S.C.R.

  38. Acknowledgements • Mariana Amaro • see her at 2-POS • RadekSachl • GokcanAydogan • Robert Vacha(Brno) MD simulations see him at 61-POS • Ilya Mikhalyov(Moscow) synthesising labelled GM1 molecules • Gerhard Groebner(Umea) for NMR on nanoheterogenities • Funding by Czech Academy of Sciences (Praemium Academiae) till 12/2017

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