Heterozygosity for neurodevelopmental disorder-associated TRIO variants yields distinct deficits in behavior, neuronal development, and synaptic transmission in mice
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Curated by eLife
eLife Assessment
This study provides useful findings about the effects of heterozygosity for Trio variants linked to neurodevelopmental and psychiatric disorders in mice. However, the strength of the evidence is limited and incomplete mainly because the experimental flow is difficult to follow, raising concerns about the conclusions' robustness. Clearer connections between variables, such as sex, age, behavior, brain regions, and synaptic measures, and more methodological detail on breeding strategies, test timelines, electrophysiology, and analysis, are needed to support their claims.
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Abstract
Genetic variants in TRIO are associated with neurodevelopmental disorders (NDDs) including schizophrenia (SCZ), autism spectrum disorder (ASD) and intellectual disability. TRIO uses its two guanine nucleotide exchange factor (GEF) domains to activate GTPases (GEF1: Rac1 and RhoG; GEF2: RhoA) that control neuronal development and connectivity. It remains unclear how discrete TRIO variants differentially impact these neurodevelopmental events. Here, we investigate how heterozygosity for NDD-associated Trio variants – +/K1431M (ASD), +/K1918X (SCZ), and +/M2145T (bipolar disorder, BPD) – impact mouse behavior, brain development, and synapse structure and function. Heterozygosity for different Trio variants impacts motor, social, and cognitive behaviors in distinct ways that align with clinical phenotypes in humans. Trio variants differentially impact head and brain size with corresponding changes in dendritic arbors of motor cortex layer 5 pyramidal neurons (M1 L5 PNs). Although neuronal structure was only modestly altered in the Trio variant heterozygotes, we observe significant changes in synaptic function and plasticity. We also identified distinct changes in glutamate synaptic release in +/K1431M and +/M2145T cortico-cortical synapses. The TRIO K1431M GEF1 domain has impaired ability to promote GTP exchange on Rac1, but +/K1431M mice exhibit increased Rac1 activity, associated with increased levels of the Rac1 GEF Tiam1. Acute Rac1 inhibition with NSC23766 rescued glutamate release deficits in +/K1431M variant cortex. Our work reveals that discrete NDD-associated Trio variants yield overlapping but distinct phenotypes in mice, demonstrates an essential role for Trio in presynaptic glutamate release, and underscores the importance of studying the impact of variant heterozygosity in vivo.
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eLife Assessment
This study provides useful findings about the effects of heterozygosity for Trio variants linked to neurodevelopmental and psychiatric disorders in mice. However, the strength of the evidence is limited and incomplete mainly because the experimental flow is difficult to follow, raising concerns about the conclusions' robustness. Clearer connections between variables, such as sex, age, behavior, brain regions, and synaptic measures, and more methodological detail on breeding strategies, test timelines, electrophysiology, and analysis, are needed to support their claims.
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Reviewer #1 (Public review):
Summary:
This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.
Strengths:
This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.
Weak…
Reviewer #1 (Public review):
Summary:
This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.
Strengths:
This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.
Weaknesses:
While the authors present extensive results, the flow of experiments is challenging to follow, raising concerns about the strength of the experimental conclusions. Additionally, the connection between sex, age, behavioral data, brain regions, synaptic transmission, and plasticity lacks clarity, making it difficult to understand the rationale behind each experiment. Clearer explanations of the purpose and connections between experiments are recommended. Furthermore, the methodology requires more detail, particularly regarding mouse breeding strategies, timelines for behavioral tests, electrophysiology conditions, and data analysis procedures.
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Reviewer #2 (Public review):
Summary:
The authors generated three mouse lines harboring ASD, Schizophrenia, and Bipolar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking, the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bipolar disorder development.
Strengths:
The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bipolar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was …
Reviewer #2 (Public review):
Summary:
The authors generated three mouse lines harboring ASD, Schizophrenia, and Bipolar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking, the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bipolar disorder development.
