Embed Size (px)
Citation preview
Alvin V. Terry, Jr., Ph.D.
Department of Pharmacology and Toxicology
Axonal Transport in Living Rats Exposed to Gulf War Relevant
Pesticides
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 74 of 279
Central Cholinergic Pathways
Human
Rat Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press
Acetylcholine
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 75 of 279
Esters of phosphoric, phosphonic & phosphinic acid
First synthesized in the 1800s Insecticide Potential Recognized in the 1930s Further developed as “Nerve Agents” by the
Germans and British during WWII
Organophosphates
HO-P-OH
OH
O
HO-P-OH
H
O
H-P-OH
H
O
phosphoric acid phosphonic acid phosphinic Acid
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 76 of 279
Insecticides (e.g., malathion, parathion, diazinon, chlorpyrifos)
Chemical Warfare (“nerve”) Agents (e.g., soman, sarin, tabun, VX)
Ophthalmic Agents (e.g., echothiophate, isoflurophate)
Antihelmintics (e.g., trichlorfon) Herbicides (e.g., tribufos, merphos) Solvents, Plasticizers, and Extreme
Pressure Additives for Lubricants
Organophosphate-Based Chemicals Found in:
Reviewed, Katz and Brooks, 2010
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 77 of 279
OH
N-CH2-CH2-O-C-CH3
CH3
CH3
H3C ++
O H3C N-CH2-CH2-OHCH3
CH3
acetylcholinesterase
acetylcholine
choline
acetic acidanionic esteratic
+
HO-C-CH3
OHO-C-CH3
O
R1-O-P-O-R2
O
R3
R1-O-P-O-R2
O
R3organophosphate
++
C3H7O P F
OH7C3
O
Sarin
Soman
DFP
O P F
CH3
O
C(CH3)3
CH3
H
C3H7O P FCH3
O
NH5C2
OP
O
OH5C2
S
ClCl
ClChlorpyrifos
NH5C2
OP
O
OH5C2
O
ClCl
Cl
Chlorpyrifos oxon
CYP 450
Nerve Agents
Insecticides
Mechanism of Action
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 78 of 279
Organophosphate Toxicity Acute
Muscarinic (postganglionic parasympathetic) “DUMB-BELS”: diaphoresis and diarrhea, urination, miosis, bradycardia, bronchospasm, emesis, lacrimation, salivation.
Nicotinic (neuromuscular junction)- muscle fasciculations, weakness, paralysis, respiratory failure; (CNS)- seizures or CNS depression/coma.
Chronic/Repeated Subthreshold Exposures* Anxiety, depression, psychotic symptoms, deficits
in short-term memory, learning, attention, information processing, eye-hand coordination and reaction time, and extrapyramidal symptoms.
* Data primarily from case reports and retrospective epidemiological studies.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 79 of 279
Different OPs can have markedly different toxicological profiles despite having similar effects on AChE activity.
Both AChE knockout mice and their wild type controls exposed to OPs exhibited similar signs of neurotoxicity.
Reports in both the human and animal literature indicate that OP toxicity (especially associated with chronic exposure) can occur in the absence of AChE inhibition.
Human studies of occupational exposures to OPs often fail to find a significant association between blood AChE activity and neurobehavioral deficits.
AChE inhibition may not be the whole story….
Reviewed in Banks and Lein, NeuroToxicology 33 575–584, 2012
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 80 of 279
Overall Objectives Determine the consequences of repeated,
“subthreshold” exposures to representative organophosphates (OPs) on cognitive function in animal models. Information processing and attention Spatial Learning Recognition Memory Working Memory
Determine the consequences of repeated, “subthreshold” exposures to representative OPs on neurobiological substrates of cognitive function Cholinergic Markers Neurotrophins Axonal Transport
Identify therapeutic targets for drug development
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 81 of 279
Exposure to one or more acetylcholinesterase inhibitors appears to offer a particularly plausible explanation for several of the neurological-based symptoms of GWI (Golomb et al., 2008)
An estimated 41,000 military personnel in the first gulf war were exposed to relatively high levels of insecticides that contained either carbamate or OP-based AChEIs (Fricker et al., 2000; US Department of Defense, 2003)
As many as 100,000 military personnel may have been exposed to low (i.e., non-acutely toxic) levels of sarin/cyclosarin following the destruction of an Iraqi munitions storage complex at Khamisiyah, Iraq, in March 1991 (Berardocco, 1997).
