Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A
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Evaluation Summary:
This paper addresses the mechanisms regulating cytoskeletal changes mediating neuronal branching and axon growth. The authors assess the role of the scaffolding protein giant ankyrin B in cortical neuron differentiation and present strong data implicating ankyrin B functions with the cell adhesion molecule/Sema3A receptor complex and an actin severing protein. With a better explanation of the function of ankyrin B in the Sema3A signaling pathway, this manuscript will be of interest to cell biologists and developmental neurobiologists working to uncover the biological mechanisms of early circuit development, and how these mechanisms relate to autism spectrum disorders.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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.
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Evaluation Summary:
This paper addresses the mechanisms regulating cytoskeletal changes mediating neuronal branching and axon growth. The authors assess the role of the scaffolding protein giant ankyrin B in cortical neuron differentiation and present strong data implicating ankyrin B functions with the cell adhesion molecule/Sema3A receptor complex and an actin severing protein. With a better explanation of the function of ankyrin B in the Sema3A signaling pathway, this manuscript will be of interest to cell biologists and developmental neurobiologists working to uncover the biological mechanisms of early circuit development, and how these mechanisms relate to autism spectrum disorders.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
This paper addresses the mechanisms regulating cytoskeletal changes mediating neuronal branching and axon growth. The authors assess the role of the scaffolding protein giant ankyrin B in cortical neuron differentiation and present strong data implicating ankyrin B functions with the cell adhesion molecule/Sema3A receptor complex and an actin severing protein. With a better explanation of the function of ankyrin B in the Sema3A signaling pathway, this manuscript will be of interest to cell biologists and developmental neurobiologists working to uncover the biological mechanisms of early circuit development, and how these mechanisms relate to autism spectrum disorders.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This paper investigates the function of the neurospecific 440-kDa ankyrin-B isoform in axonal branching. Previous work has shown that AnkB440 KO mice have increased branching and an enlarged corpus callosum. It was also previously shown that AnkB440 interacts with L1CAM. This paper expands this previous work to investigate the mechanism by which AnkB440-L1CAM mediates signalling. Since L1CAM is known to also interact with Neuropilin1 and Sema3A, the authors investigated whether AnkB440-L1CAM might mediate its effects on neuronal branching through modulating Sema3A collapse. Indeed, the authors find that AnkB440 is required for Sema3A-mediated growth cone collapse. AnkB440 does this by stabilizing the L1CAM-Nrp1 complex at the cell surface, as well as mediating F-actin disassembly during growth cone collapse. …
Reviewer #1 (Public Review):
This paper investigates the function of the neurospecific 440-kDa ankyrin-B isoform in axonal branching. Previous work has shown that AnkB440 KO mice have increased branching and an enlarged corpus callosum. It was also previously shown that AnkB440 interacts with L1CAM. This paper expands this previous work to investigate the mechanism by which AnkB440-L1CAM mediates signalling. Since L1CAM is known to also interact with Neuropilin1 and Sema3A, the authors investigated whether AnkB440-L1CAM might mediate its effects on neuronal branching through modulating Sema3A collapse. Indeed, the authors find that AnkB440 is required for Sema3A-mediated growth cone collapse. AnkB440 does this by stabilizing the L1CAM-Nrp1 complex at the cell surface, as well as mediating F-actin disassembly during growth cone collapse. In addition, the authors also identify that AnkB440 and the AnkB220 isoforms exert different effects on axonal transport and cytoskeletal changes.
A strength of the paper is the careful cell biological and molecular assays which provide convincing evidence of the authors conclusions. It is also interesting that none of the autism variants could rescue the phenotype of the AnkB440 KO neurons, indicating that each of these variants affected function.
The paper represents a confirmation and advance over previous work. Many of the results follow those previously published by Yang et al., 2019. Both papers demonstrate that AnkB440 mutant mice have an enlarged corpus callosum. It is not evident if this is a more generalized nervous system effect or specific for callosal axons. It is not clear why the corpus callosum is enlarged. Increased dendritic branching of cortical neurons was observed but axonal branching was not analyzed in vivo. Callosal axons do not usually branch within the tract at the midline and no evidence is provided to explain whether callosal axons are more spread out in the tract or whether there are more branches. Since Sema3A-Nrp1 signalling also regulates the targeting of callosal axons in the contralateral hemisphere as well as other features of cortical development, it would be interesting to identify whether AnkB440 mutations may disrupt these functions.
