Phenotypic Characterization and Brain Structure Analysis of Calcium Channel Subunit α2δ-2 Mutant (Ducky) and α2δ Double Knockout Mice.

Stefanie M Geisler,Nadia Stefanova,Christoph Schwarzer,Gerald J Obermair,Clemens L Schöpf,Arnold Schwartz,Ariane Benedetti

doi:10.3389/fnsyn.2021.634412

Abstract

Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel’s law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.

Highlights

In the central nervous system (CNS) the second messenger calcium regulates a variety of pivotal functions including neurotransmitter release, gene regulation, and synaptic plasticity (Nanou and Catterall, 2018)
The neocortex provides an intriguing brain region to further address these possibilities and shed light onto the role of α2δ subunits in the normal and diseased brain in several aspects: first, we found that the cortical volume was decreased in all three mouse models lacking the α2δ-2 isoform (Figure 4, see ducky, α2δ-1/-2 and α2δ-2/-3), which has not been described in the original reports of young ducky mice (Meier, 1968)
The reduction of cortex volume cannot be solely explained by developmental retardation, further implicating the role of neuronal α2δ subunits in the stabilization of axonal and dendritic arborization. This is the first study providing a general assessment of behavioral phenotypes and systemic analysis of different brain regions of three newly established α2δ double knockout mouse models (α2δ-1/-3, α2δ-1/-2, and α2δ-2/-3) and adult α2δ-2 mutant ducky mice

Summary

Introduction

In the central nervous system (CNS) the second messenger calcium regulates a variety of pivotal functions including neurotransmitter release, gene regulation, and synaptic plasticity (Nanou and Catterall, 2018). The entry of calcium is tightly controlled by voltage-gated calcium channels (VGCCs). Neuronal VGCCs are hetero-multimeric protein complexes consisting of a transmembrane pore-forming α1 subunit, which conducts Ca2+ upon membrane depolarization, and cytoplasmic β and extracellular α2δ subunits (Catterall, 2000; Zamponi et al, 2015). Four genes (Cacna2d1-4) encode four α2δ subunit isoforms (α2δ-1 to -4), which are post-translationally processed into highly glycosylated α2 and δ peptides linked to each other by disulfide bonds (De Jongh et al, 1990; Sandoval et al, 2004; Calderon-Rivera et al, 2012). All α2δ subunit isoforms display a related topology with a rather similar domain structure (reviewed in Geisler et al, 2015; Dolphin, 2018)

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