Gulf War Illness and OPs Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 82 of 279
Fly Bait azamethiphos
Pest Strips dichlorvos
Sprayed Liquids chlorpyrifos, diazinon, malathion
Fogs chlorpyrifos, malathion
OP-Pesticide Use in the First Gulf War
Source: http://www.gulflink.osd.mil/pest_final/index.html.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 83 of 279
The Rat, Five Choice Serial Reaction Time Task (5C-SRTT)
A H X J A K X O I Y A U B A X
Hit Lever Continuous Performance Task (CPT)
AX Type
Food Magazine
Light Stimulus Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 84 of 279
5C-SRTT Dependent Measures
Accuracy (% correct) Sustained Attention % Omissions Attention/Motivation Premature Responses Impulsivity Perseverative Responses Compulsivity Timeout Responses Compulsivity/Cognitive Flexibility Correct Latency (mean) Incorect Latency (mean) Reward Latency (mean)
Processing Speed and/or Motivation
Task Measure Behavioral Domain
Robbins TW. Psychopharmacology 163:362-380, 2002
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 85 of 279
Test Session5 10 15 20 25 30 35 40 45 50 55 60
% c
orre
ct
0
50
60
70
80
90
100
During Exposure During Washout
VehicleChlorpyrifosVehicleVehicleChlorpyrifosChlorpyrifos
* ** ************************
**** *** *
5C-SRTT-Chlorpyrifos (Alternate Day Exposures)
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 86 of 279
5C-SRTT-Chlorpyrifos (Alternate Day Exposures)
Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 87 of 279
5C-SRTT-Chlorpyrifos Experiments Conclusion
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 88 of 279
Test Session0 10 20 30 40 50 60 70
% C
orre
ct
070
80
90
100
VehicleDFP
During Exposure During Washout
*
1 8 0 0
2 0 0 0
2 2 0 0
2 4 0 0
V E H D F P
AU
C
3 0 0 0
3 2 0 0
3 4 0 0
3 6 0 0
V E H D F P
AU
C
Terry et al., Neurotoxicology and Teratology 44: 18–19, 2014
5C-SRTT-DFP 0.5 mg/kg (Alternate Day Exposures)
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 89 of 279
Terry et al., Neurotoxicology and Teratology 44: 18–19, 2014
Test Session0 10 20 30 40 50 60 70
Tim
eout
Res
p
0
5
10
15
20
VehicleDFP
During Exposure During Washout
* *
01 0 02 0 03 0 04 0 0
V E H D F PA
UC
0
1 0 0
2 0 0
3 0 0
4 0 0
V E H D F P
AU
C
5C-SRTT-DFP 0.5 mg/kg (Alternate Day Exposures)
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 90 of 279
5C-SRTT-DFP Experiments Conclusion
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 91 of 279
Water Maze Hidden Platform Test
Fig Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press
Hidden Platform
During Learning After Learning
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 92 of 279
Phase 1
Day of Testing
1 2 3 4 5
Late
ncy
to P
latfo
rm (s
ec)
0
10
20
30
40
50
60
70
VehicleCPF 10.0 mg/kgCPF 18.0 mg/kg
1 2 3 4 50
10
20
30
40
50
60
70
*
*
+
Day of Testing1 2 3 4 5
Swim
Spe
eds
(cm
/sec
)
0
10
20
30
Phase 2
Day of Testing1 2 3 4 5
Swim
Spe
eds
(cm
/sec
)
0
10
20
30
* = p<0.05 + = p<0.07
140 days post OP exposure
NH5C2
OP
O
OH5C2
S
ClCl
Cl
Terry et al., Neurotoxicology and Teratology 34 (1), 1–8, 2012.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 93 of 279
Day of Testing1 2 3 4 5
Late
ncy
to P
latfo
rm (s
ec)
0
10
20
30
40
50
60
70
VehicleDFP 0.25 mg/kgDFP 0.75 mg/kg
Phase 2
1 2 3 4 50
10
20
30
40
50
60
70
****
**
*+
Day of Testing1 2 3 4 5
Swim
