NLGN3 autism variants have distinct functional impact on synapses and sleep behavior in Drosophila
6 affiliations
- 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.
- 2Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, Texas, United States of America.
- 3Department of Neurology, Baylor College of Medicine, Houston, Texas, United States of America.
- 4Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada.
- 5Children's Hospital Research Institute of Manitoba, Winnipeg, Manitoba, Canada.
- 6Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America.
Abstract
Neuroligin-3 (NLGN3) was first identified as a X-linked risk gene associated with autism spectrum disorder (ASD). Most reported variants are identified in males who inherited their variants from an unaffected mother, but de novo variants have also been identified in both affected heterozygous females and hemizygous males. The p.R451C variant was the initial variant associating NLGN3 with ASD and has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. To determine the functional consequences of other NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to a de novo variant (p.R175W) identified in a heterozygous female proband with ASD, a maternally inherited variant (p.R451C) identified in a hemizygous male with ASD, and another maternally inherited variant identified in a male (p.R597W) associated with sleep disorder and ASD. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that the de novo NLGN3 p.R175W variant identified in a heterozygous female exhibits gain-of-function properties, while maternally inherited variants have context-dependent functional impact. Moreover, some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author summary: Autism spectrum disorder (ASD) is a common neurodevelopmental disorder. Variants in the X-linked Neuroligin-3 (NLGN3) gene are associated with ASD, but very few of these changes have been characterized in animal models. Most of these variants affect hemizygous male individuals who maternally inherited their genetic alteration from unaffected heterozygous maternal carriers; however, more rarely affected female individuals may present with a heterozygous variant that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different variants, one being identified in a heterozygous female and two variants identified in hemizygous males, affect the fly’s behavior and synapse development. We identified altered sleep patterns when some NLGN3 variants are expressed which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and pathogenic variants are not all equivalent and may fall into distinct classes of functional impact depending on context. This demonstrates the utility of assessing multiple variants simultaneously because each variant may have distinct functional significances.
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BibTeX
@article{townsley2026nlgn3,
title = {{NLGN3 autism variants have distinct functional impact on synapses and sleep behavior in Drosophila}},
author = {Rebekah E Townsley and Jonathan C Andrews and Saurabh Srivastav and Sharayu V Jangam and Shabab B Hannan and Oguz Kanca and Paul C Marcogliese and Shinya Yamamoto and Michael F Wangler},
journal = {PLOS Genetics},
year = {2026},
month = oct,
doi = {10.1371/journal.pgen.1012341},
url = {https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012341},
note = {CC BY},
}
RIS
TY - JOUR
TI - NLGN3 autism variants have distinct functional impact on synapses and sleep behavior in Drosophila
AU - Rebekah E Townsley
AU - Jonathan C Andrews
AU - Saurabh Srivastav
AU - Sharayu V Jangam
AU - Shabab B Hannan
AU - Oguz Kanca
AU - Paul C Marcogliese
AU - Shinya Yamamoto
AU - Michael F Wangler
PY - 2026
DA - 2026/10/16
JO - PLOS Genetics
DO - 10.1371/journal.pgen.1012341
UR - https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1012341
AB - Neuroligin-3 (NLGN3) was first identified as a X-linked risk gene associated with autism spectrum disorder (ASD). Most reported variants are identified in males who inherited their variants from an unaffected mother, but de novo variants have also been identified in both affected heterozygous females and hemizygous males. The p.R451C variant was the initial variant associating NLGN3 with ASD and has been heavily investigated, yet little is known about the functional consequences of other NLGN3 variants. To determine the functional consequences of other NLGN3 variants in vivo, we generated transgenic Drosophila models corresponding to a de novo variant (p.R175W) identified in a heterozygous female proband with ASD, a maternally inherited variant (p.R451C) identified in a hemizygous male with ASD, and another maternally inherited variant identified in a male (p.R597W) associated with sleep disorder and ASD. In Drosophila, loss of the fly homolog, Nlg3, altered sleep patterns, synaptic architecture, and vesicle dynamics, which were rescued by the expression of the human NLGN3Ref allele. When comparing the variants, the p.R175W variant and the maternally inherited p.R451C variant altered synapse morphology with minimal effects on vesicle dynamics, and the p.R597W variant altered sleep and vesicle dynamics with minimal impact on synapse morphology. Using overexpression models, human NLGN3Ref altered sleep patterns and synaptic morphology. Moreover, the p.R175W variant exacerbated sleep phenotypes, and the p.R175W and p.R451C variants exacerbated synapse morphology phenotypes. Together, our findings suggest that the de novo NLGN3 p.R175W variant identified in a heterozygous female exhibits gain-of-function properties, while maternally inherited variants have context-dependent functional impact. Moreover, some NLGN3 variants disrupt synaptic development, while other variants alter synaptic function, suggesting that NLGN3 variants have differential effects. These functional differences may provide insight into the heterogeneity of individuals with ASD. Author summary: Autism spectrum disorder (ASD) is a common neurodevelopmental disorder. Variants in the X-linked Neuroligin-3 (NLGN3) gene are associated with ASD, but very few of these changes have been characterized in animal models. Most of these variants affect hemizygous male individuals who maternally inherited their genetic alteration from unaffected heterozygous maternal carriers; however, more rarely affected female individuals may present with a heterozygous variant that was not identified in either of the parents. Here, we utilized the fruit fly model to investigate how three different variants, one being identified in a heterozygous female and two variants identified in hemizygous males, affect the fly’s behavior and synapse development. We identified altered sleep patterns when some NLGN3 variants are expressed which is consistent with sleep disturbances being highly comorbid with ASD. Additionally, we identified alterations in synapse development and function which is consistent with the role of NLGN3 in synapse formation and maturation. Together, our findings support that NLGN3 is important for regulating the synapse and pathogenic variants are not all equivalent and may fall into distinct classes of functional impact depending on context. This demonstrates the utility of assessing multiple variants simultaneously because each variant may have distinct functional significances.
SN - 1553-7404
SN - 1553-7390
ER -