Victor V. Uteshev

The therapeutic promise of positive allosteric modulation of nicotinic receptors.

In the central nervous system, deficits in cholinergic neurotransmission correlate with decreased attention and cognitive impairment, while stimulation of neuronal nicotinic acetylcholine receptors improves attention, cognitive performance and neuronal resistance to injury as well as produces robust analgesic and anti-inflammatory effects. The rational basis for the therapeutic use of orthosteric agonists and positive allosteric modulators (PAMs) of nicotinic receptors arises from the finding that functional nicotinic receptors are ubiquitously expressed in neuronal and non-neuronal tissues including brain regions highly vulnerable to traumatic and ischemic types of injury (e.g., cortex and hippocampus). Moreover, functional nicotinic receptors do not vanish in age-, disease- and trauma-related neuropathologies, but their expression and/or activation levels decline in a subunit- and brain region-specific manner. Therefore, augmenting the endogenous cholinergic tone by nicotinic agents is possible and may offset neurological impairments associated with cholinergic hypofunction. Importantly, because neuronal damage elevates extracellular levels of choline (a selective agonist of α7 nicotinic acetylcholine receptors) near the site of injury, α7-PAM-based treatments may augment pathology-activated α7-dependent auto-therapies where and when they are most needed (i.e., in the penumbra, post-injury). Thus, nicotinic-PAM-based treatments are expected to augment the endogenous cholinergic tone in a spatially and temporally restricted manner creating the potential for differential efficacy and improved safety as compared to exogenous orthosteric nicotinic agonists that activate nicotinic receptors indiscriminately. In this review, I will summarize the existing trends in therapeutic applications of nicotinic PAMs.

A positive allosteric modulator of α7 nAChRs augments neuroprotective effects of endogenous nicotinic agonists in cerebral ischaemia

Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type‐II positive allosteric modulators (PAMs‐II) of α7 nAChRs, such as 1‐(5‐chloro‐2,4‐dimethoxyphenyl)‐3‐(5‐methylisoxazol‐3‐yl)‐urea (PNU‐120596). This proof‐of‐concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU‐120596.

A Type-II Positive Allosteric Modulator of α7 nAChRs Reduces Brain Injury and Improves Neurological Function after Focal Cerebral Ischemia in Rats

In the absence of clinically-efficacious therapies for ischemic stroke there is a critical need for development of new therapeutic concepts and approaches for prevention of brain injury secondary to cerebral ischemia. This study tests the hypothesis that administration of PNU-120596, a type-II positive allosteric modulator (PAM-II) of α7 nicotinic acetylcholine receptors (nAChRs), as long as 6 hours after the onset of focal cerebral ischemia significantly reduces brain injury and neurological deficits in an animal model of ischemic stroke. Focal cerebral ischemia was induced by a transient (90 min) middle cerebral artery occlusion (MCAO). Animals were then subdivided into two groups and injected intravenously (i.v.) 6 hours post-MCAO with either 1 mg/kg PNU-120596 (treated group) or vehicle only (untreated group). Measurements of cerebral infarct volumes and neurological behavioral tests were performed 24 hrs post-MCAO. PNU-120596 significantly reduced cerebral infarct volume and improved neurological function as evidenced by the results of Bederson, rolling cylinder and ladder rung walking tests. These results forecast a high therapeutic potential for PAMs-II as effective recruiters and activators of endogenous α7 nAChR-dependent cholinergic pathways to reduce brain injury and improve neurological function after cerebral ischemic stroke.

α7 nicotinic ACh receptors as a ligand-gated source of Ca(2+) ions: the search for a Ca(2+) optimum.

The spatiotemporal distribution of cytosolic Ca(2+) ions is a key determinant of neuronal behavior and survival. Distinct sources of Ca(2+) ions including ligand- and voltage-gated Ca(2+) channels contribute to intracellular Ca(2+) homeostasis. Many normal physiological and therapeutic neuronal functions are Ca(2+)-dependent, however an excess of cytosolic Ca(2+) or a lack of the appropriate balance between Ca(2+) entry and clearance may destroy cellular integrity and cause cellular death. Therefore, the existence of optimal spatiotemporal patterns of cytosolic Ca(2+) elevations and thus, optimal activation of ligand- and voltage-gated Ca(2+) ion channels are postulated to benefit neuronal function and survival. Alpha7 nicotinic -acetylcholine receptors (nAChRs) are highly permeable to Ca(2+) ions and play an important role in modulation of neurotransmitter release, gene expression and neuroprotection in a variety of neuronal and non-neuronal cells. In this review, the focus is placed on α7 nAChR-mediated currents and Ca(2+) influx and how this source of Ca(2+) entry compares to NMDA receptors in supporting cytosolic Ca(2+) homeostasis, neuronal function and survival.

