Structure-function relationship of oligomers in amyloidoses

(1)

Structure-function relationship

of oligomers in amyloidoses

Thesis seminar, March 20

th

_{, 2014}

Céline Huynen

(2)

AMYLOIDOSES: PROTEIN MISFOLDING DISEASES

(3)

AMYLOIDOSES: PROTEIN MISFOLDING DISEASES

NEURODEGENERATIVE DISORDERS

Chiti and Dobson, 2006, Annu. Rev. Biochem.

Alzheimer’s disease (Aβ)

Parkinson’s disease (α-syn)

Huntington’s disease (htt)

(4)

AMYLOIDOSES: PROTEIN MISFOLDING DISEASES

NEURODEGENERATIVE DISORDERS

NON-NEUROPATHIC SYSTEMIC AMYLOIDOSES

Alzheimer’s disease (Aβ)

Parkinson’s disease (α-syn)

Huntington’s disease (htt)

Spinocerebellar ataxias (ataxin,…)

AL amyloidosis (Ig light chains)

(5)

AMYLOIDOSES: PROTEIN MISFOLDING DISEASES

NEURODEGENERATIVE DISORDERS

NON-NEUROPATHIC SYSTEMIC AMYLOIDOSES

NON-NEUROPATHIC LOCALIZED AMYLOIDOSES

Alzheimer’s disease (Aβ)

Parkinson’s disease (α-syn)

Huntington’s disease (htt)

Spinocerebellar ataxias (ataxin,…)

AL amyloidosis (Ig light chains)

Lysozyme amyloidosis

_{Type II diabetes (IAPP)}

(6)

Prevalence of Neurodegenerative Diseases

(ND)

Alzheimer’s and Parkinson’s diseases are respectively the 6

th

_{and 14}

th

_{cause of}

death in USA in 2010

Shery et al., 2010, NVSS Cause of death % _{2010 (%)}2009 to Heart diseases 24.2 - 2.0 Cancer 23.3 - 0.4 Accidents 4.9 + 1.3 Alzheimer’s disease 3.4 + 3.7 Parkinson’s disease 0.9 + 6.8

(7)

Prevalence of Neurodegenerative Diseases

(ND)

Alzheimer’s and Parkinson’s diseases are respectively the 6

th

_{and 14}

th

_{cause of}

death in 2010 in USA

Shery et al., 2010, NVSS Cause of death Heart diseases Cancer Accidents Alzheimer’s disease Parkinson’s disease

R

at

e

p

er

1

0

0 U

.S

. s

td

p

o

p

.

Year

1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 2010

More and more deaths due to ND and still no treatment

0.1 1000

10 100

(8)

Amyloidoses

Soluble functional peptide/protein

Higly organized insoluble fibrillar aggregates

extra- or intra-cellular deposits

Ex: Aβ (AD), α-synuclein (PD), huntingtin (HD),

IAPP (type II diabetes)

Amyloid fibrils

(9)

Amyloid fibrils

Soluble functional peptide/protein

Higly organized insoluble fibrillar aggregates

extra- or intra-cellular deposits

Ex: Aβ (AD), α-synuclein (PD), huntingtin (HD),

IAPP (type II diabetes)

Amyloid fibrils

No sequence identity or structural

homology

Common motif

 Fibrillar appearance (TEM + AFM)

(10)

Amyloid fibrils

Soluble functional peptide/protein

Higly organized insoluble fibrillar aggregates

extra- or intra-cellular deposits

Ex: Aβ (AD), α-synuclein (PD), huntingtin (HD),

IAPP (type II diabetes)

Amyloid fibrils

No sequence identity or structural

homology

Common motif

 Fibrillar appearance (TEM + AFM)

 Fibrils (up to 30 nm wide): 2-6 protofilaments

(2-5 nm diameter) twistest or laterally associated

Stefani and Dobson, 2003, J Mol Med

4 twisted protofilaments

(11)

Amyloid fibrils

Soluble functional peptide/protein

Higly organized insoluble fibrillar aggregates

extra- or intra-cellular deposits

Ex: Aβ (AD), α-synuclein (PD), huntingtin (HD),

IAPP (type II diabete)

