Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES. Lipid rafts in neurodegenerative diseases. Nigel M.

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1 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases Nigel M. Hooper

2 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases 1. Alzheimer s disease role of rafts in the proteolytic production of amyloid β 2. Prion protein role of rafts in endocytosis and disease conversion 3. Prion protein and Alzheimer s prion protein inhibits amyloid β production mechanism of inhibition

3 Cholesterol rich, detergent insoluble membrane rafts Hooper (1998) Current Biology 8, R114

4 Cholesterol rich, detergent insoluble membrane rafts GPI anchored proteins cluster in rafts proteins can move laterally into and out of rafts characterised by relative insolubility in Triton X 100 at 4 o C Hooper (1998) Current Biology 8, R114 Taylor & Hooper (2007) Sem. Cell Develop. Biol. 18, 638

5 Triton X 100 and rafts Live cells immunofluorescence microscopy 0 min 1 min 2 min 3 min GPI CFP VSVG YFP GPI CFP VSVG YFP Fluorescence (%) Time (s)

6 Triton X 100 and rafts Live cells immunofluorescence microscopy 0 min 1 min 2 min 3 min GPI CFP Supported bilayer atomic force microscopy 0 min 5 min 15 min 30 min 60 min 0.075% VSVG YFP 0 min 60 min GPI CFP VSVG YFP Fluorescence (%) Time (s) Rinia & Kruijff (2001) FEBS Lett. 504, 194 Garner et al. (2008) Biophys. J. 94,

7 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases 1. Alzheimer s disease role of rafts in the proteolytic production of amyloid β 2. Prion protein role of rafts in endocytosis and disease conversion 3. Prion protein and Alzheimer s prion protein inhibits amyloid β production mechanism of inhibition

8 Cause, e.g. gene defect, ageing Alzheimer s disease: the amyloid cascade hypothesis Amyloid β peptide accumulation Dementia Amyloid plaque Alzheimer s brain

9 Proteolytic processing of the Alzheimer s amyloid precursor protein (APP) Amyloid plaques Aβ

10 Proteolytic processing of the Alzheimer s amyloid precursor protein (APP) N APP Aβ C Cytosol Membrane Amyloidogenic Pathway sappβ Aβ Amyloid plaques β secretase BACE1 Aβ γ secretase Presenilins

11 Proteolytic processing of the Alzheimer s amyloid precursor protein (APP) N APP Aβ C Cytosol Membrane Amyloidogenic Pathway Non Amyloidogenic Pathway sappα α secretase ADAM10 TACE sappβ β secretase BACE1 Aβ γ secretase Presenilins Aβ

12 APP processing and cholesterol rich lipid rafts Wolozin (2004) Neuron 41, 7

13 Distribution of BACE in lipid rafts 5% 30% 40% Fraction no. WT BACE kda transmembrane Flotillin 41 kda N C Lipid raft

14 Distribution of BACE in lipid rafts 5% 30% 40% Fraction no. WT BACE kda transmembrane Flotillin 41 kda N C 5% 30% 40% Fraction no GPI anchor GPI BACE 75 kda N C Flotillin 50 kda 37 kda Lipid raft

15 Processing of APP by wild type and GPI anchored BACE Full length APP β actin Un WT GPI Density ( arbitrary units ) Untrans. WT BACE GPI BACE sappα Un WT GPI Density ( arbitrary units ) Untrans. WT BACE GPI BACE

16 Processing of APP by wild type and GPI anchored BACE Full length APP β actin Un WT GPI Density ( arbitrary units ) Untrans. WT BACE GPI BACE sappα sappβ Un WT GPI Un WT GPI Density ( arbitrary units ) Density ( arbitrary units ) Untrans. WT BACE GPI BACE APP APP Untrans. WT BACE GPI BACE

17 Amyloid β secretion from cells expressing BACE Aβ 1 40 (ng/ml) Aβ 1 42 WT BACE <0.05 <0.05 GPI BACE sappβ Aβ β secretase BACE1 Aβ γ secretase Presenilins

18 Disruption of rafts by cholesterol depletion decreases processing of APP by BACE 5% 30% 40% 5% 30% 40% GPI BACE kda Flotillin Mock treated Lovastatin Methyl β cyclodextrin 50 kda 37 kda

19 Disruption of rafts by cholesterol depletion decreases processing of APP by BACE 5% 30% 40% 5% 30% 40% GPI BACE kda Flotillin 50 kda 37 kda Mock treated Lovastatin Methyl β cyclodextrin Treatment + + sappβ WT GPI WT GPI Density ( arbitrary units ) Mock treated Mock treated Treated WT BACE sappβ GPI BACE Cordy et al. (2003) PNAS 100,

