preclinical gwi research; therapeutic targets, plasma biomarkers … · cytochrome c oxidase is the...
TRANSCRIPT
Preclinical GWI research; therapeutic
targets, plasma biomarkers and clinical
translation Fiona Crawford, Ph.D.
President and CEO
Roskamp Institute
Sarasota, Florida
Neurobehavior
Neuropathology
Develop mouse
models of exposure to
known Gulf War
agents such as
Pyridostigmine
Bromide and
Permethrin
Molecular
profiling
From mouse brain and
blood studies: Identify key
molecular functions and
blood profiles and validate
results in
human patients
Evaluate in
GWI patients
and controls
Novel Treatment
strategies and
Blood Biomarkers for
diagnosis and
prognosis of GWI
Our Approach
Characterize
laboratory models of
GWI using
Neurobehavioral
tests,
Neuropathological
and Molecular
analyses (“omics”
technology)
Identify brain molecular
pathways, and blood
molecular profiles,
correlating with GW-
agent exposure –
dependent behavioral
and pathological
changes
Translate to
human
populations with
clinical trials of
novel
therapeutics,
and biomarker
profiling in
patients
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 135 of 279
PB+PER+DEET+Stress mouse
model of GW agent exposure
Adapted from the Abdel-Rahman rat exposure paradigm
For 28 days, C57BL6 mice (15-17 weeks old, male and
female) were daily exposed to
1.3mg/kg PB orally,
0.13 mg/kg PER dermally,
40 mg/kg DEET dermally, and
5 min of restrained stress
Control mice only received vehicle
(n = 10 per group)
Exposure (days 1-28)
Open field test (day 28)
Rotarod test (days 1-7)
Morris Water Maze (days
20-28)
Euthanasia (day 42)
Post-exposure period Exposure period
Behavioral studies and timeline for
PB+PER+DEET+Stress mouse model
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 136 of 279
GFAP neuropathology in
PB+PER+DEET+Stress mouse model
ControlPB+PER+DEET+Stress
0
0.5
1.0
1.5
2.0
control PB+PER+DEET+Stress
GF
AP
are
a (
%)
± S
EM
ControlPB+PER+DEET+Stress
*
Significant increase in GFAP (astrocytosis) in exposed versus
controls
PB+PER mouse model
of GW agent exposure
Male CD1 mice (9 weeks old) exposed to
2mg/kg PB
200 mg/kg PER
Daily (i.p.) for 10 days
N = 10-11 per group
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 137 of 279
Exposure (days 1-
10)
Rotarod (days 5-9)
Rotarod test (days
1-7)
Open field test
(day 15)
Open field test (day 30)
Morris Water Maze (days 43-115)
Euthanasia (day 150)
Post-exposure period Exposure period
Behavioral studies and timeline for
PB+PER mouse model
Open field test results from PB+PER model
control
PB+PER
0
250
500
750
1000
5 10 15
0
15
30
45
60
5 10 15
minutesminutes
Tim
e sp
en
t at
the p
erim
ete
r (s
) ±
SE
M
Total distance (cm) ± SEM
To
tal
dis
tan
ce
(cm
) ±
SE
M
0
250
500
750
1000
5 10 150
15
30
45
60
5 10 15
Tim
e s
pen
t a
t th
e p
erim
eter
(s)
± S
EM
minutes minutes
Day
15
Day
30
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 138 of 279
Morris water maze test results from
PB+PER mouse model
0
15
30
45
60
51 86 115
control
PB+PER
0
8
15
23
30
51 86 115
Esc
ap
e la
ten
cy (
s) ±
SE
M
NE
qu
ad
ran
t d
ura
tio
n (
s) ±
SE
M
daysdays
0
15
30
45
60
43 44 45 46 47 48 49 50 51
La
ten
cy t
o e
scap
e (
s) ±
SE
M
control PB+PER
GFAP neuropathology in
PB+PER mouse model
GFAP
(ratio to total area) ± SEM
0
8
15
23
30
control PB+PER
ControlPB+PER
PB+PER control
*
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 139 of 279
Analysis of proteomic data
• lists of significantly regulated
proteins
• upload to Ingenuity Pathway
Analysis knowledgebase to identify
• Canonical pathways
• Biological functions
• That are significantly modulated in
response to GW-agent exposure
Brain biofunctions modulated
in response to PB+PER exposure
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 140 of 279
Plasma biofunctions modulated
in response to PB+PER exposure
PB+PER+CPF mouse model
of GW agent exposure
Pilot study (N=4 per group) on effects of OP exposure
Male C57BL/6 mice aged 24 weeks
Mice were administered
chlorpyrifos (CPF) 5mg/kg or
5mg/kg CPF + 0.7mg/kg PB + 200mg/kg PER
Daily (i.p.) for 10 days.