Amyloid fibrils

No sequence identity or structural

homology

Common motif

 Fibrillar appearance (TEM + AFM)

2-6 protofilaments

 Cross-β structure  Thioflavin T and Congo red

Stefani and Dobson, 2003, J Mol Med

(12)

Amyloid fibril formation = toxic gain of

function

Soluble functional peptide/protein

_{Amyloid fibrils}

No sequence identity or structural

homology

Common motif

Common mechanism of formation

Conserved mechanism of pathogenesis for phenotypically diverse diseases

TOXIC GAIN OF FUNCTION

Lotz and Legleiter, 2013, J Mol Med; Winklhofer et al., 2008, EMBO; Bucciantini et al., 2002, Nature, Gillam and MacPhee, 2013, J. Phys.

(13)

Time

Aggregation

Nucleation

Maturation

MF F M MM D N

Kinetics of amyloid fibril formation

What are the toxic species?

Generic property of polypetide chains, Astbury and Dickinson, 1935

(14)

Plan of the presentation

1) Fibrils versus oligomers: Who is toxic?

2) How can we study oligomers? in vivo and in vitro techniques

3) What are the different oligomeric species and their structure?

4) Why are oligomers toxic?

(15)

(16)

Mechanism of amyloid fibril formation

Lotz and Legleiter, 2013, J Mol Med, Stefani and Dobson, 2003, J Mol Med, Gillam and MacPhee, 2013, J. Phys.

 Intrinsically disordered proteins

 Native proteins in dynamic equilibrium with different

conformations (e.g., partially unfolded or misfolded species

more aggregation-prone)

Mutations, misprocessing, aberrant interactions, changes in environmental conditions,

chemical modifications, enhanced protein synthesis, reduction of protein clearance can

(17)

Mechanism of amyloid fibril formation

Lotz and Legleiter, 2013, J Mol Med

Various intermediate aggregated states,

on- or off- pathways, depending on

proteins and conditions

(18)

Amyloid fibrils = toxic species … or … ?

Amyloid fibrils and plaques

Irreversible sequestration of different

proteins  loss of their function

Jellinger, 2009, Neuronal transm

Reservoirs of soluble toxic species

(19)

Amyloid fibrils = toxic species … or … ?

Amyloid fibrils and plaques

Irreversible sequestration of different

proteins  loss of their function

Systemic or localized amyloidoses:

Accumulation of kg of fibrils in organs and tissues: steric rigidity, inelasticity  impairment and

dysfunction of tissue architecture

Chiti and Dobson, Annu Rev Biochem, 2006; Pepys, 2006, Annu Rev Med; Pepys, 2001, Phil Trans R Soc Soc Lond B

Reservoirs of soluble toxic species

(20)

Amyloid fibrils = toxic species … or … ?

Amyloid fibrils and plaques

Irreversible sequestration of different

proteins  loss of their function

Systemic or localized amyloidoses:

Accumulation of kg of fibrils in organs and tissues: steric rigidity, inelasticity  impairment and

dysfunction of tissue architecture

Neurodegenerative amyloidoses:

Aβ deposits in cerebral blood vessel walls: hemorrhages and stroke

Amyloid fibrils of Aβ = toxic to cultured neuronal cells by inducing membrane depolarization

Chiti and Dobson, Annu Rev Biochem, 2006; Pepys, 2006, Annu Rev Med; Pepys, 2001, Phil Trans R Soc Soc Lond B

Pepys, 2006, Annu Rev Med; Fändrich, 2012, J Mol Med; chiti and dobson, Annu. Rev. Biochem, 2006

Reservoirs of soluble toxic species

(21)

Amyloid fibrils = toxic species … or …

protective?

Fibrils = inert and inactive insoluble end-products sequestering soluble toxic species?

