303 - Genetic deletion of Na,K-ATPase beta1-subunit in cerebellar granule cells results in a complex neurodevelopmental phenotype with behavioral and motor deficiencies

Publication Number: 4297.303

Karen Sperle, Nemours Children's Hospital, Wilmington, DE, United States; Mikail Rasheed, Nemours Children's Hospital, New Castle, DE, United States; Mohamed Khalife, Nemours Children's Hospital, Wilmington, DE, United States; Amanda Hernan, Nemours Children's Hospital, Wilmington, DE, United States; Sigrid A. Langhans, Nemours Children's Health, Wilmington, DE, United States

Background: Genetic advances have expanded our understanding of neurodevelopmental disorders (NDDs), but risk genes overlap and the same mutation in a single gene can result in varied symptoms. This underscores the continued need to uncover biological processes that contribute to and modulate the clinical manifestations of NDDs. Na,K-ATPase is an ion pump composed of two subunits, a catalytic α-subunit and a structural β-subunit. As a pump, it generates a transmembrane ion gradient that is essential for intracellular ion homoeostasis and the resting membrane potential in excitable cells. Lesser known is that the enzyme also functions in cell signaling and as a cell adhesion molecule. Recent research implicated the α-subunit in neurological disorders but the functions of its β-subunits in neurons remain less well understood.

Objective: To test whether the Na,K-ATPase β1-isoform (β1) plays a role in cerebellar development and NDDs.

Design/Methods: We generated a knockout (KO) mouse with deletion of β1 specifically in cerebellar granule cells (CGCs). The KO was confirmed by cerebellar genomic DNA analysis and immunoblotting. Morphological changes in the cerebellum were assessed by histology, immunohistochemistry, transmission electron microscopy, and MRI. Cell annotations in mouse cerebella and cell neighborhood analysis were done by spatial transcriptomics. Motor performance was tested by Catwalk analysis and social behavior was assessed by Barnes maze tests for learning and memory and social interaction.

Results: Deletion of β1 in CGCs resulted in complex neurological deficits. Homozygous KO mice had symptoms of cerebellar ataxia that worsened with age. Their lifespan was significantly reduced. Heterozygous KO mice physically appeared normal. Functional analysis showed no apparent changes in motor function, but heterozygous KO mice exhibited deficiencies in the Barnes maze tasks. Morphologically, the cerebella had no obvious structural differences, although the cerebella of homozygous KO mice were smaller, probably due to the smaller size of the mice.

Conclusion(s): The cerebellum displays an unexpected level of functional diversity participating in motor and non-motor behaviors such as cognition, emotion, and spatial navigation. We developed a novel mouse model with CGC-specific deletion of β1. Heterozygous knockout mice exhibit social and memory deficits while homozygous knockout mice present with ataxia during early postnatal cerebellar maturation. This provides the first evidence that the β1 expression level in CGCs affects high-level functioning of the cerebellum.

Targeted deletion of the β1-subunit of Na,K-ATPase in cerebellar granule cells.
(A,B) Verification of 1 deletion. PCR amplification of genomic DNA from tail (A) and cerebellum (B). (C) Homozygous KO mice present with clasping of the hindlimbs indicative of cerebellar dysfunction. Wildtype control mice show the typical spreading of the hindlimbs. Pictures taken at postnatal day (PND) 17. (D) Size difference in adult (79 days) homozygous 1 KO mouse and control littermate.

Spatial scatter plots with annotations of cerebella from knockout mice.