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EGF Treatment Improves Motor Behavior and Cortical GABAergic Function in the R6/2 Mouse Model of Huntington’s Disease

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Abstract

Recent evidence indicates that disruption of epidermal growth factor (EGF) signaling by mutant huntingtin (polyQ-htt) may contribute to the onset of behavioral deficits observed in Huntington’s disease (HD) through a variety of mechanisms, including cerebrovascular dysfunction. Yet, whether EGF signaling modulates the development of HD pathology and the associated behavioral impairments remain unclear. To gain insight on this issue, we used the R6/2 mouse model of HD to assess the impact of chronic EGF treatment on behavior, and cerebrovascular and cortical neuronal functions. We found that bi-weekly treatment with a low dose of EGF (300 µg/kg, i.p.) for 6 weeks was sufficient to effectively improve motor behavior in R6/2 mice and diminish mortality, compared to vehicle-treated littermates. These beneficial effects of EGF treatment were dissociated from changes in cerebrovascular leakiness, a result that was surprising given that EGF ameliorates this deficit in other neurodegenerative diseases. Rather, the beneficial effect of EGF on R6/2 mice behavior was concomitant with a marked amelioration of cortical GABAergic function. As GABAergic transmission in cortical circuits is disrupted in HD, these novel data suggest a potential mechanistic link between deficits in EGF signaling and GABAergic dysfunction in the progression of HD.

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Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

aCSF:

Artificial cerebrospinal fluid

EGF:

Epidermal growth factor

GAD:

Glutamate decarboxylase

HD:

Huntington’s disease

htt:

Huntingtin protein

polyQ-htt:

Mutant htt

IHC:

Immunohistochemical

IPSC:

Inhibitory postsynaptic currents

KHC:

Kinesin heavy chain

PFC:

Prefrontal cortex

R6/2 mice:

Mice that express the 5′ end of the human HD gene containing 160 ± 5 poly Q

TBSX:

TBS containing 0.25% triton X-100

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Acknowledgments

We would like to thank Norma Hernandez for technical assistance with mouse breeding and UL1TR002003, which allowed the use of the Keyence microscope.

Funding

Supported by National Institutes of Health Grants (R01AG061114, R21AG053876, and R21AG061715 to L.M.T; R21NS096642 to G.A.M.), University of Illinois at Chicago institutional start-up funds (L.M.T), and CHDI research contract No. A-11014 (G.A.M and S.T.B).

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Author notes

  1. Felecia M. Marottoli, Mercedes Priego and Eden Flores-Barrera contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA

    Felecia M. Marottoli, Mercedes Priego, Eden Flores-Barrera, Rohan Pisharody, Steve Zaldua, Kelly D. Fan, Giri K. Ekkurthi, Scott T. Brady, Gerardo A. Morfini, Kuei Y. Tseng & Leon M. Tai

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  1. Felecia M. Marottoli
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Contributions

L.M.T. and K.Y.T. wrote the manuscript and prepared the figures. L.M.T, F.M.M., G.A.M., S.T.B., and K.Y.T. designed and supervised the study. F.M.M., L.M.T., P.M., R.P., S.Z., K.D.F., G.K.E., and N.H. conducted behavior testing, biochemical, and immunohistochemical analyses. K.Y.T. and E.F. designed and performed the electrophysiological experiments and data analyses. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Leon M. Tai.

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Research Involving Human Participants and/or Animals

This article does not contain any studies with human participants performed by any of the authors.

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All procedures follow the UIC Institutional Animal Care and Use Committee protocols.

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Electronic Supplementary Material

Supplementary Fig. 1

Sex does not modulate open-field or rotarod performance in R6/2 mice. Ten/eleven-week-old male and female, wild-type (WT), and R6/2 mice were tested for motor behavior. Compared to WT mice, R6/2 mice displayed a impaired performance in the rotarod test and b hypoactivity, lower average speed, and higher number of stops in the open-field behavioral test. Rotarod: F(2, 23) = 70.63. Open-field distance: F(2, 23) = 28.90. Open-field speed: F(2, 23) = 10.27. Open-field number of stops: F(2, 23) = 17.08. Sex did not modulate performance in R6/2 mice in open-field or rotarod tests. Data expressed as mean ± SEM. *p < 0.05 Tukey’s post hoc analysis after two-way ANOVA analysis (only genotype interactions were significant). n = 5 for male WT mice, 7 for female WT mice, 7 for male R6/2 mice, and 8 for female R6/2 mice. (AI 233 kb)