20 APP processing and cholesterol rich lipid rafts Wolozin (2004) Neuron 41, 7

21 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases 1. Alzheimer s disease role of rafts in the proteolytic production of amyloid β 2. Prion protein role of rafts in endocytosis and disease conversion 3. Prion protein and Alzheimer s prion protein inhibits amyloid β production mechanism of inhibition

22 Prion Protein Transmissible spongiform encephalopathies (TSEs), e.g. Creutzfeldt Jakob disease (CJD), BSE, scrapie, etc Cellular PrP c undergoes conformational change to infectious PrP Sc PrP C PrP Sc

23 Role of PrP C in copper and zinc homeostasis Cu/Zn endocytosis

24 Role of PrP C in copper and zinc homeostasis Surface biotinylation Cu/Zn wtprp CuSO 4 endocytosis wtprp + CuSO 4 wtprp + ZnSO 4 wtprp + MnSO 4 PrP internalised (%) Trypsin Time/min 0 + Perera & Hooper (2001) Current Biology 11, Watt & Hooper (2003) TiBS 28, CuSO 4 + CuSO 4 + ZnSO 4 Metal ion (100µM) + MnSO 4

25 What is the mechanism of copper mediated endocytosis of PrP??? Adaptor? Cu Cu Adaptor? Cu Cu Lipid raft Clathrin Caveolin/Coat protein? Endosome? Lysosome? TGN?

26 Copper causes the prion protein to move out of detergent insoluble lipid rafts 40% 30% 5% 40% 30% 5% P Fraction P Cu 2+ + Cu 2+ Flotillin 1 TfR PrP C

27 Copper causes the prion protein to move out of detergent insoluble lipid rafts 40% 30% 5% 40% 30% 5% P Fraction P Cu 2+ + Cu 2+ Flotillin 1 TfR PrP C PrP N PrP oct PrP C N ~~~ GPI PrP oct N ~~~

28 Copper causes PrP C to redistribute into detergent soluble regions of the plasma membrane Cu 2+ Cu 2+ + TX Cu 2+ + Cu 2+ + TX 100 Top of cell Individual section Pixel Intensity (Arb U) % cell surface showing PrP staining p < 0.05 * Cu 2+ Cu 2+ +Cu 2+ +TX 100 +Cu 2+ +TX 100 Cell surface Cell surface Cell surface Cell surface Taylor et al. (2005) J. Cell Sci. 118,

29 PrP C translocates out of membrane rafts prior to its clathrin mediated endocytosis PrP C + Cu 2+ Cu Cu Cu LRP 1 Cu Lipid raft Non raft region Walmsley et al. (2003) J. Biol. Chem. 278, Taylor et al. (2005) J. Cell Sci. 118, 5141 Taylor & Hooper (2007) Biochem. J. 402, AP 2 AP180 Internalised Clathrin coated pit

30 Knockdown of the raft interacting partner of PrP C promotes it endocytosis Surface biotinylation Raft partner β actin Raft partner sirna + Trypsin Raft partner sirna + + +

31 Role of cellular compartments in the conformational conversion of PrP C to PrP Sc

32 Knockdown of the raft interacting partner of PrP C reduces its conversion to PrP Sc ScN2a cells 30 Proteinase K resistant PrP PrP C PrPSc sirna +

33 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases 1. Alzheimer s disease role of rafts in the proteolytic production of amyloid β 2. Prion protein role of rafts in endocytosis and disease conversion 3. Prion protein and Alzheimer s prion protein inhibits amyloid β production mechanism of inhibition

34 Alzheimer s and prion protein a connection? Cases of co existent Alzheimer s and CJD pathology Met/Val129 polymorphism a risk factor for Alzheimer s PrP C to PrP Sc conversion and production of Aβ occurs in lipid rafts APP and PrP C shed from the cell surface by zinc metalloproteases

35 Alzheimer s and prion protein a connection? Cases of co existent Alzheimer s and CJD pathology Met/Val129 polymorphism a risk factor for Alzheimer s PrP C to PrP Sc conversion and production of Aβ occurs in lipid rafts APP and PrP C shed from the cell surface by zinc metalloproteases Aim: does PrP C influence the proteolytic processing of APP?