Jellinger, 2009, Neuronal transm; Fändrich, 2012, J Mol Med

The number of fibril plaques and inclusions do not correlate well with

symptoms and severity of the disease

Amyloid fibrils and plaques

(22)

Toxic species are soluble oligomeric

intermediates

 Soluble intermediate aggregates correlate with

neurotoxicity (e.g., cognitive impairment in AD)

(23)

Toxic species are soluble oligomeric

intermediates

 Soluble intermediate aggregates correlate with

neurotoxicity (e.g., cognitive impairment in AD)

Lue et al., 1999, Am J Pathol

 Immunization with anti-Aβ antibody reduce

memory loss in AD mouses but not amyloid plaques

(24)

Toxic species are soluble oligomeric

intermediates

 Transgenic mice with non-fibrillar α-synuclein depositions: motor deficiencies and losses of

dopaminergic neurones

Masliah et al., 2000, Science

 Soluble intermediate aggregates correlate

with neurotoxicity (e.g., cognitive impairment in

AD)

 Immunization with anti-Aβ antibody reduce

memory loss in AD mouses but not amyloid plaques

(25)

Toxic species are soluble oligomeric

intermediates

 Transgenic mice with nonfibrillar α-synuclein depositions: motor deficiencies and losses of

dopaminergic neurones

Masliah et al., 2000, Science

 Soluble intermediate aggregates correlate

with neurotoxicity (e.g., cognitive impairment in

AD)

 Immunization with anti-Aβ antibody reduce

memory loss in AD mouses but not amyloid plaques

Dodart et al., 2002, Nat Neurosci

 Prefibrillar species of non disease-associated proteins (SH3 domain from PI3 and N-ter

domain of HypF) are highly cytotoxic in vitro

(26)

2) How can we study oligomers? in vivo

and in vitro techniques

(27)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC): Size and proportion of

the different populations of species inside a protein sample (e.g. monomers – oligomers – fibrils)

Different techniques to study oligomers

Redecke et al., 2007, jsb

Monomers

Small oligomers

Large oligomers

(28)

Oligomeric species

Dynamic light scattering (DLS) + size exclusion chromatography (SEC): Size and proportion of

the different populations of species inside a protein sample (e.g. monomers – oligomers – fibrils)

Different techniques to study oligomers

Monomers

SEC

Large

particles

Native

monomers

(29)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM): size and

morphology of the species

Different techniques to study oligomers

Bartolini et al., 2011, Anal Biochem

Spherical oligomers

Celej et al., 2012, Biochem J

(30)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)

Circular dichroism (CD) and Infrared spectroscopy (FTIR): β-sheet structures

Different techniques to study oligomers

Monomers

Oligomers with β-sheet

conformation: min at 217 nm

Redecke et al., 2007, jsb

(31)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)

Circular dichroism (CD) and Infrared spectroscopy (FTIR): β-sheet structures

Different techniques to study oligomers

Oligomers

Fibrils

1695 cm

-1

_{1625 cm}

-1

ATR-FTIR: Amide I absorption band

Antiparallel β-sheet

1628 cm

-1

Parallel β-sheet

(32)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)

Circular dichroism (CD) and Infrared spectroscopy (FTIR)

ANS binding: exposure of hydrophobic patches

Different techniques to study oligomers

A N S fl u o re sc e n ce in te n si ty , 4 7 0 n m ( a. u .) [ANS] (µM)

HypF-N oligomers (A conditions)

HypF-N oligomers (B conditions)

(33)

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)

Circular dichroism (CD) and Infrared spectroscopy (FTIR)

ANS binding

Thioflavin T (ThT) + congo Red binding: amyloid character

Different techniques to study oligomers

(34)

Different techniques to study oligomers

Bucciantini et al., 2002, Nature

Dynamic light scattering (DLS) + size exclusion chromatography (SEC)

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)

Circular dichroism (CD) and Infrared spectroscopy (FTIR)

ANS binding

Thioflavin T (ThT) + congo Red binding

(35)

Different techniques to study oligomers

In vivo techniques + Ab A11

In vivo techniques: injection of fluorescently labeled oligomer species in rat brains

 Activation of microglia (inflamatory response)

 Viability of ChAT neurons

(36)

An antibody specific to oligomers to perform in

vivo and in vitro experiments

Rabbit immunization with Aβ synthetic oligomers

Kayed et al., 2003, Science

Polyclonal serum immunoreactivity

Monomers and low-MW species

Spherical oligomers (TEM)

Elongated protofibrils (TEM)

