Organophosphate Exposure and Cognition: Novel …€¦ · Neurotoxicity (OPIDN)-aggregation and accumulation of cytosketelal proteins, microtubules and neurofilaments (reviewed, Abou-Donia

Organophosphate Exposure and Cognition: Novel Mechanisms of

Neurotoxicity

Alvin V. Terry, Jr., Ph.D.Professor of Pharmacology and Toxicology

DirectorSmall Animal Behavior Core

Central Cholinergic Pathways

Human

RatSource: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press

Acetylcholine

Appendix A Presentation 3 - Terry

RAC-GWVI Meeting Minutes June 28-29, 2010 Page 71 of 214

Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press

Acetylcholine (cholinergic) Synapse

Acetylcholine

proNGF NGF

proteolysis

proNGF NGF

p75NTRSortilin

Cell Death Cell Survival

Neuronal Membrane

TrkA

Adapted from Nykjaer et al., Nature 427:843-848, 2004

Nerve Growth FactorSignaling



Growth factorKinesinDyneinVesicle

Myelin

Dendrite

ER

Mito

GANode

of Ranvier Terminal

CNS Neuron

Nucleus

“Esters of phosphoric acid”First synthesized in the 1800sInsecticide Potential Recognized in the 1930sFurther developed as “Nerve Agents”by the Germans and British during WWII

Organophosphates R1-O-P-O-R2

O

R3

R1-O-P-O-R2

R3



Improvements in Farming ProductivityControl of Vector-Borne Diseases

MalariaYellow FeverTyphusViral Encephalitis

Control of “Nuisance” PestsFlies, Ants, Roaches. Etc

Relatively inexpensive, less persistent than organochlorines

Organophosphate PesticidesAdvantages

R1-O-P-O-R2

O

R3

R1-O-P-O-R2

R3

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

Chlorpyrifosoxon

CYP 450

NerveAgents

Insecticides

Mechanism of Action



Organophosphate ToxicityAcute

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 and/or Repeated Low-Level 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.

Overall ObjectivesDetermine the consequences of repeated, “subthreshold” exposures to representative organophosphates on cognitive function in animal models.

Information processing and attentionSpatial LearningRecognition MemoryWorking Memory

Determine the consequences of repeated, low-level exposures to representative organophosphates on neurobiological substrates of cognitive function

Cholinergic MarkersNeurotrophinsAxonal Transport

Identify therapeutic targets for drug development



Study Algorithm

Learning and MemoryWater MazeNovel Object RecognitionPrepulse Inhibition5C-SRTTRadial Arm Maze

Neurotrophins (NGF)Western BlotImmunohistochemistry

OP Exposure

Axonal TransportSciatic Nerve-DIC MicroscopyMitochondrial MovementβAPP Immunohistochemistry

OP-FreeWashout

Cholinergic MarkersEnzyme AssaysReceptor AutoradiographyWestern Blot

OP Effects On Axonal Transport?Organophosphate Ester-Induced Delayed Neurotoxicity (OPIDN)-aggregation and accumulation of cytosketelal proteins, microtubules and neurofilaments (reviewed, Abou-Donia 2003)Decreased expression of presynapticcholinergic receptors in the brain (Stone et al., 2000)Disruption of tubulin polymerization & microtubule formation (Prendergast et al., 2007)Inhibited kinesin-dependent microtubule motility (Gearhart et al., 2007)OPs Covalently modify tyrosines on α and βtubulin (Grigoryan et al., 2009)



Axo

nal T

rans

port

Qua

ntity

(# v

esic

les/

view

ing

win

dow

)

0

100

200

300

400

500Anterograde Retrograde

-20%*

-20%*

Vehicl

e

Chlorpyri

fos

Vehicl

e

Chlorpyri

fos

Axo

nal T

rans

port

Qua

ntity

(# v

esic

les/

view

ing

win

dow

)

0

100

200

300

400

500Anterograde Retrograde

-30%*

-20%*

Vehicl

e

Chlorpyri

fos

Vehicl

e

Chlorpyri

fos

A B

Time after injection (hours)0 10 20 30 40 50

Axo

nal T

rans

port

Qua

ntity

(# v

esic

les/

view

ing

win

dow

)

250

300

350

400

450

-20%*

-10%*-15%*

Vehicle control

Anterograde

C D

Time after injection (hours)0 10 20 30 40 50

Axo

nal T

rans

port

Qua

ntity

(# v

esic

les/

view

ing

win

dow

)50

100

150

200

250

-33%*

-7%*

-26%*

Vehicle control

Retrograde

Axonal Transport

Chlorpyrifos 18.0 mg/kgN=3-5

Day 14 of WashoutDay 1 of Washout

Single Dose-Temporal Effect Single Dose-Temporal Effect

Terry et al., J. Pharmacol Exp Ther 322: 1117-1128, 2007.

