Naa10 regulates hippocampal neurite outgrowth via Btbd3 N-α-acetylation-mediated actin dynamics
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Curated by eLife
eLife Assessment
This valuable study suggests that Naa10, an N-α-acetyltransferase with known mutations that disrupt neurodevelopment, acetylates Btbd3, which has been implicated in neurite outgrowth and obsessive-compulsive disorder, in a manner that regulates F-actin dynamics to facilitate neurite outgrowth. While the study provides promising insights and biochemical, co-immunoprecipitation, and proteomic data that enhance our understanding of protein N-acetylation in neuronal development, the evidence supporting larger claims is incomplete. Nonetheless, the implications of these findings are noteworthy, particularly regarding neurodevelopmental and psychiatric conditions tied to altered expression of Naa10 or Btbd3.
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Abstract
Protein N-α-acetylation is widespread in eukaryotes, yet its neuronal role remains unclear. Mutations in human N-α-acetyltransferase 10 (NAA10) lead to developmental defects affecting brain function, such as intellectual disability and autism. We found that hippocampal CA1-specific Naa10 -knockout mice exhibit anxiety and reduced hippocampal dendritic complexity. Mechanistically, Naa10 promotes neurite outgrowth by acetylating BTB/POZ domain-containing protein 3 (Btbd3), crucial for the interaction of Btbd3 with filamentous actin (F-actin)-capping protein subunit beta (CapZb). Disrupting the Btbd3/CapZb interaction, either through Naa10 knockout or by expressing an N-α-acetylation-defective Btbd3 mutant, diminishes CapZb binding to F-actin and reduces neurite outgrowth. Moreover, cytochalasin D, a compound like CapZb in capping the barbed end of F-actin, rescues the Naa10 knockout-induced neurite reduction in hippocampal primary neurons. These findings unveil the role of Naa10 in enhancing hippocampal neurite outgrowth through the Btbd3-CapZb-F-actin axis, shedding light on potential mechanisms underlying X-linked Ogden syndrome resulting from human NAA10 mutations.
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Author response:
eLife Assessment
This valuable study suggests that Naa10, an N-α-acetyltransferase with known mutations that disrupt neurodevelopment, acetylates Btbd3, which has been implicated in neurite outgrowth and obsessive-compulsive disorder, in a manner that regulates F-actin dynamics to facilitate neurite outgrowth. While the study provides promising insights and biochemical, co-immunoprecipitation, and proteomic data that enhance our understanding of protein N-acetylation in neuronal development, the evidence supporting larger claims is incomplete. Nonetheless, the implications of these findings are noteworthy, particularly regarding neurodevelopmental and psychiatric conditions tied to altered expression of Naa10 or Btbd3.Thank you very much for recognizing our study, carefully reviewing our work, and providing …
Author response:
eLife Assessment
This valuable study suggests that Naa10, an N-α-acetyltransferase with known mutations that disrupt neurodevelopment, acetylates Btbd3, which has been implicated in neurite outgrowth and obsessive-compulsive disorder, in a manner that regulates F-actin dynamics to facilitate neurite outgrowth. While the study provides promising insights and biochemical, co-immunoprecipitation, and proteomic data that enhance our understanding of protein N-acetylation in neuronal development, the evidence supporting larger claims is incomplete. Nonetheless, the implications of these findings are noteworthy, particularly regarding neurodevelopmental and psychiatric conditions tied to altered expression of Naa10 or Btbd3.Thank you very much for recognizing our study, carefully reviewing our work, and providing insightful comments and constructive criticism!
Public Reviews:
Reviewer #1 (Public review):
The manuscript examines the role of Naa10 in cKO animals, in immortalized neurons, and in primary neurons. Given that Naa10 mutations in humans produce defects in nervous system function, the authors used various strategies to try to find a relevant neuronal phenotype and its potential molecular mechanism.
This work contains valuable findings that suggest that the depletion of Naa10 from CA1 neurons in mice exacerbates anxiety-like behaviors. Using neuronal-derived cell lines authors establish a link between N-acetylase activity, Btbd3 binding to CapZb, and F-actin, ultimately impinging on neurite extension. The evidence demonstrating this is in most cases incomplete, since some key controls are missing and clearly described or simply because claims are not supported by the data. The manuscript also contains biochemical, co-immunoprecipitation, and proteomic data that will certainly be of value to our knowledge of the effects of protein N--acetylation in neuronal development and function.
Thanks! It would be appreciated if the Reviewer could point out in the public review which experiment lacks a control group.