Physiological Concentrations of Choline Activate Native α7-Containing Nicotinic Acetylcholine Receptors in the Presence of PNU-120596 [1-(5-Chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea]

The use of PNU-120596 [1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea], a positive allosteric modulator of α7 nicotinic acetylcholine receptor (nAChR), may be beneficial for enhancing cholinergic therapies. However, the effects of PNU-120596 on activation of native α7-containing nAChRs by physiological concentrations of choline are not known and were investigated in this study using patch-clamp electrophysiology and histaminergic tuberomammillary neurons in hypothalamic slices. In the presence of PNU-120596, subthreshold (i.e., inactive) physiological concentrations of choline (∼10 μM) elicited repetitive step-like whole-cell responses reminiscent of single ion channel openings that were reversibly blocked by 20 nM methyllycaconitine, a selective α7 nAChR antagonist. The effects of choline and PNU-120596 were synergistic as administration of 10 to 40 μM choline or 1 to 4 μM PNU-120596 alone did not elicit responses. In voltage clamp at −60 mV, the persistent activation of α7-containing nAChRs by 10 μM choline plus 1 μM PNU-120596 was estimated to produce a sustained influx of Ca2+ ions at a rate of 8.4 pC/min (∼0.14 pA). In the presence of PNU-120596 in current clamp, transient step-like depolarizations (∼5 mV) enhanced neuronal excitability and triggered voltage-gated conductances; a single opening of an α7-containing nAChR channel appeared to transiently depolarize the entire neuron and facilitate spontaneous firing. Therefore, this study tested and confirmed the hypothesis that PNU-120596 enhances the effects of subthreshold concentrations of choline on native α7-containing nAChRs, allowing physiological levels of choline to activate these receptors and produce whole-cell responses in the absence of exogenous nicotinic agents. In certain neurological disorders, this activation may be therapeutically beneficial, more efficacious, and safer than treatments with nAChR agonists.

Mechanisms of facilitation of synaptic glutamate release by nicotinic agonists in the nucleus of the solitary tract

The nucleus of the solitary tract (NTS) is the principal integrating relay in the processing of visceral sensory information. Functional nicotinic acetylcholine receptors (nAChRs) have been found on presynaptic glutamatergic terminals in subsets of caudal NTS neurons. Activation of these receptors has been shown to enhance synaptic release of glutamate and thus may modulate autonomic sensory-motor integration and visceral reflexes. However, the mechanisms of nAChR-mediated facilitation of synaptic glutamate release in the caudal NTS remain elusive. This study uses rat horizontal brainstem slices, patch-clamp electrophysiology, and fluorescent Ca(2+) imaging to test the hypothesis that a direct Ca(2+) entrance into glutamatergic terminals through active presynaptic non-α7- or α7-nAChR-mediated ion channels is sufficient to trigger synaptic glutamate release in subsets of caudal NTS neurons. The results of this study demonstrate that, in the continuous presence of 0.3 μM tetrodotoxin, a selective blocker of voltage-activated Na(+) ion channels, facilitation of synaptic glutamate release by activation of presynaptic nAChRs (detected as an increase in the frequency of miniature excitatory postsynaptic currents) requires external Ca(2+) but does not require activation of presynaptic Ca(2+) stores and presynaptic high- and low-threshold voltage-activated Ca(2+) ion channels. Expanding the knowledge of mechanisms and pharmacology of nAChRs in the caudal NTS should benefit therapeutic approaches aimed at restoring impaired autonomic homeostasis.

High therapeutic potential of positive allosteric modulation of α7 nAChRs in a rat model of traumatic brain injury: proof-of-concept.

