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

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.

Presynaptic α 2 δ subunits are key organizers of glutamatergic synapses

“…Livecell immunolabeling of the HA-epitope allows a direct and, most importantly, comparative analysis of α 2 δ isoform surface expression. Using the same antibody (anti-HA) for quantitatively comparing distinct α 2 δ isoforms provides an important advantage over currently available α 2 δ antibodies, which either do not reliably detect the native proteins (28) or are not suitable for immunocytochemical experiments (29). Although the overall intensity of total surface expression levels differs between isoforms (α 2 δ-2 > α 2 δ-3 > α 2 δ-1), all three isoforms are localized to the somatodendritic and axonal membrane (SI Appendix, Fig.…”

“…With the exception of the α 2 δ-2 mutant mouse ducky, knockout mice for α 2 δ-1 and α 2 δ-3 display only mild neuronal phenotypes, suggesting a potential and at least partial functional redundancy (discussed above). Therefore, in order to gain insight into the functional diversity of α 2 δ subunits, we generated double-knockout mice by pairwise cross-breeding single-knockout (α 2 δ-1 and α 2 δ-3) and mutant (α 2 δ-2 du ) mice (29). While α 2 δ-1/-3 knockout mice are viable for up to 3 mo, similar to ducky mice, α 2 δ-1/-2 and α 2 δ-2/-3 knockout mice have a strongly reduced lifespan (Fig.…”

Impaired Subcortical Processing of Amplitude-Modulated Tones in Mice Deficient for Cacna2d3, a Risk Gene for Autism Spectrum Disorders in Humans

“…The α 2 δ proteins α 2 δ1 – α 2 δ4 are expressed in the nervous system, muscle and endocrine organs, with α 2 δ1, α 2 δ2, and α 2 δ3 expressed in the brain ( Cole et al, 2005 ; Ablinger et al, 2020 ; Geisler et al, 2021 ). They function as largely extracellular components of voltage-activated Ca 2+ channels and modulate the abundance and biophysical properties of these channels ( Catterall et al, 2005 ; Dolphin, 2013 ).…”

“…They function as largely extracellular components of voltage-activated Ca 2+ channels and modulate the abundance and biophysical properties of these channels ( Catterall et al, 2005 ; Dolphin, 2013 ). Moreover, α 2 δ proteins may also act independently of channel function such as (1) in organizing synapses in development; (2) trafficking Ca 2+ channels along axons; or (3) in transsynaptic coupling ( Eroglu et al, 2009 ; Kurshan et al, 2009 ; Pirone et al, 2014 ; Fell et al, 2016 ; Kadurin et al, 2016 ; Dolphin, 2018 ; Ferron et al, 2018 ; Geisler et al, 2019 , 2021 ; Ablinger et al, 2020 ; Bikbaev et al, 2020 ).…”

“…Lack of α 2 δ3 resulted in altered pain processing, increased acoustic startle response, altered auditory processing, cortical sensory cross-activation, anxiety-like behavior, and a volume decrease in corpus callosum ( Neely et al, 2010 ; Pirone et al, 2014 ; Landmann et al, 2018 ; Geisler et al, 2021 ). Of note, the lack of α 2 δ3 in α 2 δ3 −/− mice is partially rescued by α 2 δ1 and α 2 δ2, as shown in a recent study on single and double knock-out mice for the brain-specific subunits α 2 δ1, α 2 δ2, and α 2 δ3 ( Geisler et al, 2021 ).…”