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Binding of chemicals to ionotropic glutamate receptors leads to impairment of learning and memory via loss of drebrin from dendritic spines of neurons
Point of Contact
- Shihori Tanabe
- Yuko Sekino
- Evgeniia Kazymova
|Handbook Version||OECD status||OECD project|
This AOP was last modified on April 29, 2023 13:02
Revision dates for related pages
|Binding of agonist, Ionotropic glutamate receptors||September 16, 2017 10:15|
|Overactivation, NMDARs||January 04, 2023 18:39|
|Loss of drebrin||December 11, 2022 21:04|
|Synaptic dysfunction||January 04, 2023 18:47|
|Impairment, Learning and memory||March 22, 2023 16:30|
|Increased, Intracellular Calcium overload||June 26, 2020 04:45|
|Binding of agonist, Ionotropic glutamate receptors leads to Overactivation, NMDARs||November 29, 2016 20:44|
|Overactivation, NMDARs leads to Loss of drebrin||December 11, 2022 21:08|
|Overactivation, NMDARs leads to Increased, Intracellular Calcium overload||November 29, 2016 20:08|
|Loss of drebrin leads to Dysfunctional synapses||December 11, 2022 21:08|
|Dysfunctional synapses leads to Impairment, Learning and memory||December 11, 2022 21:08|
Neurotoxicity risk assessment is an important issue for regulatory agencies. Currently, chemicals with potential risks are determined by time-consuming and costly animal testing. Therefore, in vitro testing methods are needed to rapidly evaluate thousands of chemicals for which no safety data on neurotoxicity exist. In recent years, chemicals that induce learning and memory impairment are thought to increase the risk of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Therefore, such risk assessment is necessary for human safety. The existing AOP No. 6 in OECD Series on AOPs (AOP48 in the AOP-Wiki) defines a molecular initiating event (MIE) as “Binding of agonists to ionotropic glutamate receptors”, causing neuronal cell death linked to impairment of learning and memory through receptor hyperactivation(1).
Recent studies have shown that synaptic dysfunction precedes neuronal death in the early stages of dementia accompanied with the neurodegenerative diseases. Synaptic dysfunction is presumed as a decrease in the number of dendritic spines in neurons of the cerebral cortex and hippocampus, which are essential for learning and memory(2). Therefore, the risk for impairment of learning and memory can be assessed by the synaptic dysfunction, namely decreased number of dendritic spines(3).
Dendritic spines are small actin-rich projections protruding from the dendrites of neurons that form excitatory synapses in the cortex and hippocampus(4). Drebrin is an actin-binding protein that localizes to dendritic spines and is said to play a specific role in their formation(5). Drebrin is known to decrease in Alzheimer's disease with a high correlation to symptom stage(6,7). In low-density cultures of hippocampal neurons, the number of dendritic spines can be counted as the number of drebrin clusters with immunostaining.
We have developed an experimental protocol for low-density neuronal culture in 96-well plates and an algorithm that automatically counts the number of drebrin clusters by high-content imaging analysis(8). These protocols have been shown to be useful for screening chemicals that bind to the NMDA receptor. In fact, we have examined the toxicity of phencyclidine (PCP) and PCP-analogues and published results in a paper(9). We have developed not only the immunocytochemical protocol for in vitro assay using neuronal culture but also enzyme-linked immunosorbent assay (ELISA) kits to evaluate drebrin protein levels. Thus, decreased number of dendritic spines induced by chemicals can be assessed quantitatively as a loss of drebrin immunocytochemically and biochemically.
Here, we propose a new AOP with the same MIE as AOP No. 6, in which loss of drebrin as KE leads to impairment of learning and memory. Studies of genetically engineered mice have shown that drebrin deficiency is directly related to synaptic dysfunction and leads to the impairment of learning and memory, even in the absence of neuronal cell death(10,11,12). This is the most important distinction between the proposed AOP and the existing AOP. Measurement of drebrin expression levels in neurons with immunocytochemistry and/or ELISA is easy and high-reproducible. The new KE, loss of drebrin, will promote accumulation of data of chemicals for neurotoxicity. The proposed AOP is expected to contribute to the development of many in vitro test for neurotoxicity and to establish in silico prediction to evaluate safety of many substances for human and environments.
