university of manchester uk · 2017. 2. 3. · app & prp c shed from the cell surface by the...

Prion protein as a therapeutic target in Alzheimer’s diseasePrion protein as a therapeutic target in Alzheimer’s disease

Nigel Hooper

University of ManchesterUK

Prion protein and Alzheimer’s Prion protein and Alzheimer’s –– a connection?a connection?

- causative agent of transmissible spongiform encephalopathies (TSEs), e.g. Creutzfeldt-Jakob disease (CJD), BSE, scrapie, etc- cellular PrPC undergoes conformational change to infectious PrPSc

• Metal homeostasis• Anti-oxidant response• Cell signalling• Ligand/protein binding

� Cases of co-existent Alzheimer’s and CJD pathology(Hainfellner et al. 1998; Voigtlander et al. 2001)

� Met/Val129 polymorphism in PRNP a risk factor for Alzheimer’s(Dermaut et al. 2003; Riemenschneider et al. 2004)

� PrPC to PrPSc conversion & Aβ production occur in cholesterol-rich lipid rafts(Cordy et al. 2003; Ehehalt et al. 2003; Taraboulos et al. 1995)

� APP & PrPC shed from the cell surface by the zinc metalloprotease ADAM10(Parvathy et al. 1998; Parkin et al. 2004; Taylor et al. 2009)

� PrPC inhibits the β-secretase cleavage of APP(Parkin et al. 2007; Griffiths et al. 2011)

• Ligand/protein binding

Cellular prion Cellular prion protein protein ((PrPPrPCC): ): a receptor for Aa receptor for Aββ oligomersoligomers

• Screened expression library of 225,000 mouse brain cDNAs225,000 mouse brain cDNAs

• PrPC the only specific, high-affinity receptor for Aβ oligomers (kd ~ 0.4 nM)

Cisse & Mucke (2009) Nature 457, 1090-1

Aβ natively

fibrilsprotofibrils

Deposit in senile plaques

Neurotoxic forms Neurotoxic forms of of amyloidamyloid--ββ

Fibrils

unfoldedmonomer

low-n 6mer 12mer(Aβ*56)

ADDLs(>56 kDa)

APF globulomer

Bind to neurons

Memory impairments

Toxicity and cell death

Alzheimer’s Disease

Rushworth & Hooper (2011) Int. J. Alzheimers Dis. Article ID 603052

Oligomers

Biophysical and immunological characterisation of ABiophysical and immunological characterisation of Aββ11--4242

oligomers (ADDLs)oligomers (ADDLs)

Tomic et al., 2009

Conformation-dependent antibodies

6E10(Aβ)

OC (fibrillar epitope)

A11 (pre-fibrillar epitope)

100 nm

monomer oligomer

5-6 nm Ø 10

17

26

55

170

﹜kDa

Aβoligomers

< tetramer< trimer

< monomer

Conformation-dependent antibodies

(Charlie Glabe, California)

A11 (pre-fibrillar

oligomers)

OC (fibrillar

oligomers)“Levels of soluble fibrillar oligomers detected by OC antibody are significantly elevated in multiple brain regions of AD patients”

Rushworth et al. (2013) J. Biol. Chem. 288, 8935

PrPPrPCC is required for the cellular binding of is required for the cellular binding of fibrillarfibrillar AAββ oligomers oligomers and activation of Fyn kinaseand activation of Fyn kinase

Rat hippocampal neurons

Rushworth et al. (2013) J Biol Chem 288, 8935

PrPPrPCC is GPI anchored and localised is GPI anchored and localised in cholesterolin cholesterol--rich, rich, detergentdetergent--resistant lipid raftsresistant lipid rafts

Fyn

PrPC

Disruption of lipid rafts prevents Disruption of lipid rafts prevents AAββ oligomeroligomer binding and binding and activation of Fyn activation of Fyn kinasekinase

Rushworth et al. (2013) J. Biol. Chem. 288, 8935

MβCD = methyl β-cyclodextrin, sequesters cholesterol

The transmembrane LRP1 is required for the binding and The transmembrane LRP1 is required for the binding and cytotoxicity of Acytotoxicity of Aββ oligomersoligomers

LRP1 = low-density

Rushworth et al. (2013) J. Biol. Chem. 288, 8935

LRP1 = low-density lipoprotein receptor-related protein 1

RAP = receptor-associated protein

AAββ oligomersoligomers promote the clustering of promote the clustering of PrPPrPCC and LRP1and LRP1

Rushworth et al. (2013) J. Biol. Chem. 288, 8935

PrPC

LRP1

Fy

n

lipid raft

Neuroprotection?

Membrane

Neurotoxic

Fy

n

P

Fy

n

Aββββ oligomers

Metabotropic glutamate receptor 5 Metabotropic glutamate receptor 5 (mGluR5) is (mGluR5) is a a coreceptorcoreceptorwith with PrPPrPCC for Afor Aβ β oligomersoligomers

Neuron 79, 887-902

AAββ oligomeroligomer binding stimulates the clustering of specific neuronalbinding stimulates the clustering of specific neuronalreceptors into aberrant signalling platforms at the synapsereceptors into aberrant signalling platforms at the synapse

Normal Alzheimer’s disease

Rushworth & Hooper (2011) Int. J. Alzheimers Dis. Article ID 603052

PrPPrPCC as a therapeutic target in Alzheimer’s disease?as a therapeutic target in Alzheimer’s disease?