36 Prion protein inhibits the proteolytic processing of APP SH SY5Y cells 45 kda PrP 30 kda 20.1 kda APP 97 kda sappα 97 kda PrP +

37 Prion protein inhibits the proteolytic processing of APP SH SY5Y cells 45 kda PrP 30 kda 20.1 kda sappβ PrP kda APP 97 kda Relative amounts of sappβ sappα 97 kda 0 PrP + PrP + sappβ β secretase BACE1 Aβ

38 Prion protein inhibits the proteolytic processing of APP SH SY5Y cells 45 kda PrP APP sappα 30 kda 20.1 kda 97 kda 97 kda sappβ PrP + Relative amounts of sappβ PrP + 97 kda [Aβ] (ng ml 1 ) Aβ peptide Aβ1 40 Aβ1 42 n.d. PrP + PrP + sappβ β secretase BACE1 Aβ Aβ γ secretase Presenilins

39 Down regulation of prion protein increases Aβ secretion N2a cells 45 kda 30 kda PrP C * Aβ 1 40 Aβ kda 97 kda Actin APP Relative amounts of Aβ kda Control PrP Scramble sirna sirna sappα Control PrP sirna 0 Scramble sirna Control PrP sirna Scramble sirna

40 Aβ levels are increased in prion protein null mice PrP APP Control Null 129OlaPrP / 45 kda 30 kda 97 kda Relative amounts of Aβ Control ** Null * Aβ 1 40 Aβ 1 42 Parkin et al. (2007) PNAS 104,

41 Prion protein inhibits the β secretase cleavage of APP and hence Aβ production N APP Aβ C Cytosol Plasma Membrane PrP Amyloidogenic Pathway sappβ Aβ β secretase BACE1 Aβ γ secretase Presenilins

42 Cause, e.g. gene defect, ageing Prion protein Prion protein prevents Alzheimer s disease by inhibiting the production of the amyloid β peptide Amyloid β peptide accumulation Dementia Amyloid plaque Alzheimer s brain

43 Localisation of PrP C in cholesterol rich lipid rafts is required to inhibit BACE1 cleavage of APP PrP C N ~~~ GPI CTM N C 277 GPI N Untreated Triton X 100 MβCD PrP C CTM Taylor et al. (2005) J. Cell Sci. 118,

44 Localisation of PrP C in cholesterol rich lipid rafts is required to inhibit BACE1 cleavage of APP PrP C N ~~~ GPI CTM N C PrP C GPI N APP Untreated Triton X 100 MβCD sappα PrP C sappβ PG14 A116V Dpl CTM Relative amounts of sappβ Taylor et al. (2005) J. Cell Sci. 118, Control wt CTM GPI P Un PrP C CTM GPI

45 Heparin disrupts the PrP C BACE1 interaction and reverses the inhibitory effect of PrP C on APP processing Co immunoprecipitation SH SY5Y PrP + BACE1 SH SY5Y PrP 30 PrP 66 BACE1 97 APP Absorbance at 450nm Mem PrP IP PrP IP + Hep PrP IP ELISA rprp rbace Heparin +

46 Heparin disrupts the PrP C BACE1 interaction and reverses the inhibitory effect of PrP C on APP processing Co immunoprecipitation SH SY5Y PrP + BACE1 SH SY5Y PrP Exogenous heparin added to cells 97 APP 30 PrP 97 sappα 66 BACE1 97 sappβ 97 Absorbance at 450nm rprp rbace1 Mem PrP IP ELISA + PrP IP + Hep + PrP IP Heparin + + APP [Heparin] (nm) PrP Relative amounts of total sappβ [Heparin] (nm) PrP

47 Models for how PrP C inhibits BACE1 cleavage of APP 1) BACE1 cleaves APP in rafts GAGs APP BACE1 Aβ Lipid raft

48 Models for how PrP C inhibits BACE1 cleavage of APP 1) BACE1 cleaves APP in rafts GAGs APP BACE1 Aβ Lipid raft 2) PrP C interacts with BACE1 and prevents it cleaving APP PrP C Aβ

49 Models for how PrP C inhibits BACE1 cleavage of APP 1) BACE1 cleaves APP in rafts GAGs APP BACE1 Aβ Lipid raft 2) PrP C interacts with BACE1 and prevents it cleaving APP PrP C Aβ 3) PrP C sequesters BACE1 in different rafts to where it cleaves APP Aβ Hooper & Turner (2008) TiBS in press

50 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Lipid rafts in neurodegenerative diseases Conclusions 1. Alzheimer s disease rafts are the site for the production of amyloid β 2. Prion protein PrP C translocates out of rafts prior to its endocytosis disrupting the raft localisation of PrP C reduces its conversion into PrP Sc 3. Prion protein and Alzheimer s PrP C inhibits amyloid β production at the level of the β secretase (BACE1) localisation of PrP C in rafts is required for the inhibition of BACE1 Taylor & Hooper (2007) Sem. Cell Develop. Biol. 18, 638

51 Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES Proteolysis Research Group Heledd Griffiths Isobel Morten David Taylor Nicole Watt Tony Turner Jo Cordy Ashley Garner Su Perera Ed Parkin Adrian Walmsley GlaxoSmithKline, Harlow, UK Ishrut Hussain Roslin Institute, Edinburgh, UK Jean Manson Herbert Baybutt Mayo Clinic, Jacksonville, USA Chris Eckman Liz Eckman Bioimaging suite Gareth Howell