Mice were euthanized at 3 days post exposure for
neuropathological analyses
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 141 of 279
Doublecortin staining in the dentate gyrus
after exposure to CPF or CPF+PB+PER
Synaptophysin staining in the hippocampi and cortices after exposure to CPF or CPF+PB+PER
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 142 of 279
Astrocytic staining in the hippocampi and cortices after exposure to CPF or CPF+PB+PER
Quantification of pathological findings after exposure to CPF or CPF+PB+PER
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 143 of 279
Characterization of the
PB+PER Mouse Model of GWI
Pyridostigmine Bromide (PB) – prophylactic against nerve gas attack
Permethrin (PER) – pesticide extensively applied by troops
Translation to the more common C57BL6 mouse strain
Mice exposed for 10 days daily to 0.7mg/kg of PB + 200 mg/kg of PER
To mimic the experience of GW veterans, acute exposure at a young age
to GW agents and longitudinal evaluation over the mouse lifespan
10 days
exposure
to PB+PER
Cognitive Testing ; Neuropathological analyses ; “Omic” analyses
EXPOSURE EVALUATION
Time
post-exposure: acute 5 months 15/16 months 22 months
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Groups
%Area±SEM
Quan fica onofGFAPStaining-CerebralCortexYoungCohort,10dayspostexposure
Control
PB+PER
No acute differences in GFAP
staining after exposure
0
5
10
15
20
25
Groups
%Area±SEM
Quan fica onofGFAPStaining-HippocampusYoungCohort,10dayspostexposure
Control
PB+PER
NS
NS
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 144 of 279
Elevation of GFAP at 5 months post-exposure
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Groups
%Area±SEM
Quan fica onofGFAPStaining-CerebralCortexAgedCohort,141dayspostexposure
Control
PB+PER
0
5
10
15
20
25
30
35
40
45
50
Groups
%Area±SEM
Quan fica onGFAPStaining-HippocampusAgedCohort,141dayspostexposure
Control
PB+PER
P = 0.0051 P = 0.0348
Aged cohort showed significant increase in astrocytosis, and reduction in synaptic markers and measures of neurogenesis
Inflammatory dysregulation – CNS v PNS; pro- v anti-inflammatory markers
Acute reduction in synaptophysin after exposure
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 145 of 279
Reduction in synaptophysin 5 months after exposure
Cognitive performance deteriorates over time in
mice exposed to the Gulf War agents PB+PER
0
10
20
30
40
50
60
70
Distance(cm
)±SEM
Vehicle
PB+PER
5Months
*
Distance traveled to find the
target hole is significantly
further in mice exposed to
PB+PER
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 146 of 279
Proteomic profiling shows significant changes in expression of
brain proteins in the GW mouse model compared to control
at 5 (red) and 16 (blue) months post-exposure
Highlighting : Immune/inflammatory functions
Mitochondrial function
Lipid metabolism
GFAP staining as a % of control
Persistence of inflammatory (astroglial) pathology over the
mouse lifespan after exposure to GW agents
B
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 147 of 279
Disruption of mitochondrial proteins in mice
exposed to the Gulf War agents PB+PER (COX6C)
Cytochrome C Oxidase subunit VIc
Disruption of mitochondrial proteins in mice
exposed to the Gulf War agents PB+PER (UQCRC1)
Ubiquinol-Cytochrome C Reductase Core Protein I
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 148 of 279
Mitochondrial function is compromised in mice
exposed to the Gulf War agents PB+PER
*
0
50
100
150
200
250
300
350
400
Units/mL/mg±SEM
IntactmitochondrionCytochromeCOxidaseAc vity
Control
PB+PER
*
Cytochrome C oxidase is the final step in the electron transport chain –
its activity is thought to be a good indicator of metabolic capacity
required for neuronal function in neurons.
