neutrophils induce alzheimer's-like disease via ...• coexpress 3 independent transgenes...
Post on 25-Oct-2020
2 Views
Preview:
TRANSCRIPT
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
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
Zenaro et al., Nat Med 2015
WT ctrl
WT ctrl
Neutrophils (naphtol AS-D chloroacetate esterase + cells)
accumulate in the brain during early disease
Zenaro et al., Nat Med 2015
Neutrophils accumulate in the brain
during all disease phases
Zenaro et al., Nat Med 2015
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
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
Zenaro et al., Nat Med 2015
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
Contextual fear conditioning test
associative learning task
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
Contextual fear conditioning test
associative learning task
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.
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
*P<0.05
B CDepletion of Gr-1+ cells reduces tau phosphorylation in
3xTg-AD mice
*P<0.05
Tau
Zenaro et al., Nat Med 2015
B CDepletion of Gr-1+ cells restores synaptic protein
expression in 3xTg-AD mice
*P<0.05
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
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
Zenaro et al., Nat Med 2015
Neutrophil extracellular traps (NETs) are produced in the
brain of subjects with Alzheimer’s disease
Zenaro et al., Nat Med 2015
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
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
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
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
Rolling
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
RollingIntegrin-activation and arrest
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
RollingIntegrin-activation and arrest
Transmigration
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
RollingIntegrin-activation and arrest
Transmigration
Pericyte
Endothelial cell Astrocyte Tight junction Chemoattractant
Mucin SelectinIntegrin
Integrin-ligand
AbPerivascular macrophage GPCR
Ab plaque
Cytokines,
ROS,
Enzymes
Blood vessel
Brain parenchyma
Perivascular basement
membrane
Endothelial basement
membrane
Microglia
Neuron
RollingIntegrin-activation and arrest
Transmigration
Pericyte
Endothelial cell Astrocyte Tight junction Chemoattractant
Mucin SelectinIntegrin
Integrin-ligand
AbPerivascular macrophage GPCR
Ab plaque
Cytokines,
ROS,
Enzymes
Blood vessel
Brain parenchyma
Perivascular basement
membrane
Endothelial basement
membrane
Microglia
Neuron
RollingIntegrin-activation and arrest
Transmigration
Cytokines,
ROS,
Enzymes
Cytokines,
ROS,
Enzymes
Pericyte
Endothelial cell Astrocyte Tight junction Chemoattractant
Mucin SelectinIntegrin
Integrin-ligand
AbPerivascular macrophage GPCR
Ab plaque
Cytokines,
ROS,
Enzymes
Blood vessel
Brain parenchyma
Perivascular basement
membrane
Endothelial basement
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
top related