There are currently no clinically efficacious drug therapies to treat brain damage secondary to traumatic brain injury (TBI). In this proof-of-concept study, we used a controlled cortical impact model of TBI in young adult rats to explore a novel promising approach that utilizes PNU-120596, a previously reported highly selective Type-II positive allosteric modulator (α7-PAM) of α7 nicotinic acetylcholine receptors (nAChRs). α7-PAMs enhance and prolong α7 nAChR activation, but do not activate α7 nAChRs when administered without an agonist. The rational basis for the use of an α7-PAM as a post-TBI treatment is tripartite and arises from: (1) the intrinsic ability of brain injury to elevate extracellular levels of choline (a ubiquitous cell membrane-building material and a selective endogenous agonist of α7 nAChRs) due to the breakdown of cell membranes near the site and time of injury; (2) the ubiquitous expression of functional α7 nAChRs in neuronal and glial/immune brain cells; and (3) the potent neuroprotective and anti-inflammatory effects of α7 nAChR activation. Therefore, both neuroprotective and anti-inflammatory effects can be achieved post-TBI by targeting only a single player (i.e., the α7 nAChR) using α7-PAMs to enhance the activation of α7 nAChRs by injury-elevated extracellular choline. Our data support this hypothesis and demonstrate that subcutaneous administration of PNU-120596 post-TBI in young adult rats significantly reduces both brain cell damage and reactive gliosis. Therefore, our results introduce post-TBI systemic administration of α7-PAMs as a promising therapeutic intervention that could significantly restrict brain injury post-TBI and facilitate recovery of TBI patients.

The dual effect of PNU-120596 on α7 nicotinic acetylcholine receptor channels

PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea), a Type-II positive allosteric modulator of α(7) nicotinic acetylcholine receptors inhibits α(7) desensitization and robustly prolongs openings of α(7) channels. However, these effects may render α(7) channels more accessible to positively charged molecules and thus, more susceptible to voltage-dependent open-channel-block-like inhibition. To test this hypothesis, choline chloride (i.e., choline), a selective endogenous α(7) agonist, and bicuculline methochloride (i.e., bicuculline), a competitive α(7) antagonist, were used as membrane voltage-sensitive probes in whole-cell voltage-clamp recordings from hippocampal CA1 interneurons in acute brain slices in the absence and presence of PNU-120596. PNU-120596 enhanced voltage-dependent inhibition of α(7) responses by bicuculline and choline. In the presence of PNU-120596, α(7) channels favored a burst-like kinetic modality in the presence, but not absence of bicuculline and bursts of α(7) openings were voltage-dependent. These results suggest that PNU-120596 alters the pharmacology of α(7) channels by making these channels more susceptible to voltage-dependent inhibitory interactions with positively charged drugs at concentrations that do not potently inhibit α(7) channels without PNU-120596. This inhibition imitates α(7) nicotinic receptor desensitization and compromises the potentiating anti-desensitization effects of PNU-120596 on α(7) nicotinic receptors. This unexpected dual action of PNU-120596, and possibly other Type-II positive allosteric modulators of α(7) nicotinic receptors, may lead to unanticipated α(7) channel-drug interactions and misinterpretation of α(7) single-channel data.