Increased Total Brain PC and SM at
5 months post-exposure control PB+PER
0
8
15
23
30
Total PC
0
0.6
1.2
1.8
2.4
Total SM
µm
ol/
g o
f p
rote
in ±
SE
M
µm
ol/
g o
f p
rote
in ±
SE
M
* *
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 149 of 279
Catalase staining at 5 months post-exposure
PB+PERControl
0
1.8
3.5
5.3
7.0
control
PB+PER
Per
cent
area
sta
ined
± S
EM
dentate
gyrus
CA1
0
1
2
3
4
Per
cent
area
sta
ined
± S
EM
*
Lipidomic profile at 16-months post-exposure
to GW agents
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 150 of 279
Metabolomics profile at 16-months
post-exposure to GW agents
AA (an w-6 fatty acid [FA]) and DHA (an w-3 FA) - essential fatty acids primarily acquired through diet owing to the low capacity of the body to synthesize these lipids. AA metabolism initiates a pro-inflammatory cascade whereas DHA metabolism produces anti-inflammatory metabolites. Dietary intake of DHA is particularly important in aging adults to maintain cognitive function and for optimal neurotransmission. Figure from: Nature Chemical Biology 6, 401–402 (2010)
Role of omega-3 and omega-6
fatty acid-containing lipids in human health
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 151 of 279
50µm 50µm
AA and DHA imbalance
*
Pe
rce
nta
ge
of co
ntr
ol ±
SE
M
Astroglia pathology in the cortices of GWI mice
100µm
control PB+PER
control PB+PER
Pe
rce
nta
ge
of co
ntr
ol ±
SE
M
*
AA and DHA imbalance Astroglia pathology in the dentate gyri of GWI mice
PB+PER
control
PB+PER
control
100µm
A chronic imbalance of AA and DHA correlates with
astroglial pathology in mice exposed to PB+PER
0
20
40
60
80
100
120
140
Plasma PC and LPC profiles 18 days
following GW agent exposure
0
20
40
60
80
100
120
140
%contr
ol ±
SE
M
0
20
40
60
80
100
120
Total PC
control
PB+PER
%contr
ol ±
SE
M
%contr
ol ±
SE
M
* * * *
*
0
20
40
60
80
100
120
Total LPC
control
PB+PER
*
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 152 of 279
Plasma PC and LPC profiles 5-months
post-exposure to GW agents
0
20
40
60
80
100
120
140
160
180
PC
(32:2
)
PC
(32:1
)
PC
(32:0
)
eP
C(3
4:2
)
eP
C(3
4:1
)
eP
C(3
4:0
)
PC
(34:3
)
PC
(34:2
)
PC
(34:1
)
PC
(34:0
)
eP
C(3
6:4
)
eP
C(3
6:3
)
eP
C(3
6:2
)
eP
C(3
6:1
)
eP
C(3
6:0
)
PC
(36:6
)
PC
(36:5
)
PC
(36:4
)
PC
(36:3
)
PC
(36:1
)
eP
C(3
8:6
)
eP
C(3
8:5
)
eP
C(3
8:4
)
eP
C(3
8:3
)
eP
C(3
8:1
)
eP
C(3
8:0
)
PC
(38:6
)
PC
(38:5
)
PC
(38:4
)
PC
(38:3
)
PC
(38:2
)
eP
C(4
0:6
)
eP
C(4
0:3
)
eP
C(4
0:2
)
PC
(40:8
)
PC
(40:7
)
PC
(40:6
)
PC
(40:5
)
PC
(40:4
)
*
%contr
ol ±
SE
M
* *
0
20
40
60
80
100
120
Total PC
control
PB+PER
%contr
ol ±
SE
M
0
20
40
60
80
100
120
total LPC
control
PB+PER
%contr
ol ±
SE
M
0
20
40
60
80
100
120
140
%contr
ol ±
SE
M
* *
Plasma PC and LPC profiles at 16-
months post-exposure to GW agents
0
50
100
150
200
250
PC
(32:2
)P
C(3
2:1
)P
C(3
2:0
)eP
C(3
4:2
)eP
C(3
4:1
)eP
C(3
4:0
)P
C(3
4:3
)P
C(3
4:2
)P
C(3
4:1
)P
C(3
4:0
)eP
C(3
6:4
)eP
C(3
6:3
)eP
C(3
6:2
)eP
C(3
6:1
)eP
C(3
6:0
)P
C(3
6:6
)P
C(3
6:5
)P
C(3
6:4
)P
C(3
6:3
)P
C(3
6:2
)P
C(3
6:1
)eP
C(3
8:6
)eP
C(3
8:5
)eP
C(3
8:4
)eP
C(3
8:1
)eP
C(3
8:0
)P
C(3
8:6
)P
C(3
8:5
)P
C(3
8:4
)P
C(3
