Acetylcholinesterase Inhibition Leading to Neurodegeneration

• Kendra Conrow
• Karen Watanabe
• Natalia Reyero
• Priscilla Pacheco
• Dennis Sinitsyn

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Sex: The AOP is not sex-specific

Life stages: the AOP is relevant to all life stages. Immature or developing populations may be more sensitive due to their increased susceptibility to seizures and developing cholinergic systems.

Taxonomic: given that both cholinergic and glutamatergic systems are highly conserved among vertebrates, this AOP is likely to be applicable to all vertebrates.

All KEs in AOP 281 rank high for essentiality.  The provided studies demonstrate direct evidence and include experiments involving inhibition of AChE through the application of various inhibitors, gene-knockout experiments, receptor antagonist studies, and anticonvulsant treatments which are shown to result in the reduction of neurodegeneration.

• AChE Inhibition (MIE) evidence is high. This is supported by several studies that measured increases in ACh after inhibition of AChE by a variety of inhibitors (Del Pino et al., 2017, Karanth et al., 2007, Kim et al., 2003, Kosasa et al., 1999, Ray et al., 2009). Additionally, researchers have demonstrated that pretreatment with a combination of reversible AChE inhibitors, nicotinic and mAChR receptor antagonists prior to exposure to soman resulted in a significantly higher survival rate and overall reduced brain ACh levels compared to controls (Harris et al., 1980).
• ACh accumulation in synapses (KE 1) evidence is high. Blocking the effects of ACh with atropine, an mAChR antagonist, was demonstrated to significantly reduce the pathological effects and neurodegeneration associated with soman intoxication (McDonough et al., 1989).
• Activation of mAChRs (KE 2) evidence is high. M1-mAChR deficient mice through gene-knockout studies were shown to be resistant to seizures induced by pilocarpine, an mAChR agonist (Hamilton et al., 1997).
• Occurrence of Focal Seizure (KE 3) evidence is high. Treatment with diazepam, a GABA_A receptor agonist and known anticonvulsant, both prevented seizures and resulted in significantly reduced brain pathology (McDonough et al., 1989).
• Increased Glutamate (KE 4) evidence is high. Application of 500 µM of glutamate showed in reduction in neuron survival, however if NMDA antagonist MK-801 was used in conjunction with glutamate, neuron survival returned to control levels (Michaels and Rothman, 1990). Other in vivo experiments using MK-801 or ketamine demonstrated a reduction in seizure activity and reduced neurodegeneration (Borris et al., 2000, Braitman and Sparenborg, 1989, Sparenborg et al., 1992).
• Overactivation of NMDARs (KE 5) evidence is high. Multiple experiments using ketamine and MK-801, both NMDA receptor antagonists, have been demonstrated to terminate or reduce both seizure activity and neurodegeneration (Borris et al., 2000, Braitman and Sparenborg, 1989, Sparenborg et al., 1992).
• Increased Intracellular Calcium Overload (KE 6) evidence is high. Calcium chelation in zebrafish models of organophosphate exposure significantly reduced neurodegeneration (Faria et al., 2015). Additionally, Deshpande et al. (2008) demonstrated that cell death could be significantly reduced given a low extracellular calcium solution in an in vitro model of SE in rat hippocampal neurons.
• Status Epilepticus (KE 7) evidence follows that of KE 3 and is considered high. Anticonvulsant treatment using diazepam was demonstrated to significantly reduce neurodegeneration (McDonough et al., 1989).
• Cell Injury/Death (KE 8) evidence is considered high. Cell death in the context of the brain is considered a form of neurodegeneration (Przedborski et al., 2003).  Therefore, prevention of cell death directly results in the prevention of the adverse outcome.

Biological plausibility: Biological plausibility refers to the structural or functional relationship between the key events based on our fundamental understanding of "normal biology". The evidence for biological plausibility throughout this AOP from inhibition of AChE to neurodegeneration is high. It is well understood that inhibition of AChE is followed by an accumulation of ACh, which subsequently leads to activation of muscarinic acetylcholine receptors and focal seizures. The seizures then lead to increased glutamate, which binds to and overactivates NMDARs. Following that step, we find the highest biological uncertainty in the pathway, with moderate biological plausibility (from overactivation of NMDARs leading to status epilepticus to increased glutamate). The rest of the pathway is considered of high biological plausibility all the way to neurodegeneration.

Concordance of dose-response relationships: Dose response concordance considers the degree to which upstream events are shown to occur at test concentrations equal to or lower than those that cause significant effects on downstream key events, the underlying assumption being that all KEs can be measured with equal precision. There is a significant amount of quantitative data providing dose and temporal concordance for multiple species between AChE inhibitors and the resulting percent AChE inhibition and ACh concentration (Kosasa et al., 1999). Dose-response relationships have been well stablished by showing that AChE inhibition resulted in the progressive accumulation of extracellular ACh. Furthermore, the relationship between increased intracellular calcium and cell death through dose and temporal concordance has also been demonstrated. Additionally, Faria et al. (2015) demonstrated a dose-response relationship between increasing doses of the organophosphate chlorpyrifos-oxon and the prevalence of a severe phenotype marked by measurably increased necrosis.

Temporal concordance: Temporal concordance refers to the degree to which the data support the hypothesized sequence of the key events; i.e., the effect on KE1 is observed before the effect on KE2, which is observed before the effect on KE3 and so on. Temporal concordance has been shown between seizure activity and increasing levels of glutamate (KE4 and KE7). Furthermore, temporal concordance has also been established between status epilepticus and increased intracellular calcium in rats. The relationship between increased intracellular calcium and cell death through dose and temporal concordance has also been demonstrated.

Consistency: We are not aware of cases where the whole chain of key events described was observed without also observing a significant impact on neurodegeneration. Nevertheless, the final adverse outcome is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to other adverse outcomes.

Uncertainties, inconsistencies, and data gaps: The current main uncertainties within this AOP are related to seizures and the location of AChE inhibition. Even though it is well known that there are two phases of seizure activity driven by cholinergic and glutamatergic mechanisms, the transition between these phases is not well understood. Additionally, the levels of increasing glutamate post-AChE inhibition appear to be dependent on the location of inhibition as well as stressor specific.

At present, the quantitative understanding of this AOP varies by level of biological organization. While the initial KEs leading to activation of muscarinic acetylcholine receptors have a high level of quantitative understanding, following KEs leading all the way to the Adverse Outcome (Cell Injury / Death Leading to Neurodegeneration) have a much lower quantitative understanding. The exception would be KE5 (Increased Glutamate leading to Overactivation of NMDARs), that has multiple kinetic models available to evaluate quantitative relationships. Overall, better quantitative relationships need to be developed to be able to quantitively and effectively predict the adverse outcome.