Pathogenic NLGN4X variants reveal dual roles in synaptic connectivity and cortical development

Neuroligins are synaptic adhesion molecules important for neurodevelopment, and rare, highly penetrant variants underlie neurodevelopmental disorders (NDDs). Here, we characterize a rare missense variant NLGN4X G243R identified in a pedigree with NDDs and associated with severe protein trafficking deficits. NLGN4X G243R lies within a cluster of rare NLGN4X variants (amino acids 235-245), all of which impair trafficking.

Using biochemical, imaging, and electrophysiological approaches, we find that these NLGN4X variants exhibit severe trafficking defects, leading to impaired cell surface localization and synaptogenesis. Structural modeling further suggests that the NLGN4X NDD-variants destabilize protein conformation which is supported by thermal stability assessment. Notably, we uncover a previously unrecognized role for NLGN4X in regulating neuronal migration during early brain development, extending its functional scope beyond synaptogenesis.

Collectively, these results show that NDD-associated NLGN4X variants disrupt both synapse formation and neuronal migration, offering mechanistic insight into their contribution to neurodevelopmental pathology and highlighting potential therapeutic avenues. We are grateful to members of the Roche lab for technical assistance and for discussions of the project and manuscript. We thank the affected families that shared their clinical data with us.

This research was supported [in part] by the Intramural Research Program of the National Institutes of Health (NIH). The contributions of the NIH authors were made as part of their official duties as NIH federal employees, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S.

Department of Health and Human Services. This research was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (to E.H., M.R.M., J.D.B., W.L., and K.W.R.), the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (to A.K.), and the Polish National Science Center grant SONATINA 2023/48/C/NZ4/00072 (to P.B.). Receptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Eunhye Hong, John D.

Roche Synapse and Neural Circuit Research Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Miriam Reyes Mendez, Patrycja Brzdąk & Wei Lu Laboratoire de Biochimie et Biologie Moléculaire, CHU Nîmes, University of Montpellier, Nîmes, France Unité de génétique médicale 2, Hôpital L’Archet, Nice, France National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA Department of Biophysics and Neuroscience, Wroclaw Medical University, Wroclaw, Poland The authors declare no competing interests. All experimental animal procedures were approved by the NINDS Animal Care and Use Committee (protocol No. 1171) and were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Informed consent was obtained from all participants prior to their inclusion in the study.