Are positive allosteric modulators of α7 nAChRs clinically safe

Dear Editor, In a recent research article published in the May 2015 issue of this journal (Journal of Neurochemistry 2015, 133: 309– 319), Guerra-Alvarez et al. (2015) reported a cytotoxic potential of PNU120596, a Type-II positive allosteric modulator (PAMII) of a7 nicotinic acetylcholine receptors (a7 nAChRs). The authors used human SH-SY5Y neuroblastoma cells, rat organotypic hippocampal slice culture, electrophysiology and fluorescent Ca imaging to evaluate cellular responsiveness and survival after various treatments with nicotinic agonists and PNU120596. The study concluded that treatments that combine a7 agonists with PNU120596 could result in cytotoxicity via a release of Ca from intracellular stores and intracellular Ca overloading. The results are interesting and may contribute to understanding the mechanisms of cytotoxicity as related to excessive activation of a7 nAChRs in the presence of PAMIIs. However, the impact could have been considerably greater if clinicallyand physiologically relevant experimental conditions were used. The pharmacokinetics of PNU120596 and the common clinically relevant a7 agonists are available in scientific literature. Judging by the existing information, the study by Guerra-Alvarez et al. 2015 attempts to predict clinical limitations of PAMIIs from in vitro modeling of strictly overdose conditions. My skepticism about the value of this approach stems from the following illustrative comparisons of concentrations and exposure durations used in Guerra-Alvarez et al. 2015 versus previous in vivo studies: 3–10 lM PNU120596 versus estimated 0.5–1.5 lM found therapeutic in vivo (Hurst et al. 2005; McLean et al. 2012; Kalappa et al. 2013); 100 lM nicotine versus a maximum < 1 lM found in vivo (Russell et al. 1980; Gourlay and Benowitz 1997; Rose et al. 2010); and 10 lMPNU282987, a selectivea7 agonist versus a realistic < 1 lM expected in vivo (Walker et al. 2006). The drug exposure duration was set to 24 h for a7 agonists and 48 h for PNU120596 versus ~ 8 h, the half-life time of PNU120596 in vivo (McLean et al. 2012) or ~ 2 h, the halflife time of nicotine in vivo (Russell et al. 1980; Gourlay and Benowitz 1997; Rose et al. 2010). One would understandably question the rationale for extreme drug concentrations and exposure durations employed by Guerra-Alvarez et al. 2015. By purposely targeting only excessive levels of a7 nAChR activation, the study fuels the very ‘controversy’ it attempted to resolve: cytoprotection versus cytotoxicity in the presence of PAMIIs. This letter is written with the intention of facilitating discussion and efforts toward the resolution of an important question: are PAMIIs clinically safe? Clearly, the convincing answers to this question can only come from animal studies and clinical trials. Nevertheless, useful insights can be gained from the results of in vitro studies. The cytotoxic potential of PAMIIs has been recognized in the past as seen in at least two earlier in vitro studies (Ng et al. 2007; Williams et al. 2012) referenced by GuerraAlvarez et al. 2015 which have reported cytotoxicity by PNU120596 at concentrations above 3 lM. In contrast to Guerra-Alvarez et al. 2015; those previous studies used choline (a selective endogenous a7 nAChR agonist) and a broad range of drug concentrations. However these studies failed to detect cytotoxicity at a lower range of PNU120596 concentrations: i.e., 0.3–1 lM. In fact, in both studies a trend for increased cell viability was apparent for 0.3–1 lM PNU120596 [see Fig. 3 in (Ng et al. 2007) and Figs 4 and 6 in (Williams et al. 2012)]. This increase in cell viability is consistent with our own studies that demonstrated < 1 lM PNU120596 significantly reduced brain injury and neurological deficits after focal ischemia in a transient 90 min middle cerebral artery occlusion model of ischemic stroke in young adult rats (Kalappa et al. 2013; Sun et al. 2013), as well as significantly reduced brain cell damage and reactive gliosis after brain injury in a control cortical impact model of traumatic brain injury (Gatson et al. 2015). A concentration of < 1 lM PNU120596 can be achieved in rodents shortly after intravenous 1 mg/kg (Hurst et al. 2005) or subcutaneous 10–30 mg/kg (McLean et al. 2012; Kalappa et al. 2013) administration. In these in vivo studies, there was no

Projection target-specific action of nicotine in the caudal nucleus of the solitary tract.

The brainstem nucleus of the solitary tract (NTS) is the key integrating relay in the central processing of sensory information from the thoracic and from most subdiaphragmatic viscera. Modulation of neuronal excitability and synaptic activity in the NTS by nicotinic agents can have potent effects on vital physiological functions, such as feeding, digestion, respiration, and blood circulation. Caudal NTS neurons demonstrate considerable heterogeneity in projection targets, synaptic properties, and expression of nicotinic acetylcholine receptors (nAChRs). However, despite its heterogeneity, the caudal NTS may contain discrete subsets of neurons with unique projection target‐specific properties. To test this hypothesis, we used in vivo fluorescent tracing and ex vivo patch‐clamp electrophysiology to evaluate responsiveness to nicotine of anatomically identified caudal NTS neurons that project to the hypothalamic paraventricular nucleus (PVN) and the brainstem caudal ventrolateral medulla (CVLM). The results of this study demonstrate that responsiveness to nicotine correlates with where the neurons project. Specifically, PVN‐projecting caudal NTS neurons respond to nicotine only presynaptically (i.e., via activation of presynaptic nAChRs and potentiation of synaptic release of glutamate), suggesting indirect, glutamate‐dependent effects of nicotine on the PVN‐projecting NTS circuitry. By contrast, CVLM‐projecting caudal NTS neurons exhibit only limited presynaptic, but dominant somatodendritic, responsiveness to nicotine, suggesting that the effects of nicotine on the CVLM‐projecting NTS circuitry are direct and largely glutamate independent. Understanding the relationships among function‐specific brainstem/hypothalamic neuronal networks, nuclei, and individual neurons could help develop therapies targeting identifiable neuronal circuits to offset impaired autonomic homeostasis.