Fibrils

Ab

Oligomeric

species

(37)

An antibody specific to oligomers to perform in

vivo and in vitro experiments

Specific to soluble oligomer intermediates of many

amyloid proteins and peptides

 Common epitope

ο Soluble Oligomers

▲ Monomers and Low-MW species ■ Fibrils

(38)

An antibody specific to oligomers to perform in

vivo and in vitro experiments

Specific to soluble oligomer intermediates of many

amyloid proteins and peptides

Useful to detect oligomers in human AD brains

 Relevance of oligomers formed in vitro

Green

: Clusters of immunoreactive deposits: spherical and

curvilinear structure = oligomers

Red

: fibrils stained by thioflavin S

AD brains

Control brains:

Not demented

(39)

3) What are the different oligomeric

species and their stucture

(40)

Amyloid intermediates

A wide range of proteins associated with ND form soluble amyloid intermediates (oligomers):

Aβ (AD)

Tau (AD)

α-synuclein (PD)

PrP (spongiform encephalopathies)

polyQ proteins (HD and spinocerebellar ataxias)

…

And also proteins associated with non-neuropathic amyloidoses and not associated with

diseases

Metastable/transient species difficult to study

Many intermediate species: high diversity in shape, in size, and in structure

Many different conditions to study and form oligomers

 Complexicity

(41)

Structure and diversity of Aβ amyloid oligomers

From dimers to high molecular mass soluble species (< 10 to > 100 kDa)

SDS stable low-n oligomers (di- trimers to dodecamers)

Building blocks for the formation of larger soluble oligomers and

insoluble fibrils

Toxic

(42)

Structure and diversity of Aβ amyloid oligomers

From dimers to high molecular mass soluble species (< 10 to > 100 kDa)

Walsh and Selkoe, 2007, J Neurochem; Bartolini et al., 2011, Anal Biochem; Kayed and Reeves, 2013, Journal of AD; Gadad et al., 2011, Journal of AD

Spherical oligomers (10 – 25 nm diameter)

ADDL (Aβ-derived diffusible ligands) = globular

oligomers (4-5 nm diameter)

Highly neurotoxic

Kayed and Reeves, 2013, Journal of AD Bartolini et al., 2011, Anal Biochem

Spherical oligomers

AFM

(43)

Structure and diversity of Aβ amyloid oligomers

From dimers to high molecular mass soluble species (< 10 to > 100 kDa)

Annular oligomers (150 – 250 kDa, 8 – 12 outer diameter, 2.0 –

2.5 inner diameter): doughnut-like structure

Cytotoxicity: Formation of non-specific pores such as cytolytic β-barrel

pore-forming toxins

Spherical oligomers

ADDL

(44)

Structure and diversity of Aβ amyloid oligomers

From dimers to high molecular mass soluble species (< 10 to > 100 kDa)

Protofibrils (1 – 3 nm height, 5 – 10 nm width, > 110 nm length): curvilinear structure that

can dissociate into low molecular weight oligomers or form fibrils

Toxic

(45)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

Spherical oligomers

(10-60 nm diameter, β-sheet structure, cytotoxic)

Ring circularized oligomers

(annular, circular or elliptical, cytotoxic, prevent fibril

formation?)

Protofibrils

(granular, cytotoxic, precursor of fibrils)

Conway et al., 2000, PNAS Celej et al., 2012, Biochem J

Hashimoto et al., 1998, Brain Research; Daturpalli et al., 2013, J Mol Biol; Gadad et al., 2011, Journal of AD; Conway et al., 2000, PNAS; Celej et al., 2012, Biochem J

Conway et al., 2000, PNAS

Circular annular oligomers

35-55 nm diameter

Elliptical annular oligomers

35-55 nm width, 65-130 nm length

(46)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

strands, rapidely incorporated into inclusion

Soluble oligomers

(> 60 kDa, toxic, β-bodies)

(47)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

Soluble oligomers

PolyQ proteins

(Sperm whale

Myo, htt, Atx3:)

Non-fibrillar aggregates, amorphous

aggregates

(3, 80-90 mers, β-sheets not

orderly aligned)