The work nicely demonstrates that AnkB440 mutations cause increased neuronal branching by preventing Sema3A-mediated F-actin disassembly and growth cone collapse.
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Reviewer #2 (Public Review):
The ANKB gene generates through alternative spiling two isoforms whose expression and functions differ. One is ubiquitous, ANKB220 while the other one, ANKB440 is neuron-specific. ANKB is a component of the sub-membrane cytoskeleton known to play important roles during brain development. This is also highlighted by the identification of variants associated with autism spectrum disorder. In their study, the authors investigated the roles exerted by the neuron-specific ANKB 440. To distinguish them with those of ANK220, they generated a mouse model specifically deleted for the ANKB440, that retains normal ubiquitous expression of ANKB220. The study, combining phenotypic analysis of corpus callosum tract, cortical cultures, functional assays and biochemistry, reveals that ANK440 regulates the branching pattern …
Reviewer #2 (Public Review):
The ANKB gene generates through alternative spiling two isoforms whose expression and functions differ. One is ubiquitous, ANKB220 while the other one, ANKB440 is neuron-specific. ANKB is a component of the sub-membrane cytoskeleton known to play important roles during brain development. This is also highlighted by the identification of variants associated with autism spectrum disorder. In their study, the authors investigated the roles exerted by the neuron-specific ANKB 440. To distinguish them with those of ANK220, they generated a mouse model specifically deleted for the ANKB440, that retains normal ubiquitous expression of ANKB220. The study, combining phenotypic analysis of corpus callosum tract, cortical cultures, functional assays and biochemistry, reveals that ANK440 regulates the branching pattern of cortical neurons and their sensitivity to exogenous signals previously found to contribute to the wiring of cortical connectivity. This study thus provides new insights into the mechanisms that shape cortical neurons during brain development, and also highlights some novel etiological causes of autism spectrum disorder.
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Reviewer #3 (Public Review):
This manuscript shows that the neuron-specific giant ankyrin B isoform (AnkB440) is functioning to decrease excessive branching and plays an important role in the collapse response of cortical growth cones to the chemorepellent Sema3A. They combine a number of knockout or mutant mouse lines to show convincingly that AnkB440 specifically is involved Sema3A growth cone collapse through L1CAM/neuropilin-1 interactions. They also show that these responses, in contrast to previously shown interactions of AnkB440 with microtubules, occur through AnkB440 signaling through actin and cofilin. Moreover, they show that several ASD-linked AnkB440 point mutants do not rescue the Sema3A-induced growth cone collapse. Overall, the authors make a convincing case that AnkB440 is functioning through a well-known guidance cue …
Reviewer #3 (Public Review):
This manuscript shows that the neuron-specific giant ankyrin B isoform (AnkB440) is functioning to decrease excessive branching and plays an important role in the collapse response of cortical growth cones to the chemorepellent Sema3A. They combine a number of knockout or mutant mouse lines to show convincingly that AnkB440 specifically is involved Sema3A growth cone collapse through L1CAM/neuropilin-1 interactions. They also show that these responses, in contrast to previously shown interactions of AnkB440 with microtubules, occur through AnkB440 signaling through actin and cofilin. Moreover, they show that several ASD-linked AnkB440 point mutants do not rescue the Sema3A-induced growth cone collapse. Overall, the authors make a convincing case that AnkB440 is functioning through a well-known guidance cue pathway. The experiments often have positive and negative controls, show clear and robust differences and are analyzed in a rigorous fashion, with, for the most part, appropriate statistical tests. The one area in which the manuscript is lacking is putting these data together in the context of what is known about the Sem3A signaling pathway. If they were able to provide a strong model showing where AnkB440 fits into the Sema3A pathway, it would definitely strengthen the manuscript and increase the impact of the work.
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