Supplementary Fig. 2

Study design. (AI 234 kb)

Supplementary Fig. 3

The effect of EGF treatment on behavior and HD-relevant pathology in wild-type mice. Wild-type (WT) mice were treated twice per week with vehicle (Veh) or EGF (300 μg/kg) from 4/5 to 10/11 weeks of age. a EGF treatment reduced rotarod performance but b did not modulate performance in open field. c EGF levels were higher in the plasma after EGF treatment. EGF treatment did not cause changes in d fibrinogen extravasation, e vessel coverage, or f levels of GABAergic proteins in the cortex. Data expressed as mean ± SEM. *p < 0.05 by Student’s t test. n = 5 per group. In (d), green, CD31; red, fibrinogen. In (e), green, laminin. Scale bar, 50 μm. (AI 1.11 kb)

Supplementary Fig. 4

EGF treatment does not modulate HD-relevant pathology in the striatum of R6/2 mice. Wild-type (WT) or R6/2 mice were treated twice per week with vehicle (Veh) or EGF (300 μg/kg) from 4/5 to 10/11 weeks of age. a Fibrinogen levels were higher in R6/2 mice compared to wild-type (WT) mice but were unaffected by EGF treatment in the striatum (IHC analysis). F(2, 16) = 77.23. b Cortical vessel coverage (laminin staining) was higher in R6/2 mice compared to vehicle-treated R6/2 mice but was also unaltered by EGF treatment in R6/2 mice. F(2, 16) = 3.997. c Top, representative images of fibrinogen (red) and brain endothelial cell (CD31, green) staining in the striatum. Bottom, representative images of laminin (green) staining in striatum. Scale bar, 50 μm. d PSD95 and DARPP-32 levels were lower in R6/2 mice compared to WT mice, whereas GAD67 and GAD65 levels were similar when assessed by western blot analysis. PSD95: F(2, 14) = 27.61. DARPP-32: F(2, 14) = 31. EGF treatment did not alter levels of PSD95, GAD65, GAD67, or DARPP-32 in the striatum of R6/2 mice. Left, quantification of each protein when normalized to KHC as a loading control. All data were then expressed as a ratio to vehicle-treated WT mice. Right, representative blots of each protein and loading control with bands on the same gel in nonadjacent positions separated by a dashed line. Data expressed as mean ± SEM. *p < 0.05 Tukey’s post hoc analysis after one-way ANOVA analysis. In (a)–(c), n = 5 for WT mice, 6 for vehicle-treated R6/2 mice, and 8 for EGF-treated R6/2 mice. In (d), n = 5 for WT mice, 6 for vehicle-treated R6/2 mice, and 6 for EGF-treated R6/2 mice. (AI 604 kb)

Supplementary Fig. 5

EGF treatment does not alter PSD95, NR1, or NR2A levels in the cortex of R6/2 mice. Wild-type (WT) or R6/2 mice were treated twice per week with vehicle (Veh) or EGF (300 μg/kg) from 4/5 to 10/11 weeks of age. Levels of PSD95, NR1, and NR2A were lower in R6/2 mice compared to WT. PSD95: F(2, 14) = 13.71. NR1: F(2, 14) = 23.84. NR2A: F(2, 14) = 38.23. However, EGF treatment did not alter levels of these markers in the cortex when assessed by western blot analysis. a Quantification of each protein when normalized to GAPDH as a loading control. All data were then expressed as a ratio to vehicle-treated WT mice. b Representative blots of each protein and loading control, with bands on the same gel in nonadjacent positions separated by a dashed line. Data expressed as mean ± SEM. *p < 0.05 using Tukey’s post hoc test after significant one-way ANOVA. n = 5 for WT mice, 6 for vehicle-treated R6/2 mice, and 6 for EGF-treated R6/2 mice. (AI 552 kb)

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Marottoli, F.M., Priego, M., Flores-Barrera, E. et al. EGF Treatment Improves Motor Behavior and Cortical GABAergic Function in the R6/2 Mouse Model of Huntington’s Disease. Mol Neurobiol 56, 7708–7718 (2019). https://doi.org/10.1007/s12035-019-1634-y

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  • DOI: https://doi.org/10.1007/s12035-019-1634-y

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