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IRCCS Foundation “Carlo Besta” Neurological Institute, Milano

Fabrizio Tagliavini

Fondazione Casimiro Mondino «Centenario» – Pavia 2017

Alzheimer

Update on Disease Mechanisms

Italy 0.7 million

EU 7.3 million

World 35 million

Alzheimer’s disease

caused by mutation in

• Aβ precursor protein (APP)

• Presenilin 1 / Presenilin 2

� sporadic ~99 %

� familial ~ 1 %

All mutations alter APP processing and/or Aβ propertiesincreased Aβ production and/or Aβ aggregation

major genetic risk factor

• ApoE ε4

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the key players in Alzheimer disease

Braak stage VI

Sone et al. – Alzheimer’s & Dementia 2017

Amyloid and Tau imaging in early AD

Amyloid PET Tau PET

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Kang et al. - Neurobiol Aging 2017

Negative correlation between Tau signal and glucose hypometabolism in AD and MCI

secretasesβγ

Aβ

Aβ amyloiddeposits

mature amyloid plaques

glial activation: neuroinflammation

APP

misfolded Aβ tau pathology and neurotoxicity

Aβ oligomers

Aβ cascade hypothesis of ADHardy J & Higgins G (1992) Science 256: 184–185

Hardy J & Selkoe D (2002) Science 297:353-356

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Aβ pathology in ADsenile plaques and CA

Aβ

APPsAPPα

C83

APP Processingamyloidogenic non amyloidogenic

α

γ

ACID

P3

APPsAPPβ

C99ACID

Aβ 1-40Aβ 1-42Others

β

γ

1 : 5

secretases secretases

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Model of Aggregation of Misfolded Proteins

Aβ peptides assemble into oligomers and amyloid fibrils

M. Morbin

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The core consists of a dimer of

Aβ42 molecules per fibril layer, with

residues 15–42 forming a double

horseshoe–like cross–β-sheet entity.

Residues 1–14 are partially ordered and

in a β-strand conformation.

Wälti et al. - PNAS 2016, 113:E4976-84

Atomic-resolution structure of a disease-relevant

Aβ1-42 amyloid fibril

Tau pathology in ADneurofibrillary tanlges

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Microtubule-associated protein tau

Isoforms

In the brain TAU is expressed at high levels in neuronsand lower levels in oligodendrocytes and astrocytes

Microtubule-associated protein tau

properties• is higly hydrophilic, highly soluble, heat stable

• has an extendend and flexible conformation

• is a phosphoprotein which contains 38 potentialphosphorylation sites

major function• promotes assembly and mantains structure of

microtubules

• tau binding to microtubules is regulated by itsphosphorylation state

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• Normal tau contains 2-3 moles of phosphate/mole tau

• The phosphorylation level in AD is remarkably higher

Hyperphosphorylation leads to conformational change

and aggregation into oligomers and abnormal filaments

breakdown of microtubule network

ph-tau assembly into PHF and NFT formation

retrgrade degeneration and loss of synapses

death of neurons

compromised axoplasmic flow

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Staging of Alzheimer’s disease-related neurofibrillary tangles

Braak H & Braak E - Neurobiol Aging 1995

Stages I- II Stages III- IV Stages V- VI

Nature 2017, 574:185-190

PHF

SF

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Cross-sections of the PHF and SF cryo-EM structures

Fitzpatrick et al. Nature 2017, 574:185-190

PHF

SF

Both PHF and SF are composed by

2 identical protofilaments with

C-saped subunits, which adopt a

combined cross-β/β-helix structure

The core of both protofilaments is

composed by R3, R4 and 10 aa

C-terminal to the repeats, while the

N- and C-termini of tau form the

fuzzy coat

The difference between PHF and SF

is due to differences in their inter-

protofilament packing (symmetrical

in PHF, asymmetrical in SF)

The ordered core define the seed of

aggregation of tau in AD

Cryo-EM reconstruction of PHF and SF of AD

Fitzpatrick et al. Nature 2017, 574:185-190

Knowledge of the atomic coordinates of tau

filaments may be useful for the rational design of

• specific inhibitors of tau aggregation

• specific tracers

• specfic diagnostic tests (e.g., amplification of disease

specific tau isoforms from olfactory muscosa by RT-QuIC)

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Aβ cascade hypothesis of ADHardy & Selkoe Science 2002, 297:353-356

additional events are required to cause neurodegeneration

and cognitive impairment

• Genetic forms of AD are associated with mutations in APP, PS1 or PS2.

These mutations alter APP processing and/or Aβ properties leading to

increased Aβ production and/or Aβ aggregation.

• Trisomy of APP (Down syndrome) lead to AD pathology

• ApoE ε4 genotype is a risk factor for sporadic AD

in favour

• Lack of correlation between amyloid load and dementia

• Frequent amyloid deposition without significant cognitive impairment

among the elderly

against

Systemic inflammation might trigger the process

Calsolaro and Edison, Alzheimer's & Dementia 2016, 12: 719-732

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Hypothetical model of dynamic biomarkers including

neuroinflammation in AD

Calsolaro and Edison, Alzheimer's & Dementia 2016, 12: 719-732

Aβ – τConnection ?