Spe
eds
(cm
/sec
)
0
10
20
30
Phase 1
Day of Testing1 2 3 4 5
Swim
Spe
eds
(cm
/sec
)
0
10
20
30
A B
* = p<0.05 ** = p<0.01 + = p<0.07
140 days post OP exposure
C3H7O P F
OH7C3
O
Terry et al., Neurotoxicology and Teratology 34 (1), 1–8, 2012.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 94 of 279
Day of Washout
ChE
Act
ivity
(% o
f Con
trol
)
0
20
40
60
80
100
120
PlasmaBrain
0 14 30 45
DFP
Fig 1. The effects of repeated exposures to CPF 18.0 mg/kg (Left) or DFP 0.75 mg/kg (Right) on cholinesterase activity in the plasma and brain at various time points during a 45 day OP-free washout period. Data (mean ± SEM) are presented as % of vehicle-matched control levels. (N=3-6).
Terry et al., Neurotoxicology and Teratology 34:1-8, 2012
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 95 of 279
“Repeated, subthreshold exposures to CPF and DFP may lead to chronic deficits in spatial learning and memory (i.e., long after cholinesterase inhibition has abated) and that insecticide and nerve agent OPs may have differential effects depending on the cognitive domain evaluated”.
Conclusion
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 96 of 279
OP Effects On Axonal Transport?
Decreases in presynaptic cholinergic receptors in the rat brain (Stone et al., 2000)
Impairments in axonal transport of vesicles in sciatic nerves in rats (Terry at., 2003; 2007)
Disruption of tubulin polymerization & microtubule formation in vitro (Prendergast et al., 2007)
Inhibited kinesin-dependent microtubule motility in vitro (Gearhart et al., 2007)
OPs Covalently modify tyrosines on tubulin (Grigoryan et al., 2009; Bartlett et al, unpublished)
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 97 of 279
Growth factor Kinesin Dynein Vesicle
Myelin
Dendrite
ER
Mito
GA Node
of Ranvier Terminal
CNS Neuron
Nucleus
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 98 of 279
CargoCargo
+
+ Cargo
Cargo
Kinesin
Dynein
Tubulin
Anterograde Transport
Retrograde Transport
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 99 of 279
Louise Middlemore-Risher
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 100 of 279
Vehicle CPF oxon (5.0 nM)
MitoTracker® Imaging Measurements Movement = mean # moving/µm Length= average length in the ROI Number= # of mitochondria/µm
Scale bar= 100 µm
Middlemore-Risher et al., J Pharm Exper Ther 339: 341-349, 2011
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 101 of 279
Chlorpyrifos (µM)
Chlorpyrifos oxon (µM)
Movement Length Number
* *
MitoTracker® Imaging Measurements
Middlemore-Risher et al., J Pharm Exper Ther 339: 341-349, 2011
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 102 of 279
DePsipher™ Assay CPF CPO
VEH
20 µM
500 µM
Middlemore-Risher et al., J Pharm Exp Ther 339:341-349, 2011
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 103 of 279
Concentration-dependent decrease in the transport of mitochondria in axons, an increase in mitochondrial length, and a decrease in mitochondrial number (indicative of increased fusion versus fission events)
The neuronal changes occurred at OP concentrations that did not inhibit AChE, they were not blocked by cholinergic antagonists, and they did not appear to be associated with directly toxic effects on mitochondria (i.e., ATP production, mitochondrial membrane potential, superoxide production).