Summary of Previous Chlorpyrifos Studies (repeated Subthreshold exposures)

Impairments in spatial learningImpairments in Prepulse Inhibition of the auditory startle response Decreased expression of cholinergic marker proteins in the brainDecreased expression of neurotrophin-related proteins in the brainImpairments of anterograde and retrograde axonal transport ex vivo





Recent and Ongoing Chlorpyrifos Studies

NH5C2

OP

O

OH5C2

S

ClCl

Cl

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 LeverContinuous Performance Task (CPT)

AX Type

Food Magazine

Light Stimulus



5C-SRTT-Chlorpyrifos (Every Day Exposure)

Test Session5 10 15 20 25 30 35 40 45

% c

orre

ct

060

70

80

90

100

During Exposure During Washout

VehicleChlorpyrifosVehicleVehicleChlorpyrifosChlorpyrifos

* ***

* * **

* * **** *

****

*** *** ****

Louise Middlemore-Risher

Middlemore-Risher et al., Neurotoxicology and Teratology 32: 415–424, 2010

5C-SRTT-Chlorpyrifos (Every Day Exposure)




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)


5C-SRTT-Chlorpyrifos (Alternate Day Exposures)




VehicleTreated

Chlorpyrifos oxon1.0 μM

Rat Cortical Neurons (MitoTracker® Imaging)

Vehicle

Chlorpyrifosoxon

5.0 nM

MitoTracker®

Imaging



% C

ontr

ol

0

50

100

150

200

250

Mitochondrial Number

020406080

100120

Mitochondrial Length

CPF (μM)0 1 5 10 20

CPF (μM)0 1 5 10 20

% C

ontr

ol

Rat Cortical Neurons(MitoTracker® Imaging)

** *

*

* *

*

# of Mitochondria Moving

% C

ontr

ol

020406080

100120

CPF (μM)0 1 5 10 20

**

*

*

Mitochondrial Length

% C

ontr

ol

0

50

100

150

200

250

Mitochondrial Number

% C

ontr

ol

020406080

100120

CPO (μM)0 1 5 10 20

CPO (μM)0 1 5 10 20

# of Mitochondria Moving

% C

ontr

ol

020406080

100120

CPO (μM)0 1 5 10 20

Rat Cortical Neurons(MitoTracker® Imaging)****

* * * *

* * **



Fusion

Fission

AnterogradeMovement

RetrogradeMovement

Mitochondrial Morphologyand Movement

Adapted from Detmer and Chan, 2007

CellBody Terminal

Microtubules

Ongoing DFP Studies

C3H7O P F

OH7C3

O



0

60

120

0 20 100 120

dB

MSEC

0

60

120

0 20 100 120

dB

MSEC

0

60

120

0 20 100 120

dB

MSEC

0

60

120

0 20 100 120

dB

MSEC

Pulse

PulsePrepulse

White Noise

White Noise

Normal Response

InhibitedResponse

“Prepulse Inhibition”

Prepulse Inhibition (PPI)

Restraining Tube

Sound AttenuatingEnclosure

Acoustic (Startle)Source

Motion Sensor

Prepulse InhibitionVehicle Study

Mea

n St

artle

Apl

itude

(Arb

itrar

y U

nits

)

0

200

400

600

800

1000

Startle Stimulus AloneNull StimulusPrepul 5 Prepul 10 Prepul 15

*

*

* +

*#

#

% P

repu

lse

Inhi

bitio

n

0

20

40

60

80

100

75 dB 80 dB 85 dBPrepulse Levels

* *#

% PPI = [100 - (startle amplitude on prepulse-pulse trials ÷ startle amplitude on pulse alone trials) x 100]

Vehicle Study

Star

tle A

mpl

itude

(AU

)

0200400600800

1000120014001600

% P

repu

lse

Inhi

bitio

n

0

20

40

60

80

100

Dose of DFP (mg/kg)