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Tau-dependent effects of Aβ

Aβ and tau : the trigger and bullet in AD pathogenesis

Götz et al. Science 2001 τ P301L

Aβ

increase in NFT

APPSweτ

P301L +Lewis et al. Science 2001

acceleration of

NFT formation,

no effect on Aβ deposition

Tau-dependent effects of Aβ

Bloom GS – JAMA Neurol 2014, 71: 505-508

Aβ and tau : the trigger and bullet in AD pathogenesis

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Aβ initiates a pathological feedback loop with tau, driving AD progression

Aβ and tau : the trigger and bullet in AD pathogenesis

Bloom GS – JAMA Neurol 2014, 71: 505-508

phenotypic heterogeneity of AD

understanding the molecular basis

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Progressive cognitive or behavioural impairment involving ≥2 cognitive

domains, defined on clinical basis and confirmed by neuropsychological

tests

Clinical Heterogeneity

a. Amnestic presentation

b. Non amnestic presentations

• Language dysfunction: deficit in word finding

• Visuospatial dysfunction: deficit in spatial cognition

• Executive dysfunction: impaired reasoning, judgment and

problem solving

McKhann et al. Alzheimer’s & Dementia 2011

The diagnosis of dementia due Alzheimer’s

disease: recommendations from the NIA and the

Alzheimer’s Association workgroup

• FTD bv-like phenotype L113P, P117R, M139V, G184V, E280A, insR352

• Language presentation H163Y, G209V, L262F, R278I, E280A, A413E,

A434C

• Cerebellar Ataxia P117A, I143T, L166P, Y256S, L282V

• Spastic Paraparesis ∆I83/M84, L85P, Y154N, insF1, L166P, F237I,

V261F, P264L, R278K, E280G, E280Q, P284L,

∆ex9, L381V, P436Q

• Parkinsonism E120D, M146L, L250S, C410Y

AJ Larner & M Doran - J Neurol 2006

Phenotypes associated with mutations of the presenilin 1 gene

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phenotype of the P117A PS1 mutationClinical Features• early Ataxia• early Behavioural Disturbances• Epilepsy• relatively late Dementia

Neocortex

Cerebellum

INCBG. Giaccone

Aβ

AβAβ

ph-tau

KM670/NL671

KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL VMLK

D694N A692G

A713TA713V

T714I, T714A

V715M, V715A

I716V, I716T

V717I, V717F, V717G, V717L

L723P

E693GE693∆

H677R D678NK724N

β γ

(low penetrance)

AD phenotypes

KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL VMLK

D694N A692G

A713TE693Q (Dutch)

E693K (Italian)

E693G

L705V

CAA phenotypes or AD with severe CAA

Phenotypes associated with APP mutations

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sAD (ApoE ε3) APP V717F

sAD (ApoE ε4) APP A713T

APP A673V

PS1 P117A

Variability of Aβ neuropatological profiles in

sporadic and genetic AD

INCBG. Giaccone

Wei Qiang, Wai-Ming Yau, Jun-Xia Lu, John Collinge, Robert Tycko

Structural Variation in Aβ Fibrils from Alzheimer’s

Disease Clinical Subtypes

Nature 2017, 541:217-221

Correlations between AD phenotype and structural variation of Aβ40 and Aβ42

amyloid fibrils

prepared by seeded growth from extracts of AD cerebral cortex and

analysed by TEM and solid-state NMR

Alzheimer's disease clinical subtypes

� typical prolonged-duration form (t-AD)

� posterior cortical atrophy (PCA)

� rapidly progressive form (r-AD)

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Structural Variation in Aβ Fibrils from Alzheimer’s Disease

Clinical Subtypes

Wei Qiang et al. - Nature 2017, 541:217-221

• t-AD and PCA specific predominant Aβ40 fibril structure

• r-AD high proportion of additional Aβ40 fibrils

structures

• All subtypes Aβ42 fibrils structural heterogeneity

Variations in Aβ fibril structure

may correlate with variations in AD phenotype

in analogy to distinct prion strains

that are related to different conformers of PrPSc

Human Prion Diseases

• Creutzfeldt-Jakob disease- sporadic- genetic- acquired (iatrogenic, new variant)

• Gerstmann-Sträussler-Scheinker Disease- genetic

• Kuru - acquired (ritualistic cannibalism)

• Fatal Insomnia- genetic- sporadic

• Variable Protease-sensitive Prionopathy- sporadic

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Prion Diseases

Different phenotypes are associated with distinct PrPSc types

which encipher diverse biological properties

33

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18

14

8

CJDType1

CJDType2

GSSP102L

GSSA117V

GSSF198SQ217R

GSSQ212P

GSSG131V

Poggiolini et al. - Int J Cell Biol 2013

Different conformers of PrPSc are

associated with different prion strains

PrPSc

Telling et al. Science 1996

experimental transmission of FFI and CJDE200K to mice

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working hypothesis

Heterogeneity of Alzheimer disease

is related to the existence of

different Aβ “strains”