Reviewer #2 (Public review):
In this study, the authors sought to elucidate the neural mechanisms underlying the role of Naa10 in neurodevelopmental disruptions with a focus on its role in the hippocampus. The authors use an impressive array of techniques to identify a chain of events that occurs in the signaling pathway starting from Naa10 acetylating Btbd3 to regulation of F-actin dynamics that are fundamental to neurite outgrowth. They provide convincing evidence that Naa10 acetylates Btbd3, that Btbd3 facilitates CapZb binding to F-actin in a Naa10 acetylation-dependent manner, and that this CapZb binding to F-actin is key to neurite outgrowth. Besides establishing this signaling pathway, the authors contribute novel lists of Naa10 and Btbd3 interacting partners, which will be useful for future investigations into other mechanisms of action of Naa10 or Btbd3 through alternative cell signaling pathways.
Thank you very much for recognizing our study!
The evidence presented for an anxiety-like behavioral phenotype as a result of Naa10 dysfunction is mixed and tenuous, and assays for the primary behaviors known to be altered by Naa10 mutations in humans were not tested. As such, behavioral findings and their translational implications should be interpreted with caution.
(1) For the anxiety-like behavioral phenotype, we provided a paragraph titled “Naa10 and stress-induced anxiety” in the Discussion section of the text: “Our investigations revealed that hippocampal CA1-KO of Naa10 did not exhibit significant differences in the open field test (Figure S1K) but led to anxiety-like behavior in mice in the elevated plus maze (EPM) test (Figure 1A). This disparity might be attributed to the specific design of the EPM test, which is tailored to elicit a conflict between an animal's inclination to explore and its fear of open spaces and elevated areas. This distinction implies that Naa10 might play a role in stress responses within the emotional regulation circuitry, particularly in navigating potentially threatening and anxiety-provoking environments.” The open field test offers a less challenging, open environment that primarily promotes exploratory behavior. We agree that additional assays, such as the light-dark box test, would be helpful in clarifying the issue.
(2) We agree that the behavioral findings and their translational implications should be interpreted with caution. The primary neurological behaviors known to be altered by Naa10 mutations in humans include intellectual disability and autism-like syndrome with defective emotional control. These behaviors are influenced by many factors, including defects in the hippocampal CA1. Thus, we tested hippocampal CA1 Naa10-KO mice using the Y-maze, tail suspension test, open field test, and elevated plus maze (EPM). However, only the EPM results were affected, while the other tests showed no significant changes. It should be noted that our study employed a postnatal, CA1-specific Naa10 conditional knockout (cKO) model driven by Camk2a-Cre, which selectively depletes Naa10 from hippocampal CA1 neurons after birth. In contrast, Naa10 mutations in human patients involve global effects and impact multiple brain regions from the embryonic stage, leading to a broader spectrum of phenotypes. The limited disruption in our model likely explains the absence of learning and memory deficits and the incomplete recapitulation of the full range of patient phenotypes. Furthermore, Naa10 knockout may not produce the same effects as Naa10 mutations. Our current study is primarily intended to explore the physiological function of Naa10 in hippocampal function.
(3) We will replace all instances of “anxiety behavior” with “anxiety-like behavior.”
Finally, while not central to the main cell signaling pathway delineated, the characterization of brain region-specific and cell maturity of Naa10 expression patterns was presented in few to single animals and not quantified, and as such should also be interpreted with caution.
We agree that we should provide additional Naa10 immunostaining data from more than three WT and hippocampal CA1 Naa10-KO mouse brains, as well as quantify data such as the silver staining and Light Sheet Fluorescence Microscopy results presented in Figures 1C and 1D, respectively. Nevertheless, the current report presents consistent results across different mice used for various assays. For example, Figures 1B-D, with three different assays, each demonstrate that Naa10-cKO reduces neurite complexity in vivo.
On a broader level, these findings have implications for neurodevelopment and potentially, although not tested here, synaptic plasticity in adulthood, which means this novel pathway may be fundamental for brain health.
Thank you very much again for recognizing our study!
Summarized list of minor concerns
(1) The early claims of the manuscript are supported by very small sample sizes (often 1-3) and/or lack of quantification, particularly in Figures S1 and 1.
We agree that we should provide additional Naa10 immunostaining data from more than three WT and hippocampal CA1 Naa10-KO mouse brains, as well as quantify data such as the silver staining and Light Sheet Fluorescence Microscopy results presented in Figures 1C and 1D, respectively. Nevertheless, the current report presents consistent results across different mice used for various assays. For example, Figures 1B-D, with three different assays, each demonstrate that Naa10-cKO reduces neurite complexity in vivo.