Spheroids

(1, Small and globular, 5-65 nm diameter, beta sheet structure)

Annular oligomers

(2, 100 nm diameter)

Protofibrils

(4, short and curvilinear, up to 2 µm length,

not resistant to SDS denaturation)

(48)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

Soluble oligomers

PolyQ proteins

amorphous aggregates; Spheroids;

Non-fibrillar quasi aggregate,

Annular oligomers

Protofibrils

Prion protein

Small soluble and globular oligomers

(

β-sheet rich, 13.1 ± 0.4 nm hydrodynamic radius, 8.2 ± 2.4 nm radius 14 – 28 mers, 300

– 600 kDa , cytotoxic in vitro and in infected hamster brain)

Elongated-shape

oligomers

(β-sheet rich, 38.7

± 9.3 nm hydrodynamic radius, 31.1 ± 6 nm radius, ~

100 mers)

(49)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

Soluble oligomers

PolyQ proteins

amorphous aggregates; Spheroids;

Non-fibrillar quasi aggregate,

Annular oligomers

Protofibrils

Prion protein

Small soluble and globular oligomers

Elongated-shape

_oligomers

Transthyretin

Native

tetramers 

Mono-dimers

Annular oligomeric intermediates

(16 ± 2 nm diameter, octametric symmetry, ~15

nm R_h unstable)

Spheroid oligomers

(spherical, more compact , 8.3 nm R_h)

Protofibrils

(3.6 nm thickness, up to 60-300 nm length formation and growth

from spheroid oligomers)

Pires et al., 2012, PLOS ONE

(50)

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

Soluble oligomers

PolyQ proteins

amorphous aggregates; Spheroids;

Non-fibrillar quasi aggregate,

Annular oligomers

Protofibrils

Prion protein

Small soluble and globular oligomers

Elongated-shape

_oligomers

Transthyretin

Native

tetramers 

Mono-dimers

Annular oligomeric intermediates

Spheroid oligomers

Protofibrils

SH3-N47A (PI3

kinase)

Low-populated, heterogeneous, unstable, β-sheet structure, exposition of hydrophobic

patches

(51)

Campioni et al., 2010, N Chemical Bio

Structure and diversity of amyloid oligomers

α-synuclein

Di/trimers

_{Ring circularized oligomers}

Spherical oligomers

Protofibrils

PolyQ peptides

Soluble oligomers

PolyQ proteins

amorphous aggregates; Spheroids;

Non-fibrillar quasi aggregate,

Annular oligomers

Protofibrils

Prion protein

Small soluble and globular oligomers

Elongated-shape

_oligomers

Transthyretin

Native

tetramers 

Mono-dimers

Annular oligomeric intermediates

Spheroid oligomers

Protofibrils

SH3-N47A

β-sheet, hydrophobic patches exposed

HypF-N (E. coli)

2 Spherical oligomers

sheets, hydrophobic regions exposed vs in (intermolecular

(52)

Common oligomeric structures

Small oligomers: dimers – dodecamers

Spherical oligomers (10-60 nm diameter)

Annular oligomers

Protofibrils

Robertson and Bottomley, 2012, Landes Bioscience and Springer; Kayed et al., 2003, Science; Chiti and Dobson, 2006, Annu. Rev. Biochem.; Glabe, 2006, Neurobiology of aging, Hashimoto et al., 1998, Brain Research; Daturpalli et al., 2013, J Mol Biol; Gadad et al., 2011, Journal of AD; Conway et al., 2000, PNAS; Celej et al., 2012, Biochem

(53)

Common oligomeric structures

Small oligomers: dimers – dodecamers

Spherical oligomers (10-60 nm diameter)

Annular oligomers

Protofibrils

Amyloid character

Antiparallel β-sheet structure

Exposition of hydrophobic regions

Different common epitopes (A11-OC-…)

(54)

Common oligomeric structures

Small oligomers: dimers – dodecamers

Spherical oligomers (10-60 nm diameter)

Annular oligomers

Protofibrils

Amyloid character

Antiparallel β-sheet structure

Exposition of hydrophobic regions

Different common epitopes (A11-OC-…)