But… But… PrPPrPCC has several physiological roles has several physiological roles

PrPC

PrPPrPCC inhibits the inhibits the amyloidogenicamyloidogenic processing of the amyloid processing of the amyloid precursor protein (APP)precursor protein (APP)

α-secretase β-secretase

n = 24

PrPPrPCC is reduced in sporadic Alzheimer’s brain and inversely is reduced in sporadic Alzheimer’s brain and inversely correlates with correlates with BACE1 activity, ABACE1 activity, Aββ and and BraakBraak stage stage

BACE1 activity

Whitehouse et al. (2013) PLoS ONE 8: e59554

Soluble Aβ Insoluble Aβ Braak stage

Prion protein in human hippocampus declines with agePrion protein in human hippocampus declines with age

Whitehouse et al. (2010) J. Alz. Dis. 22, 1023-1031

PrPPrPCC facilitates neuronal facilitates neuronal zinc zinc uptake via AMPA receptorsuptake via AMPA receptors

PrPC

Actin

siRNA - +

30

43

Zn

Zn + siRNA0.4

0.6

0.8

1

1.2

Rat hippocampal neurons

Ne

wp

ort

gre

en

flu

ore

sce

nce (

rela

tive

in

cre

ase

)

New

port

gre

en f

luore

scence

(Rela

tive incre

ase)

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

Time (min)

Zn + CNQX

Zn

Zn + siRNA

Watt et al. (2012) Nat. Commun. 3:1134

Time (min)

Zn + siRNA

0.2

0.4

05 10 15 20 25 30

Ne

wp

ort

gre

en

flu

ore

sce

nce (

rela

tive

PrPC siRNAPrPC

Newport green staining

Time (min)

New

port

gre

en f

luore

scence

(Rela

tive incre

ase)

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

Zn

Zn + IEM-1460

GluA2-lacking, Ca2+ and Zn2+

permeable AMPAR

Potential mechanism Potential mechanism by which by which prion protein facilitates zinc prion protein facilitates zinc uptake into uptake into cells through the AMPA receptorcells through the AMPA receptor

Probe: GluA1

Probe: GluA2

Probe: PrPC

kDa

100

90

30

B

Watt et al. (2012) Nat. Commun. 3:1134 Watt et al. (2013) Prion 7, 203-208

Protein tyrosine phosphatase

A

0

100

200

300

400

500

600

700

800

900

WT PrP-/-

pM

PO

4-re

lease/µ

g/m

in

0

50

100

150

200

250

300

Un PrPC

B

**

*

pM

PO

4-re

lease/µ

g/m

in

SH-SY5Y cells PrP null mice

AβAβ oligomers

Neuronal death

Alzheimer’s Disease

IImpact of reduced prion protein on zinc metabolism in mpact of reduced prion protein on zinc metabolism in sporadic Alzheimer’s disease and ageingsporadic Alzheimer’s disease and ageing

23

3) � binding of Aβ oligomers to PrPC further � zinc uptake

2) � extracellular Zn promotes Aβ oligomer formation and neurotoxicity

1) � intracellular protein tyrosine phosphatase activity leading to neurotoxicity

Zn

Normal

PrPc

� Protein tyrosine phosphatase

Toxicity

Neuronal death and dysfunction

3

1

Zn

AMPA receptor

� Protein tyrosine phosphatase

Prion protein in Alzheimer’s diseasePrion protein in Alzheimer’s disease-- neuroprotectiveneuroprotective, neurotoxic or both!, neurotoxic or both!

Benilova & De Strooper(2010) EMBO Mol. Med. 2, 289

PrPPrPCC forms multiple cell surface, raftforms multiple cell surface, raft--based complexesbased complexes

Watt et al. (2014) Frontiers Cell Develop. Biol. 2

EGCG and resveratrol EGCG and resveratrol reduce the cellular binding of Areduce the cellular binding of Aββoligomers and their downstream cytotoxicity oligomers and their downstream cytotoxicity

EGCG [(-)-epigallocatechin

gallate]

Resveratrol

Rushworth et al. (2013) J. Biol. Chem. 288, 8935

PPrion rion protein protein as a therapeutic target in Alzheimer’s diseaseas a therapeutic target in Alzheimer’s disease-- issues issues to to considerconsider

� Targeting disease vs normal functions of PrPC

� Receptors other than PrPC

� Type (“strains”) and concentration of Aβ oligomer

“Rather than exist as separate entities, several of these candidate receptor molecules may be closely linked in a multi-protein receptor complex, whose specific members may change as a result of electrophysiological activity or damage.”Susan Catalano, Alzforum

Benilova & DeStrooper (2013) Science 341, 1354

AcknowledgementsAcknowledgements

Hooper Lab

Rob AndrewHeledd GriffithsKate KellettBeth Noble

Lizzie Glennon (Kings, London)Ed Parkin (Lancaster)Jo Rushworth (Leicester)

Bristol

Seth LovePat KehoeScott Miners

Edinburgh

Jean Manson

Vicki Lewis (Melbourne)Emma Vardy (Newcastle)Nicole Watt (Leeds)Isobel Whitehouse (Leeds)