Binding of chemicals to ionotropic glutamate receptors leads to impairment of learning and memory via loss of drebrin from dendritic spines of neurons

Yuko Sekino^1,3, Shihori Tanabe², Satoshi Yokota², Izuo Tsutsui^1,3, Noriko Koganezawa^1,3, Tomoaki Shirao³

1 the University of Tokyo, Japan

2 National Institute of Health Sciences, Japan

3 AlzMed, Inc,, Japan

Neurotoxicity risk assessment is crucial for regulatory agencies, as current methods rely on time-consuming and costly animal testing. With thousands of chemicals lacking neurotoxicity data, there is an urgent need for in vitro methods to rapidly evaluate potential risks. Chemicals that impair learning and memory are linked to neurodegenerative diseases such as Parkinson's and Alzheimer's, underscoring the importance of effective risk assessment. The proposed AOP highlights a cascade where the loss of drebrin from dendritic spines induces spine morphological abnormalities, leading to synaptic dysfunction. Notably, synaptic dysfunction alone, even in the absence of neuronal death, can result in learning and memory impairments. This provides a novel framework for evaluating neurotoxicity and developmental neurotoxicity.

Dendritic spines are specialized structures that serve as the primary sites of excitatory synaptic transmission, primarily mediated by glutamate receptors. Spine formation and functional maturation are governed by drebrin expression. Drebrin, an actin-binding protein discovered and named by Shirao's team, has two isoforms: drebrin E (DE) and drebrin A (DA). DE, a non-neuronal protein involved in cell motility and protrusion formation, is predominantly expressed during early brain development. It is gradually replaced by DA, a neuron-specific protein that stabilizes actin filaments during synaptogenesis and synaptic maturation. The protein levels and subcellular localization of drebrin reflect its distinct roles in neuronal development and synaptic function. Loss of drebrin triggers spine abnormalities and synaptic dysfunction, ultimately impairing learning and memory as the adverse outcome (AO). This AOP builds on the molecular initiating event (MIE) of AOP 48—“Binding of agonists to ionotropic glutamate receptors”—but uniquely highlights drebrin loss as a critical KE.

Empirical evidence supports this AOP. Studies show that glutamate induces drebrin loss and dendritic spine morphological changes. Compounds that directly bind to NMDA receptors, such as glutamate and NMDA, and compounds that indirectly enhance NMDA receptor activity, have been shown to induce drebrin loss, linking such exposure to synaptic dysfunction and learning impairments. The detection of drebrin gene expression levels, subcellular localization, and protein levels provides valuable insights into brain development and higher-order functions across species. To develop effective in vitro testing methods, it is essential to have biomarkers that are simple, reproducible, and allow for quantitative data analysis. We determined that drebrin is highly suitable as a biomarker for these purposes.

The proposed AOP promotes alternative testing methods aligned with the 3Rs (Replacement, Reduction, Refinement), using cryopreserved hippocampal neurons to reduce animal use. Quantification of drebrin via immunocytochemistry and ELISA enables reproducible and scalable chemical screening. This AOP provides a foundation for in vitro prediction models, advancing chemical safety evaluation for humans and the environment. By integrating dynamic drebrin expression patterns and functional properties, this AOP framework offers a robust tool for regulatory decision-making and assessing neurotoxicity risks.

Drebrin, identified by our group in 1985, has been a central focus of our research for many years. This actin-binding protein is known to exist in two isoforms: drebrin E and drebrin A. Drebrin E is expressed in both neuronal and certain non-neuronal cells, and it plays a crucial role during fetal and early postnatal stages of brain development. In neurons, drebrin E is involved in processes essential for cell motility, neurite outgrowth, and the extension of axons and dendrites, which together contribute to the establishment of neural networks. In contrast, drebrin A is a neuron-specific isoform whose expression is initiated during the synaptic formation stage. Drebrin A is indispensable for the formation and maintenance of dendritic spines, which serve as the primary sites for excitatory synaptic transmission, largely mediated by glutamate receptors. During brain development, drebrin E, which predominates in the early stages, is gradually replaced by drebrin A during synaptogenesis, reflecting their distinct roles in neuronal development, excitatory synapse formation and synaptic plasticity.