Di Fede et al., under review

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sCJDType1

sCJDType2

GSSP102L

GSSA117V

GSSF198SD202NQ217R

GSSQ212P

GSSG131V

The model of prion diseases

Targeting of the disease process and selective

vulnerability of neurons seem to be related to

biochemical composition, assembly state and

pathogenic properties of PrP isoforms

1. Different pathological phenotypes of sporadic and genetic (APP, PS1, PS2) AD

2. Biochemical profiling of Aβ species in affected brain regions and purified amyloid fractions by SELDI-TOF MS

3. Propagation of phenotypic diversity in animal models

Study design

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AD phenotypic diversity and Aβ profile

Aβ1-40

Aβ1-40

Aβ1-39Aβ1-38

Aβ1-40

Aβ1-40

Aβ1-42Aβ1-37

Aβ1-42

Aβ1-42

Aβ1-42

Aβ1-38

Aβ1-38

Aβ1-38

Aβ1-39

Aβ1-39

Aβ1-39

Aβ2-40

Aβ2-40

Aβ2-40

Aβ1-37

Aβ1-37

Aβ1-36

Aβ1-36

Aβ1-36

Aβ1-36

Aβ2-39

Aβ2-39

Aβ4-42

APP A673V

APP A713T

PS1 P117A

sAD ApoE ε4/ε4

APP A713T

sAD ApoE ε4/ε4

APP A673V

PS1 P117A

PROFILE 1sAD (n=14), fAD PS1 P117A, fAD PS2 A85V

(ApoE allele frequency: 69% Apo ε3)Predominant species

Aβ1-42, Aβ4-42,

Aβ3pE-42, Aβ11pE-42

Minor species

AβX-40 0

5

10

15

20

25

30

35

Relative%

Predominant species

Aβ1-40, Aβ1-42

Minor species

Aβ2-39, Aβ2-40, Aβ3pE-40

PROFILE 2sAD (n=5)

(ApoE allele frequency: 60% Apo ε4)

0

5

10

15

20

25

30

35

Relative%

Aβ profiles as revealed by SELDI-TOF MS

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Predominant species

Aβ1-40, Aβ1-38

Minor species

Aβ1-37, Aβ1-39

PROFILE 3fAD APP A673V, fAD APP A713T, fCAA APP E693K

(ApoE ε3/ε3)

0

10

20

30

40

50

60

70

Relative%

Predominant species

Aβ1-40, Aβ3pE-40

Minor species

Aβ1-36, Aβ2-40, Aβ3pE-42

PROFILE 4sAD-CAA (n=1)

(ApoE ε4/ε4)

0

10

20

30

40

50

Relative%

Aβ profiles as revealed by SELDI-TOF MS

Different Aβ profiles show distinctchemico-physical properties

Aggregation kinetics Seeding activity

Protease resistence

PK

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Different Aβ profiles show different propagationpatterns upon transmission to Tg mice

fAD PS1 P117A (Aβ profile 1)

Hippocampus

Thalamus

sAD-CAA ApoE ε4/ε4 (Aβ profile 4)fAD APP A713T (Aβ profile 3)

HippocampusThalamus

APPsw

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Aβ and phenotypic heyterogeneity of AD

• Different AD phenotypes show differences in biochemical

composition, assembly and targeting of Aβ species

• Distinct molecular AD subtypes show different aggregation kinetics,

seeding activity, PK resistance and propagation patterns of amyloid

pathology in animal models

• Different composition and/or aggregation state of Aβ species could

be a determinant of phenotypic diversity of AD, and account for

responders and non-responders to specific treatments targeting Aβ

• Molecular characterization of Aβ «strains» could enable the rational

design of theranostic compounds and specific diagnostic tests

NeuropathologyB. Pollo

G. Marucci

E. Maderna

A. Indaco

V. Redaelli

V. Fugnanesi

C. Calatozzolo

M. Patanè

Clinical UnitP. Tiraboschi

A.R. Giovagnoli

G. Di Fede

P. Caroppo

V. Redaelli

S. Prioni

I. Bizzozero

C. Muscio

BiochimistryF. Moda

E. Bistaffa

M. Rossi

C. De Luca

Genetics G. Di Fede

G. Rossi

M. Catania

I. D’Amato

CollaboratorsMario Salmona & Gianluigi ForloniMario Negri Institute Milano

Roberta Ghidoni, Luisa BenussiIRCCS FBF Brescia

Giuseppe LegnameSISSA Trieste

Mauro GiaccaICGEB Trieste

Claudio SotoHuston University, Texas

Bernardino GhettiIndiana University, IN

Gianluigi ZanussoUniversity of Verona

Funding• Ministero della Salute

• MIUR

• Cariplo/Telethon Foundations

• European Union

• Alzheimer Association

Neurology 5 &

Neuropathology

G. Giaccone