Antagonism of nicotinic acetycholinergic receptors by CN‐105, an apoE‐mimetic peptide reduces stroke‐induced excitotoxicity

Dear Editor: This letter describes our work identifying the neuronal targets of a clinical-stage stroke therapeutics CN105, and proposing a novel neuroprotective strategy involving nAChR antagonism. Stroke is a devastating disease with high morbidity and mortality. CN-105 was originally designed to mimic the anti-inflammatory activities of endogenous apolipoprotein E (apoE). Despite its proven efficacy in various animal models of brain injury and wellestablished safety profile in clinical trials,1,2 our understanding of CN-105’s mechanism of action remains incomplete. Early reports suggested that apoE-derived peptides and anumber of oligoarginine speciesmay interact directly with various neuronal targets including the nicotinic acetylcholine receptors (nAChR).3,4 The long-held view regarding nAChR was that its activation was neuroprotective, best exemplified by the cognitive-enhancing effects of nAChR agonists or positive allosteric modulators.5 One common role of nAChR, which received limited attention in stroke, is its potentiation of glutamate release at the presynaptic terminal.6 Glutamatergic neurotransmission is arguably the primary reason for the propagation of excitotoxicity in stroke.7,8 Although postsynaptic nAChR activated by ACh or nicotine could desensitize proximal NMDA receptors,9 we hypothesized that acute action of nAChR antagonist on presynaptic neurons may serve to downregulate the detrimental cascade associated with glutamate excitotoxicity. After given supratherapeutic dosing of CN-105 several orders of magnitude higher than clinical practice, Cynologous monkey exhibited symptoms of mydriasis and ptosis. A gradual recovery to normal activity took about two serum clearance half-lives of CN-105 (4 h, Table S1; Figure S1). In C57BL6 mice, CN-105 had an LD80 of 25 mg/kg (Figure 1A) with symptoms of spasm and respiratory suppression. Following intubation and mechanical ventilation, dosing of 50 mg/kg was not associated with mortality


