Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.

Magdalini Sachana, Sharon Munn, Anna Bal-Price

European Commission Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy

Corresponding author: anna.price@ec.europa.eu

Under physiological conditions activation of glutamate ionotropic receptors such as N-methyl-D-aspartate (NMDARs), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPARs) and kainate (KARs) is responsible for basal excitatory synaptic transmission and main forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) that are fundamental for learning and memory processes (Schrattenholz and Soskic, 2006). However, sustained (direct or indirect) over-activation of these receptors can induce excitotoxic neuronal cell death. Indeed, mainly increased Ca2+ influx through NMDARs promotes many pathways of toxicity due to generation of free radical species, reduced ATP production, endoplasmic reticulum (ER) stress and protein aggregation. Neuronal injury induced by over-activation of these receptors and the excessive Ca2+ influx is considered an early key event of excitotoxicity. Additionally, the excessive activation of NMDARs has been found to play a significant role in a variety of neurological disorders ranging from acute hypoxic-ischemic brain injury (Barenger et al., 2001) to chronic neurodegenerative diseases (Mehta et al., 2013). The proposed AOP is relevant to adult neurotoxicity testing. A molecular initiating event (MIE) has been defined as a direct binding of agonists to NMDARs or indirect, through prior activation of AMPARs and/or KARs resulting in sustained NMDARs over-activation causing excitotoxic neuronal cell death, mainly in hippocampus and cortex, two brain structures fundamental for learning and memory processes. The AOP is based on the empirical support describing (1) domoic acid (DomA) induced excitotoxicity triggered by indirect (through KARs/AMPARs) NMDARs over-activation leading to impaired learning and memory and (2) glufosinate (GLF) induced excitotoxicity that through direct binding to NMDARs causes convulsions and memory loss (Lanz et al., 2014). GLF is the methylphosphine analog of L-glutamate, used as a component of bactericidal and fungicidal herbicidal. DomA, a natural toxin that accumulates in mussels and shellfish is also an analogue of L-glutamate and among the most prominent features described after human exposure to DomA is memory impairment (Lefebvre and Robertson, 2010). DomA and GLF are described as the examples of the stressors due to large amounts of published data (especially in the case of DomA), however this AOP is relevant to any agonist that directly or indirectly cause NMDARs over-activation. Some of the known agonists selective for the NMDARs are derived from the naturally occurring amino acids such as ibotenic acid, homocysteine and l-aspartate and polyamines like spermidine.

The aim of the present AOP is to construct a linear pathway that captures the KEs and KERs that occur after binding of agonist to NMDA receptor in hippocampal and cortical neurons of adults. The majority of the KEs of the AOP are characterised by MODERATE essentiality for the AO (loss or reduction of cognitive function )or other KEs that follow. The biological plausibility in the majority of KERs is rated STRONG as there is extensive mechanistic understanding. However, the empirical support for the majority of presented KERs cannot be rated high as in most occasions the KEup and KEdown of a KER has not been investigated simultaneously, under the same experimental protocol or not in the suggested brain regions (cortex and hippocampus).

Exposure to xenobiotics can potentially affect the nervous system resulting in neurobehavioral alterations and/or neurological clinical symptoms. To assess the neurotoxic properties of compounds, current testing largely relies on neurobehavioural tests in laboratory animals, histopathological analysis, neurochemical and occasionally electrophysiological observations. Throughout the years, a significant number of methods have been developed to assess neurobehaviour in laboratory animals and a comprehensive summary of them can be found in OECD Series on testing and assessment, number 20, Guidance Document for Neurotoxicity Testing (2004). This document is considered an essential supplement to a substantial number of already existing OECD Test Guidelines that are applied to gain information on the neurotoxicity properties of chemical compounds. Namely, these are: general Test Guidelines such as single dose toxicity (e.g. OECD 402, 403, 420, 423 and 425), repeated dose toxicity (e.g. OECD 407 and 408), chronic exposure (e.g. OECD 452) as well as Test Guidelines specifically developed for the study of neurotoxicity in adult laboratory animals, such as OECD Test Guideline for Neurotoxicity (424).

Learning and memory is an important endpoint and a wide variety of tests to assess chemical effects on cognitive functions is available and used for the study of neurotoxicity. Some of these tests that allow the appreciation of cognitive function in laboratory animals are: habituation, ethologically based anxiety tests (elevated plus maze test, black and white box test, social interaction test), conditioned taste aversion (CTA), active avoidance, passive avoidance, spatial mazes (Morris water maze, Biel water maze, T-maze), conditional discrimination (simple discrimination, matching to sample), delayed discrimination (delayed matching-to-sample, delayed alternation) and eye-blink conditioning.

The present AOP can potentially provide the basis for development of a mechanistically informed IATA for neurotoxicity. The construction of IATA for predicting neurotoxic effects in adults is expected to make use of more than one AOP within an interconnected network in order to take into consideration all critical biological processes that may contribute to impairment of learning and memory in adults. Through this network, identification of KEs and KERs common across multiple AOPs can emerge that should be considered during IATA construction and that may inform also in vitro assay development. The development of alternative assays would allow screening of chemicals for potential NMDAR activators and reducing the use of in vivo studies.

Results from assays based on the KEs of this AOP can serve to interpret and accept results that derive from non-standard test methods. Omics data from toxicogenomic, transcriptomic, proteomic, and metabolomic studies can be interpreted in a structured way using this AOP that is relevant to adult neurotoxicity. Currently learning and memory testing is not required by the OECD TG 424. This AOP could serve as a base for chemical evaluation with potential to cause impairment of learning and memory. The assay development would refer to the identified in this AOP KEs that could form a testing strategy for identifying chemicals with potential to cause cognitive deficit. Finally, this AOP could provide the opportunity to group chemicals using not only chemical properties but also mechanistic information that can later inform data gap filling by read-across and predict neurotoxic properties of a target substance.

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