Strengths:
The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bipolar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was carefully examined using an impressive host of methods. The authors achieved their goal of identifying behavioral, physiological, and molecular alterations that are disorder/variant specific. The impact of this work is extremely high given the growing appreciation of TRIO dysfunction in a large number of brain-related disorders. This work is very interesting in that it begins to identify the unique and subtle ways brain function is altered in ASD, Schizophrenia, and Bipolar disorder.
Weaknesses:
(1) Most assays were performed in older animals and perhaps only capture alterations that result from homeostatic changes resulting from prodromal pathology that may look very different.
(2) Identification of upregulated (potentially compensating) genes in response to these disorder-specific Trio variants is extremely interesting. However, a functional demonstration of compensation is not provided.
(3) There are instances where data is not shown in the manuscript. See "data not shown". All data collected should be provided even if significant differences are not observed.
I consider weaknesses 1 and 2 minor. While they would very interesting to explore, these experiments might be more appropriate for a follow-up study. I would recommend that the missing data in 3 should be provided in the supplemental material.
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Author response:
eLife Assessment
This study provides useful findings about the effects of heterozygosity for Trio variants linked to neurodevelopmental and psychiatric disorders in mice. However, the strength of the evidence is limited and incomplete mainly because the experimental flow is difficult to follow, raising concerns about the conclusions' robustness. Clearer connections between variables, such as sex, age, behavior, brain regions, and synaptic measures, and more methodological detail on breeding strategies, test timelines, electrophysiology, and analysis, are needed to support their claims.
We appreciate the opportunity to address the constructive feedback provided by eLife and the reviewers. Below, we respond to the overall assessment and individual reviewers' comments, clarifying our experimental approach, addressing …
Author response:
eLife Assessment
This study provides useful findings about the effects of heterozygosity for Trio variants linked to neurodevelopmental and psychiatric disorders in mice. However, the strength of the evidence is limited and incomplete mainly because the experimental flow is difficult to follow, raising concerns about the conclusions' robustness. Clearer connections between variables, such as sex, age, behavior, brain regions, and synaptic measures, and more methodological detail on breeding strategies, test timelines, electrophysiology, and analysis, are needed to support their claims.
We appreciate the opportunity to address the constructive feedback provided by eLife and the reviewers. Below, we respond to the overall assessment and individual reviewers' comments, clarifying our experimental approach, addressing concerns, and providing additional details where necessary.
We thank the editors for highlighting the significance of our findings regarding the effects of Trio variant heterozygosity in mice. We acknowledge the feedback concerning the experimental flow and agree that clarity is paramount. To address these concerns:
(1) Connections between variables: We will revise the manuscript to explicitly outline and extend explanations and the relationships between sex, age, behavior, brain regions, and synaptic measures, ensuring that the rationale for each experiment and its relevance to the overall conclusions are improved.
(2) Methodological details: Our paper Methods section was formatted to be short with additional details provided in the Supplemental Methods section. We will merge all into an extended section to improve clarity. We will also expand on our breeding strategies, test timelines, electrophysiological protocols, and data analysis methods in the revised Methods section. These additions aim to enhance the transparency and reproducibility of our study and to ensure full support of our conclusions.
(3) Experimental flow: We will revise and extend our results, methods, and discussion sections to clarify the rationale and experimental design to guide readers through the experimental sequence and rationale.
We are confident these revisions address the concerns raised and enhance the robustness and coherence of our findings.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study explores how heterozygosity for specific neurodevelopmental disorder-associated Trio variants affects mouse behavior, brain structure, and synaptic function, revealing distinct impacts on motor, social, and cognitive behaviors linked to clinical phenotypes. Findings demonstrate that Trio variants yield unique changes in synaptic plasticity and glutamate release, highlighting Trio's critical role in presynaptic function and the importance of examining variant heterozygosity in vivo.
Strengths:
This study generated multiple mouse lines to model each Trio variant, reflecting point mutations observed in human patients with developmental disorders. The authors employed various approaches to evaluate the resulting behavioral, neuronal morphology, synaptic function, and proteomic phenotypes.
