Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders

Abstract

Mutations in hundreds of genes cause neurodevelopmental disorders with abnormal motor behavior alongside cognitive deficits. Boys with fragile X syndrome (FXS), a leading monogenic cause of intellectual disability, often display repetitive behaviors, a core feature of autism. By direct observation and manual analysis, we characterized spontaneous-motor-behavior phenotypes of Drosophila dfmr1 mutants, an established model for FXS. We recorded individual 1-day-old adult flies, with mature nervous systems and prior to the onset of aging, in small arenas. We scored behavior using open-source video-annotation software to generate continuous activity timelines, which were represented graphically and quantitatively. Young dfmr1 mutants spent excessive time grooming, with increased bout number and duration; both were rescued by transgenic wild-type dfmr1⁺. By two grooming-pattern measures, dfmr1-mutant flies showed elevated repetitions consistent with perseveration, which is common in FXS. In addition, the mutant flies display a preference for grooming posterior body structures, and an increased rate of grooming transitions from one site to another. We raise the possibility that courtship and circadian rhythm defects, previously reported for dfmr1 mutants, are complicated by excessive grooming. We also observed significantly increased grooming in CASK mutants, despite their dramatically decreased walking phenotype. The mutant flies, a model for human CASK-related neurodevelopmental disorders, displayed consistently elevated grooming indices throughout the assay, but transient locomotory activation immediately after placement in the arena. Based on published data identifying FMRP-target transcripts and functional analyses of mutations causing human genetic neurodevelopmental disorders, we propose the following proteins as candidate mediators of excessive repetitive behaviors in FXS: CaMKIIα, NMDA receptor subunits 2A and 2B, NLGN3, and SHANK3. Together, these fly-mutant phenotypes and mechanistic insights provide starting points for drug discovery to identify compounds that reduce dysfunctional repetitive behaviors.

Keywords:

• ASD
• assay
• ethogram
• FMR1
• perseveration
• VCode

Acknowledgments

Drs. Robert Kraft, Kathleen K. Siwicki, Sara A. Lewis, and A. John Clark contributed many insights to discussions about this work. LLR thanks Dr. Katalin Gothard for introducing us to timeline ethograms. Many students contributed to assay development and collection of preliminary data, especially Frank Valdes and Elise Blackmore at University of Arizona and Sean Miller at Wake Forest University. Hillary Taylor performed the lethal-phase analysis of dfmr1 mutants. The authors thank two anonymous reviewers for insightful suggestions that helped us enhance the clarity of this report.

Data Availability Statement

Supplemental videos 1-4 may be viewed at https://arizona.box.com/s/xc1k6l2xfz8a222v5o5u24fbijcb1ggr

Disclosure statement

No potential conflict of interest was reported by the author(s).

Web resources

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DSM-5, neurodevelopmental disorders. https://doi.org/10.1176/appi.books.9780890425596.dsm01

MATLAB central file exchange, Trujillo-Ortiz, A., & Hernandez-Walls, R. (2010). gmregress: Geometric mean regression (reduced major axis regression). https://www.mathworks.com/matlabcentral/fileexchange/27918-gmregress,

Johns Hopkins University. Online Mendelian Inheritance in Man, OMIM^®. https://omim.org

CASK-related disorders. https://omim.org/entry/300422; https://omim.org/entry/300749

FMR1. https://omim.org/entry/309550

FXS. https://omim.org/entry/300624

King, C., & Goffin, G. (1962). The Locomotion. Recorded by Little Eva. https://www.youtube.com/watch?v=eKpVQm41f8Y

Hagedorn, J., & Hailpern, J. (2008). VCode. http://social.cs.uiuc.edu/projects/vcode.html

U.S. National Library of Medicine,ClinicalTrials.gov database. Combining lovastatin and a parent-implemented language intervention for Fragile X Syndrome. https://clinicaltrials.gov/ct2/show/results/NCT02642653

Additional information

Funding

Funding was provided by the Center for Insect Science, University of Arizona through a pilot research grant (to LLR and JKG) and the NIH-funded Postdoctoral Excellence in Research and Teaching (PERT) training program [GM000708], which selected DRA as a fellow. Support for the behavioral analysis of CASK mutants was provided in part by an Undergraduate Research Grant to DC from the Honors College of the University of Arizona.