0.0 0.25 0.50 0.75 1.00

0.0 0.25 0.50 0.75 1.00

B

C

% P

repu

lse

Inhi

bitio

n

0102030405060708090

100 VehicleDFP 0.25 mg/kgDFP 0.50 mg/kgDFP 0.75 mg/kgDFP 1.00 mg/kg

75 dB 80 dB 85 dB

A

Prepulse Inhibition Studies

C3H7O P F

OH7C3

O

(Alternate Day Exposures)

N=11-26



Water Maze Hidden Platform Test

Fig Source: “Fundamental Neuroscience”, Second Edition, Copyright, 2003, Academic Press

Hidden Platform

During Learning After Learning

Day of Testing1 2 3 4 5 6

Dis

tanc

e to

Pla

tform

(cm

)

0

200

400

600

800

1000

1200

1400

1600

1800

2000 VehicleDFP 0.25 mg/kgDFP 0.50 mg/kgDFP 0.75 mg/kgDFP 1.0 mg/kg

*

A

Day of Testing1 2 3 4 5 6

Swim

Spe

ed (c

m/s

ec)

0

10

20

30

40

50B

0.0 0.25 0.50 0.75 1.00

% T

ime

in T

arge

t Qua

dran

t

0

10

20

30

40

Dose of DFP (mg/kg)

*

VehicleDFP 0.25 mg/kgDFP 0.50 mg/kg DFP 0.75 mg/kg DFP 1.00 mg/kg

C

*

Water Maze Studies C3H7O P F

OH7C3

O

N=11-26



Sample Trial

Choice Trial

Delay

Spontaneous NovelObject Recognition

15 Min Delay

0.0 0.25 0.50 0.75 1.00

Expl

orat

ion

Tim

e (s

ec)

0

5

10

15

20

25

30

Familar Novel

60 Min Delay

Expl

orat

ion

Tim

e (s

ec)

0

5

10

15

20

25

30

* *

* * * *

0.0 0.25 0.50 0.75 1.00Dose of DFP (mg/kg)

Dose DFP (mg/kg)

d2 In

dex

0.000.050.100.150.200.250.300.35

0.0 0.25 0.50 0.75 1.00

*†

C3H7O P F

OH7C3

O

Spontaneous Novel Object Recognition

N=11-24

d2 index = (novel - familiar)/(novel + familiar)

Averaged Across Delays



Treatment Time Point BF HIPP PFC CTX STR(mg/kg)

Vehicle Last Inj Day 12.37±0.78 4.42±0.24 3.83±0.33 3.01±0.15 8.94±0.42DFP 0.25 Last Inj Day 9.33±1.50*** 4.18±0.33 4.16±0.45 3.41±0.09 9.39±0.64DFP 0.50 Last Inj Day 9.88±1.02* 4.74±0.11 3.48±0.08 3.42±0.32 9.83±0.63DFP 0.75 Last Inj Day 8.21±1.11*** 4.69±0.35 3.82±0.37 2.94±0.20 8.34±0.39DFP 1.00 Last Inj Day 6.67±1.35*** 3.86±0.20 3.76±0.28 2.73±0.27 10.00±0.51

Vehicle WO Day 7 9.52±0.60 4.37±0.15 2.75±0.22 3.70±0.19 6.48±0.44DFP 0.25 WO Day 7 7.72±1.24§ 4.29±0.13 2.32±0.16 3.12±0.17 6.59±0.44DFP 0.50 WO Day 7 6.44±1.23** 4.40±0.35 2.58±0.07 3.26±0.12 6.23±0.76DFP 0.75 WO Day 7 8.06±0.94 4.64±0.21 2.21±0.12 3.03±0.09 6.41±0.65DFP 1.00 WO Day 7 7.88±0.61§ 4.85±0.19 2.25±0.10 3.07±0.17 7.37±0.46

Vehicle WO Day 14 9.06±2.22 4.69±0.37 4.95±0.37 2.99±0.13 8.89±0.80DFP 0.25 WO Day 14 8.04±0.60 4.57±0.72 3.78±0.16 2.76±0.26 9.26±1.02DFP 0.50 WO Day 14 8.52±1.66 4.93±0.54 3.97±0.0.63 2.66±0.20 8.27±0.83DFP 0.75 WO Day 14 8.50±2.33 4.74±0.65 3.63±0.29 3.39±0.33 8.01±0.93DFP 1.00 WO Day 14 5.61±1.55** 4.93±0.34 3.65±0.42 2.97±0.38 7.69±0.53

Brain Region

Choline Acetyltransferase (ChAT) Activity

ChAT activity expressed as pmoles of 3H-acetylcholine formed/μg of protein/min (mean ± S.E.M.) N= 5-6. *** p<0.001; **p<0.01; *p<0.05 §p<0.1 versus vehicle control.