Weaknesses:
While the authors present extensive results, the flow of experiments is challenging to follow, raising concerns about the strength of the experimental conclusions. Additionally, the connection between sex, age, behavioral data, brain regions, synaptic transmission, and plasticity lacks clarity, making it difficult to understand the rationale behind each experiment. Clearer explanations of the purpose and connections between experiments are recommended. Furthermore, the methodology requires more detail, particularly regarding mouse breeding strategies, timelines for behavioral tests, electrophysiology conditions, and data analysis procedures.
We appreciate the reviewer’s recognition of the novelty and comprehensiveness of our approach, particularly the generation of multiple mouse lines and our efforts to model Trio variant effects in vivo.
Weaknesses
(1) Experimental flow and rationale and connection between variables: We will expand on the connections between behavioral data, neuronal morphology, synaptic function, and proteomics in the Results and Discussion sections to clarify how each experiment informs the reasoning and the conclusions and to highlight the relationships between sex, age, behavior, and synaptic measures.
(2) Methodological details: Our paper Methods section was formatted to be short to fulfill word limits on the submitted version, with additional details provided in the Supplemental Methods section. We will merge our Methods and Supplemental Methods sections and expand on our breeding strategies, test timelines, electrophysiological protocols, and data analysis methods in the revised Methods section. These additions aim to enhance the transparency and reproducibility of our study and to ensure full support of our conclusions.
Reviewer #2 (Public review):
Summary:
The authors generated three mouse lines harboring ASD, Schizophrenia, and Bipolar-associated variants in the TRIO gene. Anatomical, behavioral, physiological, and biochemical assays were deployed to compare and contrast the impact of these mutations in these animals. In this undertaking, the authors sought to identify and characterize the cellular and molecular mechanisms responsible for ASD, Schizophrenia, and Bipolar disorder development.
Strengths:
The establishment of TRIO dysfunction in the development of ASD, Schizophrenia, and Bipolar disorder is very recent and of great interest. Disorder-specific variants have been identified in the TRIO gene, and this study is the first to compare and contrast the impact of these variants in vivo in preclinical models. The impact of these mutations was carefully examined using an impressive host of methods. The authors achieved their goal of identifying behavioral, physiological, and molecular alterations that are disorder/variant specific. The impact of this work is extremely high given the growing appreciation of TRIO dysfunction in a large number of brain-related disorders. This work is very interesting in that it begins to identify the unique and subtle ways brain function is altered in ASD, Schizophrenia, and Bipolar disorder.
Weaknesses:
(1) Most assays were performed in older animals and perhaps only capture alterations that result from homeostatic changes resulting from prodromal pathology that may look very different.
(2) Identification of upregulated (potentially compensating) genes in response to these disorder-specific Trio variants is extremely interesting. However, a functional demonstration of compensation is not provided.
(3) There are instances where data is not shown in the manuscript. See "data not shown". All data collected should be provided even if significant differences are not observed.
I consider weaknesses 1 and 2 minor. While they would very interesting to explore, these experiments might be more appropriate for a follow-up study. I would recommend that the missing data in 3 should be provided in the supplemental material.
We are grateful for the reviewer’s recognition of our study’s significance and methodological rigor. The acknowledgment of Trio dysfunction as a novel and impactful area of research is deeply appreciated.
Weaknesses:
We agree that focusing on older animals may limit insights into early-stage pathophysiology. However, given the goal of this study was to examine the functional impacts of Trio heterozygosity at an adolescent stage and to reveal the ultimate impact of these alleles on synaptic function, we believe the choice of animal age aligns with our objectives. We agree that future studies of earlier developmental stages will be beneficial and complement these findings.
Functional compensation: In this study, we tested functional compensation through rescue experiments in +/K1431M brain slices using a Rac1-specific inhibitor, NSC, which prevents its activation by Trio or Tiam1. Our findings strongly suggest that increased Rac1 activity, attributed to the proposed compensation, drives the deficiency in neurotransmitter release. Furthermore, this deficiency can be normalized by direct Rac1 inhibition.
Data not shown: We will incorporate all previously shown data into the Supplemental Materials, even when results are nonsignificant. We agree that this ensures full transparency and facilitates a more comprehensive evaluation of our findings.
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