Unique common structural feature of the

polypeptide backbone in amyloid soluble

oligomers, different from fibrils and

monomers

(55)

Robertson and Bottomley, 2012, Landes Bioscience and Springer; Kayed et al., 2003, Science

Similar structural organization of oligomers

(56)

(57)

Oligomer-associated cytotoxicity

Aberrant interaction and sequestration of other proteins

and cellular components (via exposition of hydrophobic

sites or polyQ domains)  impair their function

Structural features of oligomers cytotoxicity

Lotz and Legleiter, 2013, J Mol Med; Chiti and Dobson, 2006, Annu. Rev. Biochem.; Glabe, 2006, Neurobiology of aging; Frändrich 2012, JMB; Jellinger, 2009, J Neural Transm

(58)

Oligomer-associated cytotoxicity

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism (overwhelmed +

sequestration of their components)

Structural features of oligomers cytotoxicity

(59)

Oligomer-associated cytotoxicity

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism

Interaction with cell membranes: disruption of the

membrane integrity - channel hypothesis – ion permeability

Structural features of oligomers cytotoxicity

(60)

Oligomer-associated cytotoxicity

Oxidative stress and ROS production

(reactive oxygen species)

Structural features of oligomers cytotoxicity

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism

Interaction with cell membranes

(61)

Oligomer-associated cytotoxicity

Structural features of oligomers cytotoxicity

Mitochondrial dysfunction, Fragmentation

of the Golgi apparatus

Oxidative stress and ROS production

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism

Interaction with cell membranes

(62)

Oligomer-associated cytotoxicity

Structural features of oligomers cytotoxicity

Interference with the axonal transport

Mitochondrial dysfunction, Fragmentation

of the Golgi apparatus

Oxidative stress and ROS production

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism

Interaction with cell membranes

(63)

Oligomer-associated cytotoxicity

Structural features of oligomers cytotoxicity

Interrelated in a

complex vicious circle

Interference with the axonal transport

Mitochondrial dysfunction, Fragmentation

of the Golgi apparatus

Oxidative stress and ROS production

Aberrant interaction and sequestration of other proteins

and cellular components

Interference with the central protein quality

control/clearance mechanism

(64)

Oligomer-associated cytotoxicity

Interaction with cell membranes: disruption of the membrane integrity - channel

hypothesis – ion permeability (exposition of hydrophobic regions, antiparallel β-sheet 

β-barrel, similar to bacterial porins)

Extracellular

Intracellular

Ca

2+

Oligomers

Ca

2+

Cytoplasmic membrane

Influx

Leakage

[Ca

2+

_{] increase}

Intracellular membrane

(e.g. mitochondria, ER)

Oxidative stress + ROS production

Cytochrom C release

Apoptosis

Glabe, 2006, Neurobiology of aging; Guo et al., 2009, Handbook of Neurochemistry and Molecular Neurobiology; Frändrich 2012, JMB ; Campioni et al., 2010, N Chemical Bio; Hands and Wyttenbach, 2010, Acta Neuropathol; Cerf et al., 2009,

(65)

Example: Neurotoxicity of Aβ oligomers

1) Interaction with cell surface

(66)

Example: Neurotoxicity of Aβ oligomers

1) Interaction with cell surface

2) Aberrant Interaction with membrane receptors

(67)

1) Interaction with cell surface

2) Aberrant Interaction with membrane receptors

Example: Neurotoxicity of Aβ oligomers

3) Intracellular actions

Kayed and Reeves, 2013, Journal of AD

Neuronal

receptors

Impairs their endocytic trafficking: Synaptic dysfunction

(68)

4) Strategies to interfere with the toxicity

associated with oligomers

(69)

Therapeutic strategies targeting oligomerization

Stabilization of the native conformer (e.g. chaperones induction: Hsp70 vs α-syn)

Oligomerization

Gadad et al., 2011, Journal of AD; Lotz and Legleiter, 2013, J Mol Med

(70)

Therapeutic strategies targeting oligomerization

β-sheet breaker or blocker: peptide-based inhibitor, small synthetic peptides or

molecules

Oligomerization

E.g. Methylene blue vs Aβ: phase II, promising

(71)