8:3
)P
C(3
8:2
)eP
C(4
0:6
)eP
C(4
0:3
)P
C(4
0:8
)P
C(4
0:7
)P
C(4
0:6
)P
C(4
0:5
)P
C(4
0:4
)
*
*
* * * * * *
* * *
*
* *
0
20
40
60
80
100
120
Total PC
control
PB+PER
%contr
ol ±
SE
M
%contr
ol ±
SE
M
*
0
20
40
60
80
100
120
%contr
ol ±
SE
M
* * * * * * *
0
20
40
60
80
100
120
total LPC
control
PB+PER
*
0
20
40
60
80
100
120
total LPC
control
PB+PER
*
%contr
ol ±
SE
M
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 153 of 279
Summary of results
Plasma biomarkers of GW-agent exposure are evident at
chronic timepoints post-exposure
Characterization of these mouse models of GWI with
neurobehavior, neuropathology and proteomic and lipidomic
profiling have identified a number of potential therapeutic
targets for further evaluation.
Inflammatory responses
Lipid dysmetabolism
Mitochondrial dysfunction
Targeting Inflammation
Anatabine • Alkaloid derived from tobacco and
plants of solenaceae family
• 3 year history of safe use as a
dietary supplement
• Potent anti-inflammatory
• Efficacy in mouse models of AD,
TBI, EAE, Tauopathy
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 154 of 279
Treatment with the anti-inflammatory compound Anatabine
significantly improves cognitive function
in PB+PER mouse model of Gulf War Illness
*
0
20
40
60
80
100
120
140
160
180
DG(40x)
GFA
P
%Area±SEM
Vehicle
PB+PER
Vehicle+Anatabine
PB+PER+Anatabine
Anatabine treatment significantly reduces astrogliosis
in a mouse model of Gulf War Illness
Veh
icle
Vehicle+Anatabine
PB+PER
PB+PER
+Anatab
ine
*
Treatment began at 5
months post-exposure
to PB+PER, continued
for 2 months and mice
were then evaluated
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 155 of 279
Clinical studies of plasma
biomarkers of GWI
Recruitment of patients through the Boston and Bronx
VA hospitals (Drs. Krengel, Sullivan and Golier)
160 GWI veterans, 120 healthy GW veterans and
40 GW-era veterans
Additional recruitment of Ft. Devens cohort
Plasma samples for proteomic/lipidomic profiling
and targeted analyses
Correlation with clinical presentation (and response
to treatment)
Translational Research
from preclinical models
Biomarker studies
• Generation of blood protein and lipid profiles from
human GWI patients
• Correlation of human blood profiles with mouse blood
and brain profiles
Therapeutics
• Ongoing preclinical validation of therapeutic targets in
laboratory models of GWI
• Facilitated by Collaborations between Gulf War Illness
clinical and basic science research teams
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 156 of 279
Acknowledgements
Laila Abdullah, Ph.D. Thin Nguyen, B.S.
Ghania Ait-Ghezala, Ph.D. Joseph Ojo, Ph.D.
Gogce Crynen, Ph.D. Jon Reed, M.S.
Tanja Emmerich, M.S. Venkatarajan Mathura, Ph.D.
Jim Evans Miles Tweed, B.S.
Hannah Montague, B.S. Zuchra Zakirova, M.S.
Michael Mullan, M.D., Ph.D.
Funding:
• Veterans Administration
• CDMRP
• Roskamp Foundation
Collaborators/Consultants:
• Juila Golier, M.D.
• Maxine Krengel, Ph.D.
• Kim Sullivan, Ph.D.
• Ashok Shetty, Ph.D.
• Alvin Terry, Ph.D.
Appendix A Presentation 3 - Fiona Crawford
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 157 of 279