(2) Evidence is insufficient for CA1-specific knockdown of Naa10.
The Camk2a-Cre mice used in this study were derived from Dr. Susumu Tonegawa’s laboratory. According to the referenced paper, this strain restricts Cre/loxP recombination to the forebrain, with particularly high efficiency in the hippocampal CA1. Consistently, our data show that Naa10 was almost completely absent in the CA1 but partially depleted in the DG of the Naa10-cKO mice (Figure S1F in the text). Similar results were observed in a different pair of
(3) The relationship between the behaviors measured, which centered around mood, and Ogden syndrome, was not clear, and likely other behavioral measures would be more translationally relevant for this study. Furthermore, the evidence for an anxiety-like phenotype was mixed.
(1) For the anxiety-like behavioral phenotype, we provided a paragraph titled “Naa10 and stress-induced anxiety” in the Discussion section of the text: “Our investigations revealed that hippocampal CA1-KO of Naa10 did not exhibit significant differences in the open field test (Figure S1K) but led to anxiety-like behavior in mice in the elevated plus maze (EPM) test (Figure 1A). This disparity might be attributed to the specific design of the EPM test, which is tailored to elicit a conflict between an animal's inclination to explore and its fear of open spaces and elevated areas. This distinction implies that Naa10 might play a role in stress responses within the emotional regulation circuitry, particularly in navigating potentially threatening and anxiety-provoking environments.” The open field test offers a less challenging, open environment that primarily promotes exploratory behavior. We agree that additional assays, such as the light-dark box test, would be helpful in clarifying the issue.
(2) We agree that the behavioral findings and their translational implications should be interpreted with caution. The primary neurological behaviors known to be altered by Naa10 mutations in humans include intellectual disability and autism-like syndrome with defective emotional control. These behaviors are influenced by many factors, including defects in the hippocampal CA1. Thus, we tested hippocampal CA1 Naa10-KO mice using the Y-maze, tail suspension test, open field test, and elevated plus maze (EPM). However, only the EPM results were affected, while the other tests showed no significant changes. It should be noted that our study employed a postnatal, CA1-specific Naa10 conditional knockout (cKO) model driven by Camk2a-Cre, which selectively depletes Naa10 from hippocampal CA1 neurons after birth. In contrast, Naa10 mutations in human patients involve global effects and impact multiple brain regions from the embryonic stage, leading to a broader spectrum of phenotypes. The limited disruption in our model likely explains the absence of learning and memory deficits and the incomplete recapitulation of the full range of patient phenotypes. Furthermore, Naa10 knockout may not produce the same effects as Naa10 mutations. Our current study is primarily intended to explore the physiological function of Naa10 in hippocampal function.
(3) We will replace all instances of “anxiety behavior” with “anxiety-like behavior.”
(4) Btbd3 is characterized by the authors as an OCD risk gene, but its status as such is not well supported by the most recent, better-powered genome-wide association studies than the one that originally implicated Btbd3. However, there is evidence that Btbd3 expression, including selectively in the hippocampus, is implicated in OCD-relevant behaviors in mice.
Thanks for clarifying the issue!
(5) The reporting of the statistics lacks sufficient detail for the reader to deduce how experimental replicates were defined.
We believe we have provided sufficient detail for readers to deduce how experimental replicates were defined in each corresponding figure legend. It would be appreciated if the Reviewer could point out which specific figures lack sufficient details.
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eLife Assessment
This valuable study suggests that Naa10, an N-α-acetyltransferase with known mutations that disrupt neurodevelopment, acetylates Btbd3, which has been implicated in neurite outgrowth and obsessive-compulsive disorder, in a manner that regulates F-actin dynamics to facilitate neurite outgrowth. While the study provides promising insights and biochemical, co-immunoprecipitation, and proteomic data that enhance our understanding of protein N-acetylation in neuronal development, the evidence supporting larger claims is incomplete. Nonetheless, the implications of these findings are noteworthy, particularly regarding neurodevelopmental and psychiatric conditions tied to altered expression of Naa10 or Btbd3.
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Reviewer #1 (Public review):
The manuscript examines the role of Naa10 in cKO animals, in immortalized neurons, and in primary neurons. Given that Naa10 mutations in humans produce defects in nervous system function, the authors used various strategies to try to find a relevant neuronal phenotype and its potential molecular mechanism.
