neutrophils induce alzheimer's-like disease via ...• coexpress 3 independent transgenes...

Turin, December 3, 2016

Zenaro Elena, PhDDepartment of Medicine

University of Verona, Italy

NEUTROPHILS INDUCE ALZHEIMER'S-LIKE DISEASE VIA

LFA-1-INTEGRIN

Age-related neurodegenerative disorder

(over 1 million people only in Italy)

Alzheimer’s disease (AD)

• accumulation of Amyloid-beta (Aβ) peptide

• hyperphosphorylation of Tau protein

• neuronal death and synaptic loss

• neuroinflammation

Experimental models of Alzheimer’s Disease

• amyloid pathology

• coexpress 5 Familial Alzheimer’s Disease mutations on APP and PSEN1that lead to accelerated plaque formation and increased Aβ42 levels

• memory impairments in behavioral paradigms begins at 4 months of age

(Oakley et al., 2006; Ohno et al., 2006)5xFAD

• amyloid plaques and neurofibrillary pathology

• coexpress 3 independent transgenes encoding human mutated APP,PSEN1 and Tau

• significant impaired short-term memory in behavioral paradigms starts at6 months of age

3xTg-AD (Billings et al., 2005; Oddo et al., 2003)

Astrocytes

Glial basement membrane

Perivascular

macrophagePerivascular space

1. Capture and Rolling

Mucins, selectins, integrins

2. Integrin activationGPCR-dependent signaling

3. Arrestintegrins

4. Transmigration

Blood flow

Endothelial cells

Endothelial basement membrane

The multistep model of leukocyte transmigration in

CNS venules

Astrocytes

MicrogliaNeuron

Oligodendrocyte

Glial basement membrane

Perivascular

macrophagePerivascular space

1. Capture and Rolling

Mucins, selectins, integrins

2. Integrin activationGPCR-dependent signaling

3. Arrestintegrins

4. Transmigration

Blood flow

Endothelial cells

Endothelial basement membrane

The multistep model of leukocyte transmigration in

CNS venules

Brain vessels express adhesion molecules in mice with

memory loss during early disease

4 month-old

5xFAD (meninges)

WT control

Brain vessels express adhesion molecules in mice with

memory loss during early disease

4 month-old

5xFAD (meninges)

WT control

6 month-old

3xTg-AD

(hippocampus)

WT control

Brain vessels express vascular adhesion molecules in

proximity to Ab deposits in 5xFAD mice

Soluble oligomeric Aβ1-42 peptide upregulates the

expression of endothelial adhesion molecules

Zenaro et al., Nat Med 2015

Neutrophils (Ly6G+ cells) infiltrate AD-like brain

during early disease

5xFAD

3xTg-AD


WT ctrl

WT ctrl

Neutrophils (naphtol AS-D chloroacetate esterase + cells)

accumulate in the brain during early disease


Neutrophils accumulate in the brain

during all disease phases


Neutrophils:

- Phagocytosis

- Release cytokines

- Degranulate and release many types of enzymes (are called granulocytes!)

- Produce reactive oxygen species (ROS)

- Release neutrophil extracellular traps (NETs)

The power of neutrophils

Neutrophils play a role in both acute and chronic inflammatory diseases

Neutrophils generate neutrophil extracellular traps (NETs)

in the brain of Alzheimer’s disease mice

Neutrophils produce IL-17 in

the brain of Alzheimer’s disease mice

Cortex

Hippocampus

4 month-old 5xFAD mouse

Neutrophils

Blood vessels

In vivo imaging of neutrophil trafficking in the cortex of

mice with Alzheimer's like disease

Download Video

http://www.morethanneurons.com/Video/5.mov

Neutrophils adhere in blood vessels with Ab deposits

Amyloid b Neutrophils

5xFAD-YFPH mouse

Neurons

Download Video


Neutrophils migrate inside the parenchyma

in areas with Ab deposits

Amyloid b NeutrophilsNeurons

Download Video


Human neutrophils Murine neutrophils

mMmM

mM

mM

Soluble oligomeric Ab triggers rapid integrin-dependent

adhesion in vitro

Soluble oligomeric Ab triggers LFA-1 integrin

high-affinity state

LFA-1 affinity measurement


LFA-1 integrin controls neutrophil migration into the

brain of mice with Alzheimer’s-like disease

LFA-1 deficient

neutrophils

WT neutrophils

Download Video


Inhibition of neutrophil function in mice with cognitive

deficit during early disease

Months of age

% N

EU

TR

OP

HIL

S

5

4

3

2

1

0

1 2 3 4 5 6 7 8 9 10

Anti-

Gr-1

Cognitive decline

Neutrophil

peak

Behavioral tests and

neuropathological

assessment

Anti-

Ly6G

Anti-LFA-1

Depletion of Ly6G+ cells restores cognitive functions

in 3xTg-AD mice CFC

Recordings of 8 min

free exploration

Aa

Contextual fear conditioning test

associative learning task

* P<0.05

Zen

aro

et a

l., N

at

Med

20

15

Depletion of Ly6G+ cells restores cognitive functions

in 3xTg-AD mice

Y-Maze

* P<0.05 **P<0.005

a



Y-maze

spatial working memory

Recordings of 8 min

free exploration

A

CFC

Zen

aro

et a

l., N

at

Med

20

15

Depletion of Gr1+ cells restores cognitive functions

in 3xTg-AD mice

Y-Maze

* P<0.05 **P<0.005

a



Y-maze

spatial working memory

Recordings of 8 min

free exploration

A

CFC

Zen

aro

et a

l., N

at

Med

20

15

Depletion of Gr1+ or Ly6G+ cells restores cognitive

functions in 5xFAD mice

*P<0.05, **P<0.005, ***P<0.0005

! 20!