α7-nicotinic acetylcholine receptor

VEH DFP

β-actin

α7-nAChR

VEH DFP

β-actin

α7-nAChR

α7:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

α7:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

***kDa

5543

VEH DFP

β-actin

α7-nAChR

α7:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

BF

HIPP

PFC

kDa

5543

kDa

5543



proNGF

BF

VEH DFPkDa

36

proNGFβ-actin

HIPP

VEH DFP

β-actin

PFC

VEH DFP

β-actin

proNGF

proNGF

36

kDa

36

36kDa

36

36

kDa

36

36kDa

36

36

proN

GF:β -

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

proN

GF:β -

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

proN

GF:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

**

p75 neurotrophin receptor (p75NTR)

VEH DFP

β-actin

VEH DFP

β-actin

kDA

7542

kDA

7542

VEH DFP

β-actin

kDA7542

p75NTR

p75NTR

p75NTR

p75:β -

actin

Rat

io

0.0

0.2

0.4

0.6

0.8

VEH DFP

p75:β -

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

**

p75:β -

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

BF

HIPP

PFC



mNGF

BF

VEH DFP

mNGFβ-actin

HIPP

VEH DFP

mNGFβ-actin

PFC

VEH DFP

mNGFβ-actin

kDa

3614

kDa

3614

14

36

NG

F:β -

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

NG

F:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

NG

F:β-

actin

Rat

io

0.00.20.40.60.81.0

VEH DFP

*

**

TrkA Receptor

BF

VEH DFP

TrkAβ-actin

HIPP

VEH DFP

TrkAβ-actin

PFC

VEH DFP

TrkAβ-actin

TrkA

:β-a

ctin

Rat

io

0.00.20.40.60.81.0

VEH DFP

TrkA

:β-a

ctin

Rat

io

0.00.20.40.60.81.0

VEH DFP

TrkA

:β-a

ctin

Rat

io

0.00.20.40.60.81.0

VEH DFP

kDa148

36

kDa148

36

kDa148

36

kDa148

36

kDa148

36

kDa148

36

**

*



p-TrKA Receptor

BF

VEH DFP

p-TrkAβ-actin

HIPP

VEH DFP

p-TrkAβ-actin

PFC

VEH DFP

p-TrkAβ-actin

pTrk

A:β

-act

in R

atio

0.00.20.40.60.81.0

VEH DFP

pTrk

A:β

-act

in R

atio

0.00.20.40.60.81.0

VEH DFP

pTrk

A:β

-act

in R

atio

0.00.20.40.60.81.0

VEH DFP

kDa148

36

kDa148

36

kDa148

36

kDa148

36

kDa148

36

kDa148

36

*

βAPP Immunostaining



Summary/Preliminary ConclusionsRepeated, subthreshold exposures to both insecticide and alkylphosphate OPs lead to protracted impairments in the performance of attention and memory-related behavioral tasks in animals.Insecticide and alkylphosphate OPs may have differential effects on specific domains of cognition.The mechanisms underlying OP-related impairments of cognition may involve deleterious effects on mitochondrial morphology and movement, axonal transport, and neurotrophin signaling.

PharmacologyBao-Ling Adam, Ph.D.Dan BeckLouise Middlemore-RisherChristina WilsonBrandon HallScott Webster

Animal Behavior Patrick CallahanKristy BouchardSamantha WarnerLeah VandenhuerkRosann SchadeMark BeckmanNancy KilleDonna Blessing

Acknowledgements

Terry-Lab Personnel



Jerry J. Buccafusco, Ph.D.(August 20, 1949 – March 6, 2010)

Regents' Professor, Department of Pharmacology and Toxicology

Director, Alzheimer's Research Center Medical College of Georgia

In Memory of:

Other Acknowledgements

Michael Bartlett, Ph.D.Dale Sickles, Ph.D.Elizabeth Herman, Ph.D.Debra Gearhart, Ph.D.Gary Schwarz, B.S.Jacob Truan, B.S.Adviye Ergul, M.D., Ph.D.NIH/NIEHS ES012241



Documents

Organophosphate Exposure and Cognition: Novel …€¦ · Neurotoxicity (OPIDN)-aggregation and accumulation of cytosketelal proteins, microtubules and neurofilaments (reviewed, Abou-Donia