AOP Development Strategy
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Type||Event ID||Title||Short name|
|MIE||875||Binding of agonist, Ionotropic glutamate receptors||Binding of agonist, Ionotropic glutamate receptors|
|KE||388||Overactivation, NMDARs||Overactivation, NMDARs|
|KE||389||Increased, Intracellular Calcium overload||Increased, Intracellular Calcium overload|
|KE||2078||Loss of drebrin||Loss of drebrin|
|KE||1944||Synaptic dysfunction||Dysfunctional synapses|
|AO||341||Impairment, Learning and memory||Impairment, Learning and memory|
Relationships Between Two Key Events (Including MIEs and AOs)
|Binding of agonist, Ionotropic glutamate receptors leads to Overactivation, NMDARs||adjacent||High||Moderate|
|Overactivation, NMDARs leads to Increased, Intracellular Calcium overload||adjacent|
|Loss of drebrin leads to Dysfunctional synapses||adjacent||High||High|
|Dysfunctional synapses leads to Impairment, Learning and memory||adjacent||High||Moderate|
|Overactivation, NMDARs leads to Loss of drebrin||non-adjacent||Moderate||Moderate|
Life Stage Applicability
Overall Assessment of the AOP
Domain of Applicability
Essentiality of the Key Events
Known Modulating Factors
|Modulating Factor (MF)||Influence or Outcome||KER(s) involved|
Considerations for Potential Applications of the AOP (optional)
- Adverse Outcome Pathway on binding of agonists to ionotropic glutamate receptors in adult brain leading to excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment, Sachana M, et al. OECD Series on Adverse Outcome Pathways (2016) No. 6, OECD Publishing, Paris, doi: 10.1787/5jlr8vqgm630-en.
- Synapse pathology in Alzheimer’s disease, Griffiths J, Grant GN. Seminars in Cell and Developmental Biology (2022) doi: 10.1016/j.semcdb.2022.05.028. (review)
- Dopamine Restores Limbic Memory Loss, Dendritic Spine Structure, and NMDAR-Dependent LTD in the Nucleus Accumbens of Alcohol-Withdrawn Rats Cannizzaro C, et al. J Neurosci. (2019) Jan 30;39(5):929-943. doi: 10.1523/JNEUROSCI.1377-18.2018.
- Actin in dendritic spines: connecting dynamics to function, Hotulainen P, Hoogenraad C. J Cell Biol (2010) 17;189(4):619-29. doi: 10.1083/jcb. 201003008. (review)
- Role of actin cytoskeleton in dendritic spine morphogenesis. Sekino Y, et al. Neurochem Int. (2007) 51(2-4):92-104. doi: 10.1016/j.neuint.2007.04.029. (review)
- Drebrin, a dendritic spine protein, is manifold decreased in brains of patients with Alzheimer’s disease and Down syndrome Shim KS, Lubec G. Neurosci Lett. 2002 May 24;324(3):209-12. doi: 10.1016/s0304-3940(02)00210-0.
- Differential expression of synaptic proteins in the frontal and temporal cortex of elderly subjects with mild cognitive impairment, Counts SE, et al. J Neuropathol Exp Neurol. 2006 Jun;65(6):592-601. doi: 10.1097/00005072-200606000-00007.
- High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons Hanamura K, et al. J Pharmacol Toxicol Methods. (2019) 99:106607. doi: 10.1016/j.vascn.2019.106607.
- Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay Mitsuoka T, et al. J Pharmacol Toxicol Methods. (2019) 99:106583. doi: 10.1016/j.vascn.2019
- Genetic disruption of the alternative splicing of drebrin gene impairs context-dependent fear learning in adulthood, Kojima N, et al. Neuroscience. (2010) 165(1):138-50. Doi: 10.1016/j.neuroscience.2009.10.016.
- Drebrin A regulates hippocampal LTP and hippocampus-dependent fear learning in adult mice, Kojima N, et al. Neuroscience. (2016) Jun 2;324:218-26. doi: 10.1016/j.neuroscience.2016.03.015.
- Effective expression of Drebrin in hippocampus improves cognitive function and alleviates lesions of Alzheimer’s disease in APP (swe)/PS1 (ΔE9) mice, Liu Y, et al. CNS Neurosci Ther. (2017) Jul;23(7):590-604. doi: 10.1111/cns.12706.