Based on the dynamic expression patterns and functional properties of drebrin, we have proposed an Adverse Outcome Pathway (AOP) framework for assessing developmental neurotoxicity and neurotoxicity. Monitoring drebrin gene expression levels, subcellular localization in neurons, and protein levels provides valuable insights into brain development and higher-order brain functions across various species.

In 2017, we summarized our research findings in a monograph titled Drebrin: From Structure and Function to Physiological and Pathological Roles, published as part of Springer’s Advances in Experimental Medicine and Biology series.

Between 2020 and 2023, we initiated the development of an AOP for neurotoxicity and developmental neurotoxicity caused by glutamate receptor-binding agonists, supported by a three-year research grant from the Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI). This project, entitled "Proposal of a new AOP for the neurotoxicity and developmental neurotoxicity assessment of glutamate receptor binding agonists that cause learning and memory impairment," identified a novel adverse outcome (AO): learning and memory impairment. This AO is characterized by key events, including the loss of drebrin from dendritic spines, leading to thin and elongated spine morphology and subsequent synaptic dysfunction. Drebrin, as a key regulator of dendritic spine morphology, plays an essential role in the structural plasticity associated with learning and memory. Furthermore, the subcellular localization of drebrin is dynamically influenced by glutamate receptor activity.

To advance these studies, we optimized Banker’s method for low-density neuronal cultures and developed an immunocytochemical evaluation system for drebrin clusters in hippocampal neurons using a 96-well plate format and frozen embryonic hippocampal neurons from rats. In addition, we designed a high-content imaging algorithm for the quantitative analysis of neuronal parameters, including neuron count, dendrite length, and drebrin clustering, using confocal image cytometry. Notably, our brightness distribution analysis of drebrin clusters demonstrated exceptional sensitivity, allowing for precise quantification of structural changes in neurons. These methodological advancements have enabled us to establish standardized protocols (SOPs) for both neuronal culture techniques and analytical procedures.

We plan to extend this research by developing an experimental system utilizing neurons derived from human-induced pluripotent stem cells (iPSCs), thereby enhancing the relevance and applicability of our findings to human brain development and function.

Drebrin is an essential protein for the formation of dendritic spines, which are structural compartment modules of the protein network of learning and memory. Evidence from in vivo experiments and clinical observations indicates a strong association between the loss of drebrin and memory impairment, as the molecular mechanisms of learning do not function in spines lacking drebrin. By staining cultured neurons with drebrin using immunocytochemistry, the number of drebrin clusters with a certain threshold intensity can be quantified, and thus the loss of drebrin from dendritic spines can be detected. When NMDA-type glutamate receptors are activated, drebrin translocates from dendritic spines to dendritic trunks. This process is accompanied by a change in spine morphology from a mushroom shape to a thin and pointed shape. We proposed the disappearance of drebrin (KE2028) as a new KE following a molecular initiation event (MIE; Event ID 875) in which a compound binds to the glutamate NMDA-type receptor, followed by NMDA receptor overactivation (KE388) and intracellular calcium overload (KE389). Loss of drebrin leads to abnormal dendritic spine morphology (KE2242) and associated synaptic dysfunction (KE1944).

The overall assessment of AOP475 emphasizes its relevance in evaluating neurodevelopmental toxicity caused by chemical exposures. The pathway links disruption of dendritic spine morphology to impaired synaptic plasticity and cognitive functions, such as learning and memory. Its robust mechanistic framework integrates molecular, cellular, and behavioral endpoints. AOP475 serves as a critical tool for regulatory risk assessment, particularly in identifying early indicators of synaptic dysfunction.

AOP475 provides a framework useful in risk assessments of regulatory toxicology.

 KE 2078, drebrin loss, can be quantitatively measured using immunocytochemistry. Thus, targeted assays could be developed to evaluate chemicals' potential to disrupt synaptic function and cognitive performance. Quantitative measurement of the number of drebrin clusters, assessed by immunohistochemistry using neuronal cultures derived from frozen embryonic neurons, could be integrated into an IATA framework to provide a comprehensive neurotoxicity assessment, reducing reliance on animal testing.

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