Antagonism of nicotinic acetycholinergic receptors by CN-105, an apoE-mimetic peptide reduces stroke-induced excitotoxicity
Dear Editor: This letter describes our work identifying the neuronal targets of a clinical-stage stroke therapeutics CN-105, and proposing a novel neuroprotective strategy involving nAChR antagonism. Stroke is a devastating disease with high morbidity and mortality. CN-105 was originally designed to mimic the anti-inflammatory activities of endogenous apolipoprotein E (apoE). Despite its proven efficacy in various animal models of brain injury and wellestablished safety profile in clinical trials, 1,2 our understanding of CN-105's mechanism of action remains incomplete. Early reports suggested that apoE-derived peptides and a number of oligoarginine species may interact directly with various neuronal targets including the nicotinic acetylcholine receptors (nAChR). 3,4 The long-held view regarding nAChR was that its activation was neuroprotective, best exemplified by the cognitive-enhancing effects of nAChR agonists or positive allosteric modulators. 5 One common role of nAChR, which received limited attention in stroke, is its potentiation of glutamate release at the presynaptic terminal. 6 Glutamatergic neurotransmission is arguably the primary reason for the propagation of excitotoxicity in stroke. 7,8 Although postsynaptic nAChR activated by ACh or nicotine could desensitize proximal NMDA receptors, 9 we hypothesized that acute action of nAChR antagonist on presynaptic neurons may serve to downregulate the detrimental cascade associated with glutamate excitotoxicity.
After given supratherapeutic dosing of CN-105 several orders of magnitude higher than clinical practice, Cynologous monkey exhibited symptoms of mydriasis and ptosis. A gradual recovery to normal activity took about two serum clearance half-lives of CN-105 (4 h, Table S1; Figure S1). In C57BL6 mice, CN-105 had an LD 80 of 25 mg/kg ( Figure 1A) with symptoms of spasm and respiratory suppression. Following intubation and mechanical ventilation, dosing of 50 mg/kg was not associated with mortality  Figure 1A), but led to extended sedation after the removal of isoflurane anaesthesia (Movie S1).
Given apoE-peptides' antagonistic activity upon nAChR, 3,10 we hypothesized that the respiratory suppression of CN-105 was due to its inhibition of nAChRs present in various respiratory control pathways. An initial electrophysiological study using an HEK293 cell line overexpressing α7-nAChR showed that 10 μM CN-105 strongly suppressed the ACh induced current, similar to the inhibitory effect of apoE ( Figure S2). In the presence of 10 nM to 10 μM CN-105, we observed a typical antagonistic suppression of the ACh-induced currents ( Figure 1B). Such effect was sub-type specific, as the IC 50 of CN-105 differed by two orders of magnitude for α7-, α4β2-and α1β1δε-nAChR ( Figure 1C), the latter of which might be responsible for CN-105's respiratory toxicity at supratherapeutic dosage. The voltage dependency of CN-105 was measured in parallel with apoE130-149 ( Figure 1D), in which we observed maximal inhibition at -80 mV. In the primary culture of rat neurons, 1 mM ACh triggered strong calcium influx, which was significantly diminished by a priori incubation with CN-105 prior to ACh infusion ( Figure 1E). These results indicated that CN-105 may block the ACh-induced influx of Ca 2+ and the propagation of action potential (AP).
Based on the reported binding site of the apoE140-148 peptide on nAChR, 10 we constructed a model of the α7 nAChR-CN-105 complex. The putative binding site of CN-105 was proximal to the orthosteric binding site for ACh ( Figure S3A) and would most likely block the binding of ACh. Unsurprisingly, the arginine side chains of CN-105 were involved in extensive hydrogen bonds ( Figure  S3B). Poisson-Boltzmann analyses of the complex structure ( Figure 2A) showed that positive charges of CN-105 significantly weakened the negative electric field that is critical for the passage of cations, diminishing the propensity of cation inflow. The two preceding neutral residues  were vital for the nAChR activity ( Figure S3B). The peptide in which the first valine was replaced by an arginine lost most of its nAChR activity (Table S2)  We next studied whether CN-105's inhibition of nAChR and nAChR-mediated calcium influx would affect downstream release of glutamate. In rat hippocampal brain slices, evoked excitatory postsynaptic current (eEPSC) mediated by NMDA receptor and AMPA receptor were significantly lower in the presence of CN-105 than the control levels ( Figure 2B, Figure S4). CN-105 did not directly inhibit glutamate-induced currents in NMDA or AMPA receptors ( Figure S5). Rather, CN-105's attenuation of the spontaneous EPSC (sEPSC) frequency and the reversion by the cholinesterase inhibitor donepezil (Figure 3A), suggested an intercellular and most likely presynaptic action of CN-105. Confining the agonistic effect of donepezil to nAChR by adding the muscarinic AChR inhibitor atropine exhibited a similar effect to adding donepezil alone, indicative of an nAChR-dependent mechanism. When tetrodotoxin was added to block all AP firings, CN-105 was no longer able to affect the NMDA receptor-mediated EPSC ( Figure 3B). These results suggested that CN-105 could suppress the presynaptic release of glutamate in an AP-and AChR-dependent manner. That is, nAChR + glutamatergic neurons may be the primary cellular target of CN-105 in suppressing excitatory neurotransmission in the brain.
In a rat model of transient ischemic stroke, CN-105 (0.1 to 0.4 mg/kg) significantly reduced the infarct volume, similar to the group given the free-radical scavenger edaravone ( Figure 4A). At the infarct margin of the primary somatosensory cortex, we enumerated the α7-nAChR + cells which had higher density in the CN-105 treated groups than in the vehicle-treated groups ( Figure 4B). The number of β3-GABA A R + cells in this area was unaffected by CN-105 ( Figure 4C). Similar to the trend of α7-nAChR + cells, the overall density of viable neurons (as NeuN + cells) was higher in CN-105 treated groups ( Figure 4D), which suggested that specific interactions between CN-105 and neurons carrying α7-nAChRs in the somatosensory cortex may help protect neuronal tissues from the ischemicreperfusion injury. Our current understanding of CN-105's neuronal mechanism of action is depicted in Figure 4E. Although chronic inhibition of the cholinergic pathway may be detrimental, our results demonstrated that acute and selective antagonism of nAChR may actually protect the brain from excitotoxicity.
In conclusion, we demonstrated that one of the neuroprotective mechanisms of CN-105 was the dampening of presynaptic glutamate release via nAChR inhibition, arising from a unique electrostatic gating effect on the cation channel. Our current observations emphasize that, in addition to its effects on glia, the direct interaction between apoE (and the peptide derivatives) and neuronal ion channels may play an important role in mediating its neuroprotective effects. Our results also suggest that it may be worthwhile to reconsider the role of nAChR as a potential therapeutic target for stroke neuroprotection.

A C K N O W L E D G E M E N T S
The authors acknowledge the helpful discussion and suggestions from Dr. Taiyun Zhao on the pharmacological assays, Dr. Lihou Dong and Dr. Kelly Dong on the pharma-

C O N F L I C T O F I N T E R E S T
GC is an employee of Cerebtron Biotech. LY is an officer of ICE Biosciences, a contract research organization for conducting part of the electrophysiological assays. DTL is an officer and has equity in Aegis-CN. Duke University has equity and a global intellectual property stake in CN-105 and might benefit if proven effective and successful commercially. Cerebtron Biotech has equity and Chinese intellectual property stake in CN-105. Cerebtron provided the study drug and part of the research funding. However, neither Cerebtron Biotech, nor Aegis-CN had any editorial control over the study design, its execution, or the writing of this manuscript. Other authors declare that they have no competing interests.