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Motor Deficits in a Novel Optineurin Mouse for Amyotrophic Lateral Sclerosis

McKee, C.1, Bomze, H.1, Rhodes, T.2,3, Means, C.2,3, Rodriguiz, R.M.2,3, Wetsel, W.C.2,3,4, Tseng, H.C.1

1Department of Ophthalmology, 2Department of Psychiatry and Behavioral Sciences, 3Mouse Behavioral and Neuroendocrine Analysis Core Facility, 4Departments of Cell Biology and Neurobiology

Duke Eye Center and Duke University Medical Center, Durham, NC 27710

Introduction

Methods

Conclusions

Mutations in optineurin are associated with

familial glaucoma and amyotrophic lateral

sclerosis (ALS).

The pathophysiological mechanisms of both

neurodegenerative diseases are unknown.

Optineurin functions in autophagy, TNF-NFκB

signal transduction, protein trafficking, and

polyubiquitin binding.

We hypothesized that ALS-associated optineurin

mutations result in a loss-of-function disease

mechanism.

To test this, we generated an optineurin knockout

mouse and compared knockout animals to

C57BL/6J controls in motor behavioral tests.

This work is supported by a K12-EY016333, K08-

EY021520, and National Eye Institute core grant

P30EY005722 to the Duke Eye Center.

Acknowledgements

Using the Cre-LoxP system (Fig 1), “floxed”

optineurin mice were generated and backcrossed

to a C57BL/6J background for 5-6 generations.

When crossed to CMV-Cre, complete genetic

deletion of optineurin was confirmed by Western

blotting of dissected retinas (Fig. 1).

Motor/behavioral tests were performed using 3-4

month old optineurin homozygous knockout

(KO) and C57BL/6J (C57) mice.

Disclosures

The authors have no commercial interests in this work.

Results

Figure 1. Generation of optineurin

knockout (KO) mice

Lo

xP

Lo

xP

Cre

Lo

xP

Floxed OPTN

OPTN

Knockout

Exon 1

frt

frt NeoR

ATG

OPTN N-term

OPTN C-term

-/- +/- +/+

GAPDH

Although more physically active (open field

test), optineurin KO mice exhibited deficits in

balance and coordination (rotarod steady speed,

foot fault, and pole walk tests) compared to

control C57 mice.

Consistently, optineurin KO mice required

more time in stabilization gait phases (stance &

propel), spent less time in a destabilizing phase

(swing), and exhibited reduced paw print area

and angle.

Front paw grip was stronger in optineurin KO

compared to control C57 mice. The KO mice

also have weaker rear paws than front paws,

but were comparable to control mice.

Motor impairments observed in our optineurin

KO mice suggest they may be useful as animal

models to study ALS.

Diameter +/+ (C57) -/- (KO)

28 mm 0% 18.2%

18 mm 10% 63.6%a

12 mm 40% 36.4%

9 mm 30% 63.6%b

A p < 0.011, b p = 0.122

Figure 2. Open Field Tests

Figure 3. Rotarod Accelerating and Steady Speed Tests

Figure 4. Foot Fault Tests

Figure 5. Grip Strength Tests

Results Results Results

Figure 6. Pole Walk Tests

Figure 7. Forced Gait Analysis

Figure 8. Forced Gait Phases

Open Field Test Statistical Difference

Vertical Activity -/- (KO) showed more vertical postures

Repetitive Activity No difference

Lapping/ Circling No difference

Thigmotaxis No difference

Distance in center -/- (KO) more active In center

Time spent in center No difference

Percent of C57 (n=10) and KO (n=11) mice which

required more than 1 trial to walk across pole

Mean ± S.E.M, n = 10 for C57, n = 9 for KO * p < 0.05 by Repeated Measures Analyses of Variance (RMANOVA)

Mean ± S.E.M, n = 10 for C57, n = 9 for KO,

p = 0.077, ** p = 0.011

Repeated Measures Analyses of Variance (RMANOVA)

Mean ± S.E.M, n = 10 for C57, n = 9 for KO, * p = 0.005, ** p = 0.001,*** p = 0.023

Repeated Measures Analyses of Variance (RMANOVA)

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Mean ± S.E.M, n = 10 for C57, n = 9 for KO, * p < 0.001 by Repeated Measures Analyses of Variance (RMANOVA)

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Mean ± S.E.M, n = 10 for C57, n = 11 for KO, * p = 0.003, ** p = 0.001

Repeated Measures Analyses of Variance (RMANOVA)

Mean ± S.E.M, n = 10 for C57, n = 9 for KO * p = 0 .03, ** p < 0.001

Repeated Measures Analyses of Variance (RMANOVA)

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Mean ± S.E.M, n = 10 for C57, n = 9 for KO * p = 0.018, ** p = 0.009 by t-test

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