The results suggest that an underlying mechanism of OP-based alterations in neurological function might involve alterations in mitochondrial dynamics and/or their transport in axons.
Summary (CPF & CPO in Neuronal Culture) Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 104 of 279
APPDendra
APP695 linker Dendra
Hindlll Agel Notl
APPDendra
Tim
e (s
ec)
Distance (µm)
Tim
e (s
ec)
Distance (µm)
anterograde -> <- retrograde
Proximal Distal
Kymograph
APPDendra Tracking Studies
(Primary Cortical Neurons)
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 105 of 279
Tropolone alkaloid used since ancient times to treat rheumatism, swelling, and gout .
Binds tightly to tubulin, impairs tubulin polymerization, disrupts microtubule assembly and consequently impairs the ability of motor proteins to transport cargo in axons (Hastie, 1991; Uppuluri et al., 1993, Han et al., 1998)
Colchicine Colchicum autumnale
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 106 of 279
Velo
city
( m
/sec
)
0.00.10.20.30.40.50.6
vehicle colchicine 0.01nM colchicine 0.1nM colchicine 1nM colchicine 10nM colchicine 100nM colchicine 1uM colchicine 10uM
*
Colchicine(1hr Exposure)
*
* * *
*
** *
Anterograde Retrograde
*
*
* p< 0.05 versus vehicle control velocity
APPDendra Tracking Studies Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 107 of 279
APPDendra Tracking Studies (Primary Cortical Neurons)
Vehicle Colchicine 10 nM
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 108 of 279
Anterograde Retrograde
Velo
city
(m
/sec
)
0.00.10.20.30.40.50.6
Vehicle DFP 0.01nM DFP 0.1nM DFP 1nM DFP 10nM DFP 100nM DFP 1uM DFP 10uM *
DFP(1hr Exposure)
*
**
*
*
** **
*
*
* p< 0.05 versus vehicle control velocity
APPDendra Tracking Studies Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 109 of 279
APPDendra Tracking Studies (Primary Cortical Neurons)
Vehicle DFP 10 nM
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 110 of 279
Velo
city
(m
/sec
)0.0
0.1
0.2
0.3
0.4
0.5
Anterograde Retrograde
** * *
** * *
Vehicle DFP 0.01 nM DFP 0.1 nM DFP 1.0 nM DFP 10.0 nM DFP 100.0 nM
Velo
city
(m
/sec
)
0.0
0.1
0.2
0.3
0.4
0.5
*
Anterograde Retrograde
##
# ## #
* * * **
VehicleDFP 10nM ATR 50uMMEC 10uMDFP+ATRDFP+MEC
APPDendra Tracking Studies
(24 hr Exposure)
* p< 0.05 versus vehicle control velocity
# p< 0.05 versus DFP related velocity
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 111 of 279
Fig 3. Cell viability, structure andmorphology are not compromised after24 hr exposure to DFP. A. MTT assayresults indicate that 24 hr exposure toconcentrations of DFP up to 100 nM doesnot compromise neuronal cell viability. B.Pseudo-colored confocal images of culturedneurons indicate that the integrity ofcytoskeletal (Acti-stain, green), axonal (anti-doublecortin (DCX), red) an somatodendritic(anti-microtubule associated protein 2A/2B(MAP), blue) structure morphology werealso not compromised [DFP=100nM].Asterisks (*) indicate nuclei, pseudo-coloredfor contrast/clarity. VEH = vehicle
A. Cell Viability Assay
VEHcytoskeletal (actin)
axonal (DCX)
somatodendritic (MAP)
DFP
B. Cell Structure & Morphology
*
*
*% v
iabl
e ce
lls 100
20
406080
DFP (nM)0 0.01 0.1 1.0 10 100