Supplementary Figure 15

Supplementary Figure 15. Neutrophil depletion restores cognitive function in

5xFAD mice. Neutrophil depletion was carried out for 4 weeks in 5XFAD mice

starting at 4 months of age. Anti-Ly6G and anti-Gr-1 antibodies were used to deplete

neutrophils from the peripheral circulation. An isotype-matched anti-Ras antibody

was used as a treatment control and C57BL/6 littermates were used as wild-type

controls. Left panels (a, b) show the results of the contextual fear conditioning test.

Right panels (a, b) show the percent alternation performance in the Y-maze test.

Values represent the median ± SEM of mice in each group. Data are derived from

representative experiments with 12–14 mice per condition from a series of three with

similar results (*P < 0.05;

** P< 0.005 and ***P < 0.0005). Statistical analysis was

carried out using the Mann-Whitney test.


Y-MazeCFC Y-MazeCFC

anti-Gr1 Ab anti-Ly6G Ab

B CDepletion of Gr-1+ cells reduces microglial activation in

3xTg-AD mice

*P < 0.05; **P < 0.005

Iba-1 staining


B CDepletion of Gr-1+ cells reduces Ab deposition in

3xTg-AD mice

Amyloid Beta

WT ctrl Isotype Anti-Gr-1 Aβ

*P<0.05


*P<0.05

B CDepletion of Gr-1+ cells reduces tau phosphorylation in

3xTg-AD mice

*P<0.05

Tau


B CDepletion of Gr-1+ cells restores synaptic protein

expression in 3xTg-AD mice

*P<0.05


Does interference with neutrophil function during early

disease have an impact on late disease?

MONTHS of AGE

% N

EU

TR

OP

HIL

S

5

4

3

2

1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Treatment

Cognitive deficits

Neutrophil

peak

3xTg-AD

mice

Behavioral

Tests


B CTransient depletion of Gr-1+ cells during early

disease has long term beneficial effect

Zenaro et al., Nat Med 2015Amyloid Beta

Iba-1 staining

B CTreatment with an anti-LFA-1 antibody restores cognitive

function in 3xTg-AD mice

Efalizumab target: LFA-1 integrin

Improvement in psoriasis plaques

CFC Y-Maze

** P<0.005 **P<0.005


B C3xTg-AD/Itgal-/- lacking LFA-1 integrin show memory

improvement and lower microglia activation

* P<0.05 ***P<0.0005 Zenaro et al., Nat Med 2015

Y-Maze

Iba-1 staining

B CNeutrophils adhere in human brain vessels and migrate in

the parenchyma in subjects with AD

Control

Alzheimer


B CMPO+ cells accumulate in proximity of amyloid plaques

in subjects with AD

P<0.001 Zenaro et al., Nat Med 2015

B CNeutrophils accumulate in the brain in patients with

Alzheimer’s disease

**P < 0.005; ***P < 0.0005 Zenaro et al., Nat Med 2015

Neutrophils accumulate in the brain of subjects with Alzheimer’s disease

Alzheimer

Control

Alzheimer

Control

CD66b

DCAE

H&EAlzheimer


Neutrophil extracellular traps (NETs) are produced in the

brain of subjects with Alzheimer’s disease


Pericyte

Endothelial cell Astrocyte Tight junction Chemoattractant

Mucin SelectinIntegrin

Integrin-ligand

AbPerivascular macrophage GPCR

Ab plaque

Blood vessel

Brain parenchyma

Perivascular basement

membrane

Endothelial basement

membrane

Microglia

Neuron

Pericyte



Integrin-ligand


Ab plaque

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron

Rolling

Pericyte



Integrin-ligand


Ab plaque

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron

RollingIntegrin-activation and arrest

Pericyte



Integrin-ligand


Ab plaque

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron


Transmigration

Pericyte



Integrin-ligand


Ab plaque

Cytokines,

ROS,

Enzymes

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron


Transmigration

Pericyte



Integrin-ligand


Ab plaque

Cytokines,

ROS,

Enzymes

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron


Transmigration

Cytokines,

ROS,

Enzymes

Cytokines,

ROS,

Enzymes

Pericyte



Integrin-ligand


Ab plaque

Cytokines,

ROS,

Enzymes

Blood vessel

Brain parenchyma


membrane


membrane

Microglia

Neuron

Rolling

NETs release

Integrin-activation and arrest

Transmigration

Cytokines,

ROS,

Enzymes

Cytokines,

ROS,

Enzymes

Future studies on the role of peripheral leukocytes

1. Characterize the mechanisms of neutrophil-dependent damage

2. Characterize leukocyte trafficking mechanisms in AD

3. Establish the role of leukocyte subsets

4. Study the interplay between leukocyte subsets

5. Clarify the disease phase in which leukocyte subsets have a role

Acknowledgements

Enrica Pietronigro

Vittorina della Bianca

Gennj Piacentino

Simona Budui

Barbara Rossi

Stefano Angiari

Laura Marongiu

Tommaso Carlucci

Silvia Dusi

Jessica Arioli

Antonella Amoruso

Serena Ruggeri

Marco Bonani

Gabriela Constantin

Department of Medicine

University of Verona

Ermanna Turrano

Bruno Bonetti

Department of Neurological Sciences,

University of Verona

Simona

BuduiVittorina Della

Bianca

Enrica

Pietronigro

Giorgio Berton

Sara Nani

Alessio Montresor

Department of Pathology, University of Verona

Gennj

Piacentino

Funding agencies

neutrophils induce alzheimer's-like disease via ...• coexpress 3 independent transgenes...

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