Therapeutic strategies targeting oligomerization

Decrease the amount of monomers: RNAi or antisens oligonucleotide (under

investigation)

Stabilization of the native conformer

β-sheet breaker or blocker

(72)

Therapeutic strategies targeting oligomerization

Post-translational modifications (e.g. acetylation – phosphorylation – sumoylation –

ubiquitination – palmitoyaltion - …)

Interfere with aggregation propensity

and proteolysis

Stabilization of the native conformer

β-sheet breaker or blocker

(73)

Therapeutic strategies targeting oligomerization

Stabilization of the native conformer

β-sheet breaker or blocker

Decrease the amount of monomers

Post-translational modifications

Active and passive immunization

Injection of synthetic Aβ peptide in mice: reduced pathology

Ab vs Aβ decreases aggregation and pathology (cross BBB):

clinical trials (e.g. bapineuzumab,

solanezumab

,

gantenerumab)

(74)

A chaperone to block oligomer’s toxicity

Hsp90 vs A53T α-synuclein

Hsp90 forms a stable complex with A53T α-synuclein

SEC: Superdex 200 10/300 column

Hsp90 + oligomers

oligomers

Hsp90

Void

volume

P1 P2

O

Oligomers

Hsp90

Co-elution of Hsp90 + oligomers

SDS-PAGE

(75)

A chaperone to block oligomer’s toxicity

Hsp90 vs A53T α-synuclein

A53T:Hsp90 at 10:1 ratio

A53T:Hsp90 at 2:1 ratio

A53T

Hsp90 inhibits the aggregation of A53T α-synuclein

(76)

A chaperone to block oligomer’s toxicity

Hsp90 vs A53T α-synuclein

Hsp90 decreases cell death induced by A53T α-synuclein

A53T

A53T + Hsp90

Cell viability

(77)

A chaperone to block oligomer’s toxicity

Hsp90 vs A53T α-synuclein

Hsp90 forms non-specific hydrophobic interactions with A53T oligomers and

preferentially recognizes more structured oligomeric states

This interaction further prevents toxicity by shielding a potential toxic surface,

inducing a conformational rearrangement, or stabilizing a specific structure

(78)

An antibody recognizing many amyloid oligomers

Kayed and Glabe, 2006, Method in Enzymology, Vol. 413:

Identification of a common anti-oligomer antibody

(79)

An antibody recognizing many amyloid oligomers

 Detection, localization and quantification of oligomers in cell and brains  diagnostic

Good predictor for disease onset and progression

 Block oligomers toxicity in vitro  therapeutic tool via active or passive immunization

 Identify the exact role of oligomers

 Screening of compounds that prevent/reverse oligomer formation

 Identify new amyloidoses?

Kayed and Glabe, 2006, Method in Enzymology, Vol. 413:

Identification of a common anti-oligomer antibody

(80)

Conclusions

Many different peptides/proteins  One amyloid fold (amyloid fibrils and amyloid plaques)

Amyloidoses: Neurodegenerative and Non-neuropathic diseases

(81)

Conclusions

Many different peptides/proteins  One amyloid fold

Via oligomer formation = toxic species?

Non fibrillar

Amyloid-like structure

Antiparallel β-sheet structure

Exposition of hydrophobic patches

Unique common structural motif (common epitope)

Amyloidoses: Neurodegenerative and Non-neuropathic diseases

Spherical oligomers

Annular oligomers

Protofibrils

(82)

Conclusions

Many different peptides/proteins  One amyloid fold

Via oligomer formation = toxic species?

Amyloidoses: Neurodegenerative and Non-neuropathic diseases

Aberrant interaction with cellular components

Interference with the protein quality control

Membrane disruption

Oxidative stress, ROS production, mitochondrial dysfunction

Interference with the axonal transport

Apoptosis and

neurodegeneration

(83)

Conclusions

Many different peptides/proteins  One amyloid fold

Via oligomer formation = toxic species?

Amyloidoses: Neurodegenerative and Non-neuropathic diseases

Valuable targets to interfere with the toxicity associated with these pathologies

1) Oligomerization inhibition via:

Stabilization of the native state, reducing the amount of protein production, β-sheet