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 112 of 279
Conclusions APPDendra Tracking Studies
DFP-related impairments of axonal transport: Were concentration dependent. Occurred at concentrations that did not
inhibit AChE activity. Were not blocked by cholinergic receptor
antagonists. Were not associated with compromises in
cell viability.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 113 of 279
Manganese-Enhanced Magnetic Resonance Imaging
(MEMRI) Studies
Optic Nerve
Superior Colliculus
6 hrs 24 hrs
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 114 of 279
Intravitreal Injection Method (MnCl2)
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 115 of 279
MEMRI-Colchicine Validation Studies (N=3)
Nor
mal
ized
Inte
nsity
Distance (mm)
ON
Vehicle
Colchicine
VehicleColchicine
ON
0 2 4 6 8 100
6000
8000
10000
12000
*
***
Hernandez et al., Neurotoxicology 47:17-26, 2015
6 hrs
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 116 of 279
VEH
SC
SC
COL 2.5 μg
MEMRI-Colchicine Validation Studies (N=3)
VEH COL
Nor
mal
ized
Inte
nsity
0.00.80.91.01.11.21.31.4
***
Hernandez et al., Neurotoxicology 47:17-26, 2015
24 hrs
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 117 of 279
Nor
mal
ized
Inte
nsity
ON
ON
Vehicle
Chlorpyrifos
VehicleChlorpyrifos
0 2 4 6 8 100
4000
6000
8000
10000
12000
Distance (mm)
MEMRI-Acute CPF Exposure (18.0 mg/kg Injection, N=6)
6 hrs
Hernandez et al., Neurotoxicology 47:17-26, 2015
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 118 of 279
VEH CPF
SC
SC
VEH CPF
Nor
mal
ized
Inte
nsity
0.00.80.91.01.11.21.31.4
MEMRI-Acute CPF Exposure (18.0 mg/kg Injection, N=6)
24 hrs
Hernandez et al., Neurotoxicology 47:17-26, 2015
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 119 of 279
Baseline WO 1 WO 2
Nor
mal
ized
Inte
nsity
Vehicle
Chlorpyrifos 3.0 mg/kg
Chlorpyrifos 18.0 mg/kg
2 4 6 8 100
6000
8000
10000
12000
2 4 6 8 100
6000
8000
10000
12000
2 4 6 8 100
6000
8000
10000
12000
ON
ON
ON
Baseline
Washout 1Washout 2
Distance (mm)
Nor
mal
ized
Inte
nsity
Distance (mm)
Nor
mal
ized
Inte
nsity
Distance (mm)
CPF 18.0 mg/kg -14 Day Repeated Exposure Study (N=6)
6 hrs
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 120 of 279
VEH
CPF3.0
CPF18
BL WO 1 WO 2
BL WO 1 WO 2
Nor
mal
ized
Inte
nsity
0.00.80.91.01.11.21.31.4
BL WO 1 WO 2
Nor
mal
ized
Inte
nsity
0.00.80.91.01.11.21.31.4
BL WO 1 WO 2
Nor
mal
ized
Inte
nsity
0.00.80.91.01.11.21.31.4
* **
SC
SC
SC
SC
SC
SC
+
+
Chlorpyrifos (CPF)
14 Day Exposure Study
(MEMRI-Superior Colliculus)
BL= Baseline WO1= 24 hr after last Injection WO2 = 30 Days after last injection
*p<0.05; ** p<0.01 CPF- WO1 and WO2 versus BL, respectively + p< 0.05 versus vehicle at the same washout period. VEH = vehicle; CPF = chlorpyrifos. N=6.
Hernandez et al., Neurotoxicology 47:17-26, 2015
24 hrs Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 121 of 279
Conclusions CPF-MEMRI Study
Intravitreal injection of the tropolone alkaloid colchicine led to marked impairment of axonal transport of Mn2+ in the brain of rats.
A single subcutaneous injection of the commonly used insecticide, chlorpyrfos (i.e., at a subthreshold dose) did not appear to affect axonal transport in the brain of rats.