This work contains valuable findings that suggest that the depletion of Naa10 from CA1 neurons in mice exacerbates anxiety-like behaviors. Using neuronal-derived cell lines authors establish a link between N-acetylase activity, Btbd3 binding to CapZb, and F-actin, ultimately impinging on neurite extension. The evidence demonstrating this is in most cases incomplete, since some key controls are missing and clearly described or simply because claims are not supported by the data. The manuscript also contains …
Reviewer #1 (Public review):
The manuscript examines the role of Naa10 in cKO animals, in immortalized neurons, and in primary neurons. Given that Naa10 mutations in humans produce defects in nervous system function, the authors used various strategies to try to find a relevant neuronal phenotype and its potential molecular mechanism.
This work contains valuable findings that suggest that the depletion of Naa10 from CA1 neurons in mice exacerbates anxiety-like behaviors. Using neuronal-derived cell lines authors establish a link between N-acetylase activity, Btbd3 binding to CapZb, and F-actin, ultimately impinging on neurite extension. The evidence demonstrating this is in most cases incomplete, since some key controls are missing and clearly described or simply because claims are not supported by the data. The manuscript also contains biochemical, co-immunoprecipitation, and proteomic data that will certainly be of value to our knowledge of the effects of protein N--acetylation in neuronal development and function.
-
Reviewer #2 (Public review):
In this study, the authors sought to elucidate the neural mechanisms underlying the role of Naa10 in neurodevelopmental disruptions with a focus on its role in the hippocampus. The authors use an impressive array of techniques to identify a chain of events that occurs in the signaling pathway starting from Naa10 acetylating Btbd3 to regulation of F-actin dynamics that are fundamental to neurite outgrowth. They provide convincing evidence that Naa10 acetylates Btbd3, that Btbd3 facilitates CapZb binding to F-actin in a Naa10 acetylation-dependent manner, and that this CapZb binding to F-actin is key to neurite outgrowth. Besides establishing this signaling pathway, the authors contribute novel lists of Naa10 and Btbd3 interacting partners, which will be useful for future investigations into other mechanisms …
Reviewer #2 (Public review):
In this study, the authors sought to elucidate the neural mechanisms underlying the role of Naa10 in neurodevelopmental disruptions with a focus on its role in the hippocampus. The authors use an impressive array of techniques to identify a chain of events that occurs in the signaling pathway starting from Naa10 acetylating Btbd3 to regulation of F-actin dynamics that are fundamental to neurite outgrowth. They provide convincing evidence that Naa10 acetylates Btbd3, that Btbd3 facilitates CapZb binding to F-actin in a Naa10 acetylation-dependent manner, and that this CapZb binding to F-actin is key to neurite outgrowth. Besides establishing this signaling pathway, the authors contribute novel lists of Naa10 and Btbd3 interacting partners, which will be useful for future investigations into other mechanisms of action of Naa10 or Btbd3 through alternative cell signaling pathways. The evidence presented for an anxiety-like behavioral phenotype as a result of Naa10 dysfunction is mixed and tenuous, and assays for the primary behaviors known to be altered by Naa10 mutations in humans were not tested. As such, behavioral findings and their translational implications should be interpreted with caution. Finally, while not central to the main cell signaling pathway delineated, the characterization of brain region-specific and cell maturity of Naa10 expression patterns was presented in few to single animals and not quantified, and as such should also be interpreted with caution. On a broader level, these findings have implications for neurodevelopment and potentially, although not tested here, synaptic plasticity in adulthood, which means this novel pathway may be fundamental for brain health.
Summarized list of minor concerns
(1) The early claims of the manuscript are supported by very small sample sizes (often 1-3) and/or lack of quantification, particularly in Figures S1 and 1.
(2) Evidence is insufficient for CA1-specific knockdown of Naa10.
(3) The relationship between the behaviors measured, which centered around mood, and Ogden syndrome, was not clear, and likely other behavioral measures would be more translationally relevant for this study. Furthermore, the evidence for an anxiety-like phenotype was mixed.
(4) Btbd3 is characterized by the authors as an OCD risk gene, but its status as such is not well supported by the most recent, better-powered genome-wide association studies than the one that originally implicated Btbd3. However, there is evidence that Btbd3 expression, including selectively in the hippocampus, is implicated in OCD-relevant behaviors in mice.
(5) The reporting of the statistics lacks sufficient detail for the reader to deduce how experimental replicates were defined.
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