Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

Abstract

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Physical and functional convergence of the autism risk genesScn2aandAnk2in neocortical pyramidal cell dendrites

“…They consist of a 220 kDa isoform and a giant 440 kDa isoform, which arises from alternative splicing of a single large exon in the middle of the gene (Cunha et al, 2008). The 440 kDa ankyrin-B has been shown to be the primary isoform in the distal axon (Creighton et al, 2021; Lorenzo et al, 2014; Yang et al, 2019), but which splice variant functions in the dendrites is less clear. Western blot analysis of mouse neocortical lysates ranging from P0 to P30 revealed parallel increases in the expression levels of Na V 1.2 and ankyrin-B throughout development ( Figure 2B ).…”

Mechanisms underlying the role of ankyrin-B in cardiac and neurological health and disease

“…Early observations revealed neuroanatomical defects in the global Ank2 knockout mouse ( 57 ) and model organisms have highlighted homologous ankyrin roles in neuronal polarization ( 79 ). Other mainly in vitro studies point to a role for ANKB in GABAergic synaptic development ( 120 ), axonal branching ( 51 , 58 , 98 ), and voltage-gated calcium channel trafficking ( 101 , 102 ) ( Figure 3 ). These studies suggest ANKB regulates neurodevelopmental processes and could help explain its putative role in risk for ASD, as well as its association with epilepsy and seizure.…”

“…Mouse Ank2 is comprised of exons exhibiting considerable homology to those found in human ANK2 and exhibits similar tissue-specific isoform expression patterns ( 24 , 25 ). In mice, global Ank2 knockout causes neonatal death ( 57 ), while conditional Ank2 knockout in the heart and brain results in significant electrical and structural impairments and death ( 44 , 51 , 58 , 59 ). Heterozygous Ank2 knockout ( Ank2 +/− ) mice model haploinsufficiency (i.e., expression of a single wildtype Ank2 allele fails to produce a wildtype phenotype), are relatively viable, and therefore used in many preclinical studies.…”

“…In the case of loss of brain-specific giant ANKB 440-kDa, which primarily localizes to axons, mice display ectopic axon branching and connectivity, transient increase in excitatory synapses, and neurodevelopmental disorder-like behaviors such as stereotype movements and impaired social behavior ( 51 ). The impairment on axonal connectivity has been linked to ANK2's role in regulating the formation of microtubule bundles in the axon and reducing branching points enriched with F-actin by promoting growth cone collapse in response to semaphorin 3A signaling ( 58 , 98 ) ( Figure 3 ). While brain specific Ank2 knockout mice do not exhibit impairments in memory and learning ( 51 ), the identified structural and connectivity changes recapitulate some of the morphological features observed in neurodevelopmental disorders, such as ASD ( 51 , 99 , 100 ).…”

Role of Nrp1 in controlling cortical inter-hemispheric circuits

“…the sequential role of Nrp1 in the guidance, growth, and refinement during CC circuit development likely requires the contextual use of several of such molecules. For example, Sema3A shows a high-to-low lateromedial gradient of developmental expression, triggers repulsion, and the collapse of branching points ( Kitsukawa et al, 1997 ; Zhao et al, 2011 ; Zhou et al, 2013 ; Creighton et al, 2021 ). Sema3A is thus a good candidate to be involved in Nrp1 down-regulation required for terminal branching refinement in S2.…”