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

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

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

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

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

Triton X 100 and rafts Live cells immunofluorescence microscopy 0 min 1 min 2 min 3 min GPI CFP VSVG YFP 120 100 GPI CFP VSVG YFP Fluorescence (%) 80 60 40 20 0 0 20 40 60 80 100 120 140 160 180 Time (s)

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 120 100 GPI CFP VSVG YFP Fluorescence (%) 80 60 40 20 0 0 20 40 60 80 100 120 140 160 180 Time (s) Rinia & Kruijff (2001) FEBS Lett. 504, 194 Garner et al. (2008) Biophys. J. 94, 1326 1340

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

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

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

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

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

Distribution of BACE in lipid rafts 5% 30% 40% Fraction no. WT BACE 9 8 7 6 5 4 3 2 1 79 kda transmembrane Flotillin 41 kda N C Lipid raft

Distribution of BACE in lipid rafts 5% 30% 40% Fraction no. WT BACE 9 8 7 6 5 4 3 2 1 79 kda transmembrane Flotillin 41 kda N C 5% 30% 40% Fraction no. 9 8 7 6 5 4 3 2 1 GPI anchor GPI BACE 75 kda N C Flotillin 50 kda 37 kda Lipid raft

Processing of APP by wild type and GPI anchored BACE Full length APP β actin Un WT GPI 150 100 37.5 Density ( arbitrary units ) 60 50 40 30 20 10 0 Untrans. WT BACE GPI BACE sappα Un WT GPI 150 100 Density ( arbitrary units ) 80 70 60 50 40 30 20 10 0 Untrans. WT BACE GPI BACE

Processing of APP by wild type and GPI anchored BACE Full length APP β actin Un WT GPI 150 100 37.5 Density ( arbitrary units ) 60 50 40 30 20 10 0 Untrans. WT BACE GPI BACE sappα sappβ Un WT GPI Un WT GPI 150 100 150 100 Density ( arbitrary units ) Density ( arbitrary units ) 80 70 60 50 40 30 20 10 0 120 100 80 60 40 20 0 Untrans. WT BACE GPI BACE APP751+770 + 770 APP695 695 Untrans. WT BACE GPI BACE

Amyloid β secretion from cells expressing BACE Aβ 1 40 (ng/ml) Aβ 1 42 WT BACE <0.05 <0.05 GPI BACE 0.58 0.19 sappβ Aβ β secretase BACE1 Aβ γ secretase Presenilins

Disruption of rafts by cholesterol depletion decreases processing of APP by BACE 5% 30% 40% 5% 30% 40% GPI BACE 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 75 kda Flotillin Mock treated Lovastatin Methyl β cyclodextrin 50 kda 37 kda

Disruption of rafts by cholesterol depletion decreases processing of APP by BACE 5% 30% 40% 5% 30% 40% GPI BACE 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4 3 2 1 75 kda Flotillin 50 kda 37 kda Mock treated Lovastatin Methyl β cyclodextrin Treatment + + sappβ WT GPI WT GPI 150 100 Density ( arbitrary units ) 25 20 15 10 5 0 Mock treated Mock treated Treated WT BACE sappβ GPI BACE Cordy et al. (2003) PNAS 100, 11735 11740

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

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

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

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 (%) 50 40 30 20 10 Trypsin Time/min 0 + Perera & Hooper (2001) Current Biology 11, 519 523. Watt & Hooper (2003) TiBS 28, 406 410. 20 + 0 CuSO 4 + CuSO 4 + ZnSO 4 Metal ion (100µM) + MnSO 4

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?

Copper causes the prion protein to move out of detergent insoluble lipid rafts 40% 30% 5% 40% 30% 5% P 1 2 3 4 5 6 7 8 9 Fraction P 1 2 3 4 5 6 7 8 9 Cu 2+ + Cu 2+ Flotillin 1 TfR PrP C

Copper causes the prion protein to move out of detergent insoluble lipid rafts 40% 30% 5% 40% 30% 5% P 1 2 3 4 5 6 7 8 9 Fraction P 1 2 3 4 5 6 7 8 9 Cu 2+ + Cu 2+ Flotillin 1 TfR PrP C PrP N PrP oct PrP C N ~~~ GPI 23 26 51 91 231 PrP oct N ~~~ 23 26 231

Copper causes PrP C to redistribute into detergent soluble regions of the plasma membrane Cu 2+ Cu 2+ + TX 100 + Cu 2+ + Cu 2+ + TX 100 Top of cell Individual section Pixel Intensity (Arb U) % cell surface showing PrP staining 100 75 50 25 0 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, 5141 5153

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, 37241 Taylor et al. (2005) J. Cell Sci. 118, 5141 Taylor & Hooper (2007) Biochem. J. 402, 17 23 AP 2 AP180 Internalised Clathrin coated pit

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 + + +

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

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 +

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

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 +

Prion protein inhibits the proteolytic processing of APP SH SY5Y cells 45 kda PrP 30 kda 20.1 kda sappβ PrP + 120 97 kda APP 97 kda Relative amounts of sappβ 100 80 60 40 20 sappα 97 kda 0 PrP + PrP + sappβ β secretase BACE1 Aβ

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 120 100 80 60 40 20 0 PrP + 97 kda [Aβ] (ng ml 1 ) 2.25 2.00 1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 Aβ peptide Aβ1 40 Aβ1 42 n.d. PrP + PrP + sappβ β secretase BACE1 Aβ Aβ γ secretase Presenilins

Down regulation of prion protein increases Aβ secretion N2a cells 45 kda 30 kda PrP C 200 180 160 * Aβ 1 40 Aβ 1 42 45 kda 97 kda Actin APP Relative amounts of Aβ 140 120 100 80 60 40 20 97 kda Control PrP Scramble sirna sirna sappα Control PrP sirna 0 Scramble sirna Control PrP sirna Scramble sirna

Aβ levels are increased in prion protein null mice PrP APP Control Null 129OlaPrP / 45 kda 30 kda 97 kda Relative amounts of Aβ 160 140 120 100 80 60 40 20 0 Control ** Null * Aβ 1 40 Aβ 1 42 Parkin et al. (2007) PNAS 104, 11062 11067

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

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

Localisation of PrP C in cholesterol rich lipid rafts is required to inhibit BACE1 cleavage of APP PrP C N ~~~ GPI 23 26 51 91 CTM N 23 26 51 91 231 C 277 GPI N 23 26 51 91 Untreated Triton X 100 MβCD PrP C CTM Taylor et al. (2005) J. Cell Sci. 118, 5141 5153

Localisation of PrP C in cholesterol rich lipid rafts is required to inhibit BACE1 cleavage of APP PrP C N ~~~ GPI CTM 23 26 51 91 N 23 26 51 91 231 277 C PrP C GPI N 23 26 51 91 APP Untreated Triton X 100 MβCD sappα PrP C 140 120 sappβ PG14 A116V Dpl CTM Relative amounts of sappβ 100 80 60 40 20 Taylor et al. (2005) J. Cell Sci. 118, 5141 5153 0 Control wt CTM GPI P Un PrP C CTM GPI

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 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 ELISA rprp + + + rbace1 + + + Heparin +

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 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 rprp rbace1 Mem PrP IP ELISA + PrP IP + Hep + PrP IP Heparin + + APP + + + [Heparin] (nm) PrP Relative amounts of total sappβ 100 80 60 40 20 0 0 0.8 8 80 800 4000 0 + + + + + + 0 0.8 8 80 800 4000 0 + + + + + + [Heparin] (nm) PrP

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

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

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

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

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