Conversely, repeated, subthreshold exposures to the commonly used insecticide, chlorpyrfos led to prolonged impairments of axonal transport in the living rodent brain.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 122 of 279
Overall Summary/Conclusions Repeated, subthreshold exposures to both
insecticide and nerve agent OPs lead to protracted impairments of memory-related behavioral tasks in animals.
Insecticide and nerve agent OPs may have differential effects on specific domains of cognition.
One (non-cholinesterase) mechanism underlying long term (OP-related) impairments of cognition may involve deleterious effects on axonal transport.
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 123 of 279
Current/Future Studies: Proteomics
β-tubulinfragment
α-tubulinfragment
500150 200 250 300 350 400 450 550100 600 650mass-to-charge ratio (m/z)
% a
bund
ance
100
50
100
50
0
173.3
115.6
482.3 504.5
571.7
383.5 426.9
672.1
625.5
126.4157.0
173.3
355.5 487.2
536.1
569.9589.8
700
Y VPR
TGT Y R
*= alkylated tyrosine
272.7*
% a
bund
ance
272.4*
YP
291.0
0
α-tubulinβ-tubulin
trypsin α- & β-tubulin
fragments
YVPR
TGTYR
Y VPR
TGT Y R
CPO PCPO P
S
OO-P-OCPF CPO
proteolytic digestion
Type of proteinaxonal transport
neuronal plasticity
peptidefragments
OPmass spec
(size)
UHPLC(sequence)
O
OO-P-O
Mass Spec
CYP450
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 124 of 279
Current/Future Studies Septal Neuron Imaging
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 125 of 279
Current/Future Studies Specific Aim #1: Determine the consequences of
repeated subthreshold exposures to representative OPs on axonal transport in the living rat brain.
Manganese-Enhanced Magnetic Resonance Imaging (MEMRI) Studies
Specific Aim #2: Determine the consequences of
repeated subthreshold exposures to representative OPs on myelin in the living rat brain.
Diffusion tensor imaging (DTI) Black Gold II Histology
DOD-CDMRP Project “Organophosphate-Related Alterations in Myelin and Axonal Transport in the Living Mammalian Brain”
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 126 of 279
CA1
DG
CA3
Vehicle DFP
Black Gold II Histology Posterior Hippocampus
Scale Bar = 100 µm
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 127 of 279
Current/Future Studies OP Effects on Cytoskeletal Proteins
Acetylated α-Tubulin
Therapeutic Interventions in vitro Microtubule Stabilizers Selective HDAC Inhibitors
OP Effects on Signaling Proteins Involved in Neuronal Plasticity and Cognition ERK AkT CREB
Development of a Novel (Operant-Based) Test of Executive Function for Rats Attentional Set Shifting
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 128 of 279
MS HIPP
Rat Brain (Sagittal View) Mn2+ Injection Site
Target Region
Mn2+ Enhanced MRI (MEMRI) Septohippocampal Pathway
Future Studies Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 129 of 279
Pharmacology Lab
Caterina Hernandez, Ph.D.
Jie Gao, Ph.D.
Indrani Poddar, MS
Dan Beck
Sean Naughton
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 130 of 279
Small Animal Behavior Core
Samantha Sinha
Kristy Bouchard
Leah Vandenheurk
Patrick Callahan, MS
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 131 of 279
Large Animal Behavior Lab
Nancy Kille Marc Plagenhoef
Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 132 of 279
Ashley Davis
Office Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 133 of 279
Collaborators Michael Bartlett, Ph.D. Nathan Yanasak, Ph.D. Jordi Magrane, Ph.D.
Departmental Colleagues Nevin Lambert, Ph.D. Bobby Thomas, Ph.D. Navneet Kaidery, Ph.D.
Previous Lab Members Louise Middlemore-Risher, Ph.D. Bao-Ling Adam, Ph.D.
Funding NIH/NIEHS ES012241 DOD/(USAMRAA) W81XWH-12-1-0536
Additional Acknowledgements Appendix A Presentation 2 - Alvin Terry
RAC-GWVI Meeting Minutes April 